scholarly journals Mapping of epitopes for monoclonal antibodies against human platelet thrombospondin with electron microscopy and high sensitivity amino acid sequencing.

1985 ◽  
Vol 101 (4) ◽  
pp. 1434-1441 ◽  
Author(s):  
N J Galvin ◽  
V M Dixit ◽  
K M O'Rourke ◽  
S A Santoro ◽  
G A Grant ◽  
...  
Keyword(s):  
Electron Microscopy ◽  
Monoclonal Antibodies ◽  
Human Platelet ◽  
High Sensitivity ◽  
Binding Domain ◽  
Peptide Chain ◽  
Dimensional Structure ◽  
Globular Domain ◽  
Heparin Binding ◽  
Heparin Binding Domain

A panel of monoclonal antibodies (Mab's) has been raised against human platelet thrombospondin (TSP). One Mab, designated A2.5, inhibits the hemagglutinating activity of TSP and immunoprecipitates the NH2 terminal 25 kD heparin binding domain of TSP (Dixit, V.M., D. M. Haverstick, K. M. O'Rourke, S. W. Hennessy, G. A. Grant, S. A. Santoro, and W. A. Frazier, 1985, Biochemistry, in press). Another Mab, C6.7, blocks the thrombin-stimulated aggregation of live platelets and immunoprecipitates an 18-kD fragment distinct from the heparin binding domain (Dixit, V. M., D. M. Haverstick, K. M. O'Rourke, S. W. Hennessy, G. A. Grant, S. A. Santoro, and W. A. Frazier, 1985, Proc. Natl. Acad. Sci. 82: 3472-3476). To determine the relative locations of the epitopes for these Mabs in the three-dimensional structure of TSP, we have examined TSP-Mab complexes by electron microscopy of rotary-shadowed proteins. The TSP molecule is composed of three 180-kD subunits, each of which consists of a small globular domain (approximately 8 nm diam) and a larger globular domain (approximately 16 nm diam) connected by a thin, flexible strand. The subunit interaction site is on the thin connecting strands, nearer the small globular domains. Mab A2.5 binds to the cluster of three small domains, indicating that this region contains the heparin binding domain and thus represents the NH2 termini of the TSP peptide chains. Mab C6.7 binds to the large globular domains on the side opposite the point at which the connecting strand enters the domain, essentially the maximum possible distance from the A2.5 epitope. Using high sensitivity automated NH2 terminal sequencing of TSP chymotryptic peptides we have ordered these fragments within the TSP peptide chain and have confirmed that the epitope for C6.7 in fact lies near the extreme COOH terminus of the peptide chain. In combination with other data, we have been able to construct a map of the linear order of the identified domains of TSP that indicates that to a large extent, the domains are arranged co-linearly with the peptide chain.

scholarly journals Platelet thrombospondin mediates attachment and spreading of human melanoma cells.

1987 ◽  
Vol 104 (1) ◽  
pp. 131-139 ◽  
Author(s):  
D D Roberts ◽  
J A Sherwood ◽  
V Ginsburg
Keyword(s):  
Monoclonal Antibodies ◽  
Cell Attachment ◽  
Melanoma Cells ◽  
Binding Domain ◽  
Heparin Binding ◽  
Amino Terminal ◽  
Sulfated Fucan ◽  
Heparin Binding Domain ◽  
Carboxyl Terminal ◽  
Inhibition Studies

