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.     

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.     

Activation of vitronectin (serum spreading factor) binding of heparin by denaturing agents

Vitronectin (serum spreading factor), a cell-adhesive glycoprotein present in mammalian serum, has previously been the subject of conflicting reports concerning its binding to heparin. Vitronectin purified from human plasma does not bind to heparin under physiological conditions, but it does so after treatment with denaturing agents including 8 M urea or 6 M guanidine-HC1, or heating at 100 degrees C for 5 min. These treatments seem to expose a heparin-binding site in vitronectin; this finding thus resolves the conflicts concerning this function.     

The role of vitronectin as multifunctional regulator in the hemostatic and immune systems

Vitronectin (= complement S-protein) belongs to the group of structurally and functionally homologous adhesive proteins (fibrinogen, fibronectin, von Willebrand factor) which are essential in the procoagulant phase of the hemostatic system, interacting with platelets and the vessel wall. In addition to a structural motif in vitronectin responsible for this interaction (cell attachment domain) other functional domains in the protein molecule exist that contribute to its multifunctional role as regulator in the immune system (complement) as well as in fibrinolysis. These various activities and the ubiquitous distribution of vitronectin in the organism are discussed with regard to structure-function relationships of the protein molecule. Vitronectin may thus provide a conceptual molecular link between cell adhesion, humoral immune response and the hemostatic system, particularly at the blood-vessel wall interphase.     

The human vitronectin (complement S-protein) gene maps to the centromeric region of 17q

Summary Vitronectin (complement S-protein, serumspreading factor, epibolin) is a multifunctional glycoprotein that mediates cell-to-substrate adhesion, inhibits the cytolytic action of the terminal complement cascade in vitro and binds to several serine protease inhibitors of the serpin family, viz. antithrombin III, plasminogen activator inhibitor I (PAI-1) and II (PAI-2), heparin cofactor II and protease nexin. Using high resolution fluorescence in situ hybridization, we mapped the vitronectin gene to the centromeric region of the long arm of chromosome 17 corresponding to 17q11. The location was confirmed by co-hybridization with the centromerespecific alphoid probe p17H8 (D17Z1) and by chromosome banding with 4,6-diamidino-2-phenylindole-dihydrochloride (DAPI). None of the previously mapped genes that are evolutionary related to vitronectin are located on the same chromosome.     

Incorporation of vitronectin into fibrin clots. Evidence for a binding interaction between vitronectin and gamma A/gamma' fibrinogen.

Vitronectin is an abundant plasma protein that regulates coagulation, fibrinolysis, complement activation, and cell adhesion. Recently, we demonstrated that plasma vitronectin inhibits fibrinolysis by mediating the interaction of type 1 plasminogen activator inhibitor with fibrin (Podor, T. J., Peterson, C. B., Lawrence, D. A., Stefansson, S., Shaughnessy, S. G., Foulon, D. M., Butcher, M., and Weitz, J. I. (2000) J. Biol. Chem. 275, 19788-19794). The current studies were undertaken to further examine the interactions between vitronectin and fibrin(ogen). Comparison of vitronectin levels in plasma with those in serum indicates that approximately 20% of plasma vitronectin is incorporated into the clot. When the time course of biotinylated-vitronectin incorporation into clots formed from (125)I-fibrinogen is monitored, vitronectin incorporation into the clot parallels that of fibrinogen in the absence or presence of activated factor XIII. Vitronectin binds specifically to fibrin matrices with an estimated K(d) of approximately 0.6 microm. Additional vitronectin subunits are assembled on fibrin-bound vitronectin multimers through self-association. Confocal microscopy of fibrin clots reveals the globular vitronectin aggregates anchored at intervals along the fibrin fibrils. This periodicity raised the possibility that vitronectin interacts with the gamma A/gamma' variant of fibrin(ogen) that represents about 10% of total fibrinogen. In support of this concept, the vitronectin which contaminates fibrinogen preparations co-purifies with the gamma A/gamma' fibrinogen fraction, and clots formed from gamma A/gamma' fibrinogen preferentially bind vitronectin. These studies reveal that vitronectin associates with fibrin during coagulation, and may thereby modulate hemostasis and inflammation.     

Activation of the collagen-binding of endogenous serum vitronectin by heating, urea and glycosaminoglycans.

