Richard U Margolis, M.D., Ph.D.

Expression of hyaluronan and the hyaluronan-binding proteoglycans neurocan, aggrecan, and versican by neural stem cells and neural cells derived from embryonic stem cells
Abaskharoun, Mary; Bellemare, Marie; Lau, Elizabeth; Margolis, Richard U
2010 Apr 23;1327:6-15, Brain research
We have examined the expression and localization patterns of hyaluronan and hyaluronan-binding chondroitin sulfate proteoglycans in neural stem cells and differentiated neural cells derived from mouse embryonic stem cells. Expression of proteoglycans and hyaluronan was weak in the SSEA1-positive embryonic stem cells but increased noticeably after retinoic acid induction to nestin-positive neural stem cells. After subsequent plating, the hyaluronan-binding chondroitin sulfate proteoglycans aggrecan, neurocan, and versican are expressed by cells in both the astrocytic and neuronal lineages. During the time period that hyaluronan was present, it co-localized with each of the hyaluronan-binding proteoglycans studied and was found to be clearly associated with beta-III tubulin-expressing neurons and oligodendrocytes expressing the O4 sulfatide marker. Although proteoglycan expression levels increased to varying degrees following neural differentiation, they did not change noticably during the following 2 weeks in culture, but there was a significant decrease in hyaluronan expression. Our studies therefore demonstrate the expression by neural stem cells and neural cells derived from them of hyaluronan and its associated proteoglycans, thereby providing a necessary foundation for integrating their specific properties into developing strategies for therapeutic applications
— id: 109031, year: 2010, vol: 1327, page: 6, stat: Journal Article,

Glypican-1, phosphacan/receptor protein-tyrosine phosphatase-zeta/beta and its ligand, tenascin-C, are expressed by neural stem cells and neural cells derived from embryonic stem cells
Abaskharoun, Mary; Bellemare, Marie; Lau, Elizabeth; Margolis, Richard U
2010 ;2(3):e00039-e00039, ASN Neuro
The heparan sulfate proteoglycan glypican-1, the chondroitin sulfate proteoglycan phosphacan/RPTP (receptor protein-tyrosine phosphatase)-zeta/beta and the extracellular matrix protein tenascin-C were all found to be expressed by neural stem cells and by neural cells derived from them. Expression of proteoglycans and tenascin-C increased after retinoic acid induction of SSEA1-positive ES (embryonic stem) cells to nestin-positive neural stem cells, and after neural differentiation, proteoglycans and tenascin-C are expressed by both neurons and astrocytes, where they surround cell bodies and processes and in certain cases show distinctive expression patterns. With the exception of tenascin-C (whose expression may decrease somewhat), expression levels do not change noticeably during the following 2 weeks in culture. The significant expression, by neural stem cells and neurons and astrocytes derived from them, of two major heparan sulfate and chondroitin sulfate proteoglycans of nervous tissue and of tenascin-C, a high-affinity ligand of phosphacan/RPTP-zeta/beta, indicates that an understanding of their specific functional roles in stem cell neurobiology will be important for the therapeutic application of this new technology in facilitating nervous tissue repair and regeneration
— id: 111590, year: 2010, vol: 2, page: e00039, stat: Journal Article,

Inhibitors of slit protein interactions with the heparan sulphate proteoglycan glypican-1: Potential agents for the treatment of spinal cord injury
Lau, Elizabeth; Margolis, Richard U
2010 Apr;37(4):417-421, Clinical & experimental pharmacology & physiology
1. The heparan sulphate proteoglycan glypican-1 is a major high-affinity ligand of the Slit proteins. 2. Messenger RNA for both Slit-2 and glypican-1 is strongly upregulated and coexpressed in the reactive astrocytes of injured adult brain, suggesting a possible function of Slit proteins and glypican-1 in the adult central nervous system as significant components of the inhibitory environment that prevents axonal regeneration after injury. 3. Based on the hypothesis that adverse effects on axonal regeneration may be due to a glypican-Slit complex or the retention of glypican-binding C-terminal proteolytic processing fragments of Slit at the injury site, we used ELISA to examine a number of small molecules and low molecular weight heparin analogues for their ability to inhibit glypican-Slit interactions. 4. Our studies have led to the identification of several potent inhibitors with a favourable therapeutic profile that can now be tested in a spinal cord injury model. Among the most promising of these are a low molecular weight heparin produced by periodate oxidation and having no significant anticoagulant activity, the chemically sulphonated yeast-derived phosphomannan PI-88 and a number of randomly derivatized water-soluble sulphated dextrans
— id: 109197, year: 2010, vol: 37, page: 417, stat: Journal Article,

Fibrinogen triggers astrocyte scar formation by promoting the availability of active TGF-beta after vascular damage
Schachtrup, Christian; Ryu, Jae K; Helmrick, Matthew J; Vagena, Eirini; Galanakis, Dennis K; Degen, Jay L; Margolis, Richard U; Akassoglou, Katerina
2010 Apr 28;30(17):5843-5854, Journal of neuroscience
Scar formation in the nervous system begins within hours after traumatic injury and is characterized primarily by reactive astrocytes depositing proteoglycans that inhibit regeneration. A fundamental question in CNS repair has been the identity of the initial molecular mediator that triggers glial scar formation. Here we show that the blood protein fibrinogen, which leaks into the CNS immediately after blood-brain barrier (BBB) disruption or vascular damage, serves as an early signal for the induction of glial scar formation via the TGF-beta/Smad signaling pathway. Our studies revealed that fibrinogen is a carrier of latent TGF-beta and induces phosphorylation of Smad2 in astrocytes that leads to inhibition of neurite outgrowth. Consistent with these findings, genetic or pharmacologic depletion of fibrinogen in mice reduces active TGF-beta, Smad2 phosphorylation, glial cell activation, and neurocan deposition after cortical injury. Furthermore, stereotactic injection of fibrinogen into the mouse cortex is sufficient to induce astrogliosis. Inhibition of the TGF-beta receptor pathway abolishes the fibrinogen-induced effects on glial scar formation in vivo and in vitro. These results identify fibrinogen as a primary astrocyte activation signal, provide evidence that deposition of inhibitory proteoglycans is induced by a blood protein that leaks in the CNS after vasculature rupture, and point to TGF-beta as a molecular link between vascular permeability and scar formation
— id: 109662, year: 2010, vol: 30, page: 5843, stat: Journal Article,

FIBRINOGEN LINKS VASCULAR DAMAGE WITH GLIAL SCAR FORMATION THROUGH REGULATION OF THE TGF-BETA/SMAD SIGNALING PATHWAY
Schachtrup, C; Ryu, JK; Alexandrou, AA; Helmrick, MJ; Degen, JL; Margolis, RU; Akassoglou, K
2009 OCT ;57(13):S152-S152, Glia
— id: 105372, year: 2009, vol: 57, page: S152, stat: Journal Article,

Molecular cloning and characterization of rat Pomt1 and Pomt2
Manya, Hiroshi; Chiba, Atsuro; Margolis, Richard U; Endo, Tamao
2006 Sep;16(9):863-873, Glycobiology
Mammalian O-mannosylation, although an uncommon type of protein modification, is essential for normal brain and muscle development. Defective O-mannosylation causes congenital muscular dystrophy with abnormal neuronal migration [Walker-Warburg syndrome (WWS)]. Here, we have identified and cloned rat Pomt1 and Pomt2, which are homologues of human POMT1 and POMT2, with identities of 86 and 90%, respectively, at the amino acid level. Coexpression of both genes was found to be necessary for enzymatic activity, as is the case with human POMT1 and POMT2. Northern blot and reverse transcriptase polymerase chain reaction (RT-PCR) analyses revealed that rat Pomt1 and Pomt2 are expressed in all tissues but most strongly in testis. In situ hybridization histochemistry of rat brain revealed that Pomt1 and Pomt2 mRNA are coexpressed in neurons (dentate gyrus and CA1-CA3 region of the hippocampus and cerebellar Purkinje cells). Two transcription-initiation sites were observed in rat Pomt2, resulting in two forms: a testis form and a somatic form. The two forms had equal protein O-mannosyltransferase activity when coexpressed with rat Pomt1. Coexpression studies also showed that the human and rat protein O-mannosyltransferases are interchangeable, providing further evidence for the closeness of their structures
— id: 67277, year: 2006, vol: 16, page: 863, stat: Journal Article,

Expression of phosphacan and neurocan during early development of mouse retinofugal pathway
Leung, K M; Margolis, R U; Chan, S O
2004 Aug 18;152(1):1-10, Brain research. Developmental brain research
We have investigated whether the two major brain chondroitin sulfate (CS) proteoglycans (PGs), phosphacan and neurocan, are expressed in patterns that correlate to the axon order changes in the mouse retinofugal pathway. Expression of these proteoglycans was examined by polyclonal antibodies against phosphacan and N- and C-terminal fragments of neurocan. In E13-E15 mouse embryos, when most optic axons grow in the chiasm and the optic tract, phosphacan and neurocan were observed in the inner regions of the retina. In the chiasm and the tract, phosphacan but not neurocan was expressed prominently at the midline and in the deep parts of the tract. Both proteoglycans were observed on the chiasmatic neurons, which have been shown to regulate axon divergence at the chiasmatic midline and the chronotopic fiber ordering in the tract, but phosphacan appeared to be the predominant form that persists to later developmental stages. Intense staining of both proteoglycans was also observed in a strip of glial-like elements in lateral regions of the chiasm, partitioning axons in the stalk from those in the tract. We conclude that phosphacan but not neurocan is likely the major carrier of the CS glycosaminoglycans that play crucial functions in axon divergence and age-related axon ordering in the mouse optic pathway. Furthermore, localization of these carrier proteins in the optic pathway raises a possibility that these two proteoglycans regulate axon growth and patterning not only through the sulfated sugars but also by interactions of the protein parts with guidance molecules on the optic axons
— id: 44658, year: 2004, vol: 152, page: 1, stat: Journal Article,

Demonstration of mammalian protein O-mannosyltransferase activity: coexpression of POMT1 and POMT2 required for enzymatic activity
Manya, Hiroshi; Chiba, Atsuro; Yoshida, Aruto; Wang, Xiaohui; Chiba, Yasunori; Jigami, Yoshifumi; Margolis, Richard U; Endo, Tamao
2004 Jan 13;101(2):500-505, Proceedings of the National Academy of Sciences of the United States of America
Defects in O-mannosylation of alpha-dystroglycan are thought to cause certain types of congenital muscular dystrophies with neuronal migration disorders. Among these muscular dystrophies, Walker-Warburg syndrome is caused by mutations in the gene encoding putative protein O-mannosyltransferase 1 (POMT1), which is homologous to yeast protein O-mannosyltransferases. However, there is no evidence that POMT1 has enzymatic activity. In this study, we first developed a method to detect protein O-mannosyltransferase activity in mammalian cells. Then, using this method, we showed that coexpression of both POMT1 and POMT2 (another gene homologous to yeast protein O-mannosyltransferases) was necessary for the enzyme activity, but expression of either POMT1 or POMT2 alone was insufficient. The requirement of an active enzyme complex of POMT1 and POMT2 suggests that the regulation of protein O-mannosylation is complex. Further, protein O-mannosylation appears to be required for normal structure and function of alpha-dystroglycan in muscle and brain. In view of the potential importance of this form of glycosylation for a number of developmental and neurobiological processes, the ability to assay mammalian protein O-mannosyltransferase activity should greatly facilitate progress in the identification and localization of O-mannosylated proteins and the elucidation of their functional roles
— id: 42063, year: 2004, vol: 101, page: 500, stat: Journal Article,

Developmental changes of aggrecan, versican and neurocan in the retina and optic nerve
Popp, Susanna; Maurel, Patrice; Andersen, Julie S; Margolis, Richard U
2004 Sep;79(3):351-356, Experimental eye research
We have used a monoclonal antibody to neurocan and specific polyclonal antibodies to the non-homologous glycosaminoglycan attachment regions of aggrecan and mRNA splice variants of versican to compare the localization and developmental changes of these structurally related hyaluronan-binding chondroitin sulfate proteoglycans in the rat retina and optic nerve. Staining for aggrecan and versican was first seen at embryonic day 16 in the optic nerve and retina, whereas neurocan was not detected in the embryonic eye. At postnatal day 0 (P0), beta-versican staining is largely confined to the inner plexiform layer whereas alpha-versican is also apparent in the neuroblastic layer. Both aggrecan and, much more weakly, neurocan immunoreactivity is present throughout the neonatal retina. At P9, aggrecan and versican immunoreactivity is most intense in the inner and outer plexiform and ganglion cell layers, accompanied by diffuse staining in the inner and outer nuclear layers. Aggrecan and alpha-versican are also present throughout the optic nerve and disk, whereas beta-versican and neurocan are confined to the laminar beams of the optic nerve. Between P0 and P9 there is a marked increase in beta-versican expression in the inner and outer nuclear layers and in the outer plexiform layer, whereas there is only weak staining of neurocan in the inner plexiform and ganglion cell layers of P9 retina. By 1 month postnatal the staining pattern of the fully differentiated retinal layers is essentially identical to that seen in the adult, where there is strong aggrecan and alpha-versican immunoreactivity in the retina and optic nerve, whereas beta-versican has essentially disappeared from the adult retina and, similarly to neurocan, is present only in the laminar beams of the optic nerve. The marked decrease of beta-versican in the retina is consistent with >90% decrease in its concentration in brain during postnatal development, suggesting that the developmental time-course for these proteoglycans in retina parallels that seen in other areas of the central nervous system
— id: 44657, year: 2004, vol: 79, page: 351, stat: Journal Article,

Structural determinants of heparan sulfate interactions with Slit proteins
Zhang, Fuming; Ronca, Francesca; Linhardt, Robert J; Margolis, Richard U
2004 Apr 30;317(2):352-357, Biochemical & biophysical research communications
We have previously demonstrated that the Slit proteins, which are involved in axonal guidance and related processes, are high-affinity ligands of the heparan sulfate proteoglycan glypican-1. Glypican-Slit protein interactions have now been characterized in greater detail using two approaches. The ability of heparin oligosaccharides of defined structure (ranging in size from disaccharide to tetradeccasaccharide) to inhibit binding of a glypican-Fc fusion protein to recombinant human Slit-2 was determined using an ELISA. Surface plasmon resonance (SPR) spectroscopy, which measures the interactions in real time, was applied for quantitative modeling of heparin-Slit binding on heparin biochips. Heparin was covalently immobilized on these chips through a pre-formed albumin-heparin conjugate, and the inhibition of Slit binding by heparin, LMW heparin, and heparin-derived oligosaccharides (di-, tetra-, hexa-, and octa-) was examined utilizing solution competition SPR. These competition studies demonstrate that the smallest heparin oligosaccharide competing with heparin binding to Slit was a tetrasaccharide, and that in the ELISA maximum inhibition ( approximately 60% at 2microM concentration) was attained with a dodecasaccharide
— id: 42223, year: 2004, vol: 317, page: 352, stat: Journal Article,

A Nodal- and ALK4-independent signaling pathway activated by Cripto-1 through Glypican-1 and c-Src
Bianco, Caterina; Strizzi, Luigi; Rehman, Aasia; Normanno, Nicola; Wechselberger, Christian; Sun, Youping; Khan, Nadia; Hirota, Morihisa; Adkins, Heather; Williams, Kevin; Margolis, Richard U; Sanicola, Michele; Salomon, David S
2003 Mar 15;63(6):1192-1197, Cancer research
Human Cripto-1 (CR-1) is a member of the epidermal growth factor-Cripto FRL1 Cryptic family that has been shown to function as a coreceptor with the type I Activin serine-threonine kinase receptor ALK4 for the transforming growth factor beta-related peptide Nodal. However, CR-1 can also activate the mitogen-activated protein kinase and Akt pathways independently of Nodal and ALK4 by an unknown mechanism. Here, we demonstrate that CR-1 specifically binds to Glypican-1, a membrane-associated heparan sulfate proteoglycan, and activates the tyrosine kinase c-Src, triggering the mitogen-activated protein kinase and Akt signaling pathways. Finally, an active Src kinase is necessary for CR-1 to induce in vitro transformation and migration in mouse mammary epithelial cells
— id: 34128, year: 2003, vol: 63, page: 1192, stat: Journal Article,

The chondroitin sulfate proteoglycans neurocan, brevican, phosphacan, and versican are differentially regulated following spinal cord injury
Jones, Leonard L; Margolis, Richard U; Tuszynski, Mark H
2003 Aug;182(2):399-411, Experimental neurology
Chondroitin sulfate proteoglycans (CSPGs) are extracellular matrix (ECM) molecules that are widely expressed throughout the developing and adult CNS. In vitro studies demonstrate their potential to restrict neurite outgrowth, and it is believed that CSPGs also inhibit axonal regeneration after CNS injury in vivo. Previous studies demonstrated that CSPGs are generally upregulated after spinal cord injury, and more recent reports have begun to identify individual proteoglycans that may play dominant roles in limiting axonal regeneration. The current study systematically examined the extended deposition patterns after CNS injury of four putatively inhibitory CSPGs that have not been extensively investigated previously in vivo: neurocan, brevican, phosphacan, and versican. After spinal cord injury, neurocan, brevican, and versican immunolabeling increased within days in injured spinal cord parenchyma surrounding the lesion site and peaked at 2 weeks. Neurocan and versican were persistently elevated for 4 weeks postinjury, and brevican expression persisted for at least 2 months. On the other hand, phosphacan immunolabeling decreased in the same region immediately following injury but later recovered and then peaked after 2 months. Combined glial fibrillary acidic protein (GFAP) immunohistochemistry and in situ hybridization demonstrated that GFAP astrocytes constituted a source of neurocan production after spinal cord injury. Thus, the production of several CSPG family members is differentially affected by spinal cord injury, overall establishing a CSPG-rich matrix that persists for up to 2 months following injury. Optimization of strategies to reduce CSPG expression to enhance regeneration may need to target several different family members over an extended period following injury
— id: 67278, year: 2003, vol: 182, page: 399, stat: Journal Article,

Localization of aggrecan and versican in the developing rat central nervous system
Popp, Susanna; Andersen, Julie S; Maurel, Patrice; Margolis, Richard U
2003 Jun;227(1):143-149, Developmental dynamics
The localization of aggrecan and mRNA splice variants of versican in the developing rat central nervous system has been examined by using specific polyclonal antibodies to the nonhomologous glycosaminoglycan attachment regions of these hyaluronan-binding chondroitin sulfate proteoglycans. At embryonic day 16 (E16), aggrecan and versican splice variants containing either or both the alpha-and beta-domains are present in the marginal zone and subplate of the cerebral cortex and in the amygdala, internal capsule, and the optic and lateral olfactory tracts. There is strong staining of versican but not of aggrecan in the hippocampus and dentate gyrus by E19, whereas both aggrecan and alpha-versican are present in the fimbria. At E19, aggrecan is seen throughout the cerebral cortex, whereas the distribution of versican is considerably more limited, being confined essentially to the marginal zone and subplate. At 1 week postnatal, both aggrecan and versican are present in the prospective white matter and in the molecular and granule cell layers of the cerebellum, but neither proteoglycan is seen in the external granule cell layer. alpha- but not beta-versican staining is seen in Purkinje cells, and aggrecan staining of Purkinje cells is also rather minimal. In the spinal cord at E13, aggrecan is present in the dorsal root entry zone, ventral funiculus, mantle layer, and floor plate, as well as in the dorsal root ganglia and ventral roots. However, alpha-versican is confined to the dorsal root entry zone and the ependyma surrounding the spinal canal, and beta-versican is not present in spinal cord parenchyma at this developmental stage, being limited to the surrounding connective tissue. By E19, there are significant amounts of all three proteoglycans in the spinal cord. Aggrecan staining is most intense in the lateral funiculus and the fasciculi gracilis and cuneatus, where alpha-versican staining is also strong. In contrast, beta-versican is seen predominantly in the motor columns. Differences in the localization and temporal expression patterns of these chondroitin sulfate proteoglycans suggest that, like neurocan and phosphacan, they have partially complementary roles during central nervous system development
— id: 46264, year: 2003, vol: 227, page: 143, stat: Journal Article,

