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.

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 Structure-function studies on human retinol-binding protein using site-directed mutagenesis

1994 ◽  
Vol 300 (2) ◽  
pp. 437-442 ◽  
Author(s):  
A Sivaprasadarao ◽  
J B Findlay
Keyword(s):  
Cell Surface ◽  
Specific Binding ◽  
Binding Protein ◽  
Specific Interaction ◽  
Binding Activity ◽  
Site Directed Mutagenesis ◽  
Retinol Binding Protein ◽  
Cell Surface Receptor ◽  
Directed Mutagenesis ◽  
Surface Receptor

Retinol-binding protein (RBP) transports vitamin A in the plasma. It consists of eight anti-parallel beta-strands (A to H) that fold to form an orthogonal barrel. The loops connecting the strands A and B, C and D, and E and F form the entrance to the binding site in the barrel. The retinol molecule is found deep inside this barrel. Apart from its specific interaction with retinol, RBP is involved in two other molecular-recognition properties, that is it binds to transthyretin (TTR), another serum protein, and to a cell-surface receptor. Using site-directed mutagenesis, specific changes were made to the loop regions of human RBP and the resultant mutant proteins were tested for their ability to bind to retinol, to TTR and to the RBP receptor. While all the variants retained their ability to bind retinol, that in which residues 92 to 98 of the loop E-F were deleted completely lost its ability to interact with TTR, but retained some binding activity for the receptor. In contrast, the double mutant in which leucine residues at positions 63 and 64 of the loop C-D were changed to arginine and serine respectively partially retained its TTR-binding ability, but completely lost its affinity for the RBP receptor. Mutation of Leu-35 of loop A-B to valine revealed no apparent effect on any of the binding activities of RBP. However, substitution of leucine for proline at position 35 markedly reduced the affinity of the protein for TTR, but showed no apparent change in its receptor-binding activity. These results demonstrate that RBP interacts with both TTR and the receptor via loops C-D and E-F. The binding sites, however, are overlapping rather than identical. RBP also appears to make an additional contact with TTR via its loop A-B. A further implication of these results is that RBP, when bound to TTR, cannot bind simultaneously to the receptor. This observation is consistent with our previously proposed mechanism for delivery of retinol to target tissues [Sivaprasadarao and Findlay (1988) Biochem. J. 255, 571-579], according to which retinol delivery involves specific binding of RBP to the cell-surface receptor, an interaction that triggers release of retinol from RBP to the bound cell rather than internalization of retinol-RBP complex.

scholarly journals Dependence on Schwann Cell-Derived Laminin-γ1 Differentiates Early Lymphoid and Myeloid Development

Blood ◽  
2020 ◽  
Vol 136 (Supplement 1) ◽  
pp. 37-37
Author(s):  
Kristin Komnick ◽  
Jennifer May ◽  
Pouneh Kermani ◽  
Sreemanti Basu ◽  
Irene Hernandez ◽  
...  
Keyword(s):  
Bone Marrow ◽  
Nervous System ◽  
Schwann Cell ◽  
Peripheral Nerve ◽  
Peripheral Nervous System ◽  
Schwann Cells ◽  
Adrenergic Nerve ◽  
Spleen Size ◽  
Mouse Line ◽  
Myeloid Development