The present study describes that the collagen-binding activity of vitronectin in human serum increases by treatment with heparin, heating and urea. Vitronectin purified from human serum was bound to native collagen, whereas endogenous vitronectin in the serum was not. We have examined the conditions to change the collagen-binding activity of endogenous vitronectin. Endogenous vitronectin in human serum became considerably bound to collagen when the serum was boiled in 4-8 M urea for 5 min and mixed with heparin (0.5-5 micrograms/ml). Each treatment of heating, urea or heparin alone, and any combination of the two factors, inefficiently activated the binding. Dextran sulfate could substitute for heparin, but dermatan sulfate, keratan sulfate, chondroitin sulfate A and C, heparan sulfate and hyaluronan could not. Possible explanations for the activation of endogenous vitronectin are discussed.     

Involvement of vitronectin in lipopolysaccaride-induced acute lung injury

Vitronectin is present in large concentrations in serum and participates in regulation of humoral responses, including coagulation, fibrinolysis, and complement activation. Because alterations in coagulation and fibrinolysis are common in acute lung injury, we examined the role of vitronectin in LPS-induced pulmonary inflammation. Vitronectin concentrations were significantly increased in the lungs after LPS administration. Neutrophil numbers and proinflammatory cytokine levels, including IL-1beta, MIP-2, KC, and IL-6, were significantly reduced in bronchoalveolar lavage fluid from vitronectin-deficient (vitronectin(-/-)) mice, as compared with vitronectin(+/+) mice, after LPS exposure. Similarly, LPS induced increases in lung edema, myeloperoxidase-concentrations, and pulmonary proinflammatory cytokine concentrations were significantly lower in vitronectin(-/-) mice. Vitronectin(-/-) neutrophils demonstrated decreased KC-induced chemotaxis as compared with neutrophils from vitronectin(+/+) mice, and incubation of vitronectin(+/+) neutrophils with vitronectin was associated with increased chemotaxis. Vitronectin(-/-) neutrophils consistently produced more TNF-alpha, MIP-2, and IL-1beta after LPS exposure than did vitronectin(+/+) neutrophils and also showed greater degradation of IkappaB-alpha and increased LPS-induced nuclear accumulation of NF-kappaB compared with vitronectin(+/+) neutrophils. These findings provide a novel vitronectin-dependent mechanism contributing to the development of acute lung injury.     

Phosphorylation of vitronectin by a protein kinase in human plasma. Identification of a unique phosphorylation site in the heparin-binding domain

Incubation of human plasma with 27 nM [gamma-32P]ATP in the presence of 20 mM MnCl2 results in the phosphorylation of several proteins detected by sodium dodecyl sulfate-polyacrylamide gel electrophoresis and autoradiography. About 60% of the incorporated radioactivity is found in a 75-kDa protein containing [32P] phosphoserine. The amino-terminal amino acid sequence of the purified 75-kDa [32P]phosphoprotein is identical to that of vitronectin (also termed serum spreading factor or complement S protein). Rabbit antiserum against vitronectin precipitates greater than 90% of the 75-kDa [32P]phosphoprotein from plasma. Reverse phase chromatography of [32P]vitronectin degraded sequentially with CNBr and chymotrypsin yields one major labeled peptide. The sequence of the peptide, Ser-Arg-Arg-Pro-[32PO4]Ser-Arg-Ala-Thr, corresponds to residues 374-381 which are located in the heparin-binding fragment of vitronectin identified by Suzuki et al. [1984) J. Biol. Chem. 259, 15307-15314). Vitronectin could potentially be phosphorylated in vivo with ATP released from injured cells or secreted by platelets activated during hemostasis.     

Vitronectin.

Vitronectin is a multifunctional glycoprotein present in blood and in the extracellular matrix. It binds glycosaminoglycans, collagen, plasminogen and the urokinase-receptor, and also stabilizes the inhibitory conformation of plasminogen activation inhibitor-1. By its localization in the extracellular matrix and its binding to plasminogen activation inhibitor-1, vitronectin can potentially regulate the proteolytic degradation of this matrix. In addition, vitronectin binds to complement, to heparin and to thrombin-antithrombin III complexes, implicating its participation in the immune response and in the regulation of clot formation. The biological functions of vitronectin can be modulated by proteolytic enzymes, and by exo- and ecto-protein kinases present in blood. Vitronectin contains an RGD sequence, through which it binds to the integrin receptor alpha v beta 3, and is involved in the cell attachment, spreading and migration. Antibodies against alpha v beta 3 or synthetic peptides containing an RGD sequence are now being tested as therapeutic agents in the treatment of human cancers, bone diseases (e.g. osteoporosis) and in pathological disorders which involve angiogenesis.     

