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PubMed2000

Localization of urokinase type plasminogen activator to focal adhesions requires ligation of vitronectin integrin receptors.

Previous studies have shown that the adhesion protein, vitronectin, directs the localization of urokinase-type plasminogen activator (uPA) to areas of cell-substrate adhesion, where uPA is thought to regulate cell migration as well as pericellular proteolysis. In the present study, HT-1080 cell lines expressing either wild-type vitronectin or vitronectin containing a single amino-acid substitution in the integrin binding domain were used to assess whether ligation of the alphavbeta5 integrin was required for uPA localization to focal adhesions. The synthesis of wild-type vitronectin by HT-1080 cells adherent to either collagen or fibronectin resulted in the redistribution of both the alphavbeta5 integrin as well as uPA to focal adhesion structures. In contrast, cells synthesizing mutant vitronectin, containing the amino-acid substitution in the integrin binding domain, were unable to direct the redistribution of either alphavbeta5 or uPA to focal adhesions. Recombinant forms of wild-type and mutant vitronectin were prepared in a baculovirus system and compared for their ability to direct the redistribution of vitronectin integrin receptors as well as uPA on human skin fibroblasts. In the absence of vitronectin, fibroblast cells adherent to fibronectin assemble focal adhesions which contain the beta1 integrin but do not contain uPA. Addition of recombinant wild-type, but not mutant, vitronectin to fibroblasts adherent to fibronectin resulted in the redistribution of alphavbeta3, alphavbeta5, and uPA into focal adhesions. However, when cells were plated directly onto antibodies directed against either the alphavbeta3 or alphavbeta5 integrins, uPA was not localized on the cell surface. These data indicate that ligation of vitronectin integrin receptors is necessary but not sufficient for the localization of uPA to areas of cell matrix adhesion, and suggest that vitronectin may promote cell migration by recruiting vitronectin integrin receptors and components of the plasminogen activator system to areas of cell matrix contact.

Extracellular Matrix↗

PubMed1994

Vitronectin-driven human keratinocyte locomotion is mediated by the alpha v beta 5 integrin receptor.

Vitronectin is a soluble serum factor that is known to promote epiboly of keratinocytes in explant cultures and enhance cell spreading and attachment to matrix. Recently, vitronectin was demonstrated to promote human keratinocyte locomotion. The mechanism(s) by which vitronectin enhances keratinocyte migration is unknown. In this study, we quantitated the vitronectin-driven migration of human keratinocytes in the presence of antibodies to vitronectin receptors. We found that vitronectin's effect of promoting human keratinocyte migration was inhibited by antibody-directed against the alpha v beta 5 receptor. In addition, we surface-labeled human keratinocytes, chromatographed extracts of the cell membranes on a vitronectin column, and then immunoprecipitated the bound and eluted proteins with antibodies to specific vitronectin receptors. We identified the vitronectin receptors on human keratinocytes as bands of 150,000 and 100,000 daltons without reduction and as 125,000 and 110,000 daltons under reducing conditions. Immunoprecipitation with specific antibodies identified the major receptor to be the alpha v beta 5 integrin. In addition, we quantitated vitronectin-driven migration of human keratinocytes in the presence of Arg-Gly-Asp (RGD) and control peptides. We found that the presence of RGD, but not control peptide, inhibited vitronectin-driven migration of human keratinocytes. These studies demonstrate that human keratinocytes express vitronectin receptors and use the alpha v beta 5 receptor for cellular locomotion.

Antibodies↗

PubMed1985

Vitronectin--a major cell attachment-promoting protein in fetal bovine serum.

