Identification of the SV2 protein receptor-binding site of botulinum neurotoxin type E

2013 ◽  
Vol 453 (1) ◽  
pp. 37-47 ◽  
Author(s):  
Stefan Mahrhold ◽  
Jasmin Strotmeier ◽  
Consuelo Garcia-Rodriguez ◽  
Jianlong Lou ◽  
James D. Marks ◽  
...  
Keyword(s):  
Binding Site ◽  
Synaptic Vesicle ◽  
Specific Binding ◽  
Cell Surface Receptor ◽  
Vesicle Membrane ◽  
Receptor Binding Site ◽  
Binding Interface ◽  
Protein Receptor ◽  
Surface Receptor ◽  
Extended Surface

The highly specific binding and uptake of BoNTs (botulinum neurotoxins; A–G) into peripheral cholinergic motoneurons turns them into the most poisonous substances known. Interaction with gangliosides accumulates the neurotoxins on the plasma membrane and binding to a synaptic vesicle membrane protein leads to neurotoxin endocytosis. SV2 (synaptic vesicle glycoprotein 2) mediates the uptake of BoNT/A and /E, whereas Syt (synaptotagmin) is responsible for the endocytosis of BoNT/B and /G. The Syt-binding site of the former was identified by co-crystallization and mutational analyses. In the present study we report the identification of the SV2-binding interface of BoNT/E. Mutations interfering with SV2 binding were located at a site that corresponds to the Syt-binding site of BoNT/B and at an extended surface area located on the back of the conserved ganglioside-binding site, comprising the N- and C-terminal half of the BoNT/E-binding domain. Mutations impairing the affinity also reduced the neurotoxicity of full-length BoNT/E at mouse phrenic nerve hemidiaphragm preparations demonstrating the crucial role of the identified binding interface. Furthermore, we show that a monoclonal antibody neutralizes BoNT/E activity because it directly interferes with the BoNT/E–SV2 interaction. The results of the present study suggest a novel mode of binding for BoNTs that exploit SV2 as a cell surface receptor.

scholarly journals Carbohydrate recognition in the peripheral nervous system: a calcium-dependent membrane binding site for HNK-1 reactive glycolipids potentially involved in Schwann cell adhesion.

1993 ◽  
Vol 121 (2) ◽  
pp. 397-408 ◽  
Author(s):  
L K Needham ◽  
R L Schnaar
Keyword(s):  
Nervous System ◽  
Cell Adhesion ◽  
Schwann Cell ◽  
Binding Site ◽  
Peripheral Nervous System ◽  
Specific Binding ◽  
Binding Activity ◽  
Cell Surface Receptor ◽  
Calcium Dependent ◽  
Surface Receptor

The carbohydrate determinants recognized by the HNK-1 antibody are potential cell-cell recognition ligands in the peripheral nervous system (PNS). The HNK-1 reactive sulfoglucuronylneolacto (SGNL) glycolipids specifically support Schwann cell adhesion, suggesting the presence of a cell surface receptor specific for SGNL-oligosaccharides. We directly probed PNS membranes for receptors complementary to SGNL determinants using a synthetic radioligand consisting of radioiodinated serum albumin derivatized with multiple SGNL-oligosaccharides. A high-affinity, saturable, calcium-dependent binding site for this ligand was found in PNS myelin membranes. Binding activity was carbohydrate-specific (most potently inhibited by SGNL-lipids compared to other glycolipids) and PNS-specific (absent from comparable central nervous system membranes). The SGNL-specific binding activity on PNS membranes reported here may be involved in peripheral myelination or myelin stabilization.

scholarly journals Characterization of a type IV collagen major cell binding site with affinity to the alpha 1 beta 1 and the alpha 2 beta 1 integrins.