Blood cell production is regulated by peripheral nerve fibers that innervate the bone marrow. However, little is known about the development or maintenance of hematopoietic innervation. Schwann cells (SCs) are the primary axon 'support cells' of the peripheral nervous system (PNS), and abnormal SC development is sufficient to impair peripheral nerve function. SCs are also the primary repair cell for the PNS which makes them an attractive therapeutic target for normalization of drug or malignancy-induced 'hematopoietic neuropathy'. We hypothesized that neural regulation of hematopoiesis is dependent on SC development. To test this hypothesis, we used the Myelin Protein Zero-Cre (MP0-Cre); Lamc1fl/fl mouse line in which laminin-γ1 expression is deleted from SC precursors and their progeny1. Early SC maturation is dependent on autocrine SC precursor-derived molecules such as laminin-γ1. SC differentiation arrests prior to axon sorting and ensheathment in MP0-Cre; Lamc1fl/fl mice, and causes a global peripheral neuropathy that persists throughout the lifetime of the animal. Preliminary hematopoietic analysis of 'steady state' MP0-Cre; Lamc1fl/fl and littermate control mice has shown the following: (1) MP0-Cre; Lamc1fl/fl bone marrow is innervated, and Cre-mediated gene recombination occurs in cells immunophenotypically consistent with SCs throughout the peripheral nervous system, including those in the bone marrow; (2) MP0-Cre; Lamc1fl/fl mice are lymphopenic but not neutropenic; (3) MP0-Cre; Lamc1fl/fl mice have significantly reduced spleen size and cellularity; and (4) MP0-Cre; Lamc1fl/fl bone marrow has an ~50% reduction in Lin-Sca-1+Kit+(LSK) cells (measured as a percentage of the Lin- compartment of the bone marrow). These results are consistent with earlier work by our groups in which we found that global Lamc1 gene deletion in adult mice induced peripheral blood lymphopenia, reduced spleen size, and a niche-dependent reduction of lymphoid progenitor and precursor cells that was secondary to increased lymphoid precursor cell apoptosis and reduced proliferation (UBC-CreERT2; Lamc1fl/fl mouse line). As with the SC-specific laminin-γ1 deficient mice, myelopoiesis was preserved in the UBC-CreERT2; Lamc1fl/fl mice. Based on results from MP0-Cre; Lamc1fl/fl and UBC-CreERT2; Lamc1fl/fl mice, we conclude that early lymphoid but not myeloid development requires laminin-γ1 expression by MP0-Cre-targetted niche cells, i.e. Schwann Cells. Our results are consistent with reports from other labs that hematopoietic sympathetic neuropathy promotes aberrant myeloid expansion at the expense of lymphopoiesis2. Going forward, we will determine whether lymphopoietic development is dependent on global versus laminin-specific SC-derived cues, and whether these signals are transmitted directly between SCs and lymphoid biased HSPCs or indirectly via other components of the hematopoietic niche. We anticipate that this line of investigation will provide molecular insights and pharmacologic targets for prevention and or normalization of the 'hematopoietic neuropathy' induced by diabetes, aging, neurotoxic chemotherapies and myeloid malignancies. REFERENCES: 1 Yu, W. M., Feltri, M. L., Wrabetz, L., Strickland, S. & Chen, Z. L. Schwann cell-specific ablation of laminin gamma1 causes apoptosis and prevents proliferation. J Neurosci25, 4463-4472, doi:10.1523/JNEUROSCI.5032-04.2005 (2005). 2 Maryanovich, M. et al. Adrenergic nerve degeneration in bone marrow drives aging of the hematopoietic stem cell niche. Nat Med24, 782-791, doi:10.1038/s41591-018-0030-x (2018). Disclosures No relevant conflicts of interest to declare.

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.

The mouse homeodomain protein Phox2 regulates Ncam promoter activity in concert with Cux/CDP and is a putative determinant of neurotransmitter phenotype

Development ◽  
1993 ◽  
Vol 119 (3) ◽  
pp. 881-896 ◽  
Author(s):  
I. Valarche ◽  
J.P. Tissier-Seta ◽  
M.R. Hirsch ◽  
S. Martinez ◽  
C. Goridis ◽  
...  
Keyword(s):  
Nervous System ◽  
Cell Adhesion ◽  
Peripheral Nervous System ◽  
Transcriptional Activation ◽  
Promoter Activity ◽  
Regulatory Element ◽  
Homeodomain Protein ◽  
Sensory Ganglia ◽  
Homeodomain Proteins ◽  
Regulatory Cascade

Transcriptional regulation of the gene encoding the cell adhesion receptor NCAM (neural cell adhesion molecule), a putative effector molecule of a variety of morphogenetic events, is likely to involve important regulators of morphogenesis. Here we identify two mouse homeodomain proteins that bind to an upstream regulatory element in the Ncam promoter: Cux, related to Drosophila cut and human CDP, and Phox2, a novel protein with a homeodomain related to that of the Drosophila paired gene. In transient transfection experiments, Cux was found to be a strong inhibitor of Ncam promoter activity, and this inhibition could be relieved by simultaneously overexpressing Phox2. These results suggest that the Ncam gene might be a direct target of homeodomain proteins and provide a striking example of regulatory cross-talk between homeodomain proteins of different classes. Whereas the expression pattern of Cux/CDP includes many NCAM-negative sites, Phox2 expression was restricted to cells also expressing Ncam or their progenitors. The localisation data thus strongly reinforce the notion that Phox2 plays a role in transcriptional activation of Ncam in Phox2-positive cell types. In the peripheral nervous system, Phox2 was strongly expressed in all ganglia of the autonomic nervous system and more weakly in some cranial sensory ganglia, but not in the sensory ganglia of the trunk. Phox2 transcripts were detected in the primordia of sympathetic ganglia as soon as they form. Phox2 expression in the brain was confined to spatially restricted domains in the hindbrain, which correspond to the noradrenergic and adrenergic nuclei once they are identifiable. All Phox2-expressing components of the peripheral nervous system are at least transiently adrenergic or noradrenergic. In the developing brain, Phox2 was expressed at all known locations of (nor)adrenergic neurones and of their precursors. These results suggest that Phox2, in addition to regulating the NCAM gene, may be part of the regulatory cascade that controls the differentiation of neurons towards this neurotransmitter phenotype.