Bovine serum is a constituent of most media used for the culture of animal cells. The adhesion-promoting properties of serum are generally attributed to fibronectin, yet there have been frequent reports of other adhesion-promoting molecules in bovine serum. Using a technique in which adhesive proteins are visualized after separation by SDS-PAGE, we graphically confirm the presence of a second cell attachment protein in bovine serum and present the evidence that this molecule is the bovine equivalent of vitronectin. The molecular size of this protein is in the same range as the size of the adhesive human plasma protein, vitronectin. The bovine protein also shared with human vitronectin an affinity for glass, and it could be purified by a combination of glass bead and ion exchange chromatography. The isolated bovine protein had varying proportions of an 80 and a 65 kD polypeptide. It showed immunological cross-reactivity with anti-human vitronectin and with anti-human somatomedin B. Somatomedin B is a serum peptide which has a NH2-terminal sequence identical to that of human vitronectin. The identity of the bovine protein as vitronectin was established by showing that its NH2-terminal amino acid sequence is strongly homologous with those of human vitronectin and somatomedin B. Quantitation of the adhesive activities of fibronectin and vitronectin in bovine plasma and fresh serum showed that more activity is associated with vitronectin than with fibronectin. The preponderance of vitronectin was particularly clear in fetal bovine serum intended for cell culture. In various batches, cell attachment activity attributable to vitronectin was 8-16-fold greater than that of fibronectin, making vitronectin the main adhesive protein in routine cell culture media.

Amino Acid Sequence↗

PubMed1990

Diversities in animal vitronectins. Differences in molecular weight, immunoreactivity and carbohydrate chains.

Six animal plasma vitronectins, human, horse, porcine, bovine, rabbit and chicken vitronectins purified by a novel method using two successive heparin affinity columns, showed marked diversity in molecular weight, immunoreactivity and carbohydrate composition. Chicken vitronectin had a distinctly different amino acid composition from the mammalian vitronectins; and bovine vitronectin was the only one to contain N-glycolylneuraminic acid as well as N-acetylneuraminic acid. Binding studies with horseradish peroxidase-labelled lectins indicated that all the vitronectins contained complex-type, sialylated N-linked sugar chains and that only porcine vitronectin had a fucosylated sugar chain. D-Galactosamine determinations and binding studies with horseradish peroxidase-peanut lectin on native and asialovitronectins revealed that the mammalian vitronectins other than human vitronectin contained O-linked sugar chains with sialic acid, chicken vitronectin contained unsialylated chains, and human vitronectin contained neither. The results indicate that diversities in vitronectins are apparent in their molecular weights and glycosylations, especially in the number and structure of O-linked sugar chains.

Amino Acids↗

PubMed2000

Vitronectin is sequestered within human spermatozoa and liberated following the acrosome reaction.

Vitronectin plays a role in the regulation of complement and thrombin activities and in cell surface proteolysis. Vitronectin is also an intrinsic protein of human spermatozoa. Vitronectin message has been detected in whole testis poly-A mRNA and localized by in-situ reverse transcription-polymerase chain reaction to spermatocytes. The proportion of spermatozoa that express vitronectin increases following their capacitation. In this study, spermatozoa from a man of proven fertility were probed with an anti-vitronectin monoclonal antibody (VN7) before and after their permeabilization with 0.1% Triton X-100. Of fresh spermatozoa observed by confocal microscopy, 0-8% showed vitronectin staining. However, 75% of those observed displayed vitronectin following permeabilization. Serial confocal sections through the sperm head confirmed the internal localization of vitronectin. The acrosomal status of capacitated spermatozoa that expressed vitronectin was then determined. Dual colour microscopy with rhodamine-conjugated anti-vitronectin antibody and a fluorescein-conjugated antibody directed against CD46 (a complement regulatory protein expressed on the inner acrosomal membrane) revealed that only acrosome-reacted (CD46-positive) spermatozoa displayed vitronectin. Two populations of these spermatozoa were observed. Fifty-seven of 260 (22%) were CD46-positive/vitronectin-positive and 72 of 260 (28%) were CD46-positive/vitronectin-negative. No spermatozoa were CD46-negative/vitronectin-positive. These results confirm that vitronectin is released from a sequestered location within the spermatozoon following the acrosome reaction.

Acrosome Reaction↗

PubMed1986

Immunological characterization of human vitronectin and its binding to glycosaminoglycans.