1991 ◽  
Vol 113 (6) ◽  
pp. 1475-1483 ◽  
Author(s):  
P Vandenberg ◽  
A Kern ◽  
A Ries ◽  
L Luckenbill-Edds ◽  
K Mann ◽  
...  
Keyword(s):  
Binding Site ◽  
Type Iv Collagen ◽  
Cell Attachment ◽  
Cell Surface Receptor ◽  
Helical Conformation ◽  
Amino Acid Residues ◽  
Cell Binding ◽  
Type Iv ◽  
Depth Study ◽  
Surface Receptor

The aim of this investigation was to identify the domains of type IV collagen participating in cell binding and the cell surface receptor involved. A major cell binding site was found in the trimeric cyanogen bromide-derived fragment CB3, located 100 nm away from the NH2 terminus of the molecule, in which the triple-helical conformation is stabilized by interchain disulfide bridges. Cell attachment assays with type IV collagen and CB3 revealed comparable cell binding activities. Antibodies against CB3 inhibited attachment on fragment CB3 completely and on type IV collagen to 80%. The ability to bind cells was strictly conformation dependent. Four trypsin derived fragments of CB3 allowed a closer investigation of the binding site. The smallest, fully active triple-helical fragment was (150)3-amino acid residues long. It contained segments of 27 and 37 residues, respectively, at the NH2 and COOH terminus, which proved to be essential for cell binding. By affinity chromatography on Sepharose-immobilized CB3, two receptor molecules of the integrin family, alpha 1 beta 1 and alpha 2 beta 1, were isolated. Their subunits were identified by sequencing the NH2 termini or by immunoblotting. The availability of fragment CB3 will allow for a more in-depth study of the molecular interaction of a short, well defined triple-helical ligand with collagen receptors alpha 1 beta 1 and alpha 2 beta 1.

scholarly journals FcαRI binding at the IgA1 CH2–CH3 interface induces long-range conformational changes that are transmitted to the hinge region

2018 ◽  
Vol 115 (38) ◽  
pp. E8882-E8891 ◽  
Author(s):  
Monica T. Posgai ◽  
Sam Tonddast-Navaei ◽  
Manori Jayasinghe ◽  
George M. Ibrahim ◽  
George Stan ◽  
...  
Keyword(s):  
Binding Site ◽  
Long Range ◽  
Conformational Changes ◽  
Lectin Binding ◽  
Coarse Grained ◽  
Alanine Scanning Mutagenesis ◽  
Long Distance ◽  
Receptor Binding Site ◽  
Binding Interface ◽  
Functional Dynamics

IgA effector functions include proinflammatory immune responses triggered upon clustering of the IgA-specific receptor, FcαRI, by IgA immune complexes. FcαRI binds to the IgA1–Fc domain (Fcα) at the CH2–CH3 junction and, except for CH2 L257 and L258, all side-chain contacts are contributed by the CH3 domain. In this study, we used experimental and computational approaches to elucidate energetic and conformational aspects of FcαRI binding to IgA. The energetic contribution of each IgA residue in the binding interface was assessed by alanine-scanning mutagenesis and equilibrium surface plasmon resonance (SPR). As expected, hydrophobic residues central to the binding site have strong energetic contributions to the FcαRI:Fcα interaction. Surprisingly, individual mutation of CH2 residues L257 and L258, found at the periphery of the FcαRI binding site, dramatically reduced binding affinity. Comparison of antibody:receptor complexes involving IgA or its precursor IgY revealed a conserved receptor binding site at the CH2–CH3 junction (or its equivalent). Given the importance of residues near the CH2–CH3 junction, we used coarse-grained Langevin dynamics simulations to understand the functional dynamics in Fcα. Our simulations indicate that FcαRI binding, either in an asymmetric (1:1) or symmetric (2:1) complex with Fcα, propagated long-range conformational changes across the Fc domains, potentially impacting the hinge and Fab regions. Subsequent SPR experiments confirmed that FcαRI binding to the Fcα CH2–CH3 junction altered the kinetics of HAA lectin binding at the IgA1 hinge. Receptor-induced long-distance conformational transitions have important implications for the interaction of aberrantly glycosylated IgA1 with anti-glycan autoantibodies in IgA nephropathy.

scholarly journals Function of the cysteine-rich domain of the haemorrhagic metalloproteinase atrolysin A: targeting adhesion proteins collagen I and von Willebrand factor

2005 ◽  
Vol 391 (1) ◽  
pp. 69-76 ◽  
Author(s):  
Solange M. T. Serrano ◽  
Li-Guo Jia ◽  
Deyu Wang ◽  
John D. Shannon ◽  
Jay W. Fox
Keyword(s):  
Binding Site ◽  
Von Willebrand Factor ◽  
Solid Phase ◽  
Collagen I ◽  
Cell Surface Receptor ◽  
Receptor Binding Site ◽  
Rich Domain ◽  
Von Willebrand ◽  
Willebrand Factor ◽  
Cysteine Rich Domain

