scholarly journals Mapping the heparin-binding sites on type I collagen monomers and fibrils.

1994 ◽  
Vol 125 (5) ◽  
pp. 1179-1188 ◽  
Author(s):  
J D San Antonio ◽  
A D Lander ◽  
M J Karnovsky ◽  
H S Slayter
Keyword(s):  
Electron Microscopy ◽  
Extracellular Matrix ◽  
Cell Adhesion ◽  
Heparan Sulfate ◽  
Binding Site ◽  
Binding Sites ◽  
Type I Collagen ◽  
Type I ◽  
Heparin Binding ◽  
Heparin Binding Site

The glycosaminoglycan chains of cell surface heparan sulfate proteoglycans are believed to regulate cell adhesion, proliferation, and extracellular matrix assembly, through their interactions with heparin-binding proteins (for review see Ruoslahti, E. 1988. Annu. Rev. Cell Biol. 4:229-255; and Bernfield, M., R. Kokenyesi, M. Kato, M. T. Hinkes, J. Spring, R. L. Gallo, and E. J. Lose. 1992. Annu. Rev. Cell Biol. 8:365-393). Heparin-binding sites on many extracellular matrix proteins have been described; however, the heparin-binding site on type I collagen, a ubiquitous heparin-binding protein of the extracellular matrix, remains undescribed. Here we used heparin, a structural and functional analogue of heparan sulfate, as a probe to study the nature of the heparan sulfate proteoglycan-binding site on type I collagen. We used affinity coelectrophoresis to study the binding of heparin to various forms of type I collagen, and electron microscopy to visualize the site(s) of interaction of heparin with type I collagen monomers and fibrils. Using affinity coelectrophoresis it was found that heparin has similar affinities for both procollagen and collagen fibrils (Kd's approximately 60-80 nM), suggesting that functionally similar heparin-binding sites exist in type I collagen independent of its aggregation state. Complexes of heparin-albumin-gold particles and procollagen were visualized by rotary shadowing and electron microscopy, and a preferred site of heparin binding was observed near the NH2 terminus of procollagen. Native or reconstituted type I collagen fibrils showed one region of significant heparin-gold binding within each 67-nm period, present near the division between the overlap and gap zones, within the "a" bands region. According to an accepted model of collagen fibril structure, our data are consistent with the presence of a single preferred heparin-binding site near the NH2 terminus of the collagen monomer. Correlating these data with known type I collagen sequences, we suggest that the heparin-binding site in type I collagen may consist of a highly basic triple helical domain, including several amino acids known sometimes to function as disaccharide acceptor sites. We propose that the heparin-binding site of type I collagen may play a key role in cell adhesion and migration within connective tissues, or in the cell-directed assembly or restructuring of the collagenous extracellular matrix.

Heparan sulfate proteoglycans function in the binding and degradation of vitronectin by fibroblast monolayers

1996 ◽  
Vol 74 (6) ◽  
pp. 887-897 ◽  
Author(s):  
Cynthia E. Wilkins-Port ◽  
Paula J. McKeown-Longo
Keyword(s):  
Extracellular Matrix ◽  
Heparan Sulfate ◽  
Binding Site ◽  
Fibroblast Cell ◽  
Sodium Chlorate ◽  
Heparin Binding ◽  
The Matrix ◽  
Cell Layers ◽  
Dose Dependent Decrease ◽  
Dose Dependent