The cell-adhesive glycoprotein vitronectin in human plasma was characterized with a monospecific anti-vitronectin antibody. Vitronectin, a mixture of monomeric 75 and 65 kDa polypeptides, was found to have different ratios of amounts of 75 and 65 kDa polypeptides in immunoblots of sera from various healthy human donors. Two states of vitronectin were previously reported; the open state binds to heparin, but the cryptic state does not (Hayashi et al. (1985) J. Biochem. 98, 1135-1138). The anti-vitronectin antibody was suggested to react more strongly with the open state of vitronectin than with the cryptic state. To quantitate all vitronectin regardless of its state, an enzyme-linked immunosorbent assay of vitronectin was developed based on prior boiling of vitronectin-containing samples in 2% (w/v) sodium dodecyl sulfate and 40 mM dithiothreitol to destroy conformational differences. About 12-20% of the vitronectin molecules in plasma were found to bind to heparin-Sepharose under physiological conditions. Vitronectin in plasma bound 30-fold more efficiently to heparin immobilized by amino groups than by carboxyl groups. Its affinity for heparin was higher than for chondroitin sulfate A or C, or dermatan sulfate. Vitronectin was also found to contain covalently-linked small polypeptides of 15 and 13 kDa. These light chains seemed to be disulfide-bonded to the 65 kDa polypeptide, and might be endogenously derived from nicks in the carboxy-terminal portion of the 75 kDa polypeptide in plasma.

Chromatography↗

PubMed1993

Vitronectin in mouse skin: immunohistochemical demonstration of its association with cutaneous amyloid.

Vitronectin is a multifunctional glycoprotein known to be associated with the dermal elastic fiber network in human adults, with various types of human amyloid and with apoptotic keratin bodies in human skin. It has also been shown to bind to human keratin intermediate filaments and to intranuclear material in vitro. To learn more of its function and mode of tissue deposition, vitronectin in mouse skin was studied. Vitronectin was purified from mouse plasma and a polyclonal anti-vitronectin antiserum was produced. This was used in an avidin-biotin-peroxidase complex technique and in an immunofluorescence technique on biopsy sections of dorsal skin from mice. Hairless lightly pigmented mice, known to develop dermal amyloid deposits at advanced ages, were used, making it possible to study vitronectin's association not only with elastic fibers but also with amyloid. Association of vitronectin with elastotic material was investigated in mice exposed to ultraviolet radiation. No vitronectin immunoreactivity could be demonstrated in association with elastin-stained material in skin specimens from any of the mice, whether exposed to ultraviolet radiation or not. In contrast, vitronectin was constantly found to be associated with dermal amyloid deposits. Moreover, mouse vitronectin was found to bind to mouse keratinocytes when studied by immunofluorescence staining of skin sections that had been pre-incubated with mouse plasma as sources of vitronectin. The results indicate that vitronectin is associated with amyloid and can bind to intracellular structures in epidermal keratinocytes in mice as in humans, and that mouse tissue may be used for the study of vitronectin's interactions with amyloid and with intracellular structures.

Amino Acid Sequence↗

PubMed1996

Localization of urokinase to focal adhesions by human fibrosarcoma cells synthesizing recombinant vitronectin.

Cell surface plasminogen activators have been proposed to participate in cell migration and invasion by activating both intracellular signaling pathways and extracellular proteolysis. Urokinase-type plasminogen activator (uPA) is secreted from many cell types and localizes to focal contact areas when cells are seeded onto the plasma protein vitronectin. Induction of vitronectin synthesis during migration of neural crest cells and growth of certain tumors suggests that the de novo synthesis and deposition of vitronectin into the tissue matrix may remodel the matrix to provide an environment suitable for cell migration and (or) tumor invasion. To investigate the effects of vitronectin secretion and matrix deposition on the localization and activity of cell-associated uPA, HT-1080 fibrosarcoma cells were transfected with the Rc/CMV expression vector containing a vitronectin cDNA insert and stable cell lines expressing vitronectin were selected. Vitronectin-secreting cells were allowed to attach and spread on collagen- and fibronectin-coated substrates. Within 6 h, vitronectin was detected on the substrate; vitronectin synthesis was accompanied by the clustering of both the alpha v beta 5 vitronectin receptor and uPA into vinculin-containing focal adhesions. Although mock transfected cells formed small focal adhesions on both collagen and fibronectin, no co-localization of uPA or alpha v beta 5 to focal adhesions was evident in these cells. Vitronectin-secreting cells also exhibited decreased levels of plasminogen activation and increased levels of cell adhesion as compared with the mock transfected cells. These data demonstrate that the synthesis of vitronectin and its matrix association by transfected HT-1080 fibrosarcoma cells results in localization of uPA to alpha v beta 5 containing focal adhesions, decreased cell surface uPA activity, and an increase in cell adhesion.

