Human Vitronectin Research Articles

Bacterial binding to extracellular matrix proteins -- in vitro adhesion.

A binding assay was developed and used to study the binding of oral streptococcus to immobilized human fibronectin, laminin, vitronectin, fibrinogen, heparin, and collagen IV. The protein binding was dependent on the broth used for bacterial growth. The binding after growth in brain heart infusion broth, trypticase soy broth, Todd-Hewitt broth, and Dulbecco's modified Eagle's medium was examined. Most of the strains were able to bind to immobilized fibronectin and laminin, and to a minor extent vitronectin. Binding was not observed on immobilized fibrinogen, collagen IV, or heparin. Measured surface hydrophobicity correlated well with the bacterial binding strength to the proteins. Streptococcal incubation with putative inhibitors indicates multiple binding mechanisms of a lectin-like and protein nature, possibly involving protein receptors.

Vitronectin is a constituent of ocular drusen and the vitronectin gene is expressed in human retinal pigmented epithelial cells.

Age-related macular degeneration (AMD) leads to dysfunction and degeneration of retinal photoreceptor cells. This disease is characterized, in part, by the development of extracellular deposits called drusen. The presence of drusen is correlated with the development of AMD, although little is known about drusen composition or biogenesis. Drusen form within Bruch's membrane, a stratified extracellular matrix situated between the retinal pigmented epithelium and choriocapillaris. Because of this association, we sought to determine whether drusen contain known extracellular matrix constituents. Antibodies directed against a battery of extracellular matrix molecules were screened on drusen-containing sections from human donor eyes, including donors with clinically documented AMD. Antibodies directed against vitronectin, a plasma protein and extracellular matrix component, exhibit intense and consistent reactivity with drusen; antibodies to the conformationally distinct, heparin binding form of human vitronectin are similarly immunoreactive. No differences in vitronectin immunoreactivity between hard and soft drusen, or between macular and extramacular regions, have been observed. RT-PCR analyses revealed that vitronectin mRNA is expressed in the retinal pigmented epithelium (RPE)-choroidal complex and cultured RPE cells. These data document that vitronectin is a major constituent of human ocular drusen and that vitronectin mRNA is synthesized locally. Based on these data, we propose that vitronectin may participate in the pathogenesis of AMD.

Vitronectin binds to the gonococcal adhesin OpaA through a glycosaminoglycan molecular bridge.

Several bacterial pathogens including Neisseria gonorrhoeae bind the human serum glycoprotein vitronectin. We aimed at defining the gonococcal receptor for vitronectin. Ligand blots demonstrated that vitronectin bound specifically to the heparin-binding outer-membrane protein OpaA, but that coating OpaA with the sulphated polysaccharide heparin was required for the interaction to occur. Bound vitronectin could be dissociated from OpaA-heparin-vitronectin complexes by the addition of excess heparin, indicating that sulphated polysaccharides provided the main linkage between the two proteins. Binding assays with intact micro-organisms substantiated the requirement of sulphated polysaccharides such as heparin and dextran sulphate for the efficient binding of vitronectin to OpaA+ gonococci. This was underscored by the increased binding of vitronectin to gonococci that had been preincubated with saturating concentrations of dextran sulphate, as opposed to the inhibition of vitronectin binding observed when bacteria were incubated simultaneously with vitronectin and saturating concentrations of dextran sulphate. Binding assays with dextran sulphates of various sizes indicated that vitronectin binding correlated with the size of the polysaccharide rather than with the amount of OpaA produced by the bacteria. The inability of zero-length cross-linking agents to couple vitronectin to OpaA provided further evidence that sulphated polysaccharides formed the linkage between vitronectin and OpaA. Infection experiments demonstrated that proteoglycan-deficient Chinese hamster ovary cells efficiently internalized dextran sulphate/vitronectin-coated gonococci, suggesting that soluble sulphated polysaccharides could substitute for cell surface glycosaminoglycans in the internalization process. On the basis of our results, we propose a novel mechanism of vitronectin binding in which sulphated polysaccharides act as molecular bridges, linking the glycosaminoglycan-binding sites of vitronectin and gonococcal OpaA.

Characterization of the ligand binding activities of vitronectin: interaction of vitronectin with lipids and identification of the binding domains for various ligands using recombinant domains.