Characterization of Slit protein interactions with glypican-1
Ronca F; Andersen JS; Paech V; Margolis RU
2001 Aug 3;276(31):29141-29147, Journal of biological chemistry
We have demonstrated previously that the Slit proteins, which are involved in axonal guidance and related developmental processes in nervous tissue, are ligands of the glycosylphosphatidylinositol-anchored heparan sulfate proteoglycan glypican-1 in brain (Liang, Y., Annan, R. S., Carr, S. A., Popp, S., Mevissen, M., Margolis, R. K., and Margolis, R. U. (1999) J. Biol. Chem. 274, 17885--17892). To characterize these interactions in more detail, recombinant human Slit-2 protein and the N- and C-terminal portions generated by in vivo proteolytic processing were used in an enzyme-linked immunosorbent assay to measure the binding of a glypican-Fc fusion protein. Saturable and reversible high affinity binding to the full-length protein and to the C-terminal portion that is released from the cell membrane was seen, with dissociation constants in the 80-110 nm range, whereas only a relatively low level of binding to the larger N-terminal segment was detected. Co-transfection of 293 cells with Slit and glypican-1 cDNAs followed by immunoprecipitation demonstrated that these interactions also occur in vivo, and immunocytochemical studies showed colocalization in the embryonic and adult central nervous system. The binding affinity of the glypican core protein to Slit is an order of magnitude lower than that of the glycanated proteoglycan. Glypican binding to Slit was also decreased 80--90% by heparin (2 microg/ml), enzymatic removal of the heparan sulfate chains, and by chlorate inhibition of glypican sulfation. The differential effects of N- or O-desulfated heparin on glypican binding also indicate that O-sulfate groups on the heparan sulfate chains play a critical role in heparin interactions with Slit. Our data suggest that glypican binding to the releasable C-terminal portion of Slit may serve as a mechanism for regulating the biological activity of Slit and/or the proteoglycan
— id: 48160, year: 2001, vol: 276, page: 29141, stat: Journal Article,

Neurocan is upregulated in injured brain and in cytokine-treated astrocytes
Asher RA; Morgenstern DA; Fidler PS; Adcock KH; Oohira A; Braistead JE; Levine JM; Margolis RU; Rogers JH; Fawcett JW
2000 Apr 1;20(7):2427-2438, Journal of neuroscience
Injury to the CNS results in the formation of the glial scar, a primarily astrocytic structure that represents an obstacle to regrowing axons. Chondroitin sulfate proteoglycans (CSPG) are greatly upregulated in the glial scar, and a large body of evidence suggests that these molecules are inhibitory to axon regeneration. We show that the CSPG neurocan, which is expressed in the CNS, exerts a repulsive effect on growing cerebellar axons. Expression of neurocan was examined in the normal and damaged CNS. Frozen sections labeled with anti-neurocan monoclonal antibodies 7 d after a unilateral knife lesion to the cerebral cortex revealed an upregulation of neurocan around the lesion. Western blot analysis of extracts prepared from injured and uninjured tissue also revealed substantially more neurocan in the injured CNS. Western blot analysis revealed neurocan and the processed forms neurocan-C and neurocan-130 to be present in the conditioned medium of highly purified rat astrocytes. The amount detected was increased by transforming growth factor beta and to a greater extent by epidermal growth factor and was decreased by platelet-derived growth factor and, to a lesser extent, by interferon gamma. O-2A lineage cells were also capable of synthesizing and processing neurocan. Immunocytochemistry revealed neurocan to be deposited on the substrate around and under astrocytes but not on the cells. Astrocytes therefore lack the means to retain neurocan at the cell surface. These findings raise the possibility that neurocan interferes with axonal regeneration after CNS injury
— id: 33471, year: 2000, vol: 20, page: 2427, stat: Journal Article,

Postnatal development of perineuronal nets in wild-type mice and in a mutant deficient in tenascin-R
Bruckner G; Grosche J; Schmidt S; Hartig W; Margolis RU; Delpech B; Seidenbecher CI; Czaniera R; Schachner M
2000 Dec 25;428(4):616-629, Journal of comparative neurology
The extracellular matrix glycoprotein tenascin-R (TN-R), colocalizing with hyaluronan, phosphacan, and aggregating chondroitin sulphate proteoglycans in the white and grey matter, is accumulated in perineuronal nets that surround different types of neurons in many brain regions. To characterize the role of TN-R in the formation of perineuronal nets, we studied their postnatal development in wild-type mice and in a TN-R knock-out mutant by using the lectin Wisteria floribunda agglutinin and an antibody to nonspecified chondroitin sulphate proteoglycans as established cytochemical markers. We detected the matrix components TN-R, hyaluronan, phosphacan, neurocan, and brevican in the perineuronal nets of cortical and subcortical regions. In wild-type mice, lectin-stained, immature perineuronal nets were first seen on postnatal day 4 in the brainstem and on day 14 in the cerebral cortex. The staining intensity of these nets for TN-R, hyaluronan, phosphacan, neurocan, and brevican was extremely weak or not distinguishable from that of the surrounding neuropil. However, all markers showed an increase in staining intensity of perineuronal nets reaching maximal levels between postnatal days 21 and 40. In TN-R-deficient animals, the perineuronal nets tended to show a granular component within their lattice-like structure at early stages of development. Additionally, the staining intensity in perineuronal nets was reduced for brevican, extremely low for hyaluronan and neurocan, and virtually no immunoreactivity was detectable for phosphacan. The granular configuration of perineuronal nets became more predominant with advancing age of the mutant animals, indicating the continued abnormal aggregation of chondroitin sulphate proteoglycans complexed with hyaluronan. As shown by electron microscopy in the cerebral cortex, the disruption of perineuronal nets was not accompanied by apparent changes in the synaptic structure on net-bearing neurons. The regional distribution patterns and the temporal course of development of perineuronal nets were not obviously changed in the mutant. We conclude that the lack of TN-R initially and continuously disturbs the molecular scaffolding of extracellular matrix components in perineuronal nets. This may interfere with the development of the specific micromilieu of the ensheathed neurons and adjacent glial cells and may also permanently change their functional properties
— id: 33470, year: 2000, vol: 428, page: 616, stat: Journal Article,

The tissue plasminogen activator (tPA)/plasmin extracellular proteolytic system regulates seizure-induced hippocampal mossy fiber outgrowth through a proteoglycan substrate
Wu YP; Siao CJ; Lu W; Sung TC; Frohman MA; Milev P; Bugge TH; Degen JL; Levine JM; Margolis RU; Tsirka SE
2000 Mar 20;148(6):1295-1304, Journal of cell biology
Short seizure episodes are associated with remodeling of neuronal connections. One region where such reorganization occurs is the hippocampus, and in particular, the mossy fiber pathway. Using genetic and pharmacological approaches, we show here a critical role in vivo for tissue plasminogen activator (tPA), an extracellular protease that converts plasminogen to plasmin, to induce mossy fiber sprouting. We identify DSD-1-PG/phosphacan, an extracellular matrix component associated with neurite reorganization, as a physiological target of plasmin. Mice lacking tPA displayed decreased mossy fiber outgrowth and an aberrant band at the border of the supragranular region of the dentate gyrus that coincides with the deposition of unprocessed DSD-1-PG/phosphacan and excessive Timm-positive, mossy fiber termini. Plasminogen-deficient mice also exhibit the laminar band and DSD- 1-PG/phosphacan deposition, but mossy fiber outgrowth through the supragranular region is normal. These results demonstrate that tPA functions acutely, both through and independently of plasmin, to mediate mossy fiber reorganization
— id: 33472, year: 2000, vol: 148, page: 1295, stat: Journal Article,

High prevalence of 2-mono- and 2,6-di-substituted manol-terminating sequences among O-glycans released from brain glycopeptides by reductive alkaline hydrolysis
Chai W; Yuen CT; Kogelberg H; Carruthers RA; Margolis RU; Feizi T; Lawson AM
1999 Aug;263(3):879-888, European journal of biochemistry
Di- to heptasaccharides isolated from total nondialyzable brain glycopeptides after release by alkaline borohydride treatment have been subjected to mass spectrometric and nuclear magnetic resonance spectroscopic analyses supplemented by TLC-MS analyses of derived neoglycolipids. A family of Manol-terminating oligosaccharides has been revealed which includes novel sequences with a 2, 6-disubstituted Manol: In contrast to the Manol-terminating HNK-1 antigen-positive chains described previously that occur as a minor population [Yuen, C.-T., Chai, W., Loveless, R.W., Lawson, A.M., Margolis, R.U. & Feizi, T. (1997) J. Biol. Chem. 272, 8924-8931], the above oligosaccharides are abundant. The ratio of these compounds to the classical N-acetylgalactosaminitol-terminating oligosaccharides is about 1 : 3. Thus, there appears to be in higher eukaryotes a major alternative pathway related to the yeast-type protein O-mannosylation, the enzymatic basis and functional importance of which now require investigation
— id: 33473, year: 1999, vol: 263, page: 879, stat: Journal Article,

Mammalian homologues of the Drosophila slit protein are ligands of the heparan sulfate proteoglycan glypican-1 in brain
Liang Y; Annan RS; Carr SA; Popp S; Mevissen M; Margolis RK; Margolis RU
1999 Jun 18;274(25):17885-17892, Journal of biological chemistry
Using an affinity matrix in which a recombinant glypican-Fc fusion protein expressed in 293 cells was coupled to protein A-Sepharose, we have isolated from rat brain at least two proteins that were detected by SDS-polyacrylamide gel electrophoresis as a single 200-kDa silver-stained band, from which 16 partial peptide sequences were obtained by nano-electrospray tandem mass spectrometry. Mouse expressed sequence tags containing two of these peptides were employed for oligonucleotide design and synthesis of probes by polymerase chain reaction and enabled us to isolate from a rat brain cDNA library a 4.1-kilobase clone that encoded two of our peptide sequences and represented the N-terminal portion of a protein containing a signal peptide and three leucine-rich repeats. Comparisons with recently published sequences also showed that our peptides were derived from proteins that are members of the Slit/MEGF protein family, which share a number of structural features such as N-terminal leucine-rich repeats and C-terminal epidermal growth factor-like motifs, and in Drosophila Slit is necessary for the development of midline glia and commissural axon pathways. All of the five known rat and human Slit proteins contain 1523-1534 amino acids, and our peptide sequences correspond best to those present in human Slit-1 and Slit-2. Binding of these ligands to the glypican-Fc fusion protein requires the presence of the heparan sulfate chains, but the interaction appears to be relatively specific for glypican-1 insofar as no other identified heparin-binding proteins were isolated using our affinity matrix. Northern analysis demonstrated the presence of two mRNA species of 8. 6 and 7.5 kilobase pairs using probes based on both N- and C-terminal sequences, and in situ hybridization histochemistry showed that these glypican-1 ligands are synthesized by neurons, such as hippocampal pyramidal cells and cerebellar granule cells, where we have previously also demonstrated glypican-1 mRNA and immunoreactivity. Our results therefore indicate that Slit family proteins are functional ligands of glypican-1 in nervous tissue and suggest that their interactions may be critical for certain stages of central nervous system histogenesis
— id: 8500, year: 1999, vol: 274, page: 17885, stat: Journal Article,

Mice deficient for tenascin-R display alterations of the extracellular matrix and decreased axonal conduction velocities in the CNS
Weber P; Bartsch U; Rasband MN; Czaniera R; Lang Y; Bluethmann H; Margolis RU; Levinson SR; Shrager P; Montag D; Schachner M
1999 Jun 1;19(11):4245-4262, Journal of neuroscience
Tenascin-R (TN-R), an extracellular matrix glycoprotein of the CNS, localizes to nodes of Ranvier and perineuronal nets and interacts in vitro with other extracellular matrix components and recognition molecules of the immunoglobulin superfamily. To characterize the functional roles of TN-R in vivo, we have generated mice deficient for TN-R by homologous recombination using embryonic stem cells. TN-R-deficient mice are viable and fertile. The anatomy of all major brain areas and the formation and structure of myelin appear normal. However, immunostaining for the chondroitin sulfate proteoglycan phosphacan, a high-affinity ligand for TN-R, is weak and diffuse in the mutant when compared with wild-type mice. Compound action potential recordings from optic nerves of mutant mice show a significant decrease in conduction velocity as compared with controls. However, at nodes of Ranvier there is no apparent change in expression and distribution of Na+ channels, which are thought to bind to TN-R via their beta2 subunit. The distribution of carbohydrate epitopes of perineuronal nets recognized by the lectin Wisteria floribunda or antibodies to the HNK-1 carbohydrate on somata and dendrites of cortical and hippocampal interneurons is abnormal. These observations indicate an essential role for TN-R in the formation of perineuronal nets and in normal conduction velocity of optic nerve
— id: 33474, year: 1999, vol: 19, page: 4245, stat: Journal Article,

High affinity binding and overlapping localization of neurocan and phosphacan/protein-tyrosine phosphatase-zeta/beta with tenascin-R, amphoterin, and the heparin-binding growth-associated molecule
Milev P; Chiba A; Haring M; Rauvala H; Schachner M; Ranscht B; Margolis RK; Margolis RU
1998 Mar 20;273(12):6998-7005, Journal of biological chemistry
We have studied the interactions of the nervous tissue-specific chondroitin sulfate proteoglycans neurocan and phosphacan with the extracellular matrix protein tenascin-R and two heparin-binding proteins, amphoterin and the heparin-binding growth-associated molecule (HB-GAM), using a radioligand binding assay. Both proteoglycans show saturable, high affinity binding to tenascin-R with apparent dissociation constants in the 2-7 nM range. Binding is reversible, inhibited in the presence of unlabeled proteoglycan, and increased by approximately 60% following chondroitinase treatment of the proteoglycans, indicating that the interactions are mediated via the core (glyco)proteins rather than by the glycosaminoglycan chains, which may in fact partially shield the binding sites. In contrast to their interactions with tenascin-C, in which binding was decreased by approximately 75% in the absence of calcium, binding of phosphacan to tenascin-R was not affected by the absence of divalent cations in the binding buffer, although there was a small but significant decrease in the binding of neurocan. Neurocan and phosphacan are also high affinity ligands of amphoterin and HB-GAM (Kd = 0.3-8 nM), two heparin-binding proteins that are developmentally regulated in brain and functionally involved in neurite outgrowth. The chondroitin sulfate chains on neurocan and phosphacan account for at least 80% of their binding to amphoterin and HB-GAM. The presence of amphoterin also produces a 5-fold increase in phosphacan binding to the neural cell adhesion molecule contactin. Immunocytochemical studies showed an overlapping localization of the proteoglycans and their ligands in the embryonic and postnatal brain, retina, and spinal cord. These studies have therefore revealed differences in the interactions of neurocan and phosphacan with the two major members of the tenascin family of extracellular matrix proteins, and also suggest that chondroitin sulfate proteoglycans play an important role in the binding and/or presentation of differentiation factors in the developing central nervous system
— id: 7682, year: 1998, vol: 273, page: 6998, stat: Journal Article,

Differential regulation of expression of hyaluronan-binding proteoglycans in developing brain: aggrecan, versican, neurocan, and brevican
Milev P; Maurel P; Chiba A; Mevissen M; Popp S; Yamaguchi Y; Margolis RK; Margolis RU
1998 Jun 18;247(2):207-212, Biochemical & biophysical research communications
We have used a slot-blot radioimmunoassay to quantitate the levels of hyaluronan-binding chondroitin sulfate proteoglycans in developing rat brain from embryonic day 14 (E 14) to eight months postnatal. Recombinant nonhomologous regions of the core proteins were used for immunization to obtain polyclonal antibodies specific for aggrecan, the alpha and beta domains of versican mRNA splice variants, and N- and C-terminal portions of neurocan, while brevican was quantitated using a specific monoclonal antibody. The concentration of aggrecan increased steadily during brain development up to 5 months of age, when it reached a level that was 18-fold higher than at E14. Alternatively spliced versican isoforms containing the alpha domain of the glycosaminoglycan attachment region were present at a relatively low level during the late embryonic and early postnatal period, decreased by approximately 50% between 1 and 2 weeks postnatal, and then increased steadily in concentration to reach a maximum at 100 days that was 7-fold that present at 10 days postnatal. In contrast to these results, versican isoforms containing the beta domain more than doubled in concentration between E14 and birth, after which they decreased by greater than 90% to reach a low 'mature' level that remained unchanged between 2 and 8 months. The N- and C-terminal portions of neurocan (produced by a developmentally-regulated proteolytic cleavage in the middle of its chondroitin sulfate attachment region) both increased in embryonic brain during development, reached a peak in the early postnatal period, and then declined thereafter. As in the case of aggrecan, only traces of brevican were detected in embryonic brain and its concentration increased steadily after birth to reach an adult level that was approximately 14-fold higher than that present in neonatal brain. These striking and distinctive changes in the concentrations of the different members of this family of structurally related proteoglycans in developing brain, including changes in opposite directions for versican mRNA splice variants, indicate that the individual proteoglycans and their isoforms probably serve unique functions during nervous tissue histogenesis.
— id: 7681, year: 1998, vol: 247, page: 207, stat: Journal Article,

The core protein of the chondroitin sulfate proteoglycan phosphacan is a high-affinity ligand of fibroblast growth factor-2 and potentiates its mitogenic activity
Milev P; Monnerie H; Popp S; Margolis RK; Margolis RU
1998 Aug 21;273(34):21439-21442, Journal of biological chemistry
Using a radioligand binding assay we have demonstrated that phosphacan, a chondroitin sulfate proteoglycan of nervous tissue that also represents the extracellular domain of a receptor-type protein tyrosine phosphatase, shows saturable, reversible, high-affinity binding (Kd approximately 6 nM) to fibroblast growth factor-2 (FGF-2). Binding was reduced by only approximately 35% following chondroitinase treatment of the proteoglycan, indicating that the interaction is mediated primarily through the core protein rather than the glycosaminoglycan chains. Immunocytochemical studies also showed an overlapping localization of FGF-2 and phosphacan in the developing central nervous system. At concentrations of 10 microg protein/ml, both native phosphacan and the core protein obtained by chondroitinase treatment potentiated the mitogenic effect of FGF-2 (5 ng/ml) on NIH/3T3 cells by 75-90%, which is nearly the same potentiation as that produced by heparin at an equivalent concentration. Although studies on the role of proteoglycans in mediating the binding and mitogenic effects of FGF-2 have previously focused on cell surface heparan sulfate, our results indicate that the core protein of a chondroitin sulfate proteoglycan may also regulate the access of FGF-2 to cell surface signaling receptors in nervous tissue
— id: 7683, year: 1998, vol: 273, page: 21439, stat: Journal Article,

Glypican and biglycan in the nuclei of neurons and glioma cells: presence of functional nuclear localization signals and dynamic changes in glypican during the cell cycle
Liang Y; Haring M; Roughley PJ; Margolis RK; Margolis RU
1997 Nov 17;139(4):851-864, Journal of cell biology
We have investigated the expression patterns and subcellular localization in nervous tissue of glypican, a major glycosylphosphatidylinositol-anchored heparan sulfate proteoglycan that is predominantly synthesized by neurons, and of biglycan, a small, leucine-rich chondroitin sulfate proteoglycan. By laser scanning confocal microscopy of rat central nervous tissue and C6 glioma cells, we found that a significant portion of the glypican and biglycan immunoreactivity colocalized with nuclear staining by propidium iodide and was also seen in isolated nuclei. In certain regions, staining was selective, insofar as glypican and biglycan immunoreactivity in the nucleus was seen predominantly in a subpopulation of large spinal cord neurons. The amino acid sequences of both proteoglycans contain potential nuclear localization signals, and these were demonstrated to be functional based on their ability to target beta-galactosidase fusion proteins to the nuclei of transfected 293 cells. Nuclear localization of glypican beta-galactosidase or Fc fusion proteins in transfected 293 cells and C6 glioma cells was greatly reduced or abolished after mutation of the basic amino acids or deletion of the sequence containing the nuclear localization signal, and no nuclear staining was seen in the case of heparan sulfate and chondroitin sulfate proteoglycans that do not possess a nuclear localization signal, such as syndecan-3 or decorin (which is closely related in structure to biglycan). Transfection of COS-1 cells with an epitope-tagged glypican cDNA demonstrated transport of the full-length proteoglycan to the nucleus, and there are also dynamic changes in the pattern of glypican immunoreactivity in the nucleus of C6 cells both during cell division and correlated with different phases of the cell cycle. Our data therefore suggest that in certain cells and central nervous system regions, glypican and biglycan may be involved in the regulation of cell division and survival by directly participating in nuclear processes
— id: 12198, year: 1997, vol: 139, page: 851, stat: Journal Article,