Thranbospondin Promotes Cell-and Platelet-Substratum Adhesion

1987 ◽  
Author(s):  
George P Tuszynski ◽  
Vicki L Rothman ◽  
Andrew Murphy ◽  
Katherine Siegler ◽  
Linda Smith ◽  
...  
Keyword(s):  
Cell Adhesion ◽  
Cell Surface ◽  
Polyacrylamide Gel Electrophoresis ◽  
Cell Types ◽  
Preliminary Evidence ◽  
Human Platelets ◽  
Cell Surface Receptor ◽  
Binding Assays ◽  
Surface Receptor

Thrombospondin (TSP), isolated from human platelets, promotes the in vitro, calcium-specific adhesion of a variety of cells, including platelets, melanoma cells, muscle cells, endothelial cells, fibroblasts, and epithelial cells. The cell adhesion-promoting activity of TSP is species independent since human, bovine, pig, rat and mouse cells all adhered to TSP. Furthermore, the cell adhesion-promoting activity of TSP is specific and not due to a nonspecific protein effect or to contamination by fibronectin, vitronectin, or laminin. That is, neither bovine serum albumin nor TSP preparations treated with a monospecific anti-TSP antibody support cell adhesion. As analyzed by polyacrylamide-gel electrophoresis and specific antibody binding assays, the TSP preparations used in these studies contained no detectable fibronectin or laminin and less than 0.04% vitronectin. The cell surface receptor for TSP appears distinct frcm that of fibronectin since an antiserum that blocks cell adhesion to fibronectin has no effect on adhesion to TSP. In addition, The platelet cell surface receptor for TSP appears distinct, frcm that of fibrinogen since thrcmbasthenic platelets adhere to TSP as well as control platelets. Antibodies to the GPIIb-GPIIIa complex block platelet adhesion to fibrinogen but have no effect on adhesion to TSP. Initial characterization of the cell surface receptor for TSP shows it to be protein in nature since cells treated with trypsin fail to adhere to TSP. In summary, our results provide the first clear evidence that TSP specifically promotes cell-substratum adhesion of a variety of cell types independent of the animal species. Our preliminary evidence suggests that the cell-surface receptor(s) for TSP is protein and that it is distinct for the receptor for fibronectin and fibrinogen. Our data suggest that TSP may play a central role in normal adhesive events mediated by platelets and other cells, such as those involved in hemostasis and wound healing. In addition, TSP may be involved in pathological adhesive events mediated by platelets and tumor cells, such as those involved in cardiovascular disease and tumor cell metastasis.

scholarly journals Ankyrin-binding proteins related to nervous system cell adhesion molecules: candidates to provide transmembrane and intercellular connections in adult brain.

1993 ◽  
Vol 121 (1) ◽  
pp. 121-133 ◽  
Author(s):  
J Q Davis ◽  
T McLaughlin ◽  
V Bennett
Keyword(s):  
Nervous System ◽  
Cell Adhesion ◽  
Amino Acid ◽  
Amino Acid Sequence ◽  
Binding Site ◽  
Adhesion Molecules ◽  
Cell Adhesion Molecules ◽  
Binding Proteins ◽  
Cytoplasmic Domain ◽  
System Cell

A major class of ankyrin-binding glycoproteins have been identified in adult rat brain of 186, 155, and 140 kD that are alternatively spliced products of the same pre-mRNA. Characterization of cDNAs demonstrated that ankyrin-binding glycoproteins (ABGPs) share 72% amino acid sequence identity with chicken neurofascin, a membrane-spanning neural cell adhesion molecule in the Ig super-family expressed in embryonic brain. ABGP polypeptides have the following features consistent with a role as ankyrin-binding proteins in vitro and in vivo: (a) ABGPs and ankyrin associate as pure proteins in a 1:1 molar stoichiometry; (b) the ankyrin-binding site is located in the COOH-terminal 21 kD of ABGP186 which contains the predicted cytoplasmic domain; (c) ABGP186 is expressed at approximately the same levels as ankyrin (15 pmoles/milligram of membrane protein); and (d) ABGP polypeptides are co-expressed with the adult form of ankyrinB late in postnatal development and are colocalized with ankyrinB by immunofluorescence. Similarity in amino acid sequence and conservation of sites of alternative splicing indicate that genes encoding ABGPs and neurofascin share a common ancestor. However, the major differences in developmental expression reported for neurofascin in embryos versus the late postnatal expression of ABGPs suggest that ABGPs and neurofascin represent products of gene duplication events that have subsequently evolved in parallel with distinct roles. The predicted cytoplasmic domains of rat ABGPs and chicken neurofascin are nearly identical to each other and closely related to a group of nervous system cell adhesion molecules with variable extracellular domains, which includes L1, Nr-CAM, and Ng-CAM of vertebrates, and neuroglian of Drosophila. The ankyrin-binding site of rat ABGPs is localized to the C-terminal 200 residues which encompass the cytoplasmic domain, suggesting the hypothesis that ability to associate with ankyrin may be a shared feature of neurofascin and related nervous system cell adhesion molecules.

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