Vitronectin is a multifunctional plasma glycoprotein which may regulate the systems related to protease cascades such as the coagulation, fibrinolysis, and complement systems as well as cell adhesion. Solid-phase assays and affinity chromatography on immobilized glycolipids indicated that vitronectin purified under denaturing conditions bound to sulfatide (Gal(3-SO4)beta1-1ceramide), cholesterol 3-sulfate, and various phospholipids, but not gangliosides. Only the unfolded or multimeric form of vitronectin bound to sulfatide, suggesting a conformational dependency of the binding activity, while vitronectin bound to cholesterol 3-sulfate regardless of its conformational state. The recombinant domains of human vitronectin and mutants with certain domains deleted were separately expressed in E. coli as fusion proteins. Using the recombinants, sulfatide-, phosphatidylserine-, cholesterol 3-sulfate-, Type I collagen-, heparin-, and beta-endorphin-binding activities were found to be attributable to hemopexin domain 2 and hemopexin domain 1. The possibility was suggested that the presence of a somatomedin domain and/or connecting region flanking hemopexin domain 1 inactivated its heparin binding. De-N-glycosylation of plasma vitronectin significantly affected the cholesterol sulfate- and collagen-binding activities, although its effects were opposite. These findings suggest that diverse ligand-binding activities could be attributed to pexin family motifs but that the interdomain interactions and glycosylations modulate the ligand binding activities of vitronectin.

Photodynamic therapy inhibits cell adhesion without altering integrin expression

Adhesion is a primordial cell function that, among others, regulates inflammation, metastasis, and tissue repair. To understand how these events could be affected by photodynamic therapy (PDT), we studied the effects of PDT on human foreskin fibroblast (HFF) adhesion to bovine collagen type I, human vitronectin or fibronectin. PDT, using benzoporphyrin derivative monoacid ring A (verteporfin) as the photosensitizer, inhibited cell adhesion in a drug dose-dependent manner, with no significant difference among matrices. The drug dose that killed 90% of cells within 20 h post-treatment inhibited HFF adhesion by 55%–68%. However, 45 min following PDT, a time period corresponding to that of the adhesion assay, HFF membrane integrity remained unaltered. In addition, cell surface expression of integrins was not modified for at least 2 h following PDT. Western blots of cell lysates, using the anti-phosphotyrosine 4G10 monoclonal antibody, revealed that PDT prevented the adhesion-induced phosphorylation of 110–130 kDa proteins. Immunoblots of cell lysates immunoprecipitated with antibodies to focal adhesion kinase suggested that its phosphorylation was suppressed by PDT. These results demonstrate that PDT inhibits cell adhesion and affects integrin signalling without modifying cell membrane integrity or integrin expression.

Measurement of vitronectin content of human spermatozoa and vitronectin concentration within seminal fluid

Objective: Vitronectin previously has been extracted from human spermatozoa and messenger RNA (mRNA) encoding vitronectin localized by reverse transcriptase in situ polymerase chain reaction (PCR) to spermatocytes of human testis. In the present experiments, we have established ranges for the content of vitronectin in living human spermatozoa and vitronectin concentration within seminal fluid of human ejaculates. Design: Seminal fluid was obtained by centrifugation and motile sperm selected by swim-up from men with normal and abnormal ejaculates, according to World Health Organization criteria, for vitronectin determinations. Setting: Academic research environment. Main Outcome Measure(s): Seminal fluid vitronectin concentrations were measured by ELISA and sperm vitronectin content by polyacrylamide gel electrophoresis and semiquantitative Western blots. Result(s): Vitronectin seminal fluid concentration was 1.35 ± 1.0 mg/mL (mean ± SD) for normospermic samples ( n = 26) and 0.78 ± 0.4 mg/mL for azoospermic specimens ( n = 6). Vitronectin sperm content ranged from 1 to 15 ng/10 6 motile cells ( n = 20). Both high- and low-molecular-weight material was observed. Sperm content of vitronectin did not vary with sperm morphology. Conclusion(s): These results suggest that spermatozoa represent a major source of seminal fluid vitronectin, but that a secondary source exists, perhaps through transudation from serum.

Binding of vitronectin to opa-expressing Neisseria gonorrhoeae mediates invasion of HeLa cells.