Vitronectin, a 75-kDa plasma protein is also found in the extracellular matrix, where it is believed to promote cell adhesion and migration. In addition to its role in adhesion, matrix vitronectin is also believed to function as an opsonin promoting the clearance of thrombin–serpin complexes from the matrix. Vitronectin is cleared from the matrix by receptor-mediated endocytosis followed by lysosomal degradation, suggesting that cells can regulate the levels of vitronectin present in the matrix. However, the mechanism by which plasma vitronectin associates with the extracellular matrix remains unclear. Studies were conducted to define the binding site(s) for vitronectin in fibroblast cell layers. Sodium chlorate, a competitive inhibitor of proteoglycan sulfation, produced a dose-dependent decrease in both binding and degradation of vitronectin. This inhibition was reversible in that removal of chlorate returned both binding and degradation of vitronectin to near control levels within 24 h. The binding of vitronectin to cell layers was not dependent on cells because vitronectin bound directly to isolated matrix. Isolated matrices prepared from cell layers treated with sodium chlorate also exhibited a dose-dependent decrease in vitronectin binding, consistent with the binding site for vitronectin in the matrix being sulfated proteoglycans. Binding and degradation of vitronectin were also sensitive to the addition of exogenous heparin, suggesting that the heparin binding domain of vitronectin was mediating binding to the matrix. Incubating fibroblast monolayers with heparinase III resulted in a 40% decrease in binding and degradation of vitronectin. Taken together, the above findings suggest that vitronectin's binding to the matrix and its subsequent degradation are dependent on heparan sulfate proteoglycans.Key words: endocytosis, vitronectin, proteoglycan, extracellular matrix.

scholarly journals Mapping of three major heparin-binding sites on laminin and identification of a novel heparin-binding site on the B1 chain

1989 ◽  
Vol 264 (30) ◽  
pp. 17971-17978
Author(s):  
K Kouzi-Koliakos ◽  
G G Koliakos ◽  
E C Tsilibary ◽  
L T Furcht ◽  
A S Charonis
Keyword(s):  
Binding Site ◽  
Binding Sites ◽  
Heparin Binding ◽  
Heparin Binding Site

scholarly journals Expression of syndecan regulates human myeloma plasma cell adhesion to type I collagen

Blood ◽  
1993 ◽  
Vol 81 (3) ◽  
pp. 767-774 ◽  
Author(s):  
RC Ridley ◽  
H Xiao ◽  
H Hata ◽  
J Woodliff ◽  
J Epstein ◽  
...  
Keyword(s):  
Bone Marrow ◽  
Cell Adhesion ◽  
B Cells ◽  
Heparan Sulfate ◽  
Cell Lines ◽  
Type I Collagen ◽  
Myeloma Cell ◽  
Type I ◽  
Myeloma Cells ◽  
Human Myeloma Cell

The syndecans comprise a family of integral membrane proteoglycans that regulate cell behaviors by binding to extracellular matrix and binding growth factors. In mouse blood cells, syndecan expression is restricted to cells of the B-cell lineage where it is expressed by pre-B cells and plasma cells, but is absent from circulating B cells. In the present study, we examined the expression, structure, and function of syndecan on human myeloma cell lines and myeloma patient bone marrow cells. On myeloma cells, syndecan is a small (modal relative molecular mass [M(r)] = 120 Kd) heparan sulfate proteoglycan localized at the cell surface. Syndecan was detected by immunodot blotting on 7 of 10 human myeloma cell lines and by reverse transcriptase polymerase chain reaction on 10 of 14 patient samples. Cell binding assays show that myeloma cells expressing syndecan bind to type I collagen via heparan sulfate chains, while those cell lines not expressing syndecan do not bind to collagen. Furthermore, the cell lines expressing syndecan were negative for CD19 and CD45 staining, indicating that syndecan expression is restricted to tumors having a well-differentiated phenotype. We conclude that syndecan acts as a matrix receptor on human myeloma cells but is not expressed by all tumors, suggesting that syndecan may participate in regulating myeloma cell adhesion to the bone marrow stromal matrix.

scholarly journals Proteomics-based screening of the endothelial heparan sulfate interactome reveals that C-type lectin 14a (CLEC14A) is a heparin-binding protein

2020 ◽  
Vol 295 (9) ◽  
pp. 2804-2821 ◽  
Author(s):  
Daniel R. Sandoval ◽  
Alejandro Gomez Toledo ◽  
Chelsea D. Painter ◽  
Ember M. Tota ◽  
M. Osman Sheikh ◽  
...  
Keyword(s):  
Heparan Sulfate ◽  
Binding Site ◽  
Binding Proteins ◽  
Limited Proteolysis ◽  
Extracellular Proteins ◽  
Live Cells ◽  
Heparin Binding ◽  
Animal Cells ◽  
Lectin Domain ◽  
Heparin Binding Site

