Activity Of Vitronectin Research Articles

Vitronectins produced by human cirrhotic liver and CCl4-treated rats differ in their glycosylation pattern and tissue remodeling activity.

Liver cirrhosis (LC) is a disease characterized by pathological accumulation and alteration of extracellular matrix (ECM) proteins; the interaction between two such proteins, collagen and vitronectin (VN), is considered to be the key to controlling ECM remodeling in liver cirrhosis. If it is possible to control the modification of oligosaccharides on VN, it may be possible to retard progression of liver cirrhosis. In this study, we examined the relationship between changes in VN glycosylation and activity related to the remodeling of hepatic tissue in human LC and a rat model of LC generated using carbon tetrachloride (CCl4). Plasma concentrations of VN in human LC declined to approximately two‐thirds that in normal plasma, but the ratio of active VN, which has collagen‐binding activities, increased 2.8 times in LC plasma. In contrast, purified LC‐VN exhibited similar binding activities toward type I, IV, and V collagens to those of normal VN. Lectin reactivities and carbohydrate analyses of LC‐VN revealed that branching, fucosylation, and sialylation of N‐glycans were higher than those of normal VN. On the other hand, the plasma level of rat CCl4‐VN increased and the ratio of active molecules to collagen in plasma decreased. Increased fucosylation of LC‐VN was not detected in carbohydrates of CCl4‐VN. The changes in rat VN due to CCl4 treatment did not correspond to the changes in plasma levels of human VN caused by LC, the ratio of active molecules, or carbohydrate composition, thereby indicating that CCl4‐treated rats are not an appropriate model for studying VNs in human LC. Glycosidase treatment of VNs supported the hypothesis that the collagen‐binding activity of VN is modulated by alterations of glycosylation during LC, which may contribute to (a) the matrix incorporation of VN and (b) tissue fibrosis.

Sialylation of vitronectin regulates stress fiber formation and cell spreading of dermal fibroblasts via a heparin-binding site.

Vitronectin (VN) plays an important role in tissue regeneration. We previously reported that VN from partial hepatectomized (PH) rats results in a decrease of sialylation of VN and de-sialylation of VN decreases the cell spreading of hepatic stellate cells. In this study, we analyzed the mechanism how sialylation of VN regulates the properties of mouse primary cultured dermal fibroblasts (MDF) and a dermal fibroblast cell line, Swiss 3T3 cells. At first, we confirmed that VN from PH rats or de-sialylated VN also decreased cell spreading in MDF and Swiss 3T3 cells. The de-sialylation suppressed stress fiber formation in Swiss 3T3 cells. Next, we analyzed the effect of the de-sialylation of VN on stress fiber formation in Swiss 3T3 cells. RGD peptide, an inhibitor for a cell binding site of VN, did not affect the cell attachment of Swiss 3T3 cells on untreated VN but significantly decreased it on de-sialylated VN, suggesting that the de-sialylation attenuates the binding activity of an RGD-independent binding site in VN. To analyze a candidate RGD-independent binding site, an inhibition experiment of stress fiber formation for a heparin binding site was performed. The addition of heparin and treatment of cells with heparinase decreased stress fiber formation in Swiss 3T3 cells. Furthermore, de-sialylation increased the binding activity of VN to heparin, as detected by surface plasmon resonance (SPR). These results demonstrate that sialylation of VN glycans regulates stress fiber formation and cell spreading of dermal fibroblast cells via a heparin binding site.

Vitronectin in the ascites of human ovarian carcinoma acts as a potent chemoattractant for ovarian carcinoma: Implication for metastasis by cancer stem cells.

Vitronectin has been identified mainly as an adhesion protein that signals through uPAR and selected integrin receptors. In addition to its pro-adhesive properties, we identified recently vitronectin as a main chemoattractant present in diluted plasma/serum that directly stimulates migration of cancer cells. We also found that this pro-migratory activity of vitronectin can be quenched by fibrinogen. Based on this we hypothesized that this may explain preference of cancer cell to metastasize to fibrinogen-low microenvironments such as lymphatics or peritoneal cavity. Based on this, we decided to investigate a role of vitronectin in metastasis of ovarian cancer cells to peritoneal cavity. We tested migratory responsiveness of three human ovarian cancer cell lines to ascites isolated from ovarian cancer patients and characterize possible molecules involved in migration of ovarian cancer cells. The ascites samples were exposed to heat inactivation, proteinase K digested, dialyzed and charcoal stripped. We also performed cut-off filtration analysis and by employing ELISA assays to measure concentration of vitronectin in ascites fluid samples. Finally, we employed shRNA against uPAR and small molecular inhibitors of integrin receptors to assess their involvement in biological effects of vitronectin. From our studies, we found that the similarly to diluted plasma, vitronectin in absence of fibrinogen is a main chemotactic/chemokinetic protein present in ascites fluid. We also found that these pro-migratory properties of vitronectin can be quenched by addition of fibrinogen. Our studies also indicate that both uPAR and integrin receptors on ovarian cancer cells regulate migration of these cells to vitronectin gradient. In summary, we identified free soluble vitronectin as a potent direct chemoattractant for ovarian cancer cells and that its activity is suppressed after binding to fibrinogen. Since in ascites fluids vitronectin is present in free form because of a lack or low level of fibrinogen, this could explain preferences of ovarian cancer stem cells to metastasize within peritoneum. We propose that inhibitors which could sequester soluble vitronectin in similar fashion as fibrinogen, could be employed as a novel anti-metastatic drugs.