Human platelet thrombospondin adsorbed on plastic promotes attachment and spreading of human G361 melanoma cells. Attachment is rapid, and spreading is maximal by 90 min with 60-90% of the attached cells spread. In contrast, thrombospondin promotes attachment but not spreading of human C32 melanoma cells, which attach and spread only on laminin substrates. The specificity of these interactions and the regions of the thrombospondin molecule involved in attachment and spreading were examined using proteolytic fragments of thrombospondin and by inhibition studies. The sulfated fucan, fucoidan, and monoclonal antibody A2.5, which is directed against the heparin-binding domain of thrombospondin, selectively inhibit spreading but only weakly inhibit attachment. Monoclonal antibodies against some other domains of thrombospondin, however, are potent inhibitors of attachment. The amino-terminal heparin-binding domain of thrombospondin does not promote attachment. Large fragments lacking the heparin-binding domain support attachment but not spreading of G361 cells. Attachment activity is lost following removal of the 18-kD carboxyl-terminal domain. These results suggest that at least two melanoma ligands are involved in cell attachment and spreading on thrombospondin. The carboxyl-terminal region and perhaps other regions of the molecule bind to receptor(s) on the melanoma surface that promote initial attachment but not cell spreading. Interaction of the heparin-binding domain with sulfated glycoconjugates on melanoma surface proteoglycans and/or sulfated glycolipids mediates spreading. Monoclonal antibodies A2.5 and C6.7 also reverse spreading of G361 cells growing on glass culture substrates, suggesting that binding to thrombospondin mediates attachment of these melanoma cells in culture.

Overlap of IGF- and heparin-binding sites in rat IGF-binding protein-5

2000 ◽  
Vol 24 (1) ◽  
pp. 43-51 ◽  
Author(s):  
H Song ◽  
J Beattie ◽  
IW Campbell ◽  
GJ Allan
Keyword(s):  
Binding Site ◽  
Conformational Changes ◽  
Binding Protein ◽  
Binding Activity ◽  
Binding Domain ◽  
Site Directed Mutagenesis ◽  
Heparin Binding ◽  
Igf I ◽  
Heparin Binding Domain ◽  
Igf Binding

Using site-directed mutagenesis, we have undertaken a study of a potential IGF-binding site in the C-terminal domain of rat IGFBP-5, lying close to or within a previously described heparin-binding domain (residues 201-218) in this protein. After analysis of binding activity using three different methods - ligand blotting, solution phase equilibrium binding and biosensor measurement of real-time on- and off-rates - we report that the mutation of two highly conserved residues within this region (glycine 203 and glutamine 209) reduces the affinity of the binding protein for both IGF-I and IGF-II, while having no effect on heparin binding. In addition, we confirm that mutation of basic residues within the heparin-binding domain (R201L, K202E, K206Q and R214A) results in a protein that has attenuated heparin binding but shows only a small reduction in affinity for IGF-I and -II. Previous findings have described the reduction in affinity of IGFBP-5 for IGFs that occurs after complexation of the binding protein with heparin or other components of the extracellular matrix (ECM) and have postulated that such an interaction may result in conformational changes in protein structure, affecting subsequent IGF interaction. Our data suggesting potential overlap of heparin- and IGF-binding domains argue for a more direct effect of ECM modulation of the affinity of IGFBP-5 for ligand by partial occlusion of the IGF-binding site after interaction with ECM.

One-Dimensional Fluorescent Nanosized-Diamond Nanowires with Fluorescent Detection of Vitamin B12

NANO ◽  
2019 ◽  
Vol 14 (07) ◽  
pp. 1950084 ◽  
Author(s):  
Jilong Wang ◽  
Siheng Su ◽  
Jingjing Qiu ◽  
Shiren Wang
Keyword(s):  
Electron Microscopy ◽  
Photoelectron Spectroscopy ◽  
High Sensitivity ◽  
Anodic Aluminum Oxide Template ◽  
Fluorescent Detection ◽  
Dimensional Structure ◽  
Fluorescent Properties ◽  
One Dimensional ◽  
Sensitivity And Selectivity ◽  
The One