The effect of heparin on the affinity chromatography of plasminogen. Demonstration of heparin-plasminogen interaction.

Evidence is presented that heparin binds rabbit plasminogen types I and II under affinity chromatographic conditions using the single stage technique earlier described (Hatton, M.W.C. and Regoeczi, E. (1974) Biochim. Biophys. Acta 359, 55-65). Thus, the affinity of types I and II for Sepharose-lysine is markedly increased in the presence of heparin and elution by epsilon-aminohexanoic acid requires a steeper gradient to recover the plasminogen types. Furthermore by adding sufficient epsilon-aminohexanoic acid to non-heparinised plasma to suppress plasminogen affinity, the presence of heparin is shown to encourage binding of plasminogen (type II more so than type I) to the gel. However, the heparin effect is quickly reversed by washing the column with 0.5 M NaCl prior to elution by epsilon-aminohexanoic acid. No evidence of a stable plasminogen-heparin complex has been found from gel filtration studies and any interaction between plasminogen and heparin probably only takes place when heparin is bound to an affinity site. Studies with 35-S-labelled heparin have shown the mucopolysaccharide to bind to the free amino group of Sepharose-lysine and Sepharose-cadaverine and to be displaced by 0.5 M NaCl elution but not by 0.1 M epsilon-aminohexanoic acid. The plasminogen types produced from heparinised plasma are free from heparin and closely resemble preparations from non-heparinised plasma when compared by polyacrylamide gel electrophoresis, Sephadex gel filtration and arginine esterase activity after urokinase activation.     

Structural requirements for the neutralization of heparin-like saccharides by complement S protein/vitronectin

S protein, a major inhibitor of the assembly of the membrane attack complex of complement, has recently been shown to be identical to the serum spreading factor vitronectin. It also neutralizes the anticoagulant activities of heparin. We have studied the structural requirements for the heparin neutralizing properties of S protein/vitronectin using heparin, heparan sulfate, and heparin oligosaccharides with well defined anticoagulant specificities. The abilities of heparin fractions, Mr 7,800-18,800, with high affinity for antithrombin, and of the International Heparin Standard, to accelerate the inactivation of thrombin and Factor Xa by antithrombin were readily neutralized by S protein/vitronectin. Binding and neutralization of heparin by S protein/vitronectin was inhibited by heparin with low affinity for antithrombin, indicating that S protein/vitronectin can interact with a region on the heparin chain that might serve as a proteinase binding site. S protein/vitronectin efficiently neutralized oligosaccharides of Mr 2,400-7,200, unlike the two other physiologically occurring heparin neutralizing proteins histidine-rich glycoprotein and platelet factor 4. Furthermore, S protein/vitronectin neutralized the anti-Factor Xa activity of a synthetic pentasaccharide comprising the antithrombin-binding sequence of heparin. High molar excess of a synthetic tridecapeptide corresponding to part (amino acids 374-359) of the proposed glycosaminoglycan binding domain of S protein/vitronectin neutralized high affinity heparin and some oligosaccharides, but failed to neutralize the synthetic antithrombin-binding pentasaccharide. Like platelet factor 4, but unlike histidine-rich glycoprotein, S protein/vitronectin readily neutralized the anticoagulant activities of heparan sulfate of Mr approximately 20,000. These findings suggest that S protein/vitronectin may interact through its glycosaminoglycan binding domain(s) with various functional domains of the heparin (heparan sulfate) molecule, including the antithrombin-binding pentasaccharide sequence. Furthermore, the results suggest that S protein/vitronectin may be a physiologically important modulator of the anticoagulant activity of heparin-like material on or near the vascular endothelium.     