Chondroitin sulfate proteoglycans as mediators of axon growth and pathfinding
Margolis RU; Margolis RK
1997 Nov;290(2):343-348, Cell & tissue research
This review focuses primarily on studies concerning the potential roles of two nervous-tissue-specific chondroitin sulfate proteoglycans, viz., neurocan and phosphacan, in cell interactions and neurite growth in the developing central nervous system. The multiple ligands of these proteoglycans and the modulatory effects of various types of glycosylation are also considered. Other chondroitin sulfate proteoglycans, such as NG2, DSD-1, Cat-301, versican, and biglycan, are briefly discussed in relation to the functional properties that have been ascribed to them
— id: 8344, year: 1997, vol: 290, page: 343, stat: Journal Article,

The fibrinogen-like globe of tenascin-C mediates its interactions with neurocan and phosphacan/protein-tyrosine phosphatase-zeta/beta
Milev P; Fischer D; Haring M; Schulthess T; Margolis RK; Chiquet-Ehrismann R; Margolis RU
1997 Jun 13;272(24):15501-15509, Journal of biological chemistry
Two nervous tissue-specific chondroitin sulfate proteoglycans, neurocan and phosphacan (the extracellular domain of protein-tyrosine phosphatase-zeta/beta), are high-affinity ligands of tenascin-C. Using portions of tenascin-C expressed as recombinant proteins in human fibrosarcoma cells, we have demonstrated both by direct radioligand binding assays and inhibition studies that phosphacan binding is retained in all deletion variants except those lacking the fibrinogen-like globe and that phosphacan binds to this single domain with nearly the same affinity (Kd approximately 12 nM) as to native or recombinant tenascin-C. However, maximum binding of neurocan requires both the fibrinogen globe and some of the adjacent fibronectin type III repeats. Binding of phosphacan and neurocan to intact tenascin-C, and of phosphacan to the fibrinogen globe, is significantly increased in the presence of calcium. Chondroitinase treatment of the proteoglycans did not affect their binding to either native tenascin-C or to any of the recombinant proteins, demonstrating that these interactions are mediated by the proteoglycan core proteins rather than through the glycosaminoglycan chains. These results are also consistent with rotary shadowing electron micrographs that show phosphacan as a rod terminated at one end by a globular domain that is frequently seen apposed to the fibrinogen globe in mixtures of phosphacan and tenascin-C. C6 glioma cells adhere to and spread on deletion variants of tenascin-C containing only the epidermal growth factor-like domains or the fibronectin type III repeats and the fibrinogen globe. In both cases cell adhesion was inhibited by similar concentrations of phosphacan, demonstrating that the fibrinogen globe is not necessary for this effect, which is apparently mediated by a direct action of phosphacan on the cells rather than by its interaction with the proteoglycan binding site on tenascin-C
— id: 7218, year: 1997, vol: 272, page: 15501, stat: Journal Article,

Brain contains HNK-1 immunoreactive O-glycans of the sulfoglucuronyl lactosamine series that terminate in 2-linked or 2,6-linked hexose (mannose)
Yuen CT; Chai W; Loveless RW; Lawson AM; Margolis RU; Feizi T
1997 Apr 4;272(14):8924-8931, Journal of biological chemistry
The monoclonal antibody HNK-1 originally raised to an antigenic marker of natural killer cells also binds to selected regions in nervous tissue. The antigen is a carbohydrate that has attracted much interest as its expression is developmentally regulated in nervous tissue, and it is found, and proposed to be a ligand, on several of the adhesive glycoproteins of the nervous system. It is also expressed on glycolipids and proteoglycans, and is the target of monoclonal auto-antibodies that give rise to a demyelinating disease. The epitope, as characterized on glycolipids isolated from the nervous system, is expressed on 3-sulfated glucuronic acid joined by beta1-3-linkage to a neolacto backbone. Here we exploit the neoglycolipid technology, in conjunction with immunodetection and in situ liquid secondary ion mass spectrometry, to characterize HNK-1-positive oligosaccharide chains derived by reductive alkaline release from total brain glycopeptides. The immunoreactive oligosaccharides detected are tetra- to octasaccharides that are very minor components among a heterogeneous population, each representing less than 0.1% of the starting material. Their peripheral and backbone sequences resemble those of the HNK-1-positive glycolipids. An unexpected finding is that they terminate not with N-acetylgalactosaminitol but with hexitol (2-substituted and 2,6-disubstituted). In a tetrasaccharide investigated in the greatest detail, the hexitol is identified as 2-substituted mannitol
— id: 33475, year: 1997, vol: 272, page: 8924, stat: Journal Article,

Chondroitin sulfate proteoglycans in the developing central nervous system. I. cellular sites of synthesis of neurocan and phosphacan
Engel M; Maurel P; Margolis RU; Margolis RK
1996 Feb 26;366(1):34-43, Journal of comparative neurology
We have used in situ hybridization histochemistry to examine the cellular sites of synthesis of two major nervous tissue proteoglycans, neurocan and phosphacan, in embryonic and postnatal rat brain and spinal cord. Both proteoglycans were detected only in nervous tissue. Neurocan mRNA was evident in neurons, including cerebellar granule cells and Purkinje cells, and in neurons of the hippocampal formation and cerebellar nuclei. In contrast, phosphacan message was detected only in astroglia, such as the Golgi epithelial cells of the cerebellum. At embryonic day 13-16, phosphacan mRNA is largely confined to areas of active cell proliferation (e.g., the ventricular zone of the ganglionic eminence and septal area of the brain and the ependymal layer surrounding the central canal of the spinal cord) as well as being present in the roof plate. The distribution of neurocan message is more widespread, extending to the cortex, hippocampal formation, caudate putamen, and basal telencephalic neuroepithelium, and neurocan mRNA is present in both the ependymal and mantle layers of the spinal cord but not in the roof plate. The presence of neurocan mRNA in areas where the proteoglycan is not expressed suggests that the short open reading frame in the 5'-leader of neurocan may function as a cis-acting regulatory signal for the modulation of neurocan expression in the developing central nervous system
— id: 33477, year: 1996, vol: 366, page: 34, stat: Journal Article,

Neurocan and phosphacan: two major nervous tissue-specific chondroitin sulfate proteoglycans
Margolis RK; Rauch U; Maurel P; Margolis RU
1996 ;3(4):273-290, Perspectives on developmental neurobiology
Neurocan is a multidomain hyaluronan-binding chondroitin sulfate proteoglycan that is synthesized by neurons, whereas the astroglial proteoglycan phosphacan is an mRNA splice variant representing the entire extracellular portion of a receptor-type protein tyrosine phosphatase. A glycoform of phosphocan (phosphocan-KS) that contains both chondroitin sulfate and keratan sulfate is present in the postnatal rat central nervous system (CNS). The concentration of neurocan in brain increases during late embryonic development but then declines steeply during the early postnatal period together with hyaluronan, and neurocan also undergoes extensive proteolytic processing during the course of brain development. In contrast, the concentrations of both phosphocan and phosphocan-KS rise steadily after embryonic day 20 to reach a plateau at about 2 weeks postnatally. In the embryonic CNS the distribution of neurocan mRNA is more widespread than that of phosphocan, which is primarily present in regions of active cell proliferation. Neurocan mRNA is also present in areas where the proteoglycan is not expressed, and there is evidence that the short open reading frame in its 5'-leader may function as a cis-acting regulatory signal for the modulation of neurocan expression in the developing CNS. Neurocan and phosphocan bind saturably, reversibly, and with high affinity to neural cell adhesion molecules (Ng-CAM/L1, NCAM, TAG-1/axonin-1) and to tenascin-C. The proteoglycans and their ligands have overlapping localizations in the CNS, and binding of phosphocan to Ng-CAM/L1, NCAM, and tenascin-C is mediated by complex-type N-linked oligosaccharides on the proteoglycan. Neurocan and phosphocan also bind to neurons and are potent inhibitors of neuronal and glial adhesion and neurite outgrowth. Through their interactions with neural cell adhesion and extracellular matrix molecules, these proteoglycans may play a major role in modulating cell adhesion, neurite growth, and signal transduction across the plasma membrane during the development of the CNS
— id: 33478, year: 1996, vol: 3, page: 273, stat: Journal Article,

Counteradhesive properties of chondroitin sulfate proteoglycans
Margolis RU; Margolis RK
Tenascin and counteradhesive molecules of the extracellular matrix Amsterdam : Harwood Academic Publishers, 1996,
— id: 2879, year: 1996, vol: , page: 145, stat: Chapter,

Chondroitin sulfate proteoglycans in the developing central nervous system. II. Immunocytochemical localization of neurocan and phosphacan
Meyer-Puttlitz B; Junker E; Margolis RU; Margolis RK
1996 Feb 26;366(1):44-54, Journal of comparative neurology
Using immunocytochemistry, we have compared the distribution of neurocan and phosphacan in the developing central nervous system. At embryonic day 13 (E13), phosphacan surrounds the radially oriented neuroepithelial cells of the telencephalon, whereas neurocan staining of brain parenchyma is very weak. By E16-19, strong staining of both neurocan and phosphacan is seen in the marginal zone and subplate of the neocortex, and phosphacan is present in the ventricular zone and also has a diffuse distribution in other brain areas. Phosphacan is also widely distributed in embryonic spinal cord, where it is strongly expressed throughout the gray and white matter, in the dorsal and ventral nerve roots, and in the roof plate at E13, when neurocan immunoreactivity is seen only in the mesenchyme of the future spinal canal. Neurocan first begins to appear in the spinal cord at E16-19, in the region of ventral motor neurons. In early postnatal and adult cerebellum, neurocan immunoreactivity is seen in the prospective white matter and in the granule cell, Purkinje cell, and molecular layers, whereas phosphacan immunoreactivity is associated with Bergmann glial fibers in the molecular layer and their cell bodies (the Golgi epithelial cells) below the Purkinje cells. These immunocytochemical results demonstrate that the expression of neurocan and phosphacan follow different developmental time courses not only in postnatal brain (as previously demonstrated by radioimmunoassay) but also in the embryonic central nervous system. The specific localization and different temporal expression patterns of these two proteoglycans are consistent with other evidence indicating that they have overlapping or complementary roles in axon guidance, cell interactions, and neurite outgrowth during nervous tissue histogenesis
— id: 33476, year: 1996, vol: 366, page: 44, stat: Journal Article,

TAG-1/axonin-1 is a high-affinity ligand of neurocan, phosphacan/protein-tyrosine phosphatase-zeta/beta, and N-CAM
Milev P; Maurel P; Haring M; Margolis RK; Margolis RU
1996 Jun 28;271(26):15716-15723, Journal of biological chemistry
Proteoglycans appear to play an important role in modulating cell-cell and cell-matrix interactions during nervous tissue histogenesis. The nervous tissue-specific chondroitin sulfate proteoglycans neurocan and phosphacan/protein-tyrosine phosphatase-zeta/beta were found to be high-affinity ligands of the neural cell adhesion molecule TAG-1/axonin-1, with dissociation constants of 0.3 nM and 0.04 nM, respectively. Phosphacan binding was decreased by approximately 70% following chondroitinase treatment, whereas binding of neurocan was not affected. The contribution of chondroitin sulfate chains to the binding of neurocan and phosphacan to TAG-1/axonin-1 is therefore the opposite of that previously observed for their binding to two other Ig-superfamily neural cell adhesion molecules, Ng-CAM/L1 and N-CAM. Moreover, whereas phosphacan interactions with certain proteins are mediated at least in part by N-linked oligosaccharides on the proteoglycan, N-deglycosylation of phosphacan had no effect on its binding to TAG-1/axonin-1. In addition to the chondroitin sulfate proteoglycans described above, we have demonstrated that N-CAM is a high-affinity ligand of TAG-1/axonin-1 (Kd approximately 1 nM), and specific binding of TAG-1/axonin-1 to tenascin-C was also observed (Kd approximately 9 nM). Immunocytochemical studies of embryonic and early postnatal nervous tissue showed an overlapping localization of TAG-1/axonin-1 with all four of these ligands, further supporting the biological significance of their ability to interact in vitro
— id: 7018, year: 1996, vol: 271, page: 15716, stat: Journal Article,

Nucleotide sequence and molecular variants of rat receptor-type protein tyrosine phosphatase-zeta/beta
Maurel P; Meyer-Puttlitz B; Flad M; Margolis RU; Margolis RK
1995 ;5(5):323-328, DNA sequence
We have previously described the cloning of phosphacan, a chondroitin sulfate proteoglycan of nervous tissue which interacts with neurons, glia, neural cell adhesion molecules, and tenascin, and represents the extracellular domain of a receptor-type protein tyrosine phosphatase. We now report the complete cDNA and deduced amino acid sequences of the rat transmembrane phosphatase, and demonstrate that the phosphatase and the extracellular proteoglycan have different 3'-untranslated regions. Northern analysis showed three probable splice variants, comprising the extracellular proteoglycan (phosphacan) and long and short forms of the transmembrane phosphatase. PCR studies of rat genomic DNA indicated that there are no introns at the putative 5' and 3' splice sites or in the 2.6 kb segment which is deleted in the short transmembrane protein. Using variant-specific riboprobes corresponding to sequences in the 3'-untranslated region of phosphacan and in the first or second phosphatase domains of the transmembrane protein, in situ hybridization histochemistry of embryonic rat brain and spinal cord and early postnatal cerebellum demonstrated identical localizations of phosphacan and phosphatase mRNAs
— id: 6934, year: 1995, vol: 5, page: 323, stat: Journal Article,

Chondroitin sulfate and chondroitin/keratan sulfate proteoglycans of nervous tissue: developmental changes of neurocan and phosphacan
Meyer-Puttlitz B; Milev P; Junker E; Zimmer I; Margolis RU; Margolis RK
1995 Nov;65(5):2327-2337, Journal of neurochemistry
We have studied developmental changes in the structure and concentration of the hyaluronic acid-binding proteoglycan, neurocan, and of phosphacan, another major chondroitin sulfate proteoglycan of nervous tissue that represents the extracellular domain of a receptor-type protein tyrosine phosphatase. A new monoclonal antibody (designated 1F6), which recognizes an epitope in the N-terminal portion of neurocan, has been used for the isolation of proteolytic processing fragments that occur together with link protein in a complex with hyaluronic acid. Both link protein and two of the neurocan fragments were identified by amino acid sequencing. The N-terminal fragments of neurocan are also recognized by monoclonal antibodies (5C4, 8A4, and 3B1) to epitopes in the G1 and G2 domains of aggrecan and/or in the hyaluronic acid-binding domain of link protein. The presence in brain of these N-terminal fragments is consistent with the developmentally regulated appearance of the C-terminal half of neurocan, which we described previously. We have also used a slot-blot radioimmunoassay to determine the concentrations of neurocan and phosphacan in developing brain. The levels of both proteoglycans increased rapidly during early brain development, but whereas neurocan reached a peak at approximately postnatal day 4 and then declined to below embryonic levels in adult brain, the concentration of phosphacan remained essentially unchanged after postnatal day 12. Keratan sulfate on phosphacan-KS (a glycoform that contains both chondroitin sulfate and keratan sulfate chains) was not detectable until just before birth, and its peak concentration (at 3 weeks postnatal) was reached approximately 1 week later than that of the phosphacan core protein. Immunocytochemical studies using monoclonal antibodies to keratan sulfate (3H1 and 5D4) together with specific glycosidases (endo-beta-galactosidase, keratanase, and keratanase II) also showed that with the exception of some very localized areas, keratan sulfate is generally not present in the embryonic rat CNS
— id: 33479, year: 1995, vol: 65, page: 2327, stat: Journal Article,

Complex-type asparagine-linked oligosaccharides on phosphacan and protein-tyrosine phosphatase-zeta/beta mediate their binding to neural cell adhesion molecules and tenascin
Milev P; Meyer-Puttlitz B; Margolis RK; Margolis RU
1995 Oct 20;270(42):24650-24653, Journal of biological chemistry
Phosphacan, a soluble nervous tissue-specific chondroitin sulfate proteoglycan, is an alternative splicing product representing the entire extracellular domain of a transmembrane receptor-type protein-tyrosine phosphatase (RPTP zeta/beta) that also occurs as a chondroitin sulfate proteoglycan in brain. We have previously demonstrated that phosphacan binds with high affinity to neural cell adhesion molecules (Ng-CAM/L1 and N-CAM) and to the extracellular matrix protein tenascin and that it is a potent inhibitor of cell adhesion and neurite outgrowth. Tryptic digests of 125I-labeled phosphacan contain two glycopeptides that bind to Ng-CAM/L1, N-CAM, and tenascin. The larger of these (17 kDa) begins at Gln-209 near the end of the carbonic anhydrase-like domain of phosphacan/RPTP zeta/beta, whereas a 13-kDa glycopeptide begins at His-361 located in the middle of the fibronectin type III-like domain. Treatment of phosphacan with peptide N-glycosidase under nondenaturing conditions reduced its binding the neural cell adhesion molecules and tenascin by 65-75%, whereas endo-beta-N-acetylglucosaminidase H had no effect, and peptide N-glycosidase treatment both decreased the molecular sizes of the tryptic peptides to congruent to 11 kDa and abolished their binding. Based on the amino acid sequence of phosphacan, it can be concluded that each of the tryptic peptides contains one potential N-glycosylation site (at Asn-232 and Asn-381), and analyses of the isolated glycopeptides demonstrated the presence of sialylated complex-type oligosaccharides. Our results therefore indicate that the interactions of phosphacan/RPTP zeta/beta with neural cell adhesion molecules and tenascin are mediated by asparagine-linked oligosaccharides present in their carbonic anhydrase- and fibronectin type III-like domains
— id: 6845, year: 1995, vol: 270, page: 24650, stat: Journal Article,

The Alzheimer amyloid precursor proteoglycan (appican) is present in brain and is produced by astrocytes but not by neurons in primary neural cultures
Shioi J; Pangalos MN; Ripellino JA; Vassilacopoulou D; Mytilineou C; Margolis RU; Robakis NK
1995 May 19;270(20):11839-11844, Journal of biological chemistry
Recent studies showed that the Alzheimer amyloid precursor (APP) occurs as the core protein of a chondroitin sulfate proteoglycan (appican) in C6 glioma cells. In the present study we show that appican is present in both human and rat brain tissue. Cortical rat brain cell cultures were used to identify appican-producing cells. Soluble secreted and cell-associated appican was produced by mixed glial cultures but not by primary neuronal cultures. Among the three major glial cell types, astrocytes produced high levels of appican, while oligodendrocytes failed to produce any. Only low levels of this molecule were occasionally detected in microglial cultures. Expression of appican in astrocyte cultures was regulated by the composition of the growth media. N2a neuroblastoma cells also produced appican; however, treatment with dibutyryl cAMP which promotes neuronal differentiation in these cells inhibited its production without inhibiting synthesis of APP. In contrast to the restricted expression of appican, APP was present in all cultures, and its production was independent of appican synthesis. Neuronal cultures produced mainly APP695 while glial cultures produced the Kunitz type protease inhibitor containing APP. The astrocyte-specific expression of appican suggests a function distinct from the function of APP. Brain appicans may play a role in the development of Alzheimer disease neuropathology
— id: 33480, year: 1995, vol: 270, page: 11839, stat: Journal Article,

The neuronal chondroitin sulfate proteoglycan neurocan binds to the neural cell adhesion molecules Ng-CAM/L1/NILE and N-CAM, and inhibits neuronal adhesion and neurite outgrowth
Friedlander DR; Milev P; Karthikeyan L; Margolis RK; Margolis RU; Grumet M
1994 May;125(3):669-680, Journal of cell biology
We have previously shown that aggregation of microbeads coated with N-CAM and Ng-CAM is inhibited by incubation with soluble neurocan, a chondroitin sulfate proteoglycan of brain, suggesting that neurocan binds to these cell adhesion molecules (Grumet, M., A. Flaccus, and R. U. Margolis. 1993. J. Cell Biol. 120:815). To investigate these interactions more directly, we have tested binding of soluble 125I-neurocan to microwells coated with different glycoproteins. Neurocan bound at high levels to Ng-CAM and N-CAM, but little or no binding was detected to myelin-associated glycoprotein, EGF receptor, fibronectin, laminin, and collagen IV. The binding to Ng-CAM and N-CAM was saturable and in each case Scatchard plots indicated a high affinity binding site with a dissociation constant of approximately 1 nM. Binding was significantly reduced after treatment of neurocan with chondroitinase, and free chondroitin sulfate inhibited binding of neurocan to Ng-CAM and N-CAM. These results indicate a role for chondroitin sulfate in this process, although the core glycoprotein also has binding activity. The COOH-terminal half of neurocan was shown to have binding properties essentially identical to those of the full-length proteoglycan. To study the potential biological functions of neurocan, its effects on neuronal adhesion and neurite growth were analyzed. When neurons were incubated on dishes coated with different combinations of neurocan and Ng-CAM, neuronal adhesion and neurite extension were inhibited. Experiments using anti-Ng-CAM antibodies as a substrate also indicate that neurocan has a direct inhibitory effect on neuronal adhesion and neurite growth. Immunoperoxidase staining of tissue sections showed that neurocan, Ng-CAM, and N-CAM are all present at highest concentration in the molecular layer and fiber tracts of developing cerebellum. The overlapping localization in vivo, the molecular binding studies, and the striking effects on neuronal adhesion and neurite growth support the view that neurocan may modulate neuronal adhesion and neurite growth during development by binding to neural cell adhesion molecules
— id: 8072, year: 1994, vol: 125, page: 669, stat: Journal Article,