Purification of Vitronectin

This unit describes the purification of extracellular vitronectin from plasma or serum by using heparin-affinity chromatography. First, the plasma is depleted of fibronectin plus other heparin- and Sepharose-binding proteins and treated with urea to activate the heparin-binding activity of vitronectin, which is subsequently bound to a heparin affinity column and eluted. The resulting vitronectin should be ∼ 98% pure.

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)

Survival Signals of Hepatic Stellate Cells in Liver Regeneration Are Regulated by Glycosylation Changes in Rat Vitronectin, Especially Decreased Sialylation

The extracellular matrix (ECM) molecules play important roles in many biological and pathological processes. During tissue remodeling, the ECM molecules that are glycosylated are different from those of normal tissue owing to changes in the expression of many proteins that are responsible for glycan synthesis. Vitronectin (VN) is a major ECM molecule that recognizes integrin on hepatic stellate cells (HSCs). The present study attempted to elucidate how changes in VN glycans modulate the survival of HSCs, which play a critical role in liver regeneration. Plasma VN was purified from partially hepatectomized (PH) and sham-operated (SH) rats at 24 h after operation and non-operated (NO) rats. Adhesion of rat HSCs (rHSCs), together with phosphorylation of focal adhesion kinase, in PH-VN was decreased to one-half of that in NO- or SH-VN. Spreading of rHSCs on desialylated NO-VN was decreased to one-half of that of control VN, indicating the importance of sialylation of VN for activation of HSCs. Liquid chromatography/multiple-stage mass spectrometry analysis of Glu-C glycopeptides of each VN determined the site-specific glycosylation. In addition to the major biantennary complex-type N-glycans, hybrid-type N-glycans were site-specifically present at Asn(167). Highly sialylated O-glycans were found to be present in the Thr(110)-Thr(124) region. In PH-VN, the disialyl O-glycans and complex-type N-glycans were decreased while core-fucosylated N-glycans were increased. In addition, immunodetection after two-dimensional PAGE indicated the presence of hyper- and hyposialylated molecules in each VN and showed that hypersialylation was markedly attenuated in PH-VN. This study proposes that the alteration of VN glycosylation modulates the substrate adhesion to rat HSCs, which is responsible for matrix restructuring.

How Glycosylation of Vitronectin Modulates the Tissue Remodeling during Liver Regeneration

Vitronectin (VN) is a multifunctional glycoprotein present in plasma and the extracellular matrix (ECM). VN binds to various biological ligands and it plays a key role in tissue remodeling by regulating cell adhesion and fibrinolysis. The present study attempted to determine the glycan structures and how alternations of glycans modulate the biological activity of VN during liver regeneration. Plasma VN was purified from partially hepatectomized (PH) and sham-operated (SH) rats at 24 hours after operation and non-operated (NO) rats. The liquid chromatography/ multiple-stage mass spectrometry analysis of glycopeptides of each VN determined the site-specific glycosylation. With the major complex-type N-glycans, hybrid-type N-glycans were site-specifically present. The complex-type N-glycans decreased in PH-VN while the fucosylation ratio was increased. Highly sialylated O-glycans were found to be present in the Thr110-Thr124 region, which contained α2,8-linked disialic acid and dramatically decreased after PH. Further study using ultracentrifugation demonstrated that the multimer sizes of PH-VN significantly increased compared with NO-VN. In accordance with this, PH-VN exhibited remarkably enhanced collagen-binding than NO-VN. The results indicate that glycan alterations during tissue remodeling induce increased multimerization state to enhance collagen-binding of VN.Adhesion of rat hepatic stellate cells (HSCs), together with phosphorylation of focal adhesion kinase (FAK), in PH-VN was decreased to half of that in NO- or SH-VN. Adhesion of HSCs in desialylated NO-VN decreased to 1/2 of that of control VN, indicating the importance of sialylation of VN for activation of HSCs. This study proposes that the alteration of glycosylation of VN is very significant in the modulation of the biological activity of VN during many steps of tissue remodeling processes. The elucidation of the molecular mechanisms of liver regeneration from the viewpoint of alterations in glycosylation of VN may contribute to the development of a strategy to regulate matrix deposition in liver cirrhosis.