Heparin binding domain in vitronectin is required for oligomerization and thus enhances integrin mediated cell adhesion and spreading

Vitronectin is a multi-functional protein found predominantly as a monomer in blood and as an oligomer in the extracellular matrix. We have dissected the minimal regions of vitronectin protein needed for effective integrin dependent cell adhesion and spreading. A fragment of vitronectin containing the RGD integrin binding site showed similar binding affinity as that of full vitronectin protein to purified integrin αvβ3 but had diminished cell adhesion and spreading function in vivo. We demonstrate that the oligomeric state of the protein is responsible for this effect. We provide compelling evidence for the involvement of the heparin binding domain of vitronectin in the oligomerization process and show that such oligomerization reinforces the activity of vitronectin in cell adhesion and spreading.

Structured summary

MINT-7905703: Vn (uniprotkb:P04004) and Vn (uniprotkb:P04004) bind (MI:0407) by molecular sieving (MI:0071)     

The Choline-binding Protein PspC of Streptococcus pneumoniae Interacts with the C-terminal Heparin-binding Domain of Vitronectin

Adherence of Streptococcus pneumoniae is directly mediated by interactions of adhesins with eukaryotic cellular receptors or indirectly by exploiting matrix and serum proteins as molecular bridges. Pneumococci engage vitronectin, the human adhesive glycoprotein and complement inhibitor, to facilitate attachment to epithelial cells of the mucosal cavity, thereby modulating host cell signaling. In this study, we identified PspC as a vitronectin-binding protein interacting with the C-terminal heparin-binding domain of vitronectin. PspC is a multifunctional surface-exposed choline-binding protein displaying various adhesive properties. Vitronectin binding required the R domains in the mature PspC protein, which are also essential for the interaction with the ectodomain of the polymeric immunoglobulin receptor and secretory IgA. Consequently, secretory IgA competitively inhibited binding of vitronectin to purified PspC and to PspC-expressing pneumococci. In contrast, Factor H, which binds to the N-terminal part of mature PspC molecules, did not interfere with the PspC-vitronectin interaction. Using a series of vitronectin peptides, the C-terminal heparin-binding domain was shown to be essential for the interaction of soluble vitronectin with PspC. Binding experiments with immobilized vitronectin suggested a region N-terminal to the identified heparin-binding domain as an additional binding region for PspC, suggesting that soluble, immobilized, as well as cellularly bound vitronectin possesses different conformations. Finally, vitronectin bound to PspC was functionally active and inhibited the deposition of the terminal complement complex. In conclusion, this study identifies and characterizes (on the molecular level) the interaction between the pneumococcal adhesin PspC and the human glycoprotein vitronectin.     

Interaction of vitronectin with collagen

Purified human plasma vitronectin was demonstrated to bind to type I collagen immobilized on plastic as measured by enzyme-linked immunosorbent assay and by binding of 125I-radiolabeled vitronectin to a collagen-coated plastic surface. Vitronectin did not bind to immobilized laminin, fibronectin, or albumin in these assays. Vitronectin showed similar interaction with all types of collagen (I, II, III, IV, V, and VI) tested. Collagen unfolded by heat treatment bound vitronectin less efficiently than native collagen. Vitronectin-coated colloidal gold particles bound to type I collagen fibrils as shown by electron microscopy. Salt concentrations higher than physiological interfered with the binding of vitronectin to collagen, suggesting an ionic interaction between the two proteins. Binding studies conducted in the presence of plasma showed that purified vitronectin added to plasma bound to immobilized collagen, whereas the endogenous plasma vitronectin bound to collagen less well. Although fibronectin did not interfere with the binding of vitronectin to native collagen, vitronectin inhibited the binding of fibronectin to collagen. These results show that vitronectin has a collagen-binding site(s) which, unlike that of fibronectin, preferentially recognizes triple-helical collagen and that the binding between vitronectin and collagen has characteristics compatible with the occurrence of such an interaction in vivo.     

Bovine ovarian follicular fluid vitronectin content is influenced by the follicle size

Vitronectin was quantified in the follicular fluid aspirated from bovine follicles with diameters of 3 to 15 mm (as determined by ultrasonography) using a specific enzyme-linked immunosorbent assay (ELISA) validated for bovine vitronectin. The primary antibody was raised in rabbit against vitronectin purified from bovine plasma. Vitronectin quantified in serial dilutions of bovine plasma and ovarian follicular fluid was highly correlated with the volume of each sample assayed. In addition, known amounts of purified bovine vitronectin added to samples of plasma or follicular fluid were accurately recovered. Follicular fluid concentrations of vitronectin were positively correlated with the follicle diameter (r = 0.8; P < 0.01). These data indicate that bovine follicular fluid concentration of vitronectin is influenced by the stage of follicular development.     