Neisseria gonorrhoeae induces local infections in the human genitourinary tract and can disseminate to other organs to cause severe disease. Blood-derived factors present in the genital mucosa have been suggested to facilitate the spread of N. gonorrhoeae in disseminated gonococcal infections. Using gentamicin invasion assays and confocal microscopy, we observed a strong stimulatory effect of fetal calf serum (FCS) on the gonococcal invasion of HeLa cells. FCS-mediated invasion was dependent on the expression of the epithelial cell invasion-associated Opa protein (plasmid-encoded Opa50 or its chromosomal homolog Opa30), while N. gonorrhoeae expressing noninvasive Opa proteins (Opa(51-60)) or no Opa protein (Opa-) was not invasive even in the presence of FCS. Incubation of N. gonorrhoeae MS11 with biotinylated FCS revealed a 78-kDa protein as the prominent protein binding to Opa50- or Opa30-expressing gonococci. This protein was recognized by antibodies against vitronectin (VN) in Western blots. Purified human or bovine VN efficiently bound to Opa50-expressing gonococci, while binding to noninvasive Opa- or Opa52-expressing gonococci was significantly lower. Binding of VN was inhibited by heparin in a concentration-dependent manner, indicating that the heparin binding sites present in VN or Opa50 may play an essential role in this interaction. Based on gentamicin invasion assays and confocal microscopy studies, VN binding was associated with an increased invasion of Opa50- and Opa30-expressing gonococci into HeLa cells. The ability of VN to mediate entry into epithelial cells may constitute an important event in the pathogenesis of local as well as disseminated gonococcal infections.

Vitronectin-binding staphylococci enhance surface-associated complement activation.

Coagulase-negative staphylococci are well recognized in medical device-associated infections. Complement activation is known to occur at the biomaterial surface, resulting in unspecific inflammation around the biomaterial. The human serum protein vitronectin (Vn), a potent inhibitor of complement activation by formation of an inactive terminal complement complex, adsorbs to biomaterial surfaces in contact with blood. In this report, we discuss the possibility that surface-immobilized Vn inhibits complement activation and the effect of Vn-binding staphylococci on complement activation on surfaces precoated with Vn. The extent of complement activation was measured with a rabbit anti-human C3c antibody and a mouse anti-human C9 antibody, raised against the neoepitope of C9. Our data show that Vn immobilized on a biomaterial surface retains its ability to inhibit complement activation. The additive complement activation-inhibitory effect of Vn on a heparinized surface is very small. In the presence of Vn-binding strain, Staphylococcus hemolyticus SM131, complement activation on a surface precoated with Vn occurred as it did in the absence of Vn precoating. For S. epidermidis 3380, which does not express binding of Vn, complement activation on a Vn-precoated surface was significantly decreased. The results could be repeated on heparinized surfaces. These data suggest that Vn adsorbed to a biomaterial surface may serve to protect against surface-associated complement activation. Furthermore, Vn-binding staphylococcal cells may enhance surface-associated complement activation by blocking the inhibitory effect of preadsorbed Vn.

Altered cell-matrix contact: a prerequisite for breast cancer metastasis?

The integrins are receptors that regulate interaction between epithelial cells and the extracellular matrix. Previous studies have shown that a reduction in the expression of the alpha2beta1, alpha3beta1, alpha6beta1, alpha(v)beta1 and alpha(v)beta5 integrins in primary breast cancer is associated with positive nodal status. In order to assess the functional significance of altered integrin expression, primary breast cancer cells were derived from individual patients with known tumour characteristics using immunomagnetic separation. Purified human fibronectin, vitronectin, laminin and type IV collagen were used to represent the principal extracellular matrix proteins in an in vitro adhesion assay. Primary breast cancer cells from lymph node-positive patients were significantly less adhesive to each of the matrix proteins studied (P<0.001, Mann-Whitney U-test). Matrix adhesion of primary breast cancer cells from node-negative patients was inhibited by appropriate integrin monoclonal antibodies (P<0.001, paired Wilcoxon test). Adhesion to fibronectin, vitronectin and laminin, but not type IV collagen, was influenced by the inhibitor arginine-glycine-aspartate, suggesting that breast cancer cell recognition of collagen IV is mediated through alternative epitopes. Weak matrix adhesion correlated with loss of integrin expression in tissue sections from corresponding patients assessed using immunohistochemistry. This study demonstrates a link between altered integrin expression and function in primary breast cancers predisposed to metastasize.