The cysteine-rich domain of the haemorrhagic metalloproteinase atrolysin A was shown to inhibit collagen-stimulated platelet aggregation and to interact with MG-63 osteosarcoma cells via integrin α2β1 to inhibit adhesion to collagen I. In addition, we demonstrate by solid-phase binding assays that atrolysin A binds to collagen I and to vWF (von Willebrand factor) via exosites in the cysteine-rich domain. Interestingly, the binding site of the cysteine-rich domain on collagen I is distinct from the cell adhesion site, since the incubation of collagen-I-coated plates with the cysteine-rich domain did not prevent the adhesion of MG-63 cells to collagen. Finally, we show by surface plasmon resonance (BIAcore™) analyses that the cysteine-rich domain can block vWF binding to collagen I as well as the binding of collagen I to vWF. Taken together, these results indicate that this domain may function as a cell-surface-receptor-binding site and/or a substrate recognition exosite and may thus play a role in the pathologies associated with atrolysin A.

Regulation of the Urokinase Receptor by Its Plasminogen Activator

1991 ◽  
Vol 66 (06) ◽  
pp. 678-683 ◽  
Author(s):  
W Hollas ◽  
D Boyd
Keyword(s):  
Molecular Weight ◽  
Plasminogen Activator ◽  
Specific Binding ◽  
Cell Surface Receptor ◽  
Specific Cell ◽  
Acid Pretreatment ◽  
Ample Evidence ◽  
Surface Receptor ◽  
A Chain ◽  
The Uk

SummaryThere is now ample evidence that the proteolytic action of urokinase (UK) is potentiated by a specific cell surface receptor. The present study was undertaken to assess the role of UK as a modulator of its receptor. GEO colonic cells, which secrete relatively low levels of UK (≃0.1 nM/72 h per 106 cells) and display approximately 104 receptors per cell, 10% of which are "tagged" with the endogenous plasminogen activator (PA), was selected for the study. A 90% reduction in the specific binding of radioactive DFP-UK was observed for cells cultivated in the presence of two-chain (TC) UK (M r 55,000). This only partly reflected occupation of the receptors with UK supplied in the culture medium, since the specific binding of the radioligand was still reduced by 60% after an acid pretreatment, which dissociates receptor-bound UK. The reduction in radioactive DFP-UK binding to cells treated with high molecular weight UK, either in the single or two-chain form, was both concentration and time dependent. Maximum reductions (70%) were achieved by treatment of the cells for 24 h with 1 nM of the plasminogen activator. In contrast, low molecular weight UK, which lacks part of the UK A chain, had no effect on ligand binding. Attenuation of radioactive DFP-UK binding to UK treated GEO cells was a consequence of a 60% reduction in the number of binding sites. Treatment of GEO cells with an antibody, which blocks the binding of endogenous UK to its receptor, augmented radioactive DFP-UK binding by two-fold. These data indicate that for one colonic cell line, at least, UK down-regulates its own binding site subsequent to it being bound to the receptor.

scholarly journals Decreased nonspecific adhesivity, receptor-targeted therapeutic nanoparticles for primary and metastatic breast cancer

2020 ◽  
Vol 6 (3) ◽  
pp. eaax3931 ◽  
Author(s):  
Jimena G. Dancy ◽  
Aniket S. Wadajkar ◽  
Nina P. Connolly ◽  
Rebeca Galisteo ◽  
Heather M. Ames ◽  
...  
Keyword(s):  
Breast Cancer ◽  
Cell Surface ◽  
Tumor Cell ◽  
Specific Binding ◽  
Metastatic Breast ◽  
Cell Surface Receptor ◽  
Surface Receptor ◽  
Specific Uptake ◽  
Effective Development ◽  
The Brain

Development of effective tumor cell–targeted nanodrug formulations has been quite challenging, as many nanocarriers and targeting moieties exhibit nonspecific binding to cellular, extracellular, and intravascular components. We have developed a therapeutic nanoparticle formulation approach that balances cell surface receptor-specific binding affinity while maintaining minimal interactions with blood and tumor tissue components (termed “DART” nanoparticles), thereby improving blood circulation time, biodistribution, and tumor cell–specific uptake. Here, we report that paclitaxel (PTX)–DART nanoparticles directed to the cell surface receptor fibroblast growth factor–inducible 14 (Fn14) outperformed both the corresponding PTX-loaded, nontargeted nanoparticles and Abraxane, an FDA-approved PTX nanoformulation, in both a primary triple-negative breast cancer (TNBC) model and an intracranial model reflecting TNBC growth following metastatic dissemination to the brain. These results provide new insights into methods for effective development of therapeutic nanoparticles as well as support the continued development of the DART platform for primary and metastatic tumors.