Interactions with tenascin and differential effects on cell adhesion of neurocan and phosphacan, two major chondroitin sulfate proteoglycans of nervous tissue
Grumet M; Milev P; Sakurai T; Karthikeyan L; Bourdon M; Margolis RK; Margolis RU
1994 Apr 22;269(16):12142-12146, Journal of biological chemistry
We have studied interactions of tenascin with two chondroitin sulfate proteoglycans, neurocan and phosphacan. Neurocan is a multi-domain proteoglycan with a 136-kDa core protein that is synthesized by neurons and binds to hyaluronic acid, whereas the 173-kDa core protein of phosphacan, which is synthesized by glia, represents an extracellular variant of the receptor-type protein tyrosine phosphatase RPTP zeta/beta. Keratan sulfate-containing glycoforms of phosphacan (designated phosphacan-KS) are also present in brain. Immunocytochemical studies of early postnatal rat cerebellum demonstrated that the localization of neurocan, phosphacan, and phosphacan-KS all overlap extensively with that of tenascin, an extracellular matrix protein that modulates cell adhesion and migration. Binding studies using purified proteins covalently attached to fluorescent microbeads demonstrated that proteoglycan-coated beads co-aggregated with differently fluorescing beads coated with tenascin. The co-aggregation was specifically inhibited by Fab' fragments of antibodies against tenascin or the proteoglycans and by soluble neurocan, phosphacan, and tenascin. A solid phase radioligand binding assay confirmed that neurocan, phosphacan, and phosphacan-KS bind to tenascin but not to laminin and fibronectin. Chondroitinase treatment of the proteoglycans or addition of free chondroitin sulfate had no significant effect, indicating that the binding activity is mediated largely via the core glycoproteins. Scatchard analysis demonstrated high affinity binding of 125I-phosphacan, phosphacan-KS, and neurocan to a single site in tenascin, and neurocan and various glycoforms of phosphacan all inhibited binding of 125I-phosphacan to tenascin. In studies of cell adhesion to proteins adsorbed to Petri dishes, phosphacan inhibited adhesion of C6 glioma cells to tenascin whereas neurocan had no effect. Our results suggest that tenascin binds phosphacan and neurocan in vivo and that interactions between chondroitin sulfate proteoglycans and tenascin may play important roles in nervous tissue histogenesis, possibly by modulating signal transduction across the plasma membrane
— id: 6399, year: 1994, vol: 269, page: 12142, stat: Journal Article,

Immunocytochemical and in situ hybridization studies of the heparan sulfate proteoglycan, glypican, in nervous tissue
Karthikeyan L; Flad M; Engel M; Meyer-Puttlitz B; Margolis RU; Margolis RK
1994 Nov;107(Pt 11):3213-3222, Journal of cell science
Using immunocytochemistry and in situ hybridization histochemistry, we have investigated in embryonic and postnatal rat nervous tissue the localization and cellular sites of synthesis of glypican, a glycosylphosphatidylinositol-anchored heparan sulfate proteoglycan. Glypican immunoreactivity is present in the marginal layer (prospective white matter) and in the dorsal root entry zone of E13-16 spinal cord, as well as in the optic nerve and retina at this stage, but does not appear at significant levels in brain until approximately E19. The proteoglycan shows a wide distribution in grey matter and axonal projections of postnatal brain, including the hippocampal formation, the parallel fibers of cerebellar granule cells, and in the medulla and brainstem. Northern analysis demonstrated high levels of glypican mRNA in brain and skeletal muscle, and in rat PC12 pheochromocytoma cells. In situ hybridization histochemistry showed that glypican mRNA was especially prominent in cerebellar granule cells, large motor neurons in the brainstem, and CA3 pyramidal cells of the hippocampus. Our immunocytochemical and in situ hybridization results indicate that glypican is predominantly a neuronal membrane proteoglycan in the late embryonic and postnatal rat central nervous system
— id: 12875, year: 1994, vol: 107, page: 3213, stat: Journal Article,

Aggrecan-versican-neurocan family proteoglycans
Margolis RU; Margolis RK
1994 ;245:105-126, Methods in enzymology
— id: 6687, year: 1994, vol: 245, page: 105, stat: Journal Article,

Phosphacan, a chondroitin sulfate proteoglycan of brain that interacts with neurons and neural cell-adhesion molecules, is an extracellular variant of a receptor-type protein tyrosine phosphatase
Maurel P; Rauch U; Flad M; Margolis RK; Margolis RU
1994 Mar 29;91(7):2512-2516, Proceedings of the National Academy of Sciences of the United States of America
We have identified cDNA clones encoding a chondroitin sulfate proteoglycan of rat brain (previously designated 3F8 and now named phosphacan) that binds to neurons and neural cell-adhesion molecules. A sequence of 1616 amino acids deduced from a 4.8-kb open reading frame contains the N-terminal amino acid sequence of the 3F8 core glycoprotein as well as four internal CNBr, tryptic, and endoproteinase Lys-C peptide sequences from the proteoglycan. The deduced amino acid sequence, beginning with a 24-amino acid signal peptide, reveals an N-terminal domain of 255 amino acids homologous to carbonic anhydrases. The entire amino acid sequence deduced from our cDNA clones corresponds to the extracellular portion of a human receptor-type protein tyrosine phosphatase (RPTP zeta/beta) with which it has 76% identity, and the proteoglycan may represent an mRNA splicing variant of the larger transmembrane protein. RNA analysis demonstrated that a probe to the N-terminal carbonic anhydrase domain of the proteoglycan hybridizes with rat brain mRNA of 9.5, 8.4, and 6.4 kb, whereas probes to the phosphatase domains hybridize with only the 9.5-kb message and with the 6.4-kb message (which corresponds to a previously identified variant of the transmembrane protein in which half of the extracellular domain is deleted). The 30 N-terminal amino acids of the 3H1 chondroitin/keratan sulfate proteoglycan of brain are identical to those of the 3F8 proteoglycan, and six internal tryptic peptide sequences also matched those found in sequenced peptides of the 3F8 proteoglycan and/or amino acid sequences deduced from the cDNA clones. We therefore conclude that the 3H1 chondroitin/keratan sulfate proteoglycan and the 3F8 chondroitin sulfate proteoglycan represent glycosylation and possible extracellular splicing variants of a receptor-type protein tyrosine phosphatase. These proteoglycans may modulate cell interactions and other developmental processes in nervous tissue through heterophilic binding to cell-surface and extracellular matrix molecules, and by competition with ligands of the transmembrane phosphatase
— id: 6449, year: 1994, vol: 91, page: 2512, stat: Journal Article,

Interactions of the chondroitin sulfate proteoglycan phosphacan, the extracellular domain of a receptor-type protein tyrosine phosphatase, with neurons, glia, and neural cell adhesion molecules
Milev P; Friedlander DR; Sakurai T; Karthikeyan L; Flad M; Margolis RK; Grumet M; Margolis RU
1994 Dec;127(6 Pt 1):1703-1715, Journal of cell biology
Phosphacan is a chondroitin sulfate proteoglycan produced by glial cells in the central nervous system, and represents the extracellular domain of a receptor-type protein tyrosine phosphatase (RPTP zeta/beta). We previously demonstrated that soluble phosphacan inhibited the aggregation of microbeads coated with N-CAM or Ng-CAM, and have now found that soluble 125I-phosphacan bound reversibly to these neural cell adhesion molecules, but not to a number of other cell surface and extracellular matrix proteins. The binding was saturable, and Scatchard plots indicated a single high affinity binding site with a Kd of approximately 0.1 nM. Binding was reduced by approximately 15% after chondroitinase treatment, and free chondroitin sulfate was only moderately inhibitory, indicating that the phosphacan core glycoprotein accounts for most of the binding activity. Immunocytochemical studies of embryonic rat spinal phosphacan, Ng-CAM, and N-CAM have overlapping distributions. When dissociated neurons were incubated on dishes coated with combinations of phosphacan and Ng-CAM, neuronal adhesion and neurite growth were inhibited. 125I-phosphacan bound to neurons, and the binding was inhibited by antibodies against Ng-CAM and N-CAM, suggesting that these CAMs are major receptors for phosphacan on neurons. C6 glioma cells, which express phosphacan, adhered to dishes coated with Ng-CAM, and low concentrations of phosphacan inhibited adhesion to Ng-CAM but not to laminin and fibronectin. Our studies suggest that by binding to neural cell adhesion molecules, and possibly also by competing for ligands of the transmembrane phosphatase, phosphacan may play a major role in modulating neuronal and glial adhesion, neurite growth, and signal transduction during the development of the central nervous system
— id: 6692, year: 1994, vol: 127, page: 1703, stat: Journal Article,

Disaccharide composition of heparan sulfates: brain, nervous tissue storage organelles, kidney, and lung
Tekotte H; Engel M; Margolis RU; Margolis RK
1994 Mar;62(3):1126-1130, Journal of neurochemistry
We have characterized the structural properties of heparan sulfates from brain and other tissues after depolymerization with a mixture of three heparin and heparan sulfate lyases from Flavobacterium heparinum. The resulting disaccharides were separated by HPLC and identified by comparison with authentic standards. In rat, rabbit, and bovine brain, 46-69% of the heparan sulfate disaccharides are N-acetylated and unsulfated, and 17-21% contain a single sulfate residue in the form of a sulfoamino group. In rabbit, bovine, and 1-day postnatal rat brain, disaccharides containing both a sulfated uronic acid and N-sulfate account for an additional 10-14%, together with smaller and approximately equal proportions (5-9%) of mono-, di-, and trisulfated disaccharides having sulfate at the 6-position of the glucosamine residue. Kidney and lung heparan sulfates are distinguished by high concentrations of disaccharides containing 6-sulfated N-acetylglucosamine residues. In chromaffin granules, the catecholamine- and peptide-storing organelles of adrenal medulla, where heparan sulfate accounts for a minor portion (5-10%) of the glycosaminoglycans, we have determined that bovine chromaffin granule membranes contain heparan sulfate in which almost all of the disaccharides are either unsulfated (71%) or monosulfated (18%). In sympathetic nerves, norepinephrine is stored in large dense cored vesicles that in biochemical composition and properties closely resemble adrenal chromaffin granules. However, in contrast to chromaffin granules, heparan sulfate accounts for approximately 75% of the total glycosaminoglycans in large dense-cored vesicles and more closely resembles heparin, insofar as it contains only 21% unsulfated disaccharides, 10% mono- and disulfated disaccharides, and 69% trisulfated disaccharides.(ABSTRACT TRUNCATED AT 250 WORDS)
— id: 33481, year: 1994, vol: 62, page: 1126, stat: Journal Article,

Functional characterization of chondroitin sulfate proteoglycans of brain: interactions with neurons and neural cell adhesion molecules
Grumet M; Flaccus A; Margolis RU
1993 Feb;120(3):815-824, Journal of cell biology
Ng-CAM and N-CAM are cell adhesion molecules (CAMs), and each CAM can bind homophilically as demonstrated by the ability of CAM-coated beads (Covaspheres) to self-aggregate. We have found that the extent of aggregation of Covaspheres coated with either Ng-CAM or N-CAM was strongly inhibited by the intact 1D1 and 3F8 chondroitin sulfate proteoglycans of rat brain, and by the core glycoproteins resulting from chondroitinase treatment of the proteoglycans. Much higher concentrations of rat chondrosarcoma chondroitin sulfate proteoglycan (aggrecan) core proteins had no significant effect in these assays. The 1D1 and 3F8 proteoglycans also inhibited binding of neurons to Ng-CAM when mixtures of these proteins were adsorbed to polystyrene dishes. Direct binding of neurons to the proteoglycan core glycoproteins from brain but not from chondrosarcoma was demonstrated using an assay in which cell-substrate contact was initiated by centrifugation, and neuronal binding to the 1D1 proteoglycans was specifically inhibited by the 1D1 monoclonal antibody. Different forms of the 1D1 proteoglycan have been identified in developing and adult brain. The early postnatal form (neurocan) was found to bind neurons more effectively than the adult proteoglycan, which represents the C-terminal half of the larger neurocan core protein. Our results therefore indicate that certain brain proteoglycans can bind to neurons, and that Ng-CAM and N-CAM may be heterophilic ligands for neurocan and the 3F8 proteoglycan. The ability of these brain proteoglycans to inhibit adhesion of cells to CAMs may be one mechanism to modulate cell adhesion and migration in the nervous system
— id: 13273, year: 1993, vol: 120, page: 815, stat: Journal Article,

Nervous tissue proteoglycans
Margolis RK; Margolis RU
1993 May 15;49(5):429-446, Experientia
The structure, biosynthesis, localization, and possible functional roles of nervous tissue glycosaminoglycans and proteoglycans were last reviewed several years ago. Since that time, there has been an exponential increase in publications on the neurobiology of proteoglycans. This review will therefore focus on reports which have appeared in the period after 1988, and especially on those concerning the properties of individual characterized nervous tissue proteoglycans. Related areas such as the regulation of glycosaminoglycan biosynthesis and the roles of cell surface proteoglycans in adhesion and growth control are covered in other contributions to this special topic issue
— id: 33483, year: 1993, vol: 49, page: 429, stat: Journal Article,

Cloning of a major heparan sulfate proteoglycan from brain and identification as the rat form of glypican
Karthikeyan L; Maurel P; Rauch U; Margolis RK; Margolis RU
1992 Oct 15;188(1):395-401, Biochemical & biophysical research communications
We have obtained the complete coding sequence of a highly conserved heparan sulfate proteoglycan which we previously characterized biochemically after isolation from rat brain. An open reading frame of 558 amino acids encodes a protein with a molecular mass of 62 kDa containing three peptide sequences present in the isolated proteoglycan. The total sequence obtained is 3.5 kb long, including 1.6 kb of 3'-untranslated sequence and 0.2 kb of 5'-untranslated sequence. The deduced amino acid sequence and the 3'- and 5'-untranslated sequences have 89% and 66-80% identity, respectively, with those of a phosphatidylinositol-anchored human lung fibroblast heparan sulfate proteoglycan (glypican) for which mRNA is detectable in a large number of human cell lines. Our data therefore demonstrate that this major heparan sulfate proteoglycan of brain is the rat form of glypican
— id: 13394, year: 1992, vol: 188, page: 395, stat: Journal Article,

Cloning and primary structure of neurocan, a developmentally regulated, aggregating chondroitin sulfate proteoglycan of brain
Rauch U; Karthikeyan L; Maurel P; Margolis RU; Margolis RK
1992 Sep 25;267(27):19536-19547, Journal of biological chemistry
We have obtained the complete coding sequence of neurocan, a chondroitin sulfate proteoglycan of rat brain which is developmentally regulated with respect to its molecular size, concentration, carbohydrate composition, sulfation, and immunocytochemical localization. Two degenerate oligonucleotides, based on amino acid sequence data from the proteoglycan isolated from adult brain by immunoaffinity chromatography with the 1D1 monoclonal antibody, were used as sense and antisense primers in the polymerase chain reaction with a brain cDNA library as template to generate an unambiguous cDNA probe. A second probe for the N-terminal portion of the early postnatal form of the proteoglycan was obtained by reverse transcription/polymerase chain reaction. The composite sequence of overlapping cDNA clones is 5.2-kilobases (kb) long, including 1.3 kb of 3'-untranslated sequence and 76 base pairs of 5'-untranslated sequence. An open reading frame of 1257 amino acids encodes a protein with a molecular mass of 136 kDa containing 10 peptide sequences present in the adult and/or early postnatal brain proteoglycans. The deduced amino acid sequence revealed a 22-amino acid signal peptide followed by an immunoglobulin domain, tandem repeats characteristic of the hyaluronic acid-binding region of aggregating proteoglycans, and an RGDS sequence. The C-terminal portion (amino acids 951-1215) has approximately 60% identity to regions in the C termini of the fibroblast and cartilage proteoglycans, versican and aggrecan, including two epidermal growth factor-like domains, a lectin-like domain, and a complement regulatory protein-like sequence. The central 595-amino acid portion of neurocan has no homology with other reported protein sequences. The proteoglycan contains six potential N-glycosylation sites and 25 potential threonine O-glycosylation sites. In the adult form of the proteoglycan (which represents the C-terminal half of neurocan) a single 32-kDa chondroitin 4-sulfate chain is linked at serin-944, whereas three additional potential chondroitin sulfate attachment sites (only two of which are utilized) are present in the larger proteoglycan species. A probe corresponding to a region of neurocan having no homology with versican or aggrecan hybridized with a single band at approximately 7.5 kb on Northern blots of mRNA from both 4-day and adult rat brain (but not with muscle, kidney, liver, or lung mRNA), indicating that the 1D1 proteoglycan of adult brain, containing a 68-kDa core protein, is generated by a developmentally regulated in vivo proteolytic processing of the 136-kDa species which is predominant in early postnatal brain.(ABSTRACT TRUNCATED AT 400 WORDS)
— id: 13438, year: 1992, vol: 267, page: 19536, stat: Journal Article,

Immunocytochemical localization of chondroitin and chondroitin 4- and 6-sulfates in developing rat cerebellum
Flaccus A; Janetzko A; Tekotte H; Margolis RK; Margolis RU
1991 May;56(5):1608-1615, Journal of neurochemistry
Monoclonal antibodies specific for unsulfated, 4-sulfated, and 6-sulfated disaccharide 'stubs' that remain attached to the core protein after chondroitinase ABC digestion of chondroitin/dermatan sulfate proteoglycans have been used to study the localization of chondroitin and the two isomeric chondroitin sulfates in developing rat cerebellum. At 1-2 weeks postnatal, unsulfated chondroitin is present in the granule cell layer, molecular layer, and prospective white matter, but there was no staining of the external granule cell layer other than light staining of Bergmann glia fibers. By 3 weeks postnatal, staining of the molecular layer has disappeared and has diminished in the white matter, whereas in adult cerebellum only the granule cell layer remains stained. The staining pattern of chondroitin 4-sulfate is similar to that for chondroitin at 1-2 weeks postnatal, but in contrast to chondroitin, chondroitin 4-sulfate increases in the molecular layer at 3 weeks, and this becomes the most densely stained region of adult cerebellum. Chondroitin 6-sulfate is present predominantly in the prospective white matter of 1-2 week postnatal cerebellum, although significant staining of the granule cell layer is also seen. By 3 weeks postnatal the granule cell staining of chondroitin 6-sulfate has decreased, and in adult cerebellum staining is seen only in the white matter and to a lesser extent in the granule cell layer. Electron microscopy confirmed the presence of chondroitin sulfate in the cytoplasm of neurons and glia of adult brain.(ABSTRACT TRUNCATED AT 250 WORDS)
— id: 14061, year: 1991, vol: 56, page: 1608, stat: Journal Article,

