scholarly journals Fibrillin-1 and −2 contain heparin-binding sites important for matrix deposition and that support cell attachment

2003 ◽  
Vol 375 (2) ◽  
pp. 425-432 ◽  
Author(s):  
Timothy M. RITTY ◽  
Thomas J. BROEKELMANN ◽  
Claudio C. WERNECK ◽  
Robert P. MECHAM
Keyword(s):  
Extracellular Matrix ◽  
Cell Attachment ◽  
Heparan Sulphate ◽  
Cell Types ◽  
Binding Activity ◽  
Elastic Fibre ◽  
Heparin Binding ◽  
Matrix Deposition ◽  
Organ Systems ◽  
Fibrillin 1

Fibrillin-1 and −2 are large modular extracellular matrix glycoproteins found in many vertebrate organ systems and are known to be key components of the elastic fibre. In the present study, we identify a new heparin-binding region in fibrillin-2 between exons 18 and 24. Additionally, we have narrowed the location of heparin-binding activity previously identified in fibrillin-1 to the last 17 residues of the mature proteolytically processed protein. This domain demonstrated higher activity as a multimer than as a monomer. The fibrillin-1 C-terminal site supported cell attachment in each of nine cell types tested. Attachment was shown to be mediated by cell-surface heparan sulphate proteoglycans. Fibrillin-1 has been shown previously to have heparin-binding activity that is important for matrix deposition of the molecule by fibroblasts. This function in deposition was confirmed in two additional fibrillin-producing cell types (osteosarcoma and epithelial cells) for the deposition of both fibrillin-1 and −2 into the extracellular matrix.

scholarly journals Mammalian and Drosophila cells adhere to the laminin α4 LG4 domain through syndecans, but not glypicans

2004 ◽  
Vol 382 (3) ◽  
pp. 933-943 ◽  
Author(s):  
Hironobu YAMASHITA ◽  
Akira GOTO ◽  
Tatsuhiko KADOWAKI ◽  
Yasuo KITAGAWA
Keyword(s):  
Cell Adhesion ◽  
Umbilical Vein ◽  
Heparan Sulphate ◽  
Direct Interaction ◽  
Binding Activity ◽  
Main Body ◽  
Heparin Binding ◽  
Adhesion Activity ◽  
Umbilical Vein Endothelial Cells

We have previously shown that the LG4 (laminin G-like) domain of the laminin α4 chain is responsible for the significantly higher affinity of the α4 chain to heparin than found for other α chains [Yamaguchi, Yamashita, Mori, Okazaki, Nomizu, Beck and Kitagawa (2000) J. Biol. Chem. 275, 29458–29465]; four basic residues were identified to be essential for this activity [Yamashita, Beck and Kitagawa (2004) J. Mol. Biol. 335, 1145–1149]. By creating GST (glutathione S-transferase)-fused LG1, LG2, LG4 and LG5 proteins, we found that only LG4 is active for the adhesion of human HT1080 cells, human umbilical vein endothelial cells and Drosophila haemocytes Kc167 with a half-saturating concentration of 20 μg/ml. Adhesion was counteracted by treatment of the cells with heparin, heparan sulphate and heparitinase I. Upon mutating the four basic residues essential for heparin binding within LG4, the adhesion activity was abolished. Pull-down experiments using glutathione beads/GST-fusion proteins indicate a direct interaction of LG4 with syndecan-4, which might be the major receptor for cell adhesion. Neither the release of glypican-1 by treating human cells with phosphatidylinositol-specific phospholipase C nor targeted knockdown of dally or dally-like protein impaired the cell-adhesion activity. As the LG4–LG5 domain of the α4 chain is cleaved in vivo from the main body of laminin-8 (α4β1γ1), we suggest that the heparan sulphate proteoglycan-binding activity of LG4 is significant in modulating the signalling of Wnt, Decapentaplegic and fibroblast growth factors.

Heparin stimulates the proliferation of intestinal epithelial cells in primary culture

1994 ◽  
Vol 107 (2) ◽  
pp. 401-411
Author(s):  
N. Flint ◽  
F.L. Cove ◽  
G.S. Evans
Keyword(s):  
Growth Factors ◽  
Heparan Sulphate ◽  
Primary Cultures ◽  
Cell Types ◽  
Heparin Binding ◽  
Epithelial Proliferation ◽  
Inhibition Of Proliferation ◽  
Trophic Effect ◽  
Heparin Binding Growth Factors