Fibroblasts Spread on Immobilized Fibrin Monomer by Mobilizing a β1-class Integrin, Together with a Vitronectin Receptor αvβ3 on Their Surface

Human and murine fibroblasts were found to spread far more avidly on fibrin monomer monolayers than on immobilized fibrinogen, indicating that removal of fibrinopeptides by thrombin is a prerequisite for the fibrin-mediated augmentation of cell spreading. In fact, cell spreading was not efficiently augmented on monolayers of a thrombin-treated dysfibrinogen lacking the release of fibrinopeptide A due to an Aalpha Arg-16 --> Cys substitution. Since a synthetic Arg-Gly-Asp (RGD)-containing peptide inhibited the fibrin-mediated cell spreading, subsequent dissociation of the carboxyl-terminal globular domain of the Aalpha-chains appears to render the RGD segments accessible to the cell-surface integrins. In support of this, fibrin-augmented cell spreading was inhibited by an antibody recognizing a 12-kDa peptide segment with gamma Met-89 at its amino terminus, which is located in close association with the RGD segment at Aalpha 95-97 in the helical coiled-coil interdomainal connector. The fibrin-mediated augmentation of cell spreading was inhibited not only by an antibody against human vitronectin receptor (LM 609) but also by an antibody against the beta1 subunit of integrin (mAb13), suggesting that the beta1-class integrin together with a vitronectin receptor, alphavbeta3, is mobilized onto the surface of fibroblasts upon contact with the fibrin monomer monolayer.

Dietary coenzyme Q10 supplementation alters platelet size and inhibits human vitronectin (CD51/CD61) receptor expression.

Improved cardiovascular morbidity and mortality have been observed in several clinical studies of dietary supplementation with coenzyme Q10 (CoQ10, ubiquinone). Several mechanisms have been proposed to explain the effects of CoQ10, but a comprehensive explanation of its cardioprotective properties is still lacking. One attractive theory links ubiquinone with the inhibition of platelets. The effect of CoQ10 intake on platelet size and surface antigens was examined in human volunteers. Study participants received 100 mg of CoQ10 twice daily in addition to their usual diet for 20 days. Receptor expression was measured by flow cytometry with monoclonal murine anti-human antibodies CD9 (p24), CD42B (Ib), CD41b (IIb), CD61 (IIIa), CD41a (IIb/IIIa), CD49b (VLA-2), CD62p (P selectin), CD31 (PECAM-1), and CD51/CD61 (vitronectin). An increase of total serum CoQ10 level (from 0.6 +/- 0.1 to 1.8 +/- 0.3 micrograms/ml; p < 0.001) was found at protocol termination. Fluorescence intensity was higher for the large platelets when compared with the whole platelet population. Significant inhibition of vitronectin-receptor expression was observed consistently throughout ubiquinone treatment. Reduction of platelet size was observed at the end of CoQ10 supplementation. Inhibition of the platelet vitronectin receptor and a reduction of the platelet size are direct evidence of a link between dietary CoQ10 intake and platelets. These findings may not be fully explained by the known antioxidant and bioenergetic properties of CoQ10. Diminished vitronectin-receptor expression and reduced platelet size resulting from CoQ10 therapy may contribute to the observed clinical benefits in patients with cardiovascular diseases.

Binding of extracellular matrix proteins by enterococci.

Forty-four enterococcal strains isolated from human clinical specimens were investigated for binding of 125I-labeled fibronectin, vitronectin, thrombospondin, lactoferrin, and collagen type I and IV, and for cell surface hydrophobicity. Most strains expressed low binding of iodine-labeled human fibronectin, collagen I and IV, and higher binding of human vitronectin, human lactoferrin, and human thrombospondin. Bacteria grown in Todd-Hewitt broth exhibited increased binding to vitronectin and thrombospondin. In particle agglutination assays (PAA), Enterococcus faecalis strains reacted strongly with coated latex beads in contrast to E. faecium strains, which generally did not react. The ability of enterococci to bind ECM proteins was affected by heating and proteolytic digestion, suggesting that some protein-binding components become surface exposed after treatment with proteases. The binding of 125I-labeled proteins to E. faecalis strain E70 was inhibited when cells were preincubated with unlabeled proteins. Preincubating cells with sulfated polymers such as dextran sulfate (Mr 5000 and 8000), pentosan sulfate and heparin decreased binding of vitronectin, lactoferrin, and thrombospondin. The binding of lactoferrin and thrombospondin was also decreased when bacteria were preincubated with galactose, fucose, and mannosamine, but not with mannose. All of 30 E. faecalis strains expressed pronounced surface hydrophobicity, but 10 of 14 E. faecium strains showed hydrophilic cell surface.