Cell Adhesion↗

PubMed1997

Plasmin and plasminogen activator inhibitor type 1 promote cellular motility by regulating the interaction between the urokinase receptor and vitronectin.

The urokinase receptor (uPAR) coordinates plasmin-mediated cell-surface proteolysis and promotes cellular adhesion via a binding site for vitronectin on uPAR. Because vitronectin also binds plasminogen activator inhibitor type 1 (PAI-1), and plasmin cleavage of vitronectin reduces PAI-1 binding, we explored the effects of plasmin and PAI-1 on the interaction between uPAR and vitronectin. PAI-1 blocked cellular binding of and adhesion to vitronectin by over 80% (IC50 approximately 5 nM), promoted detachment of uPAR-bearing cells from vitronectin, and increased cellular migration on vitronectin. Limited cleavage of vitronectin by plasmin also abolished cellular binding and adhesion and induced cellular detachment. A series of peptides surrounding a plasmin cleavage site (arginine 361) near the carboxy-terminal end of vitronectin were synthesized. Two peptides spanning res 364-380 blocked binding of uPAR to vitronectin (IC50 approximately 8-25 microM) identifying this region as an important site of uPAR-vitronectin interaction. These data illuminate a complex regulatory scheme for uPAR-dependent cellular adhesion to vitronectin: Active urokinase promotes adhesion and also subsequent detachment through activation of plasmin or complex formation with PAI-1. Excess PAI-1 may also promote migration by blocking cellular adhesion and/or promoting detachment, possibly accounting in part for the strong correlation between PAI-1 expression and tumor cell metastasis.

Cell Adhesion↗

PubMed1992

Yolk vitronectin. Purification and differences from its blood homologue in molecular size, heparin binding, collagen binding, and bound carbohydrate.

This is the first report on a unique vitronectin molecule, yolk vitronectin, which is similar to its blood homologue in cell spreading activity but different in molecular size, bound carbohydrate, and heparin and collagen binding activity. Yolk vitronectin was purified 2,500-fold from chick egg yolk by a combination of hydroxylapatite, DEAE-cellulose, and anti-vitronectin-Sepharose column chromatographies. In SDS-polyacrylamide gel electrophoresis under reducing conditions, yolk vitronectin was separated into 54- and 45-kDa bands, which are 16 and 25 kDa smaller, respectively, than the 70-kDa major band of chick blood vitronectin. The 54-kDa band shares the same NH2-terminal sequence as chick blood vitronectin. In contrast, the NH2-terminal sequence of the 45-kDa band is somewhat homologous with the internal sequences of mammalian vitronectins beginning at the 50th amino acid from the NH2 terminus. The bound carbohydrate of the 54- and 45-kDa species of yolk vitronectin is similar to, but distinct from, that of blood vitronectin. Unlike blood vitronectin, yolk vitronectin cannot bind to either heparin or collagen.

Amino Acid Sequence↗

PubMed1989

Interaction of plasminogen activator inhibitor (PAI-1) with vitronectin.

Immobilized vitronectin was found to bind both purified plasminogen activator inhibitor type 1 (PAI-1) and the PAI-1 in conditioned culture medium of human sarcoma cells. Similarly, immobilized PAI-1 bound both purified vitronectin and vitronectin from normal human serum. These interactions were demonstrated using both enzyme immunoassay and radioiodinated proteins. Solid-phase vitronectin bound PAI-1 with Kd 1.9 x 10(-7) M, and the reverse interaction gave a Kd 5.5 x 10(-8) M. Evidence was also found for a second type of binding with a Kd below 10(-10) M. The molar ratios of the two proteins in the complex at the saturation levels were approximately one molecule of soluble PAI-1 bound per three molecules of immobilized vitronectin and approximately one molecule of soluble vitronectin being bound per one molecule of immobilized PAI-1. Binding of PAI-1 to vitronectin did not lead to an irreversible loss of the ability of PAI-1 to inhibit urokinase (u-PA) and tissue-type plasminogen activator (t-PA). Active u-PA released vitronectin-bound 125I-labeled PAI-1 radioactivity, suggesting that u-PA interacts with the complex. The Mr 50,000 urokinase cleavage product of PAI-1 also bound to vitronectin, but this bound fragment did not inhibit u-PA. Binding of PAI-1 to vitronectin did not interfere with the ability of vitronectin to promote the adhesion and spreading of cells. These results suggest that the interaction between vitronectin and PAI-1 may serve to confine pericellular u-PA activity to focal contact sites where cells use proteolysis in regional detachment.

