Konstantin Ichtchenko, Ph.D.

Neuronal targeting, internalization, and biological activity of a recombinant atoxic derivative of botulinum neurotoxin A
Pellett, Sabine; Tepp, William H; Stanker, Larry H; Band, Philip A; Johnson, Eric A; Ichtchenko, Konstantin
2011 Feb 25;405(4):673-677, Biochemical & biophysical research communications
Non-toxic derivatives of botulinum neurotoxin A (BoNT/A) have potential use as neuron-targeting delivery vehicles, and as reagents to study intracellular trafficking. We have designed and expressed an atoxic derivative of BoNT/A (BoNT/A ad) as a full-length 150 kDa molecule consisting of a 50 kDa light chain (LC) and a 100 kDa heavy chain (HC) joined by a disulfide bond and rendered atoxic through the introduction of metalloprotease-inactivating point mutations in the light chain. Studies in neuronal cultures demonstrated that BoNT/A ad cannot cleave synaptosomal-associated protein 25 (SNAP25), the substrate of wt BoNT/A, and that it effectively competes with wt BoNT/A for binding to endogenous neuronal receptors. In vitro and in vivo studies indicate accumulation of BoNT/A ad at the neuromuscular junction of the mouse diaphragm. Immunoprecipitation studies indicate that the LC of BoNT/A ad forms a complex with SNAP25 present in the neuronal cytosolic fraction, demonstrating that the atoxic LC retains the SNAP25 binding capability of the wt toxin. Toxicity of BoNT/A ad was found to be reduced approximately 100,000-fold relative to wt BoNT/A
— id: 133185, year: 2011, vol: 405, page: 673, stat: Journal Article,

Recombinant derivatives of botulinum neurotoxin A engineered for trafficking studies and neuronal delivery
Band, Philip A; Blais, Steven; Neubert, Thomas A; Cardozo, Timothy J; Ichtchenko, Konstantin
2010 May;71(1):62-73, Protein expression & purification
Work from multiple laboratories has clarified how the structural domains of botulinum neurotoxin A (BoNT/A) disable neuronal exocytosis, but important questions remain unanswered. Because BoNT/A intoxication disables its own uptake, light chain (LC) does not accumulate in neurons at detectable levels. We have therefore designed, expressed and purified a series of BoNT/A atoxic derivatives (ad) that retain the wild type features required for native trafficking. BoNT/A1ad(ek) and BoNT/A1ad(tev) are full length derivatives rendered atoxic through double point mutations in the LC protease (E(224)>A; Y(366)>A). DeltaLC-peptide-BoNT/A(tev) and DeltaLC-GFP-BoNT/A(tev) are derivatives wherein the catalytic portion of the LC is replaced with a short peptide or with GFP plus the peptide. In all four derivatives, we have fused the S6 peptide sequence GDSLSWLLRLLN to the N-terminus of the proteins to enable site-specific attachment of cargo using Sfp phosphopantetheinyl transferase. Cargo can be attached in a manner that provides a homogeneous derivative population rather than a polydisperse mixture of singly and multiply-labeled molecular species. All four derivatives contain an introduced cleavage site for conversion into disulfide-bonded heterodimers. These constructs were expressed in a baculovirus system and the proteins were secreted into culture medium and purified to homogeneity in yields ranging from 1 to 30 mg per liter. These derivatives provide unique tools to study toxin trafficking in vivo, and to assess how the structure of cargo linked to the heavy chain (HC) influences delivery to the neuronal cytosol. Moreover, they create the potential to engineer BoNT-based molecular vehicles that can target therapeutic agents to the neuronal cytoplasm
— id: 107925, year: 2010, vol: 71, page: 62, stat: Journal Article,

Protein-tyrosine Phosphatase-sigma Is a Novel Member of the Functional Family of alpha -Latrotoxin Receptors
Krasnoperov, Valery; Bittner, Mary A; Mo, Wenjun; Buryanovsky, Leonid; Neubert, Thomas A; Holz, Ronald W; Ichtchenko, Konstantin; Petrenko, Alexander G
2002 Sep 27;277(39):35887-35895, Journal of biological chemistry
Receptor-like protein-tyrosine phosphatase sigma (PTPvarsigma) is essential for neuronal development and function. Here we report that PTPvarsigma is a target of alpha-latrotoxin, a strong stimulator of neuronal exocytosis. alpha-Latrotoxin binds to the cell adhesion-like extracellular region of PTPvarsigma. This binding results in the stimulation of exocytosis. The toxin-binding site is located in the C-terminal part of the PTPvarsigma ectodomain and includes two fibronectin type III repeats. The intracellular catalytic domains of PTPvarsigma are not required for the alpha-latrotoxin binding and secretory response triggered by the toxin in chromaffin cells. These features of PTPvarsigma resemble two other previously described alpha-latrotoxin receptors, neurexin and CIRL. Thus, alpha-latrotoxin represents an unusual example of the neurotoxin that has three independent, equally potent, and yet structurally distinct targets. The known structural and functional characteristics of PTPvarsigma, neurexin, and CIRL suggest that they define a functional family of neuronal membrane receptors with complementary or converging roles in presynaptic function via a mechanism that involves cell-to-cell and cell-to-matrix interaction
— id: 32497, year: 2002, vol: 277, page: 35887, stat: Journal Article,