Animal cells express heparan sulfate proteoglycans that perform many important cellular functions by way of heparan sulfate–protein interactions. The identification of membrane heparan sulfate–binding proteins is challenging because of their low abundance and the need for extensive enrichment. Here, we report a proteomics workflow for the identification and characterization of membrane-anchored and extracellular proteins that bind heparan sulfate. The technique is based on limited proteolysis of live cells in the absence of denaturation and fixation, heparin-affinity chromatography, and high-resolution LC-MS/MS, and we designate it LPHAMS. Application of LPHAMS to U937 monocytic and primary murine and human endothelial cells identified 55 plasma membrane, extracellular matrix, and soluble secreted proteins, including many previously unidentified heparin-binding proteins. The method also facilitated the mapping of the heparin-binding domains, making it possible to predict the location of the heparin-binding site. To validate the discovery feature of LPHAMS, we characterized one of the newly-discovered heparin-binding proteins, C-type lectin 14a (CLEC14A), a member of the C-type lectin family that modulates angiogenesis. We found that the C-type lectin domain of CLEC14A binds one-to-one to heparin with nanomolar affinity, and using molecular modeling and mutagenesis, we mapped its heparin-binding site. CLEC14A physically interacted with other glycosaminoglycans, including endothelial heparan sulfate and chondroitin sulfate E, but not with neutral or sialylated oligosaccharides. The LPHAMS technique should be applicable to other cells and glycans and provides a way to expand the repertoire of glycan-binding proteins for further study.

scholarly journals Binding of soluble type I collagen to fibroblasts: effects of thermal activation of ligand, ligand concentration, pinocytosis, and cytoskeletal modifiers.

1982 ◽  
Vol 95 (3) ◽  
pp. 747-751 ◽  
Author(s):  
B D Goldberg
Keyword(s):  
Binding Site ◽  
Binding Sites ◽  
Thermal Activation ◽  
Type I Collagen ◽  
Type I ◽  
Collagen Binding ◽  
Bacterial Collagenase ◽  
Ligand Interactions ◽  
Organizing Center ◽  
High Ligand

Efficient binding of native, soluble 125I-labeled type I rat collagen to mouse 3T3 fibroblast monolayers requires prior warming of the ligand to 35-37 degrees C for 10-30 min. Decreased binding at high ligand concentrations is ascribed to ligand-ligand interactions rather than to negative cooperativity. Addition of bacterial collagenase to monolayers labeled with the 125I-ligand releases a constant fraction (80%) of the bound ligand over a 2-h interval at 37 degrees C, indicating that little of the ligand becomes inaccessible by pinocytosis. Colchicine (10(-7) M) and vinblastine (5 X 10(-8) M) do not inhibit binding by morphologically intact monolayers. Cytochalasins and concanavalin A show dose-related inhibition of binding by intact monolayers that is due to a reduction in the number of available binding sites rather than to a change in binding site affinity. The collagen binding site on the fibroblast surface is proposed as an organizing center for the assembly of periodic type I collagen fibrils.

scholarly journals Expression of syndecan regulates human myeloma plasma cell adhesion to type I collagen

Blood ◽  
1993 ◽  
Vol 81 (3) ◽  
pp. 767-774 ◽  
Author(s):  
RC Ridley ◽  
H Xiao ◽  
H Hata ◽  
J Woodliff ◽  
J Epstein ◽  
...  
Keyword(s):  
Bone Marrow ◽  
Cell Adhesion ◽  
B Cells ◽  
Heparan Sulfate ◽  
Cell Lines ◽  
Type I Collagen ◽  
Myeloma Cell ◽  
Type I ◽  
Myeloma Cells ◽  
Human Myeloma Cell