Changes in glycosylation of vitronectin modulate multimerization and collagen binding during liver regeneration

Elucidating the mechanisms and factors regulating multimerization is biologically important in order to modulate the biological activities of functional proteins, especially adhesive proteins in the extracellular matrix (ECM). Vitronectin (VN) is a multifunctional glycoprotein present in plasma and ECM. Linkage of cellular adhesion and fibrinolysis by VN plays an essential role during tissue remodeling. Our previous study determined that the collagen-binding activity of VN was markedly enhanced with the decreased glycosylation during liver regeneration. This study demonstrated how alternations of glycans modulate the biological activity of VN. Human and rat VNs were used because of their similarities in structure and activities. The binding affinity of human VN to immobilized collagen was shown to be higher at pH 4.5 than at 7.5, at 37 degrees C than at 4 degrees C. Sedimentation velocity studies indicated that the greater the multimerization of human VN, the better it bound to collagen. The results indicate that the collagen binding of VN was modulated through its multimerization. Stepwise trimming of glycan with various exoglycosidases increased both the multimer size and the collagen binding of human VN, indicating that they are modulated by changes in glycosylation. The multimer sizes of VN purified from plasma of partially hepatectomized (PH) rats and sham-operated (SH) rats increased by about 45 and 31%, respectively, compared with those of nonoperated (NO) rats. In accordance with this, PH-VN exhibited remarkably enhanced collagen binding than SH-VN and NO-VN on surface plasmon resonance. In the PH rat sera, the multimer VN was increased in both amount and size compared with those in SH- and NO-sera. The results demonstrate that glycan alterations during tissue remodeling induce increased multimerization state to enhance the biological activity of VN.

Multimerization and collagen binding of vitronectin is modulated by its glycosylation

Background: The collagen-binding activity of vitronectin (VN) was found to be enhanced by glycosylation changes in vitro and during liver regeneration after partial hepatectomy. In this study, we propose a possible mechanism by which VN is activated to bind collagen by alteration of its glycosylation. Methods: Interaction between VN and type I collagen was characterized by enzyme-linked immunosorbent assay (ELISA). The effect of pH and glycosylation on the multimerization of VN was studied by analytical ultracentrifugation. Results: Sedimentation velocity analysis indicated that the multimerization of VN was more remarkable at pH 4.5, an optimal pH for collagen binding, than at pH 7.5. Neuraminidase- or N-glycosidase-treated VNs formed multimers larger than that of control VN incubated without an enzyme. By sedimentation equilibrium analysis, the deglycosylated VNs were found to remain in a multimer form at a guanidine–HCl (GdnHCl) concentration at which control VN dissociated into monomers. Conclusions: These results suggest that deglycosylated VN increased the size and stability of the multimer to eventually enhance the collagen-binding activity by a multivalent effect.

New Insights into the Size and Stoichiometry of the Plasminogen Activator Inhibitor Type-1·Vitronectin Complex

Plasminogen activator inhibitor-type 1 (PAI-1) is the primary inhibitor of endogenous plasminogen activators that generate plasmin in the vicinity of a thrombus to initiate thrombolysis, or in the pericellular region of cells to facilitate migration and/or tissue remodeling. It has been shown that the physiologically relevant form of PAI-1 is in a complex with the abundant plasma glycoprotein, vitronectin. The interaction between vitronectin and PAI-1 is important for stabilizing the inhibitor in a reactive conformation. Although the complex is clearly significant, information is vague regarding the composition of the complex and consequences of its formation on the distribution and activity of vitronectin in vivo. Most studies have assumed a 1:1 interaction between the two proteins, but this has not been demonstrated experimentally and is a matter of some controversy since more than one PAI-1-binding site has been proposed within the sequence of vitronectin. To address this issue, competition studies using monoclonal antibodies specific for separate epitopes confirmed that the two distinct PAI-1-binding sites present on vitronectin can be occupied simultaneously. Analytical ultracentrifugation was used also for a rigorous analysis of the composition and sizes of complexes formed from purified vitronectin and PAI-1. The predominant associating species observed was high in molecular weight (M(r) approximately 320,000), demonstrating that self-association of vitronectin occurs upon interaction with PAI-1. Moreover, the size of this higher order complex indicates that two molecules of PAI-1 bind per vitronectin molecule. Binding of PAI-1 to vitronectin and association into higher order complexes is proposed to facilitate interaction with macromolecules on surfaces.