scholarly journals Inhibition of the specific binding of human lactotransferrin to human peripheral-blood phytohaemagglutinin-stimulated lymphocytes by fluorescein labelling and location of the binding site

1991 ◽  
Vol 276 (3) ◽  
pp. 733-738 ◽  
Author(s):  
D Legrand ◽  
J Mazurier ◽  
P Maes ◽  
E Rochard ◽  
J Montreuil ◽  
...  
Keyword(s):  
Binding Site ◽  
Receptor Binding ◽  
Peripheral Blood ◽  
Human Peripheral Blood ◽  
Specific Binding ◽  
Peripheral Blood Lymphocytes ◽  
Receptor Binding Site ◽  
Terminal Domain ◽  
Total Fluorescence ◽  
Domain I

Labelling of human lactotransferrin with fluorescein 5′-isothiocyanate (FITC) in an equimolar ratio inhibits the binding of the protein to phytohaemagglutinin-activated human peripheral-blood lymphocytes. Therefore it can be assumed that FITC reacts at, or near, the receptor-binding site. Three FITC-labelled peptides have been purified from a tryptic digest of the FITC-labelled lactotransferrin. The determination of their amino acid sequence and their localization on the primary structure of the protein permitted the identification of two FITC-accessible areas in the N-terminal lobe and one in the C-terminal lobe. In fact, only 10% of the total FITC was conjugated to one lysine residue (Lys579) of the C-terminal lobe, whereas most (80%) of the FITC was conjugated to three close lysine residues [Lys263 (65% of total fluorescence), Lys280 and Lys282 (15% of total fluorescence)] located in beta-turn structures, of the N-terminal domain I of human lactotransferrin. The results obtained show that the receptor-binding site should be located in the vicinity of the FITC-accessible Lys263, Lys280 and Lys282, and corroborate our preliminary results reporting the involvement of the N-terminal domain I in the binding of human lactotransferrin to mitogen-stimulated lymphocytes [Rochard, Legrand, Mazurier, Montreuil & Spik (1989) FEBS Lett. 255, 201-204]. In any case, FITC labelling is not suitable for studying the binding of lactotransferrin to activated lymphocytes and its use may lead to erroneous interpretations of cell binding experiments.

scholarly journals Decreased urokinase receptor expression by overexpression of the plasminogen activator in a colon cancer cell line

1992 ◽  
Vol 285 (2) ◽  
pp. 629-634 ◽  
Author(s):  
W Hollas ◽  
E Soravia ◽  
A Mazar ◽  
J Henkin ◽  
F Blasi ◽  
...  
Keyword(s):  
Cell Surface ◽  
Binding Site ◽  
Cell Line ◽  
Marker Gene ◽  
Receptor Expression ◽  
Cell Surface Receptor ◽  
Scatchard Analysis ◽  
Specific Cell ◽  
Ample Evidence ◽  
Surface Receptor

There is now ample evidence that the proteolytic action of urokinase (UK) is potentiated by a specific cell surface receptor. The present study was undertaken to determine the role of UK as a modulator of its binding site. GEO colonic cells, which secrete low levels of UK (approximately 2.5 ng/ml per 72 h per 10(6) cells) and display approx. 10(4) receptors per cell, the majority of which are vacant, were transfected with an exogenous UK gene driven by the RSV long terminal repeat (LTR) promoter (pRSVUK). Several UK-overexpressing pRSVUK clones were identified by an e.l.i.s.a., Northern blotting and Southern blotting, and analysed for receptor numbers after an acid pretreatment which dissociates receptor-bound UK. pRSVUK GEO clones, expressing high levels of UK, consistently bound 50-75% less radioactive di-isopropylfluorophosphate (DFP)-UK than clones harbouring the selectable marker gene neo only or control GEO cells. Cross-linking experiments with a radioactive N-terminal fragment of UK which binds to the receptor showed a decreased amount of a binding protein of approx. 51 kDa in representative pRSVUK-transfected cells. Saturation and Scatchard analysis indicated that this reduction in radioligand binding reflected a 40-70% decrease in the number of UK receptors, rather than a change in the dissociation constant. The reduction in receptor display could be accounted for by a decrease in the amount of steady-state mRNA encoding the receptor. Radioactive DFP-UK binding to pRSVUK GEO clones, which display two-thirds less receptors than their neo counterparts, could be restored to control levels (untreated cells harbouring neo) by cultivating them in the presence of an antibody which inhibits the interaction of UK with its receptor. These data suggest that for one colonic cell line at least, UK reduces the expression of its own binding site via an autocrine stimulation of its cell surface receptor.