Porcine vitronectin, the most compact form of single-chain vitronectin: the smallest molecular mass among vitronectins was ascribed to deletion and substitution of base pairs, and proteolytic trimming of the peptide.

Vitronectin is a multifunctional glycoprotein regulating the fibrinolysis, complement, and coagulation systems in plasma, besides exhibiting cell-spreading activity. Porcine vitronectin has an unusually small molecular mass among the vitronectins hitherto found, which seems to make it hard for it to retain all the known activities. In this study, the complete primary structure of porcine vitronectin was elucidated by cloned cDNA and glycoprotein analyses. A coding sequence of 459 amino acids including a signal peptide of 19 amino acids was deduced from the cDNA. The coding sequence showed 70.3% homology with that of human vitronectin, but porcine vitronectin lacked 22 amino acids in the connecting region. One amino acid substitution resulted in the loss of a potential glycosylation site in accordance with the finding on glycopeptide analyses that porcine vitronectin contained two kinds of glycosylated sequences, while human vitronectin contained three. C-Terminal analysis of porcine vitronectin indicated that an 80 amino acid fragment was completely removed from the C-terminal end on proteolytic processing. Thus, porcine vitronectin only exists in a truncated single-chain form representing the most compact functional form of vitronectin, which suggests the lack of functional necessity of the truncated C-terminal fragment.

Collagen-binding activity of plasma vitronectin in chronic liver disease

The collagen-binding activity of plasma vitronectin was measured in 15 control subjects and 64 subjects with chronic liver disease. The assay of collagen-binding vitronectin was performed by an enzyme immunoassay using a monoclonal antibody to human vitronectin and type I collagen from human placenta. The plasma collagen-binding vitronectin concentration (mean ± S.D.) was 5.6 ± 1.9 μg/ml in the controls 8.3 ± 1.1 μg/ml in chronic persistent hepatitis, 8.3 ± 2.9 μg/ml in chronic active hepatitis 7.8 ± 2.9 μg/ml in liver cirrhosis and 8.2 ± 2.1 μg/ml in hepatocellular carcinoma with cirrhosis. The percent collagen-binding vitronectin to total plasma vitronectin was 2.2 ± 0.8% in the controls, 3.9 ± 2.2% in chronic persistent hepatitis, 3.9 ± 1.2% in chronic active hepatitis, 5.8 ± 3.3% in liver cirrhosis and 4.1 ± 1.2% in hepatocellular carcinoma with cirrhosis. The plasma collagen-binding vitronectin also correlated with the serum levels of 7S collagen and hyaluronic acid. These findings suggest that vitronectin may play an important role in the progression of liver disease and/or in hepatic fibrosis through its collagen-binding domain.

The cDNA sequence of porcine vitronectin and its expression in liver and skeletal muscle of GH-supplemented pigs.

A cDNA (1555 bp) (DNA database accession number, D61396) having a homology with human vitronectin (Vn) was isolated from a porcine liver cDNA library, and its sequence was determined. The open reading frame in the cDNA was found to code a protein with 388 amino acids, then the amino acid sequence of the protein (porcine putative Vn) was aligned to the other mammalian (mouse, rabbit, and human) Vns previously reported. The alignment revealed that the functional amino acid sequences reported as the cell attachment site, the heparin binding site, the region involved in glycosylation, and plasminogen activator inhibitor I-binding domain were conserved in the porcine putative Vn. These findings together with the fact that the calculated molecular weight and the N-terminal amino acid sequence of the putative Vn agreed with those reported by biochemical analysis on porcine Vn, led us to conclude that the cDNA isolated in the present study coded for the porcine Vn. Then, a time course study was performed to examine whether the administration of growth hormone (GH) affects Vn mRNA expression in liver and skeletal muscle, since the level of Vn mRNA was reported to be affected by inflammation, and since GH was reported to be involved in inflammation. This revealed that GH has no effect on the level of liver Vn mRNA, and that Vn mRNA level in skeletal muscle seemed to be affected following GH-injection.