Purification of Vitronectin

This unit describes the purification of extracellular vitronectin from plasma by a series of affinity chromatography steps. First plasma is depleted of fibronectin and other heparin-binding proteins; the sample is then treated with urea to activate the heparin-binding activity of vitronectin. The resulting vitronectin is approximately 98% pure.

Orientation of Heparin-binding Sites in Native Vitronectin: ANALYSES OF LIGAND BINDING TO THE PRIMARY GLYCOSAMINOGLYCAN-BINDING SITE INDICATE THAT PUTATIVE SECONDARY SITES ARE NOT FUNCTIONAL

A primary heparin-binding site in vitronectin has been localized to a cluster of cationic residues near the C terminus of the protein. More recently, secondary binding sites have been proposed. In order to investigate whether the binding site originally identified on vitronectin functions as an exclusive and independent heparin-binding domain, solution binding methods have been used in combination with NMR and recombinant approaches to evaluate ligand binding to the primary site. Evaluation of the ionic strength dependence of heparin binding to vitronectin according to classical linkage theory indicates that a single ionic bond is prominent. It had been previously shown that chemical modification of vitronectin using an arginine-reactive probe results in a significant reduction in heparin binding (Gibson, A., Baburaj, K., Day, D. E., Verhamme, I. , Shore, J. D., and Peterson, C. B. (1997) J. Biol. Chem. 272, 5112-5121). The label has now been localized to arginine residues within the cyanogen bromide fragment-(341-380) that contains the primary heparin-binding site on vitronectin. One- and two-dimensional NMR on model peptides based on this primary heparin-binding site indicate that an arginine residue participates in the ionic interaction and that other nonionic interactions may be involved in forming a complex with heparin. A recombinant polypeptide corresponding to the C-terminal 129 amino acids of vitronectin exhibits heparin-binding affinity that is comparable to that of full-length vitronectin and is equally effective at neutralizing heparin anticoagulant activity. Results from this broad experimental approach argue that the behavior of the primary site is sufficient to account for the heparin binding activity of vitronectin and support an exposed orientation for the site in the structure of the native protein.

Binding Sites on Native and Multimeric Vitronectin Exhibit Similar Affinity for Heparin: The Influence of Self-Association and Multivalence on Ligand Binding

A previously accepted model for the morphoregulatory activity of vitronectin is based on the idea that the heparin-binding site is buried within the circulating, monomeric form of vitronectin and that it is exposed on conversion to the multimeric form by denaturation or incorporation into the extracellular matrix. New evidence indicates that the heparin-binding sites are similarly exposed in the two forms of vitronectin and supports an alternative model for apparently altered heparin affinity. Differences in the heparin-binding properties of circulating and matrix-associated vitronectin result from an increased number of binding sites on the multivalent matrix form. By analogy with other instances in which multivalent binding interactions increase functional affinity for carbohydrates or lectins, the self-association of vitronectin into a multimeric form allows effective neutralization of heparin at the endothelial surface in the vicinity of a thrombus.

Inhibition of rat mast cell protease 1 by vitronectin

Rat mast cell protease 1 (RMCP-1) is a chymotrypsin-like serine protease specifically expressed by connective tissue-type mast cells. The enzyme is stored in the secretary granules in a macromolecular complex with heparin proteoglycan. In the present investigation it was shown that RMCP-1 is inhibited by vitronectin (VN), an RGD-containing adhesive glycoprotein with heparin-binding properties. RMCP-1 that had been separated from heparin proteoglycan was less susceptible to inhibition than RMCP-1 present in complex with heparin proteoglycan. Pre-incubation of VN with purified heparin partially blocked the RMCP-1 inhibiting activity of VN. Plasma VN had negligible RMCP-1-inhibiting activity. However, heat treatment of plasma VN, which is known to expose the heparin-binding domain, induced RMCP-1-inhibiting activity. Affinity chromatography on immobilized VN showed that RMCP-1 bound with high affinity to VN. The binding of RMCP-1 to VN was not heparin-dependent since free RMCP-1 bound with equal affinity to the immobilized VN as RMCP-1 present in complex with heparin. The inhibition of RMCP-1 by VN was shown to be reversible.