Vitronectin binds to the head region of Moraxella catarrhalis ubiquitous surface protein A2 and confers complement‐inhibitory activity

The serum resistance of the common respiratory pathogen Moraxella catarrhalis is mainly dependent on ubiquitous surface proteins (Usp) A1 and A2 that interact with complement factor 3 (C3) and complement inhibitor C4b binding protein (C4BP) preventing the alternative and classical pathways of the complement system respectively. UspA2 also has the capacity to attract vitronectin that in turn binds C9 and hereby inhibits membrane attack complex (MAC) formation. We found UspA2 as a major vitronectin binding protein and hence the UspA2/vitronectin interaction was studied in detail. The affinity constant (K_D) for vitronectin binding to UspA2 was 2.3 × 10^?8 M, and the N‐terminal region encompassing residues UspA2 30–170 bound vitronectin with a K_D of 7.9 × 10^?8 M. Electron microscopy verified that the active binding domain (UspA2^30–177) was located at the head region of UspA2. Experiments with recombinantly expressed vitronectin also revealed that UspA2^30–177 bound to the C‐terminal region of vitronectin residues 312–396. Finally, when human serum was pre‐incubated with UspA2, bacteria showed significantly less serum resistance. Our study directly reveals the binding mode between the N‐terminal domain of UspA2 and the C‐terminal part of vitronectin and thus sheds light upon the mechanism of M. catarrhalis‐dependent serum resistance.     

Characterization of a site on PAI-1 that binds to vitronectin outside of the somatomedin B domain

Vitronectin and plasminogen activator inhibitor-1 (PAI-1) are proteins that interact in the circulatory system and pericellular region to regulate fibrinolysis, cell adhesion, and migration. The interactions between the two proteins have been attributed primarily to binding of the somatomedin B (SMB) domain, which comprises the N-terminal 44 residues of vitronectin, to the flexible joint region of PAI-1, including residues Arg-103, Met-112, and Gln-125 of PAI-1. A strategy for deletion mutagenesis that removes the SMB domain demonstrates that this mutant form of vitronectin retains PAI-1 binding (Schar, C. R., Blouse, G. E., Minor, K. M., and Peterson, C. B. (2008) J. Biol. Chem. 283, 10297-10309). In the current study, the complementary binding site on PAI-1 was mapped by testing for the ability of a battery of PAI-1 mutants to bind to the engineered vitronectin lacking the SMB domain. This approach identified a second, separate site for interaction between vitronectin and PAI-1. The binding of PAI-1 to this site was defined by a set of mutations in PAI-1 distinct from the mutations that disrupt binding to the SMB domain. Using the mutations in PAI-1 to map the second site suggested interactions between alpha-helices D and E in PAI-1 and a site in vitronectin outside of the SMB domain. The affinity of this second interaction exhibited a K(D) value approximately 100-fold higher than that of the PAI-1-somatomedin B interaction. In contrast to the PAI-1-somatomedin B binding, the second interaction had almost the same affinity for active and latent PAI-1. We hypothesize that, together, the two sites form an extended binding area that may promote assembly of higher order vitronectin-PAI-1 complexes.     

Interaction of vitronectin with Haemophilus influenzae

Eight strains of Haemophilus influenzae were tested for binding to human vitronectin. All strains adhered to vitronectin-coated glass slides but no binding was detected using soluble vitronectin, suggesting that surface association of vitronectin is a prerequisite. Vitronectin binding was not likely to be mediated by fimbriae as non-fimbriated and fimbriated isogenic strains adhered equally. Adhesion could be blocked by heparin, which is also known to block vitronectin binding to Staphylococcus aureus. However, no blocking was achieved with sialic acid-rich glycoproteins such as fetuin and mucin contrasting with Helicobacter pylori for which sialic acid seems to play an important role. With Streptococcus pneumoniae binding was detected both with soluble and surface-associated vitronectin and could not be blocked by heparin. Our results suggest that H. influenzae, Streptococcus pneumoniae and Helicobacter pylori all use distinct modes to interact with vitronectin.