Abstract The syndecans comprise a family of integral membrane proteoglycans that regulate cell behaviors by binding to extracellular matrix and binding growth factors. In mouse blood cells, syndecan expression is restricted to cells of the B-cell lineage where it is expressed by pre-B cells and plasma cells, but is absent from circulating B cells. In the present study, we examined the expression, structure, and function of syndecan on human myeloma cell lines and myeloma patient bone marrow cells. On myeloma cells, syndecan is a small (modal relative molecular mass [M(r)] = 120 Kd) heparan sulfate proteoglycan localized at the cell surface. Syndecan was detected by immunodot blotting on 7 of 10 human myeloma cell lines and by reverse transcriptase polymerase chain reaction on 10 of 14 patient samples. Cell binding assays show that myeloma cells expressing syndecan bind to type I collagen via heparan sulfate chains, while those cell lines not expressing syndecan do not bind to collagen. Furthermore, the cell lines expressing syndecan were negative for CD19 and CD45 staining, indicating that syndecan expression is restricted to tumors having a well-differentiated phenotype. We conclude that syndecan acts as a matrix receptor on human myeloma cells but is not expressed by all tumors, suggesting that syndecan may participate in regulating myeloma cell adhesion to the bone marrow stromal matrix.

scholarly journals A Triad of Lys12, Lys41, Arg78 Spatial Domain, a Novel Identified Heparin Binding Site on Tat Protein, Facilitates Tat-Driven Cell Adhesion

PLoS ONE ◽  
2008 ◽  
Vol 3 (7) ◽  
pp. e2662 ◽  
Author(s):  
Jing Ai ◽  
Xianliang Xin ◽  
Mingyue Zheng ◽  
Shuai Wang ◽  
Shuying Peng ◽  
...  
Keyword(s):  
Cell Adhesion ◽  
Binding Site ◽  
Spatial Domain ◽  
Heparin Binding ◽  
Tat Protein ◽  
Heparin Binding Site

scholarly journals Thrombospondin-2 deficiency in growing mice alters bone collagen ultrastructure and leads to a brittle bone phenotype

2015 ◽  
Vol 119 (8) ◽  
pp. 872-881 ◽  
Author(s):  
Eugene Manley ◽  
Joseph E. Perosky ◽  
Basma M. Khoury ◽  
Anita B. Reddy ◽  
Kenneth M. Kozloff ◽  
...  
Keyword(s):  
Electron Microscopy ◽  
Mechanical Properties ◽  
Transmission Electron Microscopy ◽  
Extracellular Matrix ◽  
Cortical Bone ◽  
Type I Collagen ◽  
Type I ◽  
Wild Type ◽  
Transmission Electron ◽  
Series Of Experiments

Thrombospondin-2 (TSP2) is a matricellular protein component of the bone extracellular matrix. Long bones of adult TSP2-deficient mice have increased endosteal bone thickness due to expansion of the osteoblast progenitor cell pool, and these cells display deficits in osteoblastic potential. Here, we investigated the effects of TSP2 deficiency on whole bone geometric and mechanical properties in growing 6-wk-old male and female wild-type and TSP2-knockout (KO) mice. Microcomputed tomography and mechanical testing were conducted on femora and L2 vertebrae to assess morphology and whole bone mechanical properties. In a second series of experiments, femoral diaphyses were harvested from wild-type and TSP2-KO mice. Detergent-soluble type I collagen content was determined by Western blot of right femora. Total collagen content was determined by hydroxyproline analysis of left femora. In a third series of experiments, cortical bone was dissected from the anterior and posterior aspects of the femoral middiaphysis and imaged by transmission electron microscopy to visualize collagen fibrils. Microcomputed tomography revealed minimal structural effects of TSP2 deficiency. TSP2 deficiency imparted a brittle phenotype on cortical bone. Femoral tissue mineral density was not affected by TSP2 deficiency. Instead, transmission electron microscopy revealed less intensely stained collagen fibrils with altered morphology in the extracellular matrix assembled by osteoblasts on the anterior surface of TSP2-KO femora. Femoral diaphyseal bone displayed comparable amounts of total collagen, but the TSP2-KO bones had higher levels of detergent-extractable type I collagen. Together, our data suggest that TSP2 is required for optimal collagen fibrillogenesis in bone and thereby contributes to normal skeletal tissue quality.

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