Ligand binding regions in the receptor for urokinase-type plasminogen activator.

The interaction between urokinase plasminogen activator (uPA) and its cellular receptor (uPAR) is a key event in cell surface-associated plasminogen activation, relevant for cell migration and invasion. In order to define receptor recognition sites for uPA, we have expressed uPAR fragments as fusion products with the minor coat protein on the surface of M13 bacteriophages. Sequence analysis of cDNA fragments encoding uPA-binding peptides indicated the existence of a composite uPA-binding structure including all three uPAR domains. This finding was confirmed by experiments using an overlapping 15-mer peptide array covering the entire uPAR molecule. Four regions within the uPAR sequence were found to directly bind to uPA: two distinct regions containing amino acids 13--20 and amino acids 74--84 of the uPAR domain I, and regions in the putative loop 3 of the domains II and III. All the uPA-binding fragments from the three domains were shown to have an agonistic effect on uPA binding to immobilized uPAR. Furthermore, uPAR-(154--176) increased uPAR-transfected BAF3-cell adhesion on vitronectin in the presence of uPA, whereas uPAR-(247--276) stimulated the cell adhesion both in the absence or presence of uPA. The latter fragment was also able to augment the binding of vitronectin to uPAR in a purified system, thereby mimicking the effect of uPA on this interaction. These results indicate that uPA binding can take place to particular part(s) on several uPAR molecules and that direct uPAR-uPAR contacts may contribute to receptor activation and ligand binding.

The hemopexin-type repeats of human vitronectin are recognized by Streptococcus pyogenes.

The specific binding of vitronectin to Streptococcus pyogenes is believed to play an important role in the infection process by mediating adherence of the bacteria to host cells. The domain of vitronectin involved in the interaction with S. pyogenes is unknown. In the present study, we constructed a vitronectin random epitope phage display library, which was used to pan against intact cells of S. pyogenes. Several phage-displayed vitronectin peptides containing a hydrophobic pentapeptide motif within the hemopexin-type repeats were found to bind to streptococci. These data were supported by competition experiments, in which a representative 23-amino acid synthetic vitronectin peptide comprising part of a hemopexin-type repeat inhibited binding of the bacteria to vitronectin, while a control peptide with identical amino acid composition but a scrambled sequence had no effect. Moreover, cells of S. pyogenes were shown to bind to the synthetic peptide as well as to immobilized hemopexin, whose structural homology to the hemopexin-type repeats in the vitronectin molecule has long been underlined. Soluble vitronectin could inhibit streptococcal binding to immobilized hemopexin. These results provide first evidence for a biological role of hemopexin itself and respective repeats in vitronectin in bacterial binding, suggesting that during an infection process these or other hemopexin-type repeat-containing proteins could be potential targets for bacterial attachment and subsequent colonization.

Identification of novel heparin-binding domains of vitronectin.

Vitronectin is a multifunctional serum protein which provides a unique regulatory link between cell adhesion, humoral defense mechanism and the hemostatic system, and the heparin-binding properties of vitronectin are thought to have participated in various functional aspects. In addition to the carboxy-terminal glycosaminoglycan-binding motif, we report on two novel heparin-binding domains which were identified using phage display technique. One heparin-binding domain is located between amino acids Asp82 and Cys137 at the end of the connector region, while the other is in the second hemopexin-type repeat, between amino acids Lys175 and Asp219 of the vitronectin molecule. Our findings may shed new light to the activities of vitronectin and its binding to cells, which could not be explained solely on the basis of the known heparin-binding domain.

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 degrees 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.

Evidence that the heparin-binding consensus sequence of vitronectin is recognized by Staphylococcus aureus.

Binding of heparin-binding form of vitronectin to Staphylococcus aureus was inhibited completely by heparin or by the same form of vitronectin. The binding was inhibited only to about 50% by the non-heparin-binding form of vitronectin, indicating an apparent involvement of the heparin-binding properties in the interaction between vitronectin and S. aureus. This was supported by experiments in which a synthetic peptide (Ala347-Arg361, comprising heparin-binding consensus sequences) was found to partly inhibit bacterial adherence to immobilized vitronectin. A bacterial cell surface protein could bind to the quinquedecapeptide, but not to the highly charged peptides consisting entirely of arginine or lysine, immobilized on microtiter plates and the binding could be competitively inhibited by an excess of soluble peptide. Direct binding of radiolabeled peptide to bacterial cells was also demonstrated, which was rapid, saturable, and pH-dependent. Furtherly a bacterial surface protein having molecular mass of 60 kDa was isolated by affinity chromatography on a quinquedecapeptide-HiTrap-NHS column. Our data suggest that the heparin-binding properties of vitronectin play a role in bacterial recognition.

Multiple interactions between human vitronectin and Staphylococcus aureus.