The Cell Attachment and Spreading Activity of Vitronectin Is Dependent on the Arg-Gly-Asp Sequence: Analysis by Construction of RGD and Domain Deletion Mutants

The cell attachment activity of vitronectin has been ascnbed to an Arg-Gly-Asp (RGD) sequence near the amino terminus. To verify the importance of the RGD sequence for cell binding, we created RAD and RGE vitronectin mutants and also deleted either the somatomedin B (ΔS-rVN) or heparin (ΔH-rVN) binding domains. These mutants were expressed as fusion proteins, purified using Ni +2 affinity chromatography, and assayed for cell attachment. EAhy.926 cells bound equally well to wild-type, ΔS-rVN, and to ΔH-rVN, but binding to RAD-rVN and RGE-rVN was inhibited by more than 90%. We therefore conclude that the RGD sequence of vitronectin is the most important cell recognition site and that neither the somatomedin B nor heparin domains contribute signficantly to the cell adhesive activity of vitronectin.

Heat and autoclave resistance of cell-spreading activity of vitronectin

We have investigated the heat- and autoclave-resistant properties of the cell-spreading activity of vitronectin, a cell-spreading glycoprotein in animal blood plasma. Vitronectin heated at 100°C for 10 min or autoclaved at 121°C at 1.2 kg/cm 2 for 20 min retained the same cell-spreading activity as native vitronectin. In contrast, fibronectin and type-I collagen treated in the same way lost their activity almost completely. GRGDSP remarkably inhibited the cell-spreading activity of native, heated and autoclaved vitronectins. GRGESP did not inhibit the activity of native vitronectin, but, unexpectedly, partially inhibited the activity of both heated and autoclaved vitronectins. In SDS-polyacrylamide gel analysis under reducing conditions, vitronectin heated at 100°C migrated mainly as a monomer, but autoclaved vitronectin migrated at both the top and front of the gel instead of at the position of the monomer. The change in molecular size during the heat- and autoclave treatments was partially prevented by adding 10mM dithiothreitol or 2% 2-mercaptoethanol to the protein solution.

Activation of the collage-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.

Limited Proteolysis of Vitronectin by Plasmin Destroys Heparin Binding Activity

Vitronectin (VN) stabilizes plasminogen activator inhibitor type 1 (PAI-1) activity and prevents the fibrin(ogen)-induced acceleration of plasminogen activation by t-PA. These antifibrinolytic activities as well as other functions are mediated by the glycosaminoglycan (GAG) binding domain of VN. Since the GAG binding region is rich in arginyl and lysyl residues, it is a potential target for enzymes such as plasmin. In this paper, the dose and time-dependent proteolysis of VN by plasmin is demonstrated. The addition of urokinase or streptokinase (200 units/ml) to plasma also produced proteolysis of VN. With minimal proteolysis, the 75 kDa band was degraded to a 62-65 kDa form of VN. This minimal proteolysis destroyed the binding of [3H]-heparin to VN and reversed the neutralization of heparin by VN. Thus, the plasmin-mediated proteolysis of the GAG binding activity of VN could destroy the antifibrinolytic activity of VN during physiologic conditions and during thrombolytic therapy. Furthermore, other functions of VN in complement and coagulation systems that are mediated by the GAG binding domain may be destroyed by plasmin proteolysis.

Novel purification of vitronectin from human plasma by heparin affinity chromatography.

The glycoprotein vitronectin (also called S-protein, serum spreading factor, or epibolin) promotes spreading of a variety of cultured cells, inhibits the cytotoxicity of membrane attack complex C5b-9, and modulates thrombin-antithrombin III activity. We developed a strikingly simple method to purify vitronectin from human plasma by heparin affinity chromatography. Serum was obtained from plasma by adding calcium and then centrifuging. The heparin-binding activity of vitronectin in human serum was activated with 8 M urea. The activated vitronectin specifically bound to heparin-Sepharose in 8 M urea and was eluted with 0.5 M NaCl containing 8 M urea. This procedure resulted in an approximately 250-fold purification of vitronectin with a 15-30% recovery; 3-6 mg of pure vitronectin were obtained from 100 ml human plasma within 2 days. The purified vitronectin preparations promoted spreading of BHK fibroblastic cells on substrates with a half-maximal activity at only 0.1 microgram/ml. This new method is very simple, rapid, inexpensive, and flexible. It could probably be readily scaled up for commercial applications.

A Preliminary Comparison of the Thrombogenic Activity of Vitronectin and Other RGD‐containing Proteins When Bound to Surfacesa

A Preliminary Comparison of the Thrombogenic Activity of Vitronectin and Other RGD‐containing Proteins When Bound to Surfacesa