Effects of beta-xylosides on proteoglycan biosynthesis and morphology of PC12 pheochromocytoma cells and primary cultures of rat cerebellum
Margolis RK; Goossen B; Tekotte H; Hilgenberg L; Margolis RU
1991 Jun;99 ( Pt 2)(22):237-246, Journal of cell science
We have examined the effects of beta-xylosides, which act as exogenous acceptors for glycosaminoglycan chain initiation, on the morphology and proteoglycan biosynthesis of PC12 pheochromocytoma cells, and on monolayer, aggregate and explant cultures of early postnatal rat cerebellum. PC12 cells cultured for 13 days in the presence of nerve growth factor (NGF) and beta-xyloside, and labeled during days 11-13 with sodium [35S]sulfate, showed an 8- to 11-fold increase in [35S]sulfate-labeled macromolecules released into the culture medium. Most of the increase was accounted for by chondroitin sulfate, which was in the form of free glycosaminoglycan chains, which were not acid-precipitable. The presence of beta-xyloside also led to a 65-115% increase in [35S]sulfate incorporation into cell-associated glycosaminoglycans and glycoproteins of untreated and NGF-treated PC12 cells, respectively. beta-Xyloside treatment reduced the size of the chondroitin sulfate chains in both the cells and medium from approximately 34,000 to 10,000 Mr, but had much less effect on heparan sulfate, which decreased in size from 16,000 to 13,000-14,500 Mr (in the medium and cells, respectively). beta-Xyloside inhibition of proteoglycan biosynthesis was accompanied by significant morphological effects in NGF-treated PC12 cells, consisting of an increase in length and decrease in the branching, diameter and adhesion to the collagen substratum of the PC12 cell processes. p-Nitrophenyl- and 4-methylumbelliferyl-beta-D-xylosides produced similar effects, which were not seen with p-nitrophenyl-beta-D-galactoside. beta-Xylosides also produced distinct alterations in the adhesion and morphology of monolayer, aggregate, and explant cultures of early postnatal rat cerebellum, which occurred together with inhibition of chondroitin sulfate proteoglycan biosynthesis and a decrease in glycosaminoglycan chain size. These studies indicate that chondroitin sulfate (and probably also heparan sulfate) proteoglycans play a significant role in modulating cell-cell and cell-matrix interactions in nervous tissue development and differentiation
— id: 33485, year: 1991, vol: 99 ( Pt 2), page: 237, stat: Journal Article,

Isolation and characterization of developmentally regulated chondroitin sulfate and chondroitin/keratan sulfate proteoglycans of brain identified with monoclonal antibodies
Rauch U; Gao P; Janetzko A; Flaccus A; Hilgenberg L; Tekotte H; Margolis RK; Margolis RU
1991 Aug 5;266(22):14785-14801, Journal of biological chemistry
A panel of monoclonal antibodies prepared to the chondroitin sulfate proteoglycans of rat brain was used for their immunocytochemical localization and isolation of individual proteoglycan species by immunoaffinity chromatography. One of these proteoglycans (designated 1D1) consists of a major component with an average molecular size of 300 kDa in 7-day brain, containing a 245-kDa core glycoprotein and an average of three 22-kDa chondroitin sulfate chains. A 1D1 proteoglycan of approximately 180 kDa with a 150-kDa core glycoprotein is also present at 7 days, and by 2-3 weeks postnatal this becomes the major species, containing a single 32-kDa chondroitin 4-sulfate chain. The concentration of 1D1 decreases during development, from 20% of the total chondroitin sulfate proteoglycan protein (0.1 mg/g brain) at 7 days postnatal to 6% in adult brain. A 45-kDa protein which is recognized by the 8A4 monoclonal antibody to rat chondrosarcoma link protein copurifies with the 1D1 proteoglycan, which aggregates to a significant extent with hyaluronic acid. A chondroitin/keratan sulfate proteoglycan (designated 3H1) with a size of approximately 500 kDa was isolated from rat brain using monoclonal antibodies to the keratan sulfate chains. The core glycoprotein obtained after treatment of the 3H1 proteoglycan with chondroitinase ABC and endo-beta-galactosidase decreases in size from approximately 360 kDa at 7 days to approximately 280 kDa in adult brain. In 7-day brain, the proteoglycan contains three to five 25-kDa chondroitin 4-sulfate chains and three to six 8.4-kDa keratan sulfate chains, whereas the adult brain proteoglycan contains two to four chondroitin 4-sulfate chains and eight to nine keratan sulfate chains, with an average size of 10 kDa. The concentration of 3H1 increases during development from 3% of the total soluble proteoglycan protein at 7 days to 11% in adult brain, and there is a developmental decrease in the branching and/or sulfation of the keratan sulfate chains. A third monoclonal antibody (3F8) was used to isolate a approximately 500-kDa chondroitin sulfate proteoglycan comprising a 400-kDa core glycoprotein and an average of four 28-kDa chondroitin sulfate chains. In the 1D1 and 3F8 proteoglycans of 7-day brain, 20 and 33%, respectively, of the chondroitin sulfate is 6-sulfated, whereas chondroitin 4-sulfate accounts for greater than 96% of the glycosaminoglycan chains in the adult brain proteoglycans.(ABSTRACT TRUNCATED AT 400 WORDS)
— id: 13941, year: 1991, vol: 266, page: 14785, stat: Journal Article,

Association of the HNK-1 epitope with 5'-nucleotidase from Torpedo marmorata (electric ray) electric organ
Vogel M; Kowalewski HJ; Zimmermann H; Janetzko A; Margolis RU; Wollny HE
1991 Aug 15;278 ( Pt 1)(27):199-202, Biochemical journal
5'-Nucleotidase isolated from the electric organ of the electric ray (Torpedo marmorata) has a molecular mass of 62 kDa and, on two-dimensional electrophoresis, separates into up to 13 isoforms within a pI range of 5.9-6.7. The N-terminal sequence data show a 71% identity over 17 amino acids with that previously published for the rat liver enzyme. All forms of 5'-nucleotidase are recognized by the HNK-1 monoclonal antibody. HNK-1 immunoreactivity is found at the surface of the Schwann-cell processes covering the synaptic terminals and in this respect corresponds to that of 5'-nucleotidase in the same tissue. Since a number of glycoproteins involved in cell recognition and cell adhesion carry the HNK-1 epitope, 5'-nucleotidase may play a role in cell-cell or cell-extracellular matrix interaction in addition to its activity as an enzyme
— id: 33484, year: 1991, vol: 278 ( Pt 1), page: 199, stat: Journal Article,

Chromaffin granule and PC12 cell chondroitin sulfate proteoglycans and their relation to chromogranin A
Gowda DC; Hogue-Angeletti R; Margolis RK; Margolis RU
1990 Sep;281(2):219-224, Archives of biochemistry & biophysics. ABB
Two major proteoglycans, which appear to be structurally closely related, were isolated from bovine chromaffin granule matrix proteins by ion-exchange chromatography. On sodium dodecyl sulfate-polyacrylamide gel electrophoresis they have apparent average molecular sizes of 35-40 kDa (range of 23-75 kDa) and generate a 14-kDa core glycoprotein after chondroitinase treatment. Previous studies demonstrated that these two major chromaffin granule proteoglycans are very similar in terms of their peptide mapping patterns and carbohydrate composition (having a high proportion of tri- and tetraantennary N-glycosidic oligosaccharides, and O-glycosidic oligosaccharides consisting predominantly of disialyl derivatives of galactosyl(beta 1-3)N-acetylgalactosamine), and that they differed in these respects from the chromogranins. By using antisera to five synthetic peptide fragments of chromogranin A to stain immunoblots of purified chromaffin granule proteoglycans before and after chondroitinase treatment, we have now shown that these major proteoglycans are not immunochemically related to chromogranin A. However, it has recently been reported that some chromogranin A-immunoreactive material disappears after chondroitinase treatment, and our studies demonstrate that approximately 1-2% of the chromogranin A occurs in the form of a 110-kDa proteoglycan, which is converted to a 95-kDa core glycoprotein after chondroitinase treatment. Similar chromogranin A proteoglycans could be detected in rat PC12 pheochromocytoma cells, where they have a molecular size of 115-145 kDa and yield a 105-kDa core protein after chondroitinase treatment. Studies using antibodies to synthetic peptide fragments of chromogranin B (secretogranin I) did not provide any evidence that this related protein occurs in a proteoglycan form
— id: 33486, year: 1990, vol: 281, page: 219, stat: Journal Article,

Chondroitin sulfate and heparan sulfate proteoglycans of PC12 pheochromocytoma cells
Gowda DC; Goossen B; Margolis RK; Margolis RU
1989 Jul 5;264(19):11436-11443, Journal of biological chemistry
We have isolated and characterized the cell-associated and secreted proteoglycans synthesized by a clonal line of rat adrenal medullary PC12 pheochromocytoma cells, which have been extensively employed for the study of a wide variety of neurobiological processes. Chondroitin sulfate accounts for 70-80% of the [35S] sulfate-labeled proteoglycans present in PC12 cells and secreted into the medium. Two major chondroitin sulfate proteoglycans were detected with molecular sizes of 45,000-100,000 and 120,000-190,000, comprising 14- and 105-kDa core proteins and one or two chondroitin sulfate chains with an average molecular size of 34 kDa. In contrast to the chondroitin sulfate proteoglycans, one major heparan sulfate proteoglycan accounts for most of the remaining 20-30% of the [35S] sulfate-labeled proteoglycans present in the PC12 cells and medium. It has a molecular size of 95,000-170,000, comprising a 65-kDa core protein and two to six 16-kDa heparan sulfate chains. Both the chondroitin sulfate and heparan sulfate proteoglycans also contain O-glycosidically linked oligosaccharides (25-28% of the total oligosaccharides) and predominantly tri- and tetraantennary N-glycosidic oligosaccharides. Proteoglycans produced by the original clone of PC12 cells were compared with those of two other PC12 cell lines (B2 and F3) that differ from the original clone in morphology, adhesive properties, and response to nerve growth factor. Although the F3 cells (a mutant line derived from B2 and reported to lack a cell surface heparan sulfate proteoglycan) do not contain a large molecular size heparan sulfate proteoglycan species, there was no significant difference between the B2 and F3 cells in the percentage of total heparan sulfate released by mild trypsinization, and both the B2 and F3 cells synthesized cell-associated and secreted chondroitin sulfate and heparan sulfate proteoglycans having properties very similar to those of the original PC12 cell line but with a reversed ratio (35:65) of chondroitin sulfate to heparan sulfate
— id: 10547, year: 1989, vol: 264, page: 11436, stat: Journal Article,

Relation of the amyloid beta protein precursor to heparan sulfate proteoglycans
Gowda DC; Margolis RK; Frangione B; Ghiso J; Larrondo-Lillo M; Margolis RU
1989 May 19;244(4906):826-828, Science
— id: 9434, year: 1989, vol: 244, page: 826, stat: Journal Article,

Presence of the HNK-1 epitope on poly(N-acetyllactosaminyl) oligosaccharides and identification of multiple core proteins in the chondroitin sulfate proteoglycans of brain
Gowda DC; Margolis RU; Margolis RK
1989 May 16;28(10):4468-4474, Biochemistry
The chondroitin sulfate proteoglycans of brain contain several core proteins bearing HNK-1 antibody epitopes. Endo-beta-galactosidase treatment resulted in the almost complete disappearance of HNK-1 staining of proteoglycan immunoblots, indicating that a significant portion of the 3-sulfated sugar residues recognized by this antibody are present on poly(N-acetyllactosaminyl) oligosaccharides. However, after treatment with chondroitinase ABC followed by endo-beta-galactosidase, several proteoglycan species showed HNK-1 reactivity, presumably due to the presence of this epitope on other oligosaccharides which are both resistant to endo-beta-galactosidase and inaccessible to the antibody in the native proteoglycan. Immunostaining of the endo-beta-galactosidase degradation products after separation by thin-layer chromatography demonstrated that HNK-1 reactivity was confined to a minor population of large oligosaccharides. Only a relatively small portion of the native chondroitin sulfate proteoglycans of brain enter a 6-12% SDS-polyacrylamide gel. However, after treatment of the proteoglycans with chondroitinase ABC (or chondroitinase and endo-beta-galactosidase) in the presence of protease inhibitors, seven bands with molecular sizes ranging from 80 to 200 kDa appear in Coomassie Blue stained gels, and two additional bands with molecular sizes of 67 and 350-400 kDa are apparent in fluorographs of sodium [35S]sulfate labeled proteoglycans. Most of these components probably represent individual proteoglycan species rather than different degrees of nonchondroitin sulfate/keratan sulfate glycosylation of a single protein core, since [35S]methionine-labeled proteins of comparable molecular size were synthesized by an in vitro translation system. These findings suggest that chondroitin sulfate proteoglycans which differ in molecular size and composition may be specific to particular cell types in brain
— id: 10618, year: 1989, vol: 28, page: 4468, stat: Journal Article,

Structure and localization of glycoproteins and proteoglycans
Margolis RK; Margolis RU
Neurobiology of glycoconjugates New York : Plenum Press, 1989,
— id: 2846, year: 1989, vol: , page: 85, stat: Chapter,

Nervous tissue proteoglycans
Margolis RU; Margolis RK
1989 ;11(4-5):276-288, Developmental neuroscience
— id: 10844, year: 1989, vol: 11, page: 276, stat: Journal Article,

Properties of nervous tissue proteoglycans relevant to studies on Alzheimer's disease
Margolis RU; Margolis RK
1989 Sep-Oct;10(5):500-502, Neurobiology of aging
Our comments concern certain properties of nervous tissue proteoglycans which were not emphasized in the review by Snow and Wight, with particular attention to the proposed relation of the amyloid beta protein precursor to a heparan sulfate proteoglycan core protein
— id: 10516, year: 1989, vol: 10, page: 500, stat: Journal Article,

Neurobiology of glycoconjugates
Margolis, Renee K.; Margolis, Richard U
New York : Plenum Press, c1989,
— id: 87, year: 1989, vol: , page: , stat: ,

Structural properties of the heparan sulfate proteoglycans of brain
Ripellino JA; Margolis RU
1989 Mar;52(3):807-812, Journal of neurochemistry
The heparan sulfate proteoglycans present in a deoxycholate extract of rat brain were purified by ion exchange chromatography, affinity chromatography on lipoprotein lipase agarose, and gel filtration. Heparitinase treatment of the heparan sulfate proteoglycan fraction (containing 86% heparan sulfate and 10% chondroitin sulfate) that was eluted from the lipoprotein lipase affinity column with 1 M NaCl led to the appearance of a major protein core with a molecular size of 55,000 daltons, as determined by sodium dodecyl sulfate-polyacrylamide gel electrophoresis. Comparison of the effects of heparinase and heparitinase treatment revealed that the heparan sulfate proteoglycans of brain contain a significant proportion of relatively short N-sulfoglucosaminyl 6-O-sulfate [or N-sulfoglucosaminyl](alpha 1-4)iduronosyl 2-O-sulfate(alpha 1-4) repeating units and that the portions of the heparan sulfate chains in the vicinity of the carbohydrate-protein linkage region are characterized by the presence of D-glucuronic acid rather than L-iduronic acid. After chondroitinase treatment of a proteoglycan fraction that contained 62% chondroitin sulfate and 21% heparan sulfate (eluted from lipoprotein lipase with 0.4 M NaCl), the charge and density of a portion of the heparan sulfate-containing proteoglycans decreased significantly. These results indicate that a population of 'hybrid' brain proteoglycans exists that contain both chondroitin sulfate and heparan sulfate chains covalently linked to a common protein core
— id: 10715, year: 1989, vol: 52, page: 807, stat: Journal Article,

Immunoelectron microscopic localization of hyaluronic acid-binding region and link protein epitopes in brain
Ripellino JA; Margolis RU; Margolis RK
1989 May;108(5):1899-1907, Journal of cell biology
The 1C6 monoclonal antibody to the hyaluronic acid-binding region weakly stained a 65-kD component in immunoblots of the chondroitin sulfate proteoglycans of brain, and the 8A4 monoclonal antibody, which recognizes two epitopes in the polypeptide portion of link protein, produced strong staining of a 45-kD component present in the brain proteoglycans. These antibodies were utilized to examine the localization of hyaluronic acid-binding region and link protein epitopes in rat cerebellum. Like the chondroitin sulfate proteoglycans themselves and hyaluronic acid, hyaluronic acid-binding region and link protein immunoreactivity changed from a predominantly extracellular to an intracellular (cytoplasmic and intra-axonal) location during the first postnatal month of brain development. The cell types which showed staining of hyaluronic acid-binding region and link protein, such as granule cells and their axons (the parallel fibers), astrocytes, and certain myelinated fibers, were generally the same as those previously found to contain chondroitin sulfate proteoglycans and hyaluronic acid. Prominent staining of some cell nuclei was also observed. In agreement with earlier conclusions concerning the localization of hyaluronic acid and chondroitin sulfate proteoglycans, there was no intracellular staining of Purkinje cells or nerve endings or staining of certain other structures, such as oligodendroglia and synaptic vesicles. The similar localizations and coordinate developmental changes of chondroitin sulfate proteoglycans, hyaluronic acid, hyaluronic acid-binding region, and link protein add further support to previous evidence for the unusual cytoplasmic localization of these proteoglycans in mature brain. Our results also suggest that much of the chondroitin sulfate proteoglycan of brain may exist in the form of aggregates with hyaluronic acid
— id: 10667, year: 1989, vol: 108, page: 1899, stat: Journal Article,

Oligosaccharide composition, localization, and developmental changes of a CNS-specific (F3-87-8) glycoprotein
Ripellino JA; Margolis RU; Margolis RK
1989 Oct;53(4):1103-1108, Journal of neurochemistry
The F3-87-8 glycoprotein was isolated from rat brain by immunoaffinity chromatography after biosynthetic labeling by intracerebral administration of [3H]glucosamine, and the oligosaccharide composition of pronase-derived glycopeptides was determined by sequential lectin affinity chromatography and alkali treatment. Triantennary complex oligosaccharides (65%) and O-glycosidic oligosaccharides (18%) were the predominant types present, accompanied by 7-10% each of biantennary and high-mannose oligosaccharides. Twenty-two percent of the complex oligosaccharides had a fucose residue linked to the proximal N-acetylglucosamine of the chitobiose units. No poly(N-acetyllactosaminyl) or hybrid oligosaccharides were detected. Immunocytochemical studies on the localization of this glycoprotein in developing rat brain demonstrated that in 1-week postnatal cerebellum, there is light staining of the internal granule cell layer and surrounding the Purkinje cells. By 2 weeks, an intense staining of myelinating fiber tracts appears, accompanied by much lighter staining in the granule cell layer and at the base of the molecular layer. Staining of the white matter remains strong at 3 weeks postnatal, together with significant staining throughout the molecular layer, and then decreases in both areas by 1 month. In adult brain there is relatively uniform staining of approximately equal intensity in the white matter, granule cell layer, and molecular layer, whereas the Purkinje cell bodies appear unstained throughout development. In agreement with a previously reported immunochemical analysis, no staining was seen in other tissues, confirming the CNS-specific localization of this glycoprotein
— id: 10469, year: 1989, vol: 53, page: 1103, stat: Journal Article,

Hyaluronate accumulation in human epidermis treated with retinoic acid in skin organ culture
Tammi R; Ripellino JA; Margolis RU; Maibach HI; Tammi M
1989 Mar;92(3):326-332, Journal of investigative dermatology
Retinoic acid (RA) has been shown to retard the differentiation of epidermal keratinocytes by several morphologic and biochemical criteria. In this study, the epidermal content and localization of hyaluronate (HA), as well as its synthesis and disappearance in human skin organ culture, were characterized to test the idea that some of the RA influences on epidermal differentiation are associated with keratinocyte HA metabolism. RA stimulated the incorporation of 3H-glucosamine into HA by up to 60% at concentrations between 50 nM and 5 microM, while pulse-chase experiments revealed little change in its disappearance rate from epidermis. After 5 d in culture, the chemically quantified HA was more than doubled in the treated epidermis. The accumulation of HA was substantiated by light and electron microscopy with a specific probe prepared from the HA binding region of cartilage proteoglycan. The staining was particularly enhanced between the upper spinous cell layers, where the terminal differentiation into corneocytes normally takes place. A patchy, discontinuous staining was also seen in stratum granulosum and corneum layers, which are not stained at all in control cultures. The present study demonstrates that RA leads to an accumulation of HA in the superficial layers of epidermis by stimulating its synthesis in keratinocytes. This may account for the delay in terminal differentiation, and the weakened cohesion of the keratinocytes previously observed both in vivo and vitro
— id: 33487, year: 1989, vol: 92, page: 326, stat: Journal Article,