Heparin is a sulphated glycosaminoglycan derived from mast cells and has a number of functions including the inhibition of proliferation in several cell types and interactions with a range of heparin-binding growth factors. We report that heparin is a trophic factor in primary cultures of rat small intestinal epithelium. Heparin elicits a dose-dependent increase in epithelial proliferation and inhibits the growth of associated mesenchyme. The trophic effect of this molecule is not reproduced by other glycosaminoglycans including heparan sulphate but is dependent upon extensive molecular sulphation. Highly sulphated polysaccharides that are structurally unrelated to heparin (e.g. dextran sulphate and pentosan polysulphate) also stimulate epithelial proliferation in primary cultures. Heparin may act by the potentiation of mesenchyme-derived heparin-binding growth factors and these data suggest an in vivo role for mast cell-derived heparin in mucosal wound regeneration.

scholarly journals Structure and binding properties of collagen type XIV isolated from human placenta.

1993 ◽  
Vol 120 (2) ◽  
pp. 557-567 ◽  
Author(s):  
J C Brown ◽  
K Mann ◽  
H Wiedemann ◽  
R Timpl
Keyword(s):  
Human Placenta ◽  
Matrix Protein ◽  
Cell Attachment ◽  
Chondroitin Sulphate ◽  
Heparan Sulphate ◽  
Extracellular Matrix Protein ◽  
Neutral Salt ◽  
Heparin Binding ◽  
Collagen Vi ◽  
Sulphate Proteoglycan

Collagen XIV was isolated from neutral salt extracts of human placenta and purified by several chromatographic steps including affinity binding to heparin. The same procedures also led to the purification of a tissue form of fibronectin. Collagen XIV was demonstrated by partial sequence analysis of its Col1 and Col2 domains and by electron microscopy to be a disulphide-linked molecule with a characteristic cross-shape. The individual chains had a size of approximately 210 kD, which was reduced to approximately 180 kD (domain NC3) after treatment with bacterial collagenase. Specific antibodies mainly to NC3 epitopes were obtained by affinity chromatography and used in tissue and cell analyses by immunoblotting and radioimmunoassays. Two sequences from NC3 were identified on fragments obtained after trypsin cleavage. They were identical to cDNA-derived sequences of undulin, a noncollagenous extracellular matrix protein. This suggests that collagen XIV and undulin may be different splice variants from the same gene. Heparin binding was confirmed in ligand assays with a large basement membrane heparan sulphate proteoglycan. This binding could be inhibited by heparin and heparan sulphate but not by chondroitin sulphate. In addition, collagen XIV bound to the triple helical domain of collagen VI. The interactions with heparin sulphate proteoglycan and collagen VI were not shared by the NC3 domain, or by reduced and alkylated collagen XIV. No or only low binding was observed for collagens I-V, pN-collagens I and III, and several noncollagenous matrix proteins, including laminin, recombinant nidogen, BM-40/osteonectin, plasma and tissue fibronectin, vitronectin, and von Willebrand factor. Insignificant activity was also shown in cell attachment assays with nine established cell lines.

scholarly journals Fibulin1C peptide induces cell attachment and extracellular matrix deposition in lung fibroblasts

2015 ◽  
Vol 5 (1) ◽  
Author(s):  
Qi Ge ◽  
Ling Chen ◽  
Jade Jaffar ◽  
William Scott Argraves ◽  
Waleed O. Twal ◽  
...  
Keyword(s):  
Extracellular Matrix ◽  
Cell Attachment ◽  
Lung Fibroblasts ◽  
Matrix Deposition ◽  
Extracellular Matrix Deposition

scholarly journals Characterization of multiple adhesive and counteradhesive domains in the extracellular matrix protein cytotactin.

1992 ◽  
Vol 119 (3) ◽  
pp. 663-678 ◽  
Author(s):  
A L Prieto ◽  
C Andersson-Fisone ◽  
K L Crossin
Keyword(s):  
Extracellular Matrix ◽  
Cell Attachment ◽  
Cell Types ◽  
Expression Vectors ◽  
Extracellular Matrix Protein ◽  
Dependent Manner ◽  
Type Iii ◽  
Fibronectin Type Iii ◽  
Fibronectin Type ◽  
Better Than