Glycoproteins↗

PubMed1996

The binding protein for globular heads of complement C1q, gC1qR. Functional expression and characterization as a novel vitronectin binding factor.

A binding protein for the globular head domains of complement component C1q, designated gC1qR, recently described to be present on vascular and blood cells (Ghebrehiwet, B., Lim, B.-L., Peerschke, E. I. B., Willis, A. C., and Reid, K. B. M. (1994) J. Exp. Med. 179, 1809-1821 was expressed in recombinant form in bacteria to investigate its functional and structural properties. The recombinant gC1qR was found to be functional because tetramerization of the 24.3-kDa polypeptide occurred as described for the native protein, and the binding of the ligand C1q by recombinant gC1qR was indistinguishable from binding shown by gC1qR isolated from Raji cells. Recombinant gC1qR immobilized to microspheres was used to search for additional binding proteins unrelated to C1q. Surprisingly, it was found that vitronectin or complexes containing vitronectin were retained from plasma or serum, and subsequent analysis revealed the specific binding of the ternary vitronectin-thrombin-antithrombin complex to gC1qR. Because the thrombin-antithrombin complex was unable to interact with gC1qR, direct binding with vitronectin was investigated in a purified system. The heparin binding multimeric form of vitronectin but not the plasma form of vitronectin was found to bind specifically to gC1qR isolated from Raji cell membrane as well as to recombinant gC1qR. This interaction was saturable (KD approximately 20 nM) and inhibitable by glycosaminoglycans such as heparin but not by chondroitin sulfate. C1q and vitronectin did not compete with each other for binding to gC1qR, and both ligands seem to interact with different parts of the gC1qR because a truncated version of recombinant gC1qR lacking the N-terminal 22-amino acid portion hardly interacted with vitronectin but bound C1q as well as the intact gC1qR. These findings establish gC1qR as a novel vitronectin-binding protein that may participate in the clearance of vitronectin-containing complexes or opsonized particles or cooperate with vitronectin in the inhibition of complement-mediated cytolysis.

Blood Proteins↗

PubMed1994

Stimulation of migration of human aortic smooth muscle cells by vitronectin: implications for atherosclerosis.

OBJECTIVE: Migration of smooth muscle cells into the neointima has been implicated in atherogenesis. Vitronectin, a serum factor that promotes cell spreading and attachment, accumulates in atherosclerotic human tissues. The aim of this study was to determine the role of vitronectin and its receptor (integrin alpha V beta 3) in migration of smooth muscle cells. METHODS: Human aortic smooth muscle cell migration was studied in modified Boyden chambers. Expression of vitronectin receptor was determined by northern blotting of receptor mRNA and immunoprecipitation of receptor protein. RESULTS: Vitronectin dose dependently increased smooth muscle cell migration by an amount comparable to that induced by platelet derived growth factor, (PDGF)-BB. Antiserum to alpha V beta 3 diminished vitronectin driven migration. Northern blot analysis showed low constitutive expression of alpha V and beta 3 mRNA by smooth muscle cell and rapid induction with transforming growth factor beta (TGF-beta) and thrombin. Immunoprecipitation confirmed increased synthesis of the alpha V beta 3 vitronectin receptor complex by TGF-beta or thrombin. Smooth muscle cells pretreated with TGF-beta or thrombin showed increased vitronectin driven migration. cAMP suppressed induction of migration, but inhibition of protein kinase C increased it. CONCLUSIONS: These results show that vitronectin-induced human aortic smooth muscle cell migration is mediated by alpha V beta 3 vitronectin receptor and expression of the receptor is induced by TGF-beta and thrombin, which in turn induce vitronectin driven, vitronectin receptor modulated smooth muscle cell migration.