Lipid composition of PC12 pheochromocytoma cells: characterization of globoside as a major neutral glycolipid
Ariga T; Macala LJ; Saito M; Margolis RK; Greene LA; Margolis RU; Yu RK
1988 Jan 12;27(1):52-58, Biochemistry
We have studied the lipid composition of PC12 pheochromocytoma cells cultured in the presence and absence of nerve growth factor (NGF). Neutral and acidic lipid fractions were isolated by column chromatography on DEAE-Sephadex and analyzed by high-performance thin-layer chromatography (HPTLC). The total lipid concentration was approximately 220 micrograms/mg of protein, and the concentration of neutral glycolipids was 1.6-1.8 microgram/mg of protein for both NGF-treated and untreated cells. The neutral glycolipid fraction contained a major component, which accounted for approximately 80% of the total and which was characterized as globoside on the basis of HPTLC mobility, carbohydrate analysis, fast atom bombardment mass spectrometry, and mild acid hydrolysis. The major fatty acids of globoside were C16:0 (10%), C18:0 (16%), C22:0 (23%), C24:1 (17%), and C24:0 (24%). C18 sphingenine accounted for almost all of the long-chain bases. The other neutral glycolipids were tentatively identified as glucosylceramide (15%), lactosylceramide (4%), and globotriosylceramide (4.5%). The concentration of ganglioside sialic acid was approximately 0.34 and 0.18 microgram/mg of protein for cells grown in the presence and absence of NGF, respectively. Although there was an increase in ganglioside concentration in NGF-treated cells, NGF did not produce any differential effects on the relative proportions of the individual gangliosides. Several of the gangliosides appear to contain fucose, and one of these was tentatively identified as fucosyl-GM1. Brain-type gangliosides of the ganglio series were also detected by an HPTLC-immunostaining method. However, the fatty acid and long chain base compositions of PC12 cell gangliosides (and their TLC mobility) differ from those of brain gangliosides.(ABSTRACT TRUNCATED AT 250 WORDS)
— id: 33490, year: 1988, vol: 27, page: 52, stat: Journal Article,

Phosphatidylinositol-anchored glycoproteins of PC12 pheochromocytoma cells and brain
Margolis RK; Goossen B; Margolis RU
1988 May 3;27(9):3454-3458, Biochemistry
PC12 pheochromocytoma cells and cultures of early postnatal rat cerebellum were labeled with [3H]glucosamine, [3H]fucose, [3H]leucine, [3H]ethanolamine, or sodium [35S]sulfate and treated with a phosphatidylinositol-specific phospholipase C. Enzyme treatment of [3H]glucosamine- or [3H]fucose-labeled PC12 cells led to a 15-fold increase in released glycoproteins. On sodium dodecyl sulfate-polyacrylamide gel electrophoresis, most of the released material migrated as a broad band with an apparent molecular size of 32,000 daltons (Da), which was specifically immunoprecipitated by a monoclonal antibody to the Thy-1 glycoprotein. A second glycoprotein, with an apparent molecular size of 158,000 Da, was also released. After treatment with endo-beta-galactosidase, 40-45% of the [3H]glucosamine or [3H]fucose radioactivity in the phospholipase-released glycoproteins was converted to products of disaccharide size, and the molecular size of the 158-kDa glycoprotein decreased to 145 kDa, demonstrating that it contains fucosylated poly-(N-acetyllactosaminyl) oligosaccharides. The phospholipase also released labeled Thy-1 and the 158-kDa glycoprotein from PC12 cells cultured in the presence of [3H]ethanolamine, which specifically labels this component of the phosphatidylinositol membrane-anchoring sequence, while in the lipid-free protein residue of cells not treated with phospholipase, Thy-1 and a doublet at 46/48 kDa were the only labeled proteins. At least eight early postnatal rat brain glycoproteins also appear to be anchored to the membrane by phosphatidylinositol. Sulfated glycoproteins of 155, 132/134, 61, and 21 kDa are the predominant species released by phospholipase, which does not affect a major 44-kDa protein seen in [3H]ethanolamine-labeled brain cultures. The 44-48- and 155/158-kDa proteins may be common to both PC12 cells and brain
— id: 33488, year: 1988, vol: 27, page: 3454, stat: Journal Article,

Structural properties of neuronal surface macromolecules
Margolis RU
Current issues in neural regeneration research New York : Liss, 1988,
— id: 2845, year: 1988, vol: , page: 189, stat: Chapter,

Poly(N-acetyllactosaminyl) oligosaccharides of chromaffin granule membrane glycoproteins
Margolis RU; Fischer-Colbrie R; Margolis RK
1988 Dec;51(6):1819-1824, Journal of neurochemistry
Poly(N-acetyllactosaminyl) oligosaccharides have been identified, on the basis of their susceptibility to endo-beta-galactosidase, in a large-molecular-size glycopeptide fraction derived from chromaffin granule membrane glycoproteins. The glycoproteins containing poly(N-acetyl-lactosaminyl) oligosaccharides were selectively labeled by treatment of chromaffin granule membranes with endo-beta-galactosidase to expose N-acetylglucosamine residues, followed by incubation with galactosyltransferase and UDP-[14C]galactose. Sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) and fluorography demonstrated specific labeling in the 41-47 kilodalton (kD) region and in a distinct band at 90 kDa. Two-dimensional SDS-PAGE revealed that the poly(N-acetyllactosaminyl) oligosaccharides are predominantly present in glycoprotein IV, together with lesser labeling of glycoproteins II and III, whereas they are absent from dopamine beta-hydroxylase and carboxypeptidase H, which are the major glycoproteins of chromaffin granule membranes
— id: 10874, year: 1988, vol: 51, page: 1819, stat: Journal Article,

Light and electron microscopic studies on the localization of hyaluronic acid in developing rat cerebellum
Ripellino JA; Bailo M; Margolis RU; Margolis RK
1988 Mar;106(3):845-855, Journal of cell biology
The hyaluronic acid-binding region was prepared by trypsin digestion of chondroitin sulfate proteoglycan aggregate from the Swarm rat chondrosarcoma, and biotinylated in the presence of hyaluronic acid and link protein. After isolation by gel filtration and HPLC in 4 M guanidine HCl, the biotinylated hyaluronic acid-binding region was used, in conjunction with avidin-peroxidase, as a specific probe for the light and electron microscopic localization of hyaluronic acid in developing and mature rat cerebellum. At 1 w postnatal, there is strong staining of extracellular hyaluronic acid in the presumptive white matter, in the internal granule cell layer, and as a dense band at the base of the molecular layer, surrounding the parallel fibers. This staining moves progressively towards the pial surface during the second postnatal week, and extracellular staining remains predominant through postnatal week three. In adult brain, there is no significant extracellular staining of hyaluronic acid, which is most apparent in the granule cell cytoplasm, and intra-axonally in parallel fibers and some myelinated axons. The white matter is also unstained in adult brain, and no staining was seen in Purkinje cell bodies or dendrites at any age. The localization of hyaluronic acid and its developmental changes are very similar to that previously found in immunocytochemical studies of the chondroitin sulfate proteoglycan in nervous tissue (Aquino, D. A., R. U. Margolis, and R. K. Margolis. 1984. J. Cell Biol. 99:1117-1129; Aquino, D. A., R. U. Margolis, and R. K. Margolis. J. Cell Biol. 99:1130-1139), and to recent results from studies using monoclonal antibodies to the hyaluronic acid-binding region and link protein. The presence of brain hyaluronic acid in the form of aggregates with chondroitin sulfate proteoglycans would be consistent with their similar localizations and coordinate developmental changes
— id: 11179, year: 1988, vol: 106, page: 845, stat: Journal Article,

Localization of epidermal hyaluronic acid using the hyaluronate binding region of cartilage proteoglycan as a specific probe
Tammi R; Ripellino JA; Margolis RU; Tammi M
1988 Mar;90(3):412-414, Journal of investigative dermatology
Hyaluronate is actively synthesized by cultured epidermis and dermis, but no direct histological data have been available about its localization in normal human skin. A hyaluronate-specific biotinylated probe, prepared from the hyaluronate binding region of cartilage proteoglycan, was applied to human skin sections and visualized using the biotin-avidin-peroxidase system. The specificity of this staining was confirmed by hyaluronidase predigestion and by hyaluronate-derived oligosaccharides added to the staining solution. All dermis showed diffuse binding of the probe, but the highest staining intensity was observed in the epidermal intercellular spaces. The stainability extended from basal cells to the middle layers of the epidermis, whereas the granular layer and stratum corneum were completely negative. Also, the basal side of basal cells (basement membrane) did not bind the hyaluronate probe. The abundance of hyaluronate on surfaces and intercellular spaces of the spinous cells is suggested to have an important role in the physiology of human epidermis
— id: 33489, year: 1988, vol: 90, page: 412, stat: Journal Article,

Structural studies on sialylated and sulphated O-glycosidic mannose-linked oligosaccharides in the chondroitin sulphate proteoglycan of brain
Krusius T; Reinhold VN; Margolis RK; Margolis RU
1987 Jul 1;245(1):229-234, Biochemical journal
We have previously described the structures of neutral and sialylated O-glycosidic mannose-linked tetrasaccharides and keratan sulphate polysaccharide chains in the chondroitin sulphate proteoglycan of brain. The present paper provides information on a series of related sialylated and/or sulphated tri- to penta-saccharides released by alkaline-borohydride treatment of the proteoglycan glycopeptides. The oligosaccharides were fractionated by ion-exchange chromatography and gel filtration, and their structural properties were studied by methylation analysis and fast-atom-bombardment mass spectrometry. Five fractions containing [35S]sulphate-labelled oligosaccharides were obtained by ion-exchange chromatography, each of which was eluted from Sephadex G-50 as two well-separated peaks. The apparent Mr values of both the large- and small-molecular-size fractions increased with increasing acidity (and sulphate labelling) of the oligosaccharides. The larger-molecular-size fractions contained short mannose-linked keratan sulphate chains of Mr 3000-4500, together with some asparagine-linked oligosaccharides. The smaller tri- to penta-saccharides, of Mr 800-1400, appear to have a common GlcNac(beta 1-3)Manol core, and to contain one to two residues of sialic acid and/or sulphate
— id: 33492, year: 1987, vol: 245, page: 229, stat: Journal Article,

Occurrence of the HNK-1 epitope (3-sulfoglucuronic acid) in PC12 pheochromocytoma cells, chromaffin granule membranes, and chondroitin sulfate proteoglycans
Margolis RK; Ripellino JA; Goossen B; Steinbrich R; Margolis RU
1987 Jun 30;145(3):1142-1148, Biochemical & biophysical research communications
After biosynthetic labeling of sulfated glycoproteins in rat and goldfish brain and PC12 pheochromocytoma cells with sodium [35S]sulfate, it was observed that all of the bands reactive with the HNK-1 antibody on immunoblots of sodium dodecyl sulfate-polyacrylamide gels corresponded with sulfate-labeled proteins detected by fluorography. These results support data from other studies, which indicate that the HNK-1 epitope is a 3-sulfo-glucuronic acid residue. In addition to its presence in a wide range of nervous tissue glycoproteins, the HNK-1 epitope was also detected in chromaffin granule membranes, chondroitinase ABC, and in chondroitin sulfate proteoglycans of brain, cartilage, and chondrosarcoma. However, it is not present in the heparan sulfate proteoglycan of brain, or in either of two chondroitin sulfate/dermatan sulfate proteoglycans in the chromaffin granule matrix
— id: 33493, year: 1987, vol: 145, page: 1142, stat: Journal Article,

Effects of nerve growth factor-induced differentiation on the heparan sulfate of PC12 pheochromocytoma cells and comparison with developing brain
Margolis RK; Salton SR; Margolis RU
1987 Aug 15;257(1):107-114, Archives of biochemistry & biophysics. ABB
We have examined the size, charge, and sulfation pattern of heparan sulfate in the cell-soluble fraction, membranes, and culture medium of PC12 pheochromocytoma cells cultured in the presence and absence of nerve growth factor (NGF) and compared the structural features of PC12 cell heparan sulfate to that of rat brain at several stages of early postnatal development. Nitrous acid degradation studies revealed significant differences in the distribution of N-sulfate and N-acetyl groups in heparan sulfate present in the PC12 cell-soluble fraction, membranes, and medium and demonstrated that NGF treatment led to an increased proportion of N-sulfated segments in the cell-associated heparan sulfate, although no such change was seen in that released into the culture medium. There was very little change in the N-sulfation of brain heparan sulfate during the first 30 days after birth. In brain, most of the heparan sulfate glucosamine residues are N-sulfated and yield predominantly di- and tetrasaccharide nitrous acid degradation products, whereas PC12 cell heparan sulfate contains large blocks of N-acetylglucosamine residues. There was very little difference in the overall charge or size (approximately 15,000 Da) of heparan sulfate chains between the different PC12 cell fractions or brain, although NGF treatment led to a decrease in the proportion of less-charged chains in the PC12 cell membranes and a small increase in molecular size. Our studies therefore demonstrate the presence in PC12 cells of several pools of heparan sulfate having different structural properties, and that significant alterations in the charge, size, and sulfation pattern of PC12 cell heparan sulfate accompany NGF-induced differentiation and neurite outgrowth
— id: 33491, year: 1987, vol: 257, page: 107, stat: Journal Article,

Complex carbohydrate composition of large dense-cored vesicles from sympathetic nerve
Margolis RU; Ledeen RW; Sbaschnig-Agler M; Byrne MC; Klein RL; Douglas BH 2d; Margolis RK
1987 Dec;49(6):1839-1844, Journal of neurochemistry
Highly purified noradrenergic, large, dense-cored vesicles were isolated from bovine sympathetic nerve endings by sucrose-D2O density gradient centrifugation. Their concentration of glycoprotein hexosamine and sialic acid was 6.6 and 3.9 mumol/100 mg lipid-free dry weight, respectively, values which are similar to those previously found in bovine chromaffin granules. However, whereas chromaffin granule glycoproteins are characterized by their high proportion of N-acetylgalactosamine-containing O-glycosidically-linked oligosaccharides (present in the chromogranins), such oligosaccharides accounted for only 17% of those in noradrenergic synaptic vesicle glycoproteins. Fractionation of N-3H-acetylated glycopeptides by sequential lectin affinity chromatography demonstrated that approximately two-thirds of the oligosaccharides were of the tri- and tetraantennary complex type, accompanied by 14% biantennary oligosaccharides and 3% high-mannose oligosaccharides. The vesicles had a relatively low concentration of chondroitin sulfate (less than 5% of that in chromaffin granules) but significant amounts of heparan sulfate (0.4 mumol N-acetylglucosamine/100 mg lipid-free dry weight). No hyaluronic acid was detected. The concentration of ganglioside sialic acid in the noradrenergic vesicles was approximately 1 mumol/100 mg lipid-free dry weight, which is significantly higher than that of a crude membrane mixture from which the vesicles were prepared; the ratio of N-acetyl- to N-glycolylneuraminic acid was 0.8. Several molecular species of gangliosides were detected by thin-layer chromatography, but most of these did not exactly comigrate with bovine brain gangliosides. Cholera toxin binding indicated that approximately half or less of the gangliosides belong to the gangliotetraose series
— id: 11301, year: 1987, vol: 49, page: 1839, stat: Journal Article,

Cell surface and extracellular matrix glycoproteins and proteoglycans in nervous tissue
Margolis RU; Ripellino JA; Margolis RK
Mesenchymal-epithelial interactions in neural development Berlin : Springer-Verlag, 1987,
— id: 2878, year: 1987, vol: , page: 65, stat: Chapter,

Identification of an O-glycosidic mannose-linked sialylated tetrasaccharide and keratan sulfate oligosaccharides in the chondroitin sulfate proteoglycan of brain
Krusius T; Finne J; Margolis RK; Margolis RU
1986 Jun 25;261(18):8237-8242, Journal of biological chemistry
The chondroitin sulfate proteoglycan of rat brain was digested with Pronase, and after removal of glycosaminoglycans, the resulting glycopeptides were treated with alkaline borohydride to release O-glycosidically linked oligosaccharides. These were fractionated by ion exchange chromatography, gel filtration, and preparative thin layer chromatography, and their structural properties were studied by specific enzymatic degradations, methylation analysis, and gas-liquid chromatography-mass spectrometry of disaccharides as their trimethylsilylated and permethylated derivatives. In addition to the previously characterized N-acetyl-galactosamine-linked oligosaccharides and neutral mannitol-containing oligosaccharides [GlcNAc(beta 1-3) Manol and Gal(beta 1-4)[Fuc(alpha 1-3)]GlcNAc(beta 1-3)Manol] (where Fuc is fucose), we have now identified the sialylated tetrasaccharide NeuAc(alpha 2-3)Gal(beta 1-4)GlcNAc (beta 1-3)Manol, which accounts for approximately 20% of the mannitol-containing oligosaccharides. The proteoglycan also contains mannose-linked keratan sulfate chains (with a molecular size of 3,000 to 10,000 Da) composed of disaccharide repeating units consisting of Gal(beta 1-4)GlcNAc-6-O-SO4(beta 1-3), with a small proportion of branch points at C-6 of galactose residues. There is approximately one keratan sulfate chain per four chondroitin sulfate chains of 18,000-19,000 Da. After alkaline borohydride treatment of the neutral and monosialyl glycopeptide fractions, the combined decrease in mannose and N-acetylgalactosamine was very close to the observed destruction of serine + threonine and was accompanied by an equimolar increase in alanine and alpha-aminobutyric acid. One half of the mannose was destroyed by alkaline borohydride treatment of the glycopeptides and stoichiometrically converted to mannitol, while there were only small changes in the relative amounts of the other sugars and amino acids. The data demonstrate that over half of the carbohydrate-peptide linkages in the proteoglycan are of the mannosyl-O-serine/threonine type
— id: 33494, year: 1986, vol: 261, page: 8237, stat: Journal Article,

Poly(N-acetyllactosaminyl) oligosaccharides in glycoproteins of PC12 pheochromocytoma cells and sympathetic neurons
Margolis RK; Greene LA; Margolis RU
1986 Jun 3;25(11):3463-3468, Biochemistry
Endo-beta-galactosidase treatment of glycopeptides derived from the trypsinate and membranes of PC12 pheochromocytoma cells and cultured sympathetic neurons demonstrated the presence of poly(N-acetyllactosaminyl) units on tri- and tetraantennary oligosaccharides, some of which have a core fucose residue and a 2,6-substituted alpha-linked mannose residue. Nerve growth factor induced differentiation of the PC12 cells led to a small but significant decrease in the proportion of these oligosaccharides. Poly(N-acetyllactosaminyl) oligosaccharides were also identified in a major 230 000-Da cell-surface glycoprotein (the nerve growth factor inducible large external, or NILE, glycoprotein) of PC12 cells and appear to account for much or all of the difference in size between this glycoprotein as compared to the immunochemically cross-reactive 205 000-Da species present in postnatal brain. Glycoproteins containing poly(N-acetyllactosaminyl) oligosaccharides were selectively labeled by treatment of PC12 cells with endo-beta-galactosidase to expose N-acetylglucosamine residues, followed by incubation with galactosyltransferase and UDP-[14C]galactose. Sodium dodecyl sulfate-polyacrylamide gel electrophoresis and fluorography revealed the presence of a number of distinct PC12 cell glycoproteins that contain these oligosaccharides and have apparent molecular weights in the range of 25 000-250 000. Treatment of PC12 cells with nerve growth factor (NGF) altered the relative labeling of several of the glycoprotein bands, with a time course similar to the effects of NGF on neurite outgrowth.(ABSTRACT TRUNCATED AT 250 WORDS)
— id: 33495, year: 1986, vol: 25, page: 3463, stat: Journal Article,

Structure and localization of nervous tissue proteoglycans
Margolis RU; Aquino DA; Klinger MM; Ripellino JA; Margolis RK
1986 ;481(11):46-54, Annals of the New York Academy of Sciences
— id: 33496, year: 1986, vol: 481, page: 46, stat: Journal Article,

Isolation and characterization of the heparan sulfate proteoglycans of brain. Use of affinity chromatography on lipoprotein lipase-agarose
Klinger MM; Margolis RU; Margolis RK
1985 Apr 10;260(7):4082-4090, Journal of biological chemistry
Heparan sulfate proteoglycans were extracted from rat brain microsomal membranes or whole forebrain with deoxycholate and purified from accompanying chondroitin sulfate proteoglycans and membrane glycoproteins by ion-exchange chromatography, affinity chromatography on lipoprotein lipase-Sepharose, and gel filtration. The proteoglycan has a molecular size of approximately 220,000, containing glycosaminoglycan chains of Mr = 14,000-15,000. In [3H]glucosamine-labeled heparan sulfate proteoglycans, approximately 22% of the radioactivity is present in glycoprotein oligosaccharides, consisting predominantly of N-glycosidically linked tri- and tetraantennary complex oligosaccharides (60%, some of which are sulfated) and O-glycosidic oligosaccharides (33%). Small amounts of chondroitin sulfate (4-6% of the total glycosaminoglycans) copurified with the heparan sulfate proteoglycan through a variety of fractionation procedures. Incubation of [35S]sulfate-labeled microsomes with heparin or 2 M NaCl released approximately 21 and 13%, respectively, of the total heparan sulfate, as compared to the 8-9% released by buffered saline or chondroitin sulfate and the 82% which is extracted by 0.2% deoxycholate. It therefore appears that there are at least two distinct types of association of heparan sulfate proteoglycans with brain membranes
— id: 33498, year: 1985, vol: 260, page: 4082, stat: Journal Article,