The extracellular matrix molecule cytotactin is a multidomain protein that plays a role in cell migration, proliferation, and differentiation during development. To analyze the structure-function relationships of the different domains of this glycoprotein, we have prepared a series of fusion constructs in bacterial expression vectors. Results obtained using a number of adhesion assays suggest that at least four independent cell binding regions are distributed among the various cytotactin domains. Two of these are adhesive; two others appear to be counteradhesive in that they inhibit cell attachment to otherwise favorable substrates. The adhesive regions were mapped to the fibronectin type III repeats II-VI and the fibrinogen domain. The morphology of the cells plated onto these adhesive fragments differed; the cells spread on the fibronectin type III repeats as they do on fibronectin, but remained round on the fibrinogen domain. The counteradhesive properties of the molecule were mapped to the EGF-like repeats and the last two fibronectin type III repeats, VII-VIII. The latter region also contained a cell attachment activity that was observed only after proteolysis of the cells. Several cell types were used in these analyses, including fibroblasts, neurons, and glia, all of which are known to bind to cytotactin. The different domains exert their effects in a concentration-dependent manner and can be inhibited by an excess of the soluble molecule, consistent with the hypothesis that the observed properties are mediated by specific receptors. Moreover, it appears that some of these receptors are restricted to particular cell types. For example, glial cells bound better than neurons to the fibrinogen domain and fibroblasts bound better than glia and neurons to the EGF fragment. These results provide a basis for understanding the multiple activities of cytotactin and a framework for isolating different receptors that mediate the various cellular responses to this molecule.

Complete sequence, recombinant analysis and binding to laminins and sulphated ligands of the N-terminal domains of laminin α3B and α5 chains

2002 ◽  
Vol 362 (2) ◽  
pp. 213-221 ◽  
Author(s):  
Jörg H. O. GARBE ◽  
Walter GÖHRING ◽  
Karlheinz MANN ◽  
Rupert TIMPL ◽  
Takako SASAKI
Keyword(s):  
Self Assembly ◽  
Heparan Sulphate ◽  
Complete Sequence ◽  
Binding Activity ◽  
Heparin Binding ◽  
Hek 293 ◽  
Mouse Tissues ◽  
Folded Proteins ◽  
Domain V ◽  
Laminin 1

The N-terminal sequences of mouse laminin α3B and α5 chains have been completed and demonstrate the presence of a signal peptide followed by a complete laminin N-terminal (LN) module (domain VI). These signal peptides were released after recombinant production of larger fragments comprising domains VI/V (45–65kDa) from this region yielding properly folded proteins, which were secreted from HEK-293—EBNA cells. Pepsin digestion of these fragments yielded products of 25–35kDa, which consisted only of domain V. The αVI/V fragments were able to inhibit self-assembly of laminin-1, with those from the α3B and α5 chains being more active than those from α1 and α2 chains. Domain V fragments, however, showed a reduced activity, indicating the major contribution of the LN module in inhibition. These interactions were confirmed by surface-plasmon-resonance assays demonstrating moderate affinities (Kd = 0.02 to > 6μM) for the binding to laminin-1. This indicated that laminins containing α3B or α5 chains should also be able to form non-covalent networks by polymerization. The LN modules also showed heparin binding in affinity chromatography, which was strongest for α1/α2, moderate for α3B, whereas no binding was observed for α5. They all bound to heparan sulphate chains of perlecan and to sulphatides, with a lower variability in binding activity. Specific antibodies were raised against α3BVI/V and α5VI/V and were shown to stain basement membrane zones in various mouse tissues. These antibodies also allowed the identification of a new laminin assembly form 5B consisting of α3B, β3 and γ2 chains.

scholarly journals Heparin binding lectin of human placenta as a tool for histochemical ligand localization and isolation.

1991 ◽  
Vol 39 (9) ◽  
pp. 1249-1256 ◽  
Author(s):  
H J Gabius ◽  
B Kohnke-Godt ◽  
M Leichsenring ◽  
A Bardosi
Keyword(s):  
Human Placenta ◽  
Binding Sites ◽  
Specific Binding ◽  
Lectin Binding ◽  
Polyacrylamide Gel Electrophoresis ◽  
Cell Types ◽  
Binding Activity ◽  
Full Term ◽  
Heparin Binding ◽  
Specific Binding Sites

Biotinylated heparin has been used to detect the presence of specific binding sites in sections of human placenta, which has prompted demonstration of expression of lectin activity for this proteoglycan. Purification of this lectin from full-term placenta facilitates the synthesis of its biotinylated derivative, using biotin-amidocaproyl hydrazide, without affecting its activity. It also enables immunization to obtain antibodies. The labeled lectin is shown to bind specifically to nuclear and cytoplasmic locations in various cell types of human placenta, nuclear expression of lectin binding sites being more pronounced at the full-term stage than after 8 weeks of development. The structurally related histone H2B exhibits obvious differences in its binding pattern. The presence of ligands accessible to the lectin whose binding activity can be inhibited by addition of an excess of heparin correlates in most instances with the level of lectin expression detected immunohistochemically. Biochemical information on the nature of the glycohistochemically inferred lectin-specific ligand(s) is obtained by affinity chromatography on resin-immobilized lectin. It leads to isolation of a proteoglycan with similar electrophoretic mobility in agarose-polyacrylamide gel electrophoresis relative to the independently purified heparan sulfate-containing fibronectin binding proteoglycan from human placenta. Both fractions inhibit binding of heparin to the lectin and contain immunologically detected co-purified lectin, emphasizing their ligand properties. Application of labeled tissue lectins in conjunction with lectin-specific antibodies is proposed to obtain valuable insights into the expression of the receptor as well as the ligand part of protein-carbohydrate recognition.

scholarly journals Human fibronectin contains distinct adhesion- and motility-promoting domains for metastatic melanoma cells.