Arteriosclerosis↗

PubMed1994

Distribution of vitronectin in plasma and liver tissue: relationship to chronic liver disease.

To clarify the clinical significance of vitronectin, we compared the concentration of plasma vitronectin with serum fibrous markers and liver function test values in patients with chronic liver diseases. We also evaluated the vitronectin content in the liver by means of enzyme-linked immunosorbent assay and the localization of vitronectin in liver tissue with enzyme immunohistochemistry. In chronic liver disease, the concentration of plasma vitronectin was significantly lower than that in healthy controls, being related to the severity of liver disease. The plasma levels of vitronectin showed no correlation to fibrous markers but a significant correlation with those of serum albumin and prothrombin time. On the other hand, the content of vitronectin in liver tissue was significantly increased in chronic liver disease compared with that in normal controls. In the normal liver, vitronectin was observed in the portal area by light microscopy. In chronic hepatitis and cirrhosis, vitronectin was found in the connective tissue around the portal and central veins and in the areas of piecemeal and focal necrosis. These findings suggested that vitronectin is deposited in injured tissue through the process of repair and fibrosis and plays an important role as an adhesive protein. Moreover, the lower levels of plasma vitronectin in chronic liver disease may be due to its decreased synthesis, deposition or both in injured tissue.

Adult↗

PubMed1993

Identification of a role of the vitronectin receptor and protein kinase C in the induction of endothelial cell vascular formation.

When cultured on a basement membrane substratum, endothelial cells undergo a rapid series of morphological and functional changes which result in the formation of histotypic tube-like structures, a process which mimics in vivo angiogenesis. Since this process is probably dependent on several cell adhesion and cell signaling phenomena, we examined the roles of integrins and protein kinase C in endothelial cell cord formation. Polyclonal antisera directed against the entire vitronectin (alpha v beta 3) and fibronectin (alpha 5 beta 1) receptors inhibited cord formation. Subunit-specific monoclonal antibodies to alpha v, beta 3, and beta 1 integrin subunits inhibited cord formation, while monoclonal antibodies to alpha 5 did not, which implicated the vitronectin receptor, and not the fibronectin receptor, in vascular formation. Protein kinase C inhibitors inhibited cord formation, while phorbol 12-myristate 13-acetate (PMA) caused endothelial cells to form longer cords. Since the vitronectin receptor has been shown to be phosphorylated in an in vitro system by protein kinase C, the possible functional link between the vitronectin receptor and protein kinase C during cellular morphogenesis was examined. The vitronectin receptor was more highly phosphorylated in cord-forming endothelial cells on basement membrane than in monolayer cells on vitronectin. Furthermore, this phosphorylation was inhibited by protein kinase C inhibitors, and PMA was required to induce vitronectin receptor phosphorylation in endothelial cells cultured on vitronectin. Colocalization studies were also performed using antisera to the vitronectin receptor and antibodies to protein kinase C. Although no strict colocalization was found, protein kinase C was localized in the cytoskeleton of endothelial cells initially plated on basement membrane or on vitronectin, and it translocated to the plasma membrane of C-shaped cord-forming cells on basement membrane. Thus, both the vitronectin receptor and protein kinase C play a role in in vitro cord formation.

Antibodies, Monoclonal↗

PubMed2001

Certain high molecular weight heparin chains have high affinity for vitronectin.

Vitronectin is a 70-kDa protein that is found in both the extracellular matrix as well as serum. Vitronectin is one of the few proteins that regulates both the complement and the coagulation systems. Heparin is known to bind to vitronectin. Review of the literature reveals apparently conflicting outcomes of the interaction of heparin, vitronectin, and the complement system. Previous studies demonstrated that heparin diminishes vitronectin inhibition of complement activity. Numerous studies have also demonstrated that heparin exerts a net inhibitory effect on complement. We used two dimensional affinity resolution electrophoresis (2DARE) to examine this apparent paradox. 2DARE allowed simultaneous determination of binding affinity of heparin for vitronectin as well as the M(r) of the heparin species. In the 2DARE experiment, the interaction of heparin with vitronectin caused retardation of the movement of the heparin through the tube gel in the first dimension. The degree of the retardation of movement was used to calculate the approximate K(d) of that interaction. The heparin from the tube gel was then subjected to a second dimension electrophoresis to determine the M(r) of the heparin. 2DARE analysis of the interaction of heparin with vitronectin clearly demonstrated that a sub-population of heparin chains with M(r) > 8000 bound vitronectin with high affinity whereas most high M(r) chains and all lower M(r) chains showed little to no affinity for vitronectin. Our findings are consistent with the hypothesis that a unique binding domain exists in certain heparin chains for vitronectin.