Phenotypic characterization of Streptococcus dysgalactiae isolates from bovine mastitis by their binding to host derived proteins

The binding of 80 Streptococcus dysgalactiae mastitis isolates from 51 farms to plasma and connective tissue proteins fibronectin (29 kDa N-terminal fragment), vitronectin, collagen type I, fibrinogen, α 2-macroglobulin, IgG, and albumin was studied. All isolates bound the bovine 29 kDa fibronectin fragment and the binding of bovine fibrinogen, caprine albumin, bovine α 2-macroglobulintrypsin complexes and caprine IgG was also very frequent (92.5, 92.5, 72.5% and 87.5%, respectively). Binding to human vitronectin was observed in 55% of the isolates, whereas only 20% of the isolates bound human type I collagen. None of the isolates bound native α 2-macroglobulin. Nearly all isolates (91%) bound more than 3 ligands. The bacterial binding sites for these proteins (termed here receptors) occurred in different combinations of which the combination fibronectin-, albumin-, fibrinogen-, vitronectin-, α 2-macroglobulin-and IgG-receptor was the most common. More than one isolate was obtained from 10 farms. The isolates from 5 farms showed close similarity of binding profiles within the farm, indicating that they were of similar origin and suggesting that the binding characteristics were relatively stable. Wider variation among the isolates obtained from other 5 farms was detected. The different isolates of the same farm origin varied mostly in the binding of albumin, IgG and fibrinogen. Interestingly, a difference in the number of receptors between isolates from two different sampling areas was observed. The binding profiles offer a new phenotypic method for epidemiological studies and may also when combined with genetical studies provide more insight both into the role of the bacterial plasma and connective tissue protein receptors in the infection process and the regulation of receptor expression.

Vitronectin receptor expression and distribution at the photoreceptor-retinal pigment epithelial interface.

Laser scanning confocal microscopy was employed to map the distribution of integrin immunoreactivity at the photoreceptor-retinal pigment epithelial (RPE) interface of the primate retina, and to determine its relationship to the actin cytoskeleton. Immunolabeling using a polyclonal antibody to the human vitronectin receptor (VnR), a heterodimer containing the alpha v subunit in combination with either the beta 3 or beta 5 subunits, is detected primarily on the apical surface of the retinal pigment epithelium (RPE) in vivo and in vitro. It is also associated with the photoreceptor inner and outer segment cell surfaces. In contrast, immunolabeling using a polyclonal antibody to the human fibronectin receptor (FnR), a heterodimer containing the alpha 5 and beta 1 subunits, is detected principally on the basolateral surface of the RPE and is virtually absent in photoreceptors. A partial three-dimensional reconstruction of the anti-VnR labeling pattern in cone photoreceptors reveals cell surface labeling that originates at the level of the myoid just distal to the outer limiting membrane. It extends distally toward the ellipsoid and terminates at the level of the cone outer segment. Approximately 20-22 immunoreactive foci are distributed evenly around the perimeter of the cone ellipsoid. These foci correspond in number and location to the calycal processes that protrude from the distal portion of the ellipsoid. A double-labeling procedure, employing VnR antibody and a fluorescently labeled phallotoxin (phalloidin), was used to identify regions of VnR co-distribution with filamentous actin (F-actin). One such region includes the VnR-immunoreactive foci at the margins of the cone inner segments and the actin cables that course through the photoreceptor ellipsoid and terminate within the calycal processes. A second zone of co-distribution coincides with the actin-containing, circumferential bundle at the lateral borders of the RPE cells, and a third zone is associated with the apical microvilli of the RPE that ensheath cone outer segments. In order to help identify the specific subunits underlying VnR (alpha v beta 3/5) immunoreactivity, Northern blots of retinal-RPE RNA were probed with alpha 32P-cDNAs to the human alpha v, beta 3, and beta 5 subunits and additional immunolocalization studies were performed using integrin human alpha or beta subunit-specific antisera. The results from these studies strongly suggest that one or more integrins, containing the alpha v and/or beta 5 subunits, are expressed by the photoreceptors and RPE.(ABSTRACT TRUNCATED AT 400 WORDS)