1986 ◽  
Vol 102 (1) ◽  
pp. 179-188 ◽  
Author(s):  
J B McCarthy ◽  
S T Hagen ◽  
L T Furcht
Keyword(s):  
Cell Adhesion ◽  
Tumor Cells ◽  
Cell Attachment ◽  
Metastatic Tumor ◽  
Melanoma Cells ◽  
Binding Activity ◽  
Heparin Binding ◽  
Tryptic Fragment ◽  
Synthetic Cell

The active migration of tumor cells through extracellular matrices has been proposed to play a role in certain aspects of metastasis. Metastatic tumor cells migrate in vitro in response to substratum-bound adhesive glycoproteins such as fibronectin. The present studies use affinity-purified proteolytic fragments of fibronectin to determine the nature of adhesion- and/or motility-promoting domains within the protein. Two distinct fragments were identified with cell adhesion-promoting activities. By a number of criteria, the adhesive activity promoted by these two fragments was distinct. One fragment, a 75-kD tryptic fragment purified by monoclonal antibody chromatography, promoted the adhesion, spreading, and haptotactic motility of melanoma cells. Experiments using a synthetic cell attachment peptide in solution indicated that at least part of the attachment activity exhibited by the 75-kD fragment is mediated by the sequence arg-gly-asp-ser. It was not possible to demonstrate migration-stimulating activity using a small (11.5 kD) peptic fragment containing this sequence (Pierschbacher, M.D., E. G. Hayman, and E. Ruoslahti, 1981, Cell, 26:259-267) suggesting that another cell-binding activity within the 75 kD fragment distinct from arg-gly-asp-ser might be required for motility. The second fragment that stimulated melanoma adhesion was a 33-kD tryptic/catheptic carboxyl-terminal heparin-binding fragment, which is localized to the A chain of fibronectin. This fragment promotes adhesion and spreading but not the motility of these cells. Melanoma adhesion to this heparin-binding fragment was sensitive to the effects of cycloheximide, which contrasted adhesion to the haptotaxis-promoting fragment. Importantly, these studies illustrate that haptotaxis in response to fibronectin is not due to simple adhesion gradients of this protein. The results are discussed in light of a model for multiple distinct cell surface constituents mediating cell adhesion and motility on fibronectin.

scholarly journals Role of the extracellular matrix protein thrombospondin in the early development of the mouse embryo.

1990 ◽  
Vol 111 (6) ◽  
pp. 2713-2723 ◽  
Author(s):  
K S O'Shea ◽  
L H Liu ◽  
L H Kinnunen ◽  
V M Dixit
Keyword(s):  
Extracellular Matrix ◽  
Matrix Protein ◽  
Inner Cell Mass ◽  
Cell Mass ◽  
Cell Types ◽  
Extracellular Matrix Protein ◽  
Heparin Binding ◽  
Cell Stage ◽  
Binding Domains ◽  
Heparin Binding Domain

The distribution of the extracellular matrix protein thrombospondin (TSP) in cleavage to egg cylinder staged mouse embryos and its role in trophoblast outgrowth from cultured blastocysts were examined. TSP was present within the cytoplasm of unfertilized eggs; in fertilized one- to four-cell embryos; by the eight-cell stage, TSP was also densely deposited at cell-cell borders. In the blastocyst, although TSP was present in all three cell types; trophectoderm, endoderm, and inner cell mass (ICM), it was enriched in the ICM and at the surface of trophectoderm cells. Hatched blastocysts grown on matrix-coated coverslips formed extensive trophoblast outgrowths on TSP, grew slightly less avidly on laminin, or on a 140-kD fragment of TSP containing its COOH terminus and putative cell binding domains. There was little outgrowth on the NH2 terminus heparin-binding domain. Addition of anti-TSP antibodies (but not GRGDS) to blastocysts growing on TSP strikingly inhibited outgrowth. Consistent with its early appearance and presence in trophoblast cells during implantation, TSP may play an important role in the early events involved in mammalian embryogenesis.

Sign in / Sign up

Export Citation Format

Share Document