In this paper, a novel and facile method to achieve fluorescent nanosized-diamond based nanowire (NW) is reported. One-dimensional (1D) organic NW has received tremendous attention due to its superior chemical functionality and size-, shape-, and material-dependent properties. In addition, nanosized-diamond is comprehensively studied and investigated due to superior tunable fluorescent properties, cost-effectiveness, facile manufacturing and high biocompatibility. Through thermal treatment, sulfur-modified nanosized-diamond was fabricated by mixing oxidized nanosized-diamond and dibenzyl disulfide at 900∘C. Fourier transform infrared spectroscopy (FTIR), X-ray photoelectron spectroscopy (XPS) and zeta potential were employed to characterize sulfur-modified nanosized-diamond. After that, porous anodic aluminum oxide template-assisted cathodic electrophoretic deposition method was used to achieve sulfur-modified nanosized-diamond NW. Scanning electron microscopy and transmission electron microscopy were applied to present the one-dimensional structure of the NWs. The optical properties of sulfur nanosized-diamond NW were characterized via ultraviolet-visible spectroscopy and photoluminescence spectroscopy. Finally, the as-synthesized sulfur-modified nanosized-diamond NW-based optical sensor was fabricated to detect vitamin B[Formula: see text] with high sensitivity and selectivity.

C-terminal heparin-binding domain of fibronectin regulates integrin-mediated cell spreading but not the activation of mitogen-activated protein kinase

2001 ◽  
Vol 360 (1) ◽  
pp. 239-245 ◽  
Author(s):  
Jungyean KIM ◽  
Innoc HAN ◽  
Yeonhee KIM ◽  
Seungin KIM ◽  
Eok-Soo OH
Keyword(s):  
Cell Spreading ◽  
Binding Domain ◽  
Mitogen Activated Protein Kinase ◽  
Heparin Binding ◽  
Cell Binding ◽  
Rat Embryo ◽  
Mapk Activation ◽  
Mitogen Activated Protein ◽  
Heparin Binding Domain ◽  
Cell Binding Domain

Fibronectin (FN) stimulates multiple signalling events including mitogen-activated protein kinase (MAPK) activation. During cell spreading, both the cell-binding domain and the C-terminal heparin-binding domain (HepII) of FN co-operatively regulate cytoskeleton organization. However, in comparison with the large number of studies on the functions of cell-binding domain, there is little information about the role of HepII. We therefore investigated the effect of HepII on integrin-mediated cell spreading and adhesion on FN and MAPK activation. In contrast with cells on FN substrates, rat embryo fibroblasts on FN120, which lacks HepII, were less spread, had weaker adhesion to FN and failed to form focal adhesions and actin stress fibres. Phosphotyrosine was present in the focal contacts of rat embryo fibroblasts on FN within 30min but was absent from cells on FN120. Overall, tyrosine phosphorylation was much less in cell lysates from cells on FN120, with decreased phosphorylation of focal adhesion kinase (‘pp125FAK’) on tyrosine-397, implying additional regulation of tyrosine phosphorylation by HepII. Nevertheless, adhesion-mediated MAPK activity was similar in cells on FN and on FN120. Furthermore, cells spread on FN and on FN120 substrates showed similar MAPK activation in response to treatment with epidermal growth factor and with platelet-derived growth factor. Consistently, overexpression of syndecan-4, which binds to HepII, enhanced cell spreading and adhesion on FN but did not affect integrin-mediated MAPK activation. We therefore conclude that both HepII and syndecan-4 regulate integrin-mediated cell spreading but not MAPK activation.

scholarly journals Polyelectrolyte Complex for Heparin Binding Domain Osteogenic Growth Factor Delivery

10.3791/54202 ◽  
2016 ◽  
Author(s):  
Raymond Wing Moon Lam ◽  
Sunny Akogwu Abbah ◽  
Wang Ming ◽  
Mathanapriya Naidu ◽  
Felly Ng ◽  
...  
Keyword(s):  
Growth Factor ◽  
Polyelectrolyte Complex ◽  
Binding Domain ◽  
Growth Factor Delivery ◽  
Heparin Binding ◽  
Heparin Binding Domain
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