Interaction of Single-chain Urokinase and Plasminogen Activator Inhibitor Type 1

Urokinase (u-PA) is synthesized and secreted as a single-chain polypeptide (single-chain u-PA, scu-PA), which has such little enzymatic activity in solution that it has been considered essentially enzymatically inert. We found that plasminogen activator inhibitor type 1 (PAI-1), the major PAI in plasma, demonstrated concentration-dependent inhibition of this solution-phase scu-PA enzymatic activity. 125I-scu-PA formed complexes with PAI-1 in a concentration- and time-dependent manner, as detected by SDS-polyacrylamide gel electrophoresis under reducing conditions. Among a given population of scu-PA molecules, all measurable enzymatic activity was inhibited by a 10-fold molar excess of PAI-1. However, at this stoichiometry, only a minority of 125I-scu-PA molecules formed SDS-stable complexes with PAI-1 (i.e. complexes that formed a covalent bond upon denaturation), even though the uncomplexed PAI-1 molecules remained competent to inhibit u-PA enzymatic activity. Neither the extent nor the time course of complex formation was altered by using PAI-1 that had been pre-incubated with native human vitronectin, compared with native PAI-1 alone. 125I-scu-PA.PAI-1 complexes that would form a covalent bond if denatured were reversible and existed in equilibrium with either non-complexed or loosely complexed reactants. These data suggest that scu-PA has more enzyme-like properties than previously appreciated and raises the possibility that it resembles single-chain tissue type-plasminogen activator in lacking a complete zymogen conformation.

Isolation and characterisation of a vitronectin-binding surface protein from Staphylococcus aureus

In a previous study we demonstrated that cells of Staphylococcus aureus strain V8 bind 125I-labelled vitronectin in a receptor-ligand type of interaction, and a protein having a molecular mass of 60 kDa was identified as a putative high-affinity staphylococcal vitronectin-binding protein (Liang, O.D. et al. (1993) Biochim. Biophys. Acta 1225, 57–63). In the present communication we report on the isolation and preliminary characterisation of the 60 kDa vitronectin-binding protein. The bacterial cell surface proteins were released by stirring bacteria with 1 M LiCl at 37°C for 2 h and separated on an FPLC Mono-Q column with a gradient of 0–0.5 M NaCl in 20 mM Tris buffer at pH 9.0. Fractions containing vitronectin-binding activity, assayed on microtiter plates with immobilised human vitronectin, were collected and SDS-PAGE analysis showed the content to be a single protein band at the 60 kDa position. In Western blot experiments the protein transblotted onto nitrocellulose membranes could bind soluble vitronectin. Its amino-terminal amino acid sequences showed a striking similarity with those of a 60 kDa heparan sulfate-binding protein from the same staphylococcal strain (Liang, O.D. et al. (1992) Infect. Immun. 60, 899–906), suggesting that they are identical molecules. This was supported by ligand blotting experiments where both vitronectin and heparan sulfate were shown to bind to the same protein band in parallel strips.

Structures of the N-linked oligosaccharides on human plasma vitronectin.

The structures of N-linked oligosaccharides present on human plasma vitronectin were elucidated. Oligosaccharides were released from the vitronectin by N-glycosidase F digestion and tagged with 2-aminopyridine; the pyridylamino-oligosaccharides were then fractionated by anion-exchange and reverse-phase HPLC. Ten major pyridylamino-oligosaccharides were isolated. The linkages and locations of sialic acid residues were determined by desialylation with Salmonella sialidase in combination with acid. The asialo forms were then analyzed by two-dimensional sugar mapping, component sugar analysis and 400-MHz 1H-NMR spectroscopy. The major oligosaccharides of human vitronectin were of the diantennary N-acetyllactosamine type, with a lesser amount of the tri- and a small amount of the mono-antennary type, to which 1-3 mol sialic acid residues were linked, mostly through alpha 2-6 linkages, although alpha 2-3 linkages were also present. The possibility that several binding activities of vitronectin can be ascribed to its glycan moiety was discussed, based on the specific features of the N-linked oligosaccharides on human vitronectin revealed here.