Electrophoresis, Gel, Two-Dimensional↗

PubMed1997

Differential modulation of cell adhesion by interaction between adhesive and counter-adhesive proteins: characterization of the binding of vitronectin to osteonectin (BM40, SPARC).

Heparin-binding forms of vitronectin, a multifunctional adhesive glycoprotein, are associated with the extracellular matrix (ECM) at different locations in the body and serve to promote cell adhesion and the regulation of pericellular proteolysis at sites of angiogenesis. In the present study we characterized the interactions of vitronectin with the counter-adhesive protein osteonectin (also termed SPARC or BM40). Osteonectin and vitronectin were both found associated with the ECM of cultured endothelial cells and were localized in vessel wall sections of kidney tissue. In vitro, the heparin-binding multimeric isoform of vitronectin bound to immobilized osteonectin in a saturable manner with half-maximal binding at 30-40 nM. Preincubation of plasma vitronectin with plasminogen activator inhibitor 1 (PAI-1), which provoked multimer formation, induced the binding of vitronectin to osteonectin. Binding was optimal at physiological ionic strength, and binary complexes were stabilized by tissue transglutaminase-mediated cross-linking. In a concentration-dependent fashion, PAI-1, CaCl2, heparin and heparan sulphate, but not other glycosaminoglycans, interfered with the binding of vitronectin to osteonectin. Using vitronectin-derived synthetic peptides as well as mutant forms of recombinant osteonectin, we found that the heparin-binding region of vitronectin interacted with the C-terminal region of osteonectin that contains a high-affinity Ca2+-binding site with counter-adhesive properties. Adhesion of cultured endothelial cells was partly abrogated by osteonectin and was correspondingly reversed by vitronectin in a concentration-dependent manner. These results indicate that specific interactions between vitronectin and osteonectin modulate cell adhesion and might thereby regulate endothelial cell function during angiogenesis.

Amino Acid Sequence↗

PubMed1999

Vitronectin interaction with glycosaminoglycans. Kinetics, structural determinants, and role in binding to endothelial cells.

Vitronectin (VN) is a high affinity heparin-binding protein. The physiological role of this binding has hitherto received little attention, and its molecular determinants are subject to controversy. In this study, we characterized vitronectin interaction with heparin, heparin analogues, bacterial extracts, and cell surface glycosaminoglycans. As assessed by (i) fluorescence assays, (ii) precipitation with heparin-Sepharose beads, or (iii) Western blotting with antibodies against VN(347-361) (the heparin-binding site), we demonstrate an exposure of the VN heparin-binding site in multimeric but not monomeric vitronectin. Through its heparin-binding site, vitronectin also bound other glycosaminoglycans and Staphylococcus aureus extracts. The kinetics of heparin binding to vitronectin were complex. After a fast association phase (tau = 0.3 s), a slow conversion of an unstable to a stable heparin-vitronectin complex (tau = 180 s) occurred. Heparin binding kinetics and transition to a stable complex were mimicked by VN(347-361), demonstrating that this area is the fully functional heparin-binding site of vitronectin. Multimeric vitronectin bound to endothelial cells. This binding was blocked by soluble heparin and was not observed when endothelial cells were pretreated with glycosaminoglycan-removing enzymes. Glycosaminoglycan-dependent interaction of endothelial cells with multimeric vitronectin might be a relevant mechanism for removal of multimeric vitronectin from plasma. Conversion of an unstable to a stable glycosaminoglycan-vitronectin complex is likely to be relevant for association with endothelial cells under flow conditions.

Cells, Cultured↗