scholarly journals Exogenous Cyclic AMP, Cholera Toxin, and Endotoxin Induce Expression of the Lipopolysaccharide Receptor CD14 in Murine Bone Marrow Cells: Role of Purinoreceptors

1999 ◽  
Vol 6 (6) ◽  
pp. 885-890 ◽  
Author(s):  
Thierry Pedron ◽  
Robert Girard ◽  
Richard Chaby
Keyword(s):  
Bone Marrow ◽  
Cyclic Amp ◽  
Cholera Toxin ◽  
Bone Marrow Cells ◽  
Specific Binding ◽  
Cell Surface Receptor ◽  
Intracellular Camp ◽  
Purine Derivatives ◽  
Murine Bone Marrow ◽  
Surface Receptor

ABSTRACT Little is known about the mechanisms of lipopolysaccharide (LPS) signaling in immature cells that do not express the LPS receptor CD14 yet. Bone marrow granulocytes do not constitutively express CD14 but can be stimulated by low doses of LPS in the absence of serum and then express an inducible form of LPS receptor (iLpsR). We show that in addition to LPS, cholera toxin (CT) and various cyclic AMP (cAMP) analogs can also induce the expression of iLpsR, which was identified as CD14. Induction was independent of intracellular cAMP. The hypothesis that cAMP analogs act via a cell surface receptor was suggested by the unresponsiveness of trypsin-treated cells to these inducers and by the specific binding of [3H]cAMP to the cells. This binding was not inhibited by LPS or CT but was inhibited by various purine derivatives. However, the receptor involved is not a conventional purinoreceptor since both an agonist and an antagonist of such receptors were able to induce iLpsR expression. The results suggest that cAMP analogs and other purine derivatives induce iLpsR after interaction with an unconventional, trypsin-sensitive, purinoreceptor distinct from LPS and CT receptors.

scholarly journals Human Copper-Containing Amine Oxidases in Drug Design and Development

Molecules ◽  
2020 ◽  
Vol 25 (6) ◽  
pp. 1293 ◽  
Author(s):  
Serhii Vakal ◽  
Sirpa Jalkanen ◽  
Käthe M. Dahlström ◽  
Tiina A. Salminen
Keyword(s):  
Amine Oxidase ◽  
Inflammatory Diseases ◽  
Specific Binding ◽  
Mammalian Species ◽  
Model Organism ◽  
Cell Surface Receptor ◽  
Inhibitor Design ◽  
Surface Receptor ◽  
Targeted Inhibitors ◽  
Vascular Adhesion

Two members of the copper-containing amine oxidase family are physiologically important proteins: (1) Diamine oxidase (hDAO; AOC1) with a preference for diamines is involved in degradation of histamine and (2) Vascular adhesion protein-1 (hVAP-1; AOC3) with a preference for monoamines is a multifunctional cell-surface receptor and an enzyme. hVAP-1-targeted inhibitors are designed to treat inflammatory diseases and cancer, whereas the off-target binding of the designed inhibitors to hDAO might result in adverse drug reactions. The X-ray structures for both human enzymes are solved and provide the basis for computer-aided inhibitor design, which has been reported by several research groups. Although the putative off-target effect of hDAO is less studied, computational methods could be easily utilized to avoid the binding of VAP-1-targeted inhibitors to hDAO. The choice of the model organism for preclinical testing of hVAP-1 inhibitors is not either trivial due to species-specific binding properties of designed inhibitors and different repertoire of copper-containing amine oxidase family members in mammalian species. Thus, the facts that should be considered in hVAP-1-targeted inhibitor design are discussed in light of the applied structural bioinformatics and structural biology approaches.

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