The hyaluronic acid binding region as a specific probe for the localization of hyaluronic acid in tissue sections. Application to chick embryo and rat brain
Ripellino JA; Klinger MM; Margolis RU; Margolis RK
1985 Oct;33(10):1060-1066, Journal of histochemistry & cytochemistry
The hyaluronic acid binding region was prepared by clostripain digestion of chondroitin sulfate proteoglycan isolated from the Swarm rat chondrosarcoma, and biotinylated in the presence of associated hyaluronic acid and link protein. After removal of hyaluronic acid by gel filtration in 4 M guanidine HCl, the biotinylated binding region-link protein complex was used as a specific histochemical probe in conjunction with avidin-peroxidase. Its utility was initially evaluated by comparison with Alcian blue staining of the axial region of 2 to 5 day chick embryos, where staining was seen in the dorsolateral area between the neural tube and the ectoderm, in the perichordal mesenchyme, and in developing limb buds. Light and electron microscopic studies of early postnatal rat cerebellum indicate that hyaluronic acid is primarily localized in the extracellular space of immature brain. Staining specificity was demonstrated by the ability of hyaluronic acid oligosaccharides of appropriate size to block the staining reaction, and by the absence of staining after treatment of tissue sections with protease-free Streptomyces hyaluronidase, which degrades only this glycosaminoglycan
— id: 33497, year: 1985, vol: 33, page: 1060, stat: Journal Article,

Immunocytochemical localization of a chondroitin sulfate proteoglycan in nervous tissue. I. Adult brain, retina, and peripheral nerve
Aquino DA; Margolis RU; Margolis RK
1984 Sep;99(3):1117-1129, Journal of cell biology
Monospecific antibodies were prepared to a previously characterized chondroitin sulfate proteoglycan of brain and used in conjunction with the peroxidase-antiperoxidase technique to localize the proteoglycan by immunoelectron microscopy. The proteoglycan was found to be exclusively intracellular in adult cerebellum, cerebrum, brain stem, and spinal cord. Some neurons and astrocytes (including Golgi epithelial cells and Bergmann fibers) showed strong cytoplasmic staining. Although in the central nervous system there was heavy axoplasmic staining of many myelinated and unmyelinated fibers, not all axons stained. Staining was also seen in retinal neurons and glia (ganglion cells, horizontal cells, and Muller cells), but several central nervous tissue elements were consistently unstained, including Purkinje cells, oligodendrocytes, myelin, optic nerve axons, nerve endings, and synaptic vesicles. In sympathetic ganglion and peripheral nerve there was no staining of neuronal cell bodies, axons, myelin, or Schwann cells, but in sciatic nerve the Schwann cell basal lamina was stained, as was the extracellular matrix surrounding collagen fibrils. Staining was also observed in connective tissue surrounding the trachea and in the lacunae of tracheal hyaline cartilage. These findings are consistent with immunochemical studies demonstrating that antibodies to the chondroitin sulfate proteoglycan of brain also cross-react to various degrees with certain connective tissue proteoglycans
— id: 33499, year: 1984, vol: 99, page: 1117, stat: Journal Article,

Immunocytochemical localization of a chondroitin sulfate proteoglycan in nervous tissue. II. Studies in developing brain
Aquino DA; Margolis RU; Margolis RK
1984 Sep;99(3):1130-1139, Journal of cell biology
In contrast to the intracellular (cytoplasmic) localization of chondroitin sulfate proteoglycans in adult brain (Aquino, D. A., R. U. Margolis, and R. K. Margolis, 1984, J. Cell Biol. 99:940-952), immunoelectron microscopic studies in immature (7 d postnatal) rat cerebellum demonstrated almost exclusively extracellular staining in the granule cell and molecular layers. Staining was also extracellular and/or associated with plasma membranes in the region of the presumptive white matter. Axons, which are unmyelinated at this age, generally did not stain, although faint intracellular staining was present in some astrocytes. At 10 and 14 d postnatal there was a significant decrease in extracellular space and staining, and by 21 d distinct cytoplasmic staining of neurons and astrocytes appeared. This intracellular staining further increased by 33 d so as to closely resemble the pattern seen in adult brain. Analyses of the proteoglycans isolated from 7-d-old and adult brain demonstrated that they have essentially identical biochemical compositions, immunochemical reactivity, size, charge, and density. These findings indicate that the antibodies used in this study recognize the same macromolecule in both early postnatal and adult brain, and that the localization of this proteoglycan changes progressively from an extracellular to an intracellular location during brain development
— id: 33500, year: 1984, vol: 99, page: 1130, stat: Journal Article,

Structural studies on glycoprotein oligosaccharides of chromaffin granule membranes and dopamine beta-hydroxylase
Margolis RK; Finne J; Krusius T; Margolis RU
1984 Feb 1;228(2):443-449, Archives of biochemistry & biophysics. ABB
Dopamine beta-hydroxylase present in the soluble matrix of bovine adrenal medullary chromaffin granules contains biantennary complex oligosaccharides and high-mannose oligosaccharides in a molar ratio of approximately 2:1. The high-mannose oligosaccharides contain an average of six mannose residues. The largest biantennary oligosaccharides (40% of the total) have two complete peripheral branches consisting of sialic acid-galactose-N-acetylglucosamine, but an equal proportion lack sialic acid on one branch and the remainder lack N-acetylglucosamine and/or galactose. Affinity chromatography on lentil lectin-agarose demonstrated that 84% of the dopamine beta-hydroxylase biantennary oligosaccharides are substituted by fucose on the core N-acetylglucosamine which is linked to asparagine. Based on carbohydrate concentration and the proportions of biantennary and high-mannose oligosaccharides, it would appear that the four dopamine beta-hydroxylase subunits of Mr congruent to 75,000 are not identical with respect to their oligosaccharide moieties. In chromaffin granule membranes, high-mannose and biantennary oligosaccharides comprise 20 and 35%, respectively, of the glycoprotein carbohydrate. Almost 40% is present in the form of large complex oligosaccharides with three or more antennas, less than 3% of which have both a core fucose residue and a 2,6-substituted alpha-linked mannose residue. Chromaffin granule membranes also contain a small proportion (approximately 6%) of O-glycosidically linked glycoprotein oligosaccharides which are predominantly monosialyl derivatives of galactosyl-N-acetylgalactosamine. The ratio of N-acetyl- to N-glycolylneuraminic acid in dopamine beta-hydroxylase and the glycoproteins of chromaffin granule membranes is approximately 1.5:1, which is within the same range as that previously found in membrane gangliosides and in the chromogranins isolated from the soluble granule matrix
— id: 33502, year: 1984, vol: 228, page: 443, stat: Journal Article,

Fucosyl gangliosides of PC12 pheochromocytoma cells
Margolis RU; Mazzulla M; Greene LA; Margolis RK
1984 Jul 9;172(2):339-342, FEBS letters
Three monosialogangliosides are highly labeled when PC12 pheochromocytoma cells are cultured in the presence of L-[3H]fucose, and two additional monosialogangliosides are labeled to a lesser extent. In contrast, neither of the two disialogangliosides of PC12 cells contains fucose residues. Removal of sialic acid and fucose by formic acid hydrolysis demonstrated the presence of 3 major 'core' structures in the monosialogangliosides, and a single asialo derivative of the disialogangliosides which has the same chromatographic mobility as one of the monosialoganglioside hydrolysis products. None of the major formic acid hydrolysis products of the PC12 cell gangliosides corresponds to asialo-GM1, supporting our previous conclusion that PC12 cells do not contain significant amounts of brain-type gangliosides
— id: 33501, year: 1984, vol: 172, page: 339, stat: Journal Article,

Distribution and characteristics of polysialosyl oligosaccharides in nervous tissue glycoproteins
Margolis RK; Margolis RU
1983 Nov 15;116(3):889-894, Biochemical & biophysical research communications
Large polysialosyl glycopeptides can be derived from the glycoproteins of both mature as well as early postnatal brain, although their concentration (in terms of sialic acid content) decreases by approximately 75 percent during the first month in rat brain. These glycopeptides were found to contain O-sulfate residues, and subcellular fractionation studies indicated that most of the glycoproteins having polysialosyl units are present in non-synaptic plasma membranes. Large polysialosyl glycopeptides can also be obtained from the membranes of PC12 pheochromocytoma cells, demonstrating that this type of structure is not unique to brain
— id: 33504, year: 1983, vol: 116, page: 889, stat: Journal Article,

Glycoproteins and proteoglycans
Margolis RK; Margolis RU
Handbook of neurochemistry New York : Plenum Press, 1982-1985,
— id: 2844, year: 1983, vol: , page: 177, stat: Chapter,

Complex carbohydrates of cultured PC12 pheochromocytoma cells. Effects of nerve growth factor and comparison with neonatal and mature rat brain
Margolis RK; Salton SR; Margolis RU
1983 Apr 10;258(7):4110-4117, Journal of biological chemistry
The composition and biosynthesis of glycoproteins, proteoglycans, and gangliosides have been studied in a clonal line of rat pheochromocytoma (PC12) cells. Glycoproteins account for approximately 78% of the glucosamine-labeled complex carbohydrates found in the culture medium, together with 17% chondroitin sulfate and 5% heparan sulfate. 10% of the glycoproteins but less than 1% of the proteoglycans are released by trypsin treatment of the cells, whose complex carbohydrates are composed of 93% glycoproteins, 1.3% chondroitin sulfate, 3.4% heparan sulfate, and 2.6% of mono- and disialogangliosides. Sequential lectin affinity chromatography and alkali treatment of glycopeptides prepared from the medium, trypsin-releasable, membrane, and cell-soluble glycoproteins demonstrated that in all of the subfractions large tri- and tetraantennary complex oligosaccharides account for 82 to 97% of those present in PC12 cell glycoproteins. Biantennary oligosaccharides account for approximately 2-6% of those in medium and trypsinate, as compared to 10-13% in the membrane and cell soluble glycoproteins, and there were large differences (ranging from 7 to 60%) in the proportions of biantennary oligosaccharides which are substituted by fucose on the core N-acetylglucosamine which is linked to asparagine. High mannose oligosaccharides are present predominantly in the cell membrane and soluble glycoproteins, where they account for 4 to 5% of the total glycoprotein labeling. In response to nerve growth factor (NGF), the PC12 cells extend long processes and acquire other properties similar to those of differentiated sympathetic neurons. Significant alterations were also observed in the complex carbohydrates of NGF-treated cells, the most striking of which were an almost 3-fold increase in labeled gangliosides and a 75% increase in trypsin-releasable glycoproteins. Cellular heparan sulfate decreased by 70% in response to NGF and increased by an equivalent amount in the culture medium, whereas an NGF-induced increase in chondroitin sulfate labeling occurred specifically in the cell membranes
— id: 33506, year: 1983, vol: 258, page: 4110, stat: Journal Article,

Structural features of the nerve growth factor inducible large external glycoprotein of PC12 pheochromocytoma cells and brain
Margolis RK; Salton SR; Margolis RU
1983 Dec;41(6):1635-1640, Journal of neurochemistry
We have examined the oligosaccharide structure of a major Mr = 230,000 cell surface glycoprotein from rat PC12 pheochromocytoma cells, and of the immunochemically cross-reactive species present in brain. In response to nerve growth factor (NGF) the PC12 cells extend long processes and acquire other properties similar to those of differentiated sympathetic neurons. These morphological changes are accompanied by a 3- to 5-fold increase in the concentration and labeling of this cell surface glycoprotein, which has previously been named the NGF-inducible large external, or NILE, glycoprotein. Tri-and tetraantennary complex oligosaccharides are the predominant carbohydrate units present in the NILE glycoprotein predominant carbohydrate units present in the NILE glycoprotein from both brain and PC12 cells, where they represent 77-90% of the biosynthetically labeled oligosaccharides. Most of these are not substituted by fucose on the core N-acetylglucosamine which is linked to asparagine, and are accompanied by smaller proportions of biantennary and high-mannose oligosaccharides. Sequential lectin-agarose affinity chromatography employing concanavalin A, lentil lectin, and the leukoagglutinating lectin of Phaseolus vulgaris, together with neuraminidase treatment of the fractionated glycopeptides, demonstrated a moderate degree of microheterogeneity among the predominant tri- and tetraantennary oligosaccharide units with respect to the presence of core fucose, outer galactose and sialic acid residues, and the substitution positions on the alpha-linked mannose residues.(ABSTRACT TRUNCATED AT 250 WORDS)
— id: 33503, year: 1983, vol: 41, page: 1635, stat: Journal Article,

Release of chromaffin granule glycoproteins and proteoglycans from potassium-stimulated PC12 pheochromocytoma cells
Salton SR; Margolis RU; Margolis RK
1983 Oct;41(4):1165-1170, Journal of neurochemistry
Cultured PC12 pheochromocytoma cells were labeled with [3H]glucosamine, and the glycoproteins and proteoglycans released following potassium-induced depolarization were fractionated and characterized. Exposure of PC12 cells for 20 min to a high concentration of potassium (51.5 mM in Krebs-Ringers-HEPES buffer) results in an approximately sixfold increase in the release of labeled glycoproteins and proteoglycans, compared to incubation in physiological levels of potassium (6 mM). The released complex carbohydrates include chromogranins, dopamine beta-hydroxylase, and two chondroitin sulfate/heparan sulfate proteoglycan fractions, which together account for 7.4% of the soluble cell radioactivity. The chromogranins contained galactosyl(beta 1 leads to 3)N-acetylgalactosamine, as well as several mono- and disialyl O-glycosidically-linked oligosaccharides, and the tetrasaccharide AcNeu(alpha 2 leads to 3)Gal(beta 1 leads to 3)[AcNeu(alpha 2 leads to 6)] GalNAcol, obtained by alkaline borohydride treatment of the chromogranin glycopeptides, accounted for almost half of the total chromogranin labeling. The proteoglycan fractions varied in their relative proportions of chondroitin sulfate (23-68%), heparan sulfate (16-23%), and glycoprotein oligosaccharides (16-54%), which are of the tri- and tetraantennary and O-glycosidic types. As previously found in the case of proteoglycans from bovine chromaffin granules, the more acidic species has a considerably higher proportion of carbohydrate in the form of sulfated glycosaminoglycans
— id: 33505, year: 1983, vol: 41, page: 1165, stat: Journal Article,

Peptide mapping studies of the chromogranins and of two chromaffin granule proteoglycans
Banerjee S; Margolis RU
1982 Dec;39(6):1700-1703, Journal of neurochemistry
The chromogranins, a family of related acidic glycoproteins, and two chondroitin sulfate/dermatan sulfate proteoglycans were isolated from the soluble contents of bovine adrenal chromaffin granules by chromatography on DEAE-cellulose. These chromaffin granule matrix glycoconjugates were treated with trypsin, and the resulting peptides were fractionated by HPLC. The two proteoglycans, which differ in their concentration of glycosaminoglycans and glycoprotein oligosaccharides, yielded almost identical peptide patterns and would both appear to have the same protein moiety. The peptide profile of the proteoglycans differs, however, from that of the chromogranins, which they closely resemble in terms of amino acid composition. The various chromogranin fractions obtained by gel filtration were also found to have significant differences in the chromatographic patterns of their tryptic peptides
— id: 33507, year: 1982, vol: 39, page: 1700, stat: Journal Article,

Glycoproteins and proteoglycans of the chromaffin granule matrix
Kiang WL; Krusius T; Finne J; Margolis RU; Margolis RK
1982 Feb 25;257(4):1651-1659, Journal of biological chemistry
— id: 33509, year: 1982, vol: 257, page: 1651, stat: Journal Article,

Gangliosides associated with microsomal subfractions of brain: comparison with synaptic plasma membranes
Skrivanek JA; Ledeen RW; Margolis RU; Margolis RK
1982 Mar;13(2):95-106, Journal of neurobiology
To study ganglioside distribution within subcellular components and test the hypothesis that they are localized at the nerve ending, microsomes and synaptic plasma membranes were isolated from young adult rat brains and compared with respect to ganglioside composition. These were shown to be heterogeneous preparations by fractionation on a discontinuous sucrose gradient into subfractions which had differing ganglioside concentrations. The highest ganglioside concentrations occurred in membranes banding at the 0.8M/1.0M and 1.0M/1.3M interfaces for both microsomes and synaptic plasma membranes. These subfractions had closely similar ganglioside concentrations and pattern distributions. In addition, the kinetics of ganglioside labeling following administration of [3H]-glucosamine were similar for the two preparations. The fact that microsomal subfractions representing heterogeneous mixtures of brain cell membranes showed close similarity to synaptosomal plasma membranes argues against localization of gangliosides at the nerve ending. These results, together with other lines of evidence, support the concept that gangliosides are distributed over large portions of the neuron (and perhaps other brain cells). Data concerning the labeling of gangliosides in different microsomal subfractions indicated a movement of label over time from the more dense to the less dense membranes, as was also noted for the glycoproteins in the same subfractions. Specific radioactivity of the gangliosides increased relative to that of the glycoproteins with time
— id: 33508, year: 1982, vol: 13, page: 95, stat: Journal Article,

Fractionation and properties of a chondroitin sulfate proteoglycan and the soluble glycoproteins of brain
Kiang WL; Margolis RU; Margolis RK
1981 Oct 25;256(20):10529-10537, Journal of biological chemistry
— id: 33510, year: 1981, vol: 256, page: 10529, stat: Journal Article,

Sialic acid residues inhibit proteolytic degradation of dopamine beta-hydroxylase
Aquino D; Wong R; Margolis RU; Margolis RK
1980 Apr 7;112(2):195-198, FEBS letters
— id: 33513, year: 1980, vol: 112, page: 195, stat: Journal Article,

Molecular organization of prolactin granules. III. Intracellular transport of sulfated glycosaminoglycans and glycoproteins of the bovine prolactin granule matrix
Giannattasio G; Zanini A; Rosa P; Meldolesi J; Margolis RK; margolis RU
1980 Jul;86(1):273-279, Journal of cell biology
— id: 33512, year: 1980, vol: 86, page: 273, stat: Journal Article,

Complex carbohydrates and the intercellular matrix
Margolis RU
1980 ;18(2):260-260, Neurosciences research program bulletin
— id: 70461, year: 1980, vol: 18, page: 260, stat: Journal Article,

Molecular organization of prolactin granules. II. Characterization of glycosaminoglycans and glycoproteins of the bovine prolactin matrix
Zanini A; Giannattasio G; Nussdorfer G; Margolis RK; Margolis RU; Meldolesi J
1980 Jul;86(1):260-272, Journal of cell biology
Prolactin (PRL) granules can be isolated from the anterior pituitary gland of adult cows in nearly 50% yield by use of a procedure previously developed for the fractionation of the rat pituitary. Treatment of the isolated bovine granules with 0.2% Lubrol PX results in the solubilization of most membranes present in the fractin but has only a limited effect on the matrices, which remain aggregated and can be recovered and purified by gradient centrifugation. These membraneless PRL granules, studied in detail by morphological and biochemical techniques, were found to contain only small amounts of contaminants (primarily growth hormone granules and small membrane fragments). SDS polyacrylamide gel electrophoresis revealed that, in comparison with other fractions isolated from the bovine pituitary, the membraneless granules have a simpler polypeptide composition including PRL (approximately 85%), growth hormone (approximately 8%), as well as approximately 13 minor bands with apparent mol wt ranging from 80,000 go 45,000. Many of these minor bands are accounted for by glycoproteins, as revealed by their binding of 125I-concanavalin A, and two of these are also stained blue by the stains-all procedure, a reaction specific for acidic glycoconjugates. Chemical analyses of the membraneless granule fractin revealed the presence of a heterogeneous mixture of complex carbohydrates. Among glycosaminoglycans, the major component is heparan sulfate, while hyaluronic acid and chondroitin sulfate ar present in smaller amounts. Moreover, some of the glycoproteins are sulfated and account for over 50% of the nondialyzable 35S radioactivity found in the fraction isolated from labeled slices. Although the concentration of glycosaminoglycans and glycoproteins is relatively low in membraneless granules, the possibility that their presence in the fraction is largely due to cross-contamination and/or artifactual adsorption could be excluded on two grounds. These are: (a) electron microscope radiautography of preparations obtained from [35S]sulfate- and D-[6-3H]glucosamine-labeled slices showed a significant labeling of PRL granules in both intact cells and membraneless granule pellets, and (b) a mixing experiment showed that membraneless granules contain very little macromolecular sulfate radiactivity adsorbed from the soluble glycoconjugates present in the pituitary homogenate
— id: 33511, year: 1980, vol: 86, page: 260, stat: Journal Article,