Post-translational proteolytic processing of the calcium-independent receptor of alpha-latrotoxin (CIRL), a natural chimera of the cell adhesion protein and the G protein-coupled receptor. Role of the G protein-coupled receptor proteolysis site (GPS) motif
Krasnoperov, Valery; Lu, Yun; Buryanovsky, Leonid; Neubert, Thomas A; Ichtchenko, Konstantin; Petrenko, Alexander G
2002 Nov 29;277(48):46518-46526, Journal of biological chemistry
The calcium-independent receptor of alpha-latrotoxin (CIRL), a neuronal cell surface receptor implicated in the regulation of exocytosis, is a natural chimera of the cell adhesion protein and the G protein-coupled receptor (GPCR). In contrast with canonic GPCRs, CIRL consists of two heterologous non-covalently bound subunits, p120 and p85, due to endogenous proteolytic processing of the receptor precursor in the endoplasmic reticulum. Extracellularly oriented p120 contains hydrophilic cell adhesion domains, whereas p85 resembles a generic GPCR. We determined that the site of the CIRL cleavage is located within a juxtamembrane Cys- and Trp-rich domain of the N-terminal extracellular region of CIRL. Mutations in this domain make CIRL resistant to the cleavage and impair its trafficking. Therefore, we have named it GPS for G protein-coupled receptor proteolysis site. The GPS motif is found in homologous adhesion GPCRs and thus defines a novel receptor family. We postulate that the proteolytic processing and two-subunit structure is a common characteristic feature in the family of GPS-containing adhesion GPCRs
— id: 33173, year: 2002, vol: 277, page: 46518, stat: Journal Article,

Structure of the LDL receptor extracellular domain at endosomal pH
Rudenko, Gabby; Henry, Lisa; Henderson, Keith; Ichtchenko, Konstantin; Brown, Michael S; Goldstein, Joseph L; Deisenhofer, Johann
2002 Dec 20;298(5602):2353-2358, Science
The low-density lipoprotein receptor mediates cholesterol homeostasis through endocytosis of lipoproteins. It discharges its ligand in the endosome at pH < 6. In the crystal structure at pH = 5.3, the ligand-binding domain (modules R2 to R7) folds back as an arc over the epidermal growth factor precursor homology domain (the modules A, B, beta propeller, and C). The modules R4 and R5, which are critical for lipoprotein binding, associate with the beta propeller via their calcium-binding loop. We propose a mechanism for lipoprotein release in the endosome whereby the beta propeller functions as an alternate substrate for the ligand-binding domain, binding in a calcium-dependent way and promoting lipoprotein release
— id: 95601, year: 2002, vol: 298, page: 2353, stat: Journal Article,

The functional family of alpha-latrotoxin receptors
Krasnoperov, V; Ichtchenko, K; Petrenko, AG
2001 JUN ;77(1-2):6-7, Journal of neurochemistry
— id: 55017, year: 2001, vol: 77, page: 6, stat: Journal Article,

A novel ubiquitously expressed alpha-latrotoxin receptor is a member of the CIRL family of G-protein-coupled receptors
Ichtchenko K; Bittner MA; Krasnoperov V; Little AR; Chepurny O; Holz RW; Petrenko AG
1999 Feb 26;274(9):5491-5498, Journal of biological chemistry
Poisoning with alpha-latrotoxin, a neurotoxic protein from black widow spider venom, results in a robust increase of spontaneous synaptic transmission and subsequent degeneration of affected nerve terminals. The neurotoxic action of alpha-latrotoxin involves extracellular binding to its high affinity receptors as a first step. One of these proteins, CIRL, is a neuronal G-protein-coupled receptor implicated in the regulation of secretion. We now demonstrate that CIRL has two close homologs with a similar domain structure and high degree of overall identity. These novel receptors, which we propose to name CIRL-2 and CIRL-3, together with CIRL (CIRL-1) belong to a recently identified subfamily of large orphan receptors with structural features typical of both G-protein-coupled receptors and cell adhesion proteins. Northern blotting experiments indicate that CIRL-2 is expressed ubiquitously with highest concentrations found in placenta, kidney, spleen, ovary, heart, and lung, whereas CIRL-3 is expressed predominantly in brain similarly to CIRL-1. It appears that CIRL-2 can also bind alpha-latrotoxin, although its affinity to the toxin is about 14 times less than that of CIRL-1. When overexpressed in chromaffin cells, CIRL-2 increases their sensitivity to alpha-latrotoxin stimulation but also inhibits Ca2+-regulated secretion. Thus, CIRL-2 is a functionally competent receptor of alpha-latrotoxin. Our findings suggest that although the nervous system is the primary target of low doses of alpha-latrotoxin, cells of other tissues are also susceptible to the toxic effects of alpha-latrotoxin because of the presence of CIRL-2, a low affinity receptor of the toxin
— id: 7354, year: 1999, vol: 274, page: 5491, stat: Journal Article,

The CIRLs, a novel family of two-subunit G protein-coupled receptors with structural features of cell adhesion proteins
Petrenko, AG; Krasnoperov, V; Ichtchenko, K
1999 APR 23 ;13(7):A1578-A1578, FASEB journal
— id: 53942, year: 1999, vol: 13, page: A1578, stat: Journal Article,