Novel mannitol-containing oligosaccharides obtained by mild alkaline borohydride treatment of a chondroitin sulfate proteoglycan from brain
Finne J; Krusius T; Margolis RK; Margolis RU
1979 Oct 25;254(20):10295-10300, Journal of biological chemistry
Mannitol-containing oligosaccharides have been isolated from a rat brain proteoglycan after mild alkaline borohydride treatment under conditions which prevent 'peeling.' Their structural properties were studied by gas-liquid chromatography-mass spectrometry of disaccharides as their trimethylsilylated and permethylated derivatives, methylation, analysis, specific degradations, and CrO3 oxidation. The following components were identified: Gal(beta 1 leads to 4) [Fuc(alpha 1 leads to 3)]GlcNAc(beta 1 leads to 3)Manol,GlcNAc(beta 1 leads to 3)Manol, and Manol. Evidence was also obtained for the occurrence of a sialylated oligosaccharide and another (possibly sulfated) acidic oligosaccharide, both having the sequence GlcNAc(beta 1 leads to 3)Manol at their proximal ends. These mannitol-containing oligosaccharides constitute a novel group of alkali-labile oligosaccharides in mammalian glycoconjugates. The origin of the oligosaccharides and the possible occurrence of a carbohydrate-peptide linkage involving mannose are discussed
— id: 33514, year: 1979, vol: 254, page: 10295, stat: Journal Article,

Structure and distribution of glycoproteins and glycosaminoglycans
Margolis RK; Margolis RU
Complex carbohydrates of nervous tissue New York : Plenum Press, 1979,
— id: 2843, year: 1979, vol: , page: 45, stat: Chapter,

Presence of chondroitin sulfate in the neuronal cytoplasm
Margolis RK; Thomas MD; Crockett CP; Margolis RU
1979 Apr;76(4):1711-1715, Proceedings of the National Academy of Sciences of the United States of America
The distribution of glycosaminoglycans and glycoproteins has been studied in cytoplasmic and particulate fractions of neurons isolated in bulk from rat cerebrum. Lysis of the neurons in 25 mM sodium phosphate buffer at pH 7.5 released 20% of the protein and over 90% of the lactate dehydrogenase in a soluble form. Eighty-two percent of the chondroitin sulfate was also released, together with 55% of the heparan sulfate and 24-25% of the hyaluronic acid and glycoproteins. The chondroitin sulfate remaining in the membranes was completely depolymerized to disaccharides after treatment with chondroitinase ABC, and treatment of the neuronal membranes with 0.1% trypsin removed 55-63% of the chondroitin sulfate and heparan sulfate but only 25% of the sulfated glycoproteins. The results reported here support our previous conclusion that the soluble chondroitin sulfate proteoglycan of brain is largely a cytoplasmic constitutent of neurons (and astrocytes) and is not primarily present in nervous tissue as an extracellular ground substance
— id: 33515, year: 1979, vol: 76, page: 1711, stat: Journal Article,

Heparan sulfate and related complex carbohydrates of nervous tissue
Margolis RU; Margolis RK
Haparin, structure, cellular functions, and clinical applications New York : Academic Press, 1979,
— id: 2842, year: 1979, vol: , page: 227, stat: Chapter,

Soluble proteoglycans and glycoproteins of brain
Margolis RU; Margolis RK; Kiang W-L; Crockett CP
Glycoconjugate research New York : Academic Press, 1979,
— id: 2877, year: 1979, vol: , page: ?, stat: Chapter,

Complex carbohydrates of nervous tissue
Margolis, Richard U.; Margolis, Renee K
New York : Plenum Press, c1979,
— id: 171, year: 1979, vol: , page: , stat: ,

Glycosaminoglycans and glycoproteins associated with microsomal subfractions of brain and liver
Kiang WL; Crockett CP; Margolis RK; Margolis RU
1978 Sep 5;17(18):3841-3848, Biochemistry
— id: 33517, year: 1978, vol: 17, page: 3841, stat: Journal Article,

Structural features of microsomal, synaptosomal, mitochondrial, and soluble glycoproteins of brain
Krusius T; Finne J; Margolis RU; Margolis RK
1978 Sep 5;17(18):3849-3854, Biochemistry
— id: 33516, year: 1978, vol: 17, page: 3849, stat: Journal Article,

Dissociative extraction of brain proteoglycans
Margolis RK; Crockett CP; Margolis RU
1978 May;30(5):1177-1179, Journal of neurochemistry
— id: 33518, year: 1978, vol: 30, page: 1177, stat: Journal Article,

Isolation and characterization of proteoglycans from human chondrosarcomas
Pal S; Strider W; Margolis R; Gallo G; Lee-Huang S
1978 Feb 25;253(4):1279-1289, Journal of biological chemistry
— id: 15101, year: 1978, vol: 253, page: 1279, stat: Journal Article,

Composition and biogenesis of complex carbohydrates of ox adrenal chromaffin granules
Geissler D; Martinek A; Margolis RU; Margolis RK; Skrivanek JA; Ledeen R; Konig P; Winkler H
1977 ;2(5):685-693, Neuroscience
— id: 33519, year: 1977, vol: 2, page: 685, stat: Journal Article,

Metabolism and function of glycoproteins and glycosaminoglycans in nervous-tissue
Margolis, RU; Margolis, RK
1977 ;8(2):85-91, International journal of biochemistry
— id: 29620, year: 1977, vol: 8, page: 85, stat: Journal Article,

Gangliosides of the neuron: localization and origin
Ledeen RW; Skrivanek JA; Tirri LJ; Margolis RK; Margolis RU
1976 ;71(2):83-103, Advances in experimental medicine & biology
— id: 33522, year: 1976, vol: 71, page: 83, stat: Journal Article,

Glycosaminoglycans and glycoproteins associated with rat brain nuclei
Margolis RK; Crockett CP; Kiang WL; Margolis RU
1976 Dec 21;451(2):465-469, Biochimica & biophysica acta
The concentration, composition and sulfate labeling of glycosaminoglycans and glycoproteins have been studied in purified nuclei isolated in bulk from rat brain. The concentration of total glycosaminoglycans is 0.142 mumol hexosamine/100 mg protein, comprising 57% chondroitin 4-sulfate, 7% chondroitin 6-sulfate, 29% hyaluronic acid and 7% heparan sulfate. Control experiments demonstrated that less than 5% of the sulfated glycosaminoglycans associated with nuclei could be accounted for by the nonspecific adsorption of soluble acidic proteoglycans to basic nuclear proteins. Glycoprotein carbohydrate is present at a level of 206 mug/100 mg protein, and has an average composition of 30% N-acetylglucosamine, 29% mannose, 19% N-acetylneuraminic acid, 15% galactose, 4% N-acetylgalactosamine, and 3% fucose. Labeling studies also indicated the presence of ester sulfate residues on the glycoprotein oligosaccharides
— id: 33520, year: 1976, vol: 451, page: 465, stat: Journal Article,

Developmental changes in brain glycoproteins
Margolis RK; Preti C; Lai D; Margolis RU
1976 Aug 13;112(2):363-369, Brain research
During the postnatal development of rat brain there are large increases in the concentration of brain glycoproteins. Between 1 and 30 days the greatest changes (70-100%) take place in the levels of glycoprotein mannose, galactose and glucosamine, accompanied by smaller increases (35-55%) in sialic acid and fucose. By 30 days of age levels of brain glycoproteins are within 5% of the adult values. Analyses of the molecular size and composition of glycopeptides prepared from brains of 1- and 30-day-old rats lead to the conclusion that during postnatal brain development there is a preferential synthesis of a distinct population of glycoproteins containing oligosaccharides consisting predominantly of glucosamine, mannose and galactose. These oligosaccharides therefore have a large 'core' segment and a relative deficiency of the characteristically terminal sugars, fucose and sialic acid. In very young rat brain there are also large amounts of a metabolically stable form of glycogen or limit dextrin which accompanies the glycopeptides through the usual methods involved in their preparation from brain glycoproteins. The concentration of this glucose polymer decreases by 93% within 30 days after birth, but its presence even in adult brain is a likely explanation for the numerous reports of small amounts of glucose in brain glycopeptides and glycoproteins
— id: 33521, year: 1976, vol: 112, page: 363, stat: Journal Article,

Isolation and properties of a soluble chondroitin sulfate proteoglycan from brain
Margolis RU; Lalley K; Kiang WL; Crockett C; Margolis RK
1976 Dec 20;73(4):1018-1024, Biochemical & biophysical research communications
A proteoglycan in which the glycosaminoglycans are predominantly chondroitin sulfate has been isolated from the soluble fraction of rat brain by ion exchange chromatography and gel filtration. Glycoprotein oligosaccharides are also present, and result in adsorption of the proteoglycan by Concanavalin A-Sepharose. The proteoglycan-glycoprotein complex eluted from the affinity column by alpha-methylglucoside floats near the top of a cesium chloride density gradient run under dissociative conditions (4 M guanidine), but after beta-elimination of the chondroitin sulfate polysaccharide chains from their low buoyant density glycoprotein complex they sediment to the bottom of the gradient. These results suggest that relatively few polysaccharide chains are covalently linked to a large protein core in the dissociated chondroitin sulfate proteoglycan 'subunit' from brain, and that the proteoglycans are closely associated with soluble glycoproteins
— id: 48049, year: 1976, vol: 73, page: 1018, stat: Journal Article,

Proteoglycans from bovine proximal humeral articular-cartilage - structural basis for polydispersity of proteoglycan subunit
Rosenberg, L; Wolfensteintodel, C; Margolis, R; Pal, S; Strider, W
1976 ;251(20):6439-6444, Journal of biological chemistry
— id: 29413, year: 1976, vol: 251, page: 6439, stat: Journal Article,

Distribution and metabolism of glycoproteins and glycosaminoglycans in subcellular fractions of brain
Margolis RK; Margolis RU; Preti C; Lai D
1975 Nov 4;14(22):4797-4804, Biochemistry
The distribution, carbohydrate composition, and metabolism of glycoproteins have been studied in mitochondria, microsomes, axons, and whole rat brain, as well as in various synaptosomal subfractions, including the soluble protein, mitochondria, and synaptic membranes. Approximately 90% of the brain glycoproteins occur in the particulate fraction, and they are present in particularly high amounts in synaptic and microsomal membranes, where the concentration of glycoprotein carbohydrate is 2-3% of the lipid-free dry weight. Treatment of purified synaptic membranes with 0.2% Triton X-100 extracted 70% of the glycoprotein carbohydrate but only 35% of the lipid-free protein residue, and the resulting synaptic membrane subfractions differed significantly in carbohydrate composition. The glycoproteins which are not extracted by Triton X-100 also have a more rapid turnover, as indicated by the 80-155% higher specific activity of hexosamine and sialic acid 1 day after labeling with [3H]glucosamine in vivo. The specific activity of sialic acid in the synaptosomal soluble glycoproteins 2 hr after labeling was greater than 100 times that of the synaptosomal particulate fraction, whereas the difference in hexosamine specific activity in these two fractions was only twofold, and by 22 hr there was little or no difference in the specific activities of sialic acid and hexosamine in synaptosomal soluble as compared to membrane glycoproteins. These data indicate that sialic acid may be added locally to synaptosomal soluble glycoproteins before there is significant labeling of nerve ending glycoproteins by axoplasmic transport. Fifty to sixty percent of the hyaluronic acid and heparan sulfate of brain is located in the various membranes comprising the microsomal fraction, whereas half of the chondroitin sulfate is soluble and only one-third is in microsomal membranes. When microsomes are subfractionated on a discontinuous density gradient over half of the hyaluronic acid and chondroitin sulfate are found in membranes with a density less than that of 0.5 M sucrose (representing a six- to sevenfold enrichment over their concentrations in the membranes applied to the gradient), whereas half of the heparan sulfate is present in membranes with a density greater than that of 0.8 M
— id: 33523, year: 1975, vol: 14, page: 4797, stat: Journal Article,

Metabolism of the protein moiety of brain glycoproteins
Margolis RK; Preti C; Chang L; Margolis RU
1975 Nov;25(5):707-709, Journal of neurochemistry
— id: 33524, year: 1975, vol: 25, page: 707, stat: Journal Article,

Studies on mucopolysaccharides in brain
Margolis RU
1975 Apr;11(2):51-54, Psychopharmacology bulletin
— id: 33525, year: 1975, vol: 11, page: 51, stat: Journal Article,

Glycosaminoglycans of brain during development
Margolis RU; Margolis RK; Chang LB; Preti C
1975 Jan 14;14(1):85-88, Biochemistry
The concentration of hyaluronic acid, chondroitin sulfate, and heparan sulfate was measured in rat brain at 2-day intervals from birth to 1 month of age, and in 40-day-old and adult animals. The levels of all three glycosaminoglycans increased after birth to reach a peak at 7 days after which they declined steadily, attaining by 30 days concentrations within 10% of those present in adult brain. The greatest change was seen in hyaluronic acid, which decreased by 50% in 3 days, and declined to adult levels (28% of the peak concentration) by 18 days of age. Only heparan sulfate showed a significant change in metabolic activity during development (a fourfold increase in the relative specific activity of glucosamine), most of which occurred after 1 week of age. In 7-day-old rats almost 90% of the hyaluronic acid in brain is extractable by water alone, as compared to only 15% in adult animals, and this large amount of soluble hyaluronic acid in young rat brain is relatively inactive metabolically. On the basis of our data we propose that the higher amounts of hyaluronic acid found in very young brain may be responsible for the higher water content of brain at these ages, and that the hydrated hyaluronic acid serves as a matrix through which neuronal migration and differentiation may take place during early brain development
— id: 33526, year: 1975, vol: 14, page: 85, stat: Journal Article,

Organization of extracellular matrix in bovine articular cartilages
Rosenberg L; Margolis R; Wolfenstein-Todel C; Pal S; Strider W
Extracellular matrix influences on gene expression New York : Academic Press, 1975,
— id: 2880, year: 1975, vol: , page: 415, stat: Chapter,

Distribution and metabolism of mucopolysaccharides and glycoproteins in neuronal perikarya, astrocytes, and oligodendroglia
Margolis RU; Margolis RK
1974 Jul 2;13(14):2849-2852, Biochemistry
— id: 33527, year: 1974, vol: 13, page: 2849, stat: Journal Article,

Stimulation by acetylcholine of sulfated mucopolysaccharide release from the perfused cat adrenal gland
Margolis RK; Jaanus SD; Margolis RU
1973 Jul;9(4):590-594, Molecular pharmacology
— id: 33529, year: 1973, vol: 9, page: 590, stat: Journal Article,

Alkali-labile oligosaccharides of brain glycoproteins
Margolis RK; Margolis RU
1973 Apr 28;304(2):421-429, Biochimica & biophysica acta
— id: 33530, year: 1973, vol: 304, page: 421, stat: Journal Article,

Extractability of glycoproteins and mucopolysaccharides of brain
Margolis RK; Margolis RU
1973 Apr;20(4):1285-1288, Journal of neurochemistry
— id: 33532, year: 1973, vol: 20, page: 1285, stat: Journal Article,

The turnover of hexosamine and sialic acid in glycoproteins and mucopolysaccharides of brain
Margolis RK; Margolis RU
1973 Apr 28;304(2):413-420, Biochimica & biophysica acta
— id: 33531, year: 1973, vol: 304, page: 413, stat: Journal Article,

Isolation of chondroitin sulfate and glycopeptides from chromaffin granules of adrenal medulla
Margolis RU; Margolis RK
1973 Sep 1;22(17):2195-2197, Biochemical pharmacology
— id: 33528, year: 1973, vol: 22, page: 2195, stat: Journal Article,

Disposition of fucose in brain
Margolis RK; Margolis RU
1972 Apr;19(4):1023-1030, Journal of neurochemistry
— id: 33538, year: 1972, vol: 19, page: 1023, stat: Journal Article,

The heparan sulfate of rat brain
Margolis RU; Atherton DM
1972 Jul 19;273(2):368-373, Biochimica & biophysica acta
— id: 33535, year: 1972, vol: 273, page: 368, stat: Journal Article,

Mucopolysaccahrides and glycoporteins
Margolis RU; Margolis RK
1972 ;1:249-284, Research methods in neurochemistry
— id: 33785, year: 1972, vol: 1, page: 249, stat: Journal Article,

Sulfate turnover in mucopolysaccharides and glycoproteins of brain
Margolis RU; Margolis RK
1972 May 16;264(3):426-431, Biochimica & biophysica acta
— id: 33536, year: 1972, vol: 264, page: 426, stat: Journal Article,

Carbohydrate-peptide linkages in glycoproteins and mucopolysaccharides from brain
Margolis RU; Margolis RK; Atherton DM
1972 Oct;19(10):2317-2324, Journal of neurochemistry
— id: 33534, year: 1972, vol: 19, page: 2317, stat: Journal Article,

The hyaluronidase of brain
Margolis RU; Margolis RK; Santella R; Atherton DM
1972 Oct;19(10):2325-2332, Journal of neurochemistry
— id: 33533, year: 1972, vol: 19, page: 2325, stat: Journal Article,

Increased fructose production by the brain in alloxan diabetes
Margolis RU; Press R; Altszuler N
1972 Mar 24;38(2):371-375, Brain research
— id: 28897, year: 1972, vol: 38, page: 371, stat: Journal Article,

Inositol production by the brain in normal and alloxan-diabetic dogs
Margolis, R U; Press, R; Altszuler, N; Stewart, M A
1971 May 21;28(3):535-539, Brain research
— id: 28902, year: 1971, vol: 28, page: 535, stat: Journal Article,

Sulfated glycopeptides from rat brain glycoproteins
Margolis RK; Margolis RU
1970 Oct 27;9(22):4389-4396, Biochemistry
— id: 33539, year: 1970, vol: 9, page: 4389, stat: Journal Article,

Ribonuclease and deoxyribonuclease activity in hyaluronidase preparations
Margolis RU; Margolis RK
1970 May;35(1):77-81, Analytical biochemistry
— id: 33540, year: 1970, vol: 35, page: 77, stat: Journal Article,

Mucopolysaccharides
Margolis RU
Handbook of neurochemistry New York Plenum Press, 1969-1972,
— id: 2841, year: 1969, vol: , page: 245, stat: Chapter,

Glycoproteins in myelin
Margolis RU
1968 Jul;16(7):486-486, Journal of histochemistry & cytochemistry
— id: 33541, year: 1968, vol: 16, page: 486, stat: Journal Article,

Effect of intracisternally administered insulin-131-I in normal and vagotomized dogs
Margolis RU; Altszuler N
1968 Apr;127(4):1122-1125, Proceedings of the Society for Experimental Biology & Medicine
— id: 28910, year: 1968, vol: 127, page: 1122, stat: Journal Article,

Acid mucopolysaccharides and proteins of bovine whole brain, white matter and myelin
Margolis RU
1967 Jun 13;141(1):91-102, Biochimica & biophysica acta
— id: 33542, year: 1967, vol: 141, page: 91, stat: Journal Article,

Insulin in the cerebrospinal fluid
Margolis RU; Altszuler N
1967 Sep 23;215(108):1375-1376, Nature
— id: 28914, year: 1967, vol: 215, page: 1375, stat: Journal Article,

The effect of cholinergic and other pharmacologic agents on brain monophosphoinositide turnover in vivo
Margolis RU; Heller A
1966 Feb;151(2):307-312, Journal of pharmacology & experimental therapeutics
— id: 64606, year: 1966, vol: 151, page: 307, stat: Journal Article,

THE INCORPORATION OF MYO (3H2) INOSITOL INTO RAT-BRAIN MONOPHOSPHOINOSITIDE IN VIVO
MARGOLIS, R U; HELLER, A
1965 Apr 5;98:438-441, Biochimica & biophysica acta
— id: 102482, year: 1965, vol: 98, page: 438, stat: Journal Article,

A comparison between the incorporation of 14C from glucose into N-acetyly-L-aspartic acid and aspartic acid in brain perfusion experiments
MARGOLIS, R U; BARKULIS, S S; GEIGER, A
1960 Jun;5:379-382, Journal of neurochemistry
— id: 102483, year: 1960, vol: 5, page: 379, stat: Journal Article,