Multiple interactions between human vitronectin and Staphylococcus aureus strain V8 were observed. An upward-curved Scatchard plot indicated both high-affinity binding (Kd1 = 7.4 x 10(-10) M) with 260 binding sites per bacterial cell and moderate-affinity binding (Kd2 = 7.4 x 10(-8) M) with 5240 copies per cell. Negative cooperativity of this binding was characterized by its Hill coefficient of less than unity (0.70 +/- 0.08). Up to 60% of the vitronectin-bacteria interaction was unaffected by high ionic strength (i.e., 2.4 M NaCl), and was not inhibited by highly-charged heparin oligosaccharides. Various oligosaccharides (4-20 monosaccharide units) generated by partial deaminative cleavage of heparin were found to affect vitronectin binding to S. aureus. Short-chain-length oligosaccharides increase and long oligosaccharides inhibit vitronectin binding, in accordance with direct association of these saccharides with multimeric vitronectin. A protein having a molecular mass of 60 kDa was identified as a putative high-affinity staphylococcal vitronectin-binding protein. These results indicate that interaction of multimeric vitronectin, mostly present at extracellular matrix sites with multiple recognition sites on the S. aureus surface, may contribute to bacterial colonisation.

Binding of collagen, fibronectin, lactoferrin, laminin, vitronectin and heparan sulphate to Staphylococcus aureus strain V8 at various growth phases and under nutrient stress conditions.

We have examined how Staphylococcus aureus strain V8 cells interact with 125I-labelled extracellular matrix (ECM) and serum proteins (collagen type I and IV), fibronectin, lactoferrin, laminin, vitronectin, and heparan sulphate at various phases of the growth cycle. Maximal binding of these glycoproteins and heparan sulphate to the bacteria occurred after 17 to 20 h in the late stationary phase except for fibronectin-binding, which was maximal after 12 to 14 h. Binding of the glycoproteins and heparan sulphate to S. aureus V8 under nutrient stress conditions exhibited complex patterns based on different starving conditions and various binding ligands. In general, bacteria starved in distilled water and 0.02 M potassium phosphate buffer (pH 7.2) at room temperature showed high susceptibility to all binding ligands within the first 18 h, followed by entering a lower binding period (except for collagen-binding which still remained high). The binding was not correlated to cell surface charge or hydrophobicity of the bacteria. Furthermore, extracellular and cell-associated proteolytic activity of starved cells against ECM and serum proteins was found to be greater than for non-starved cells. Thus, S. aureus could sustain its ability to bind various connective tissue and cell surface components during a long period of time even in the absence of energy-yielding substrates.

Vitronectin-binding surface proteins of Staphylococcus aureus.

S. aureus strain ISP 546 was selected (of 55 strains tested) to define optimal conditions for expression of vitronectin binding. High binding was expressed when the strain was grown on blood agar and in Todd-Hewitt broth. Binding was optimal in the 6.0 to 7.2 pH range and was unaffected by divalent cations and ionic strength. Binding was partially inhibited by D-mannose, heparin, types I and IV collagen, fibronectin, fibrinogen and vitronectin, but was not affected by other carbohydrates or glycoproteins tested. Cell surface binding components were extracted with the aid of 1 M LiCl (pH 5.0) from strain ISP 546 grown in Todd Hewitt broth. Vitronectin binding proteins were purified by affinity chromatography on heparin-Sepharose. Fractions inhibiting binding of 125I-labelled vitronectin to strain ISP 546 were eluted by 0.01 M NaOH, dialysed, concentrated and subjected to SDS-PAGE. Silver staining revealed one major band (70 kDa) and two minor bands (34 and 36 kDa).

Binding of heparan sulfate to Staphylococcus aureus.

Heparan sulfate binds to proteins present on the surface of Staphylococcus aureus cells. Binding of 125I-heparan sulfate to S. aureus was time dependent, saturable, and influenced by pH and ionic strength, and cell-bound 125I-heparan sulfate was displaced by unlabelled heparan sulfate or heparin. Other glycosaminoglycans of comparable size (chondroitin sulfate and dermatan sulfate), highly glycosylated glycoprotein (hog gastric mucin), and some anionic polysaccharides (dextran sulfate and RNA) inhibited heparan sulfate binding to various extents. Heat treatment (80 degrees C for 10 min) and treatment of the bacteria with pronase E, proteinase K, pepsin, and chymotrypsin considerably reduced their ability to bind 125I-heparan sulfate, but treatment with trypsin and neuraminidase did not affect binding. Scatchard plot analysis indicated the presence of cell surface components with low affinity (Kd = 3 x 10(-5) M) for heparan sulfate. Cell surface components were released by stirring bacteria with 1 M LiCl at 37 degrees C for 2 h. Proteins of this extract that competitively inhibited binding of 125I-heparan sulfate to S. aureus were isolated by affinity chromatography on heparin-Sepharose. Two proteins having molecular masses of approximately 66 and 60 kDa and the ability to bind 125I-heparan sulfate were obtained. The first 9 amino-terminal amino acid residues of the 66-kDa protein are Asp-Trp-Thr-Gly-Trp-Leu-Ala-Ala-Ala, and the first 4 amino-terminal amino acid residues of the 60-kDa protein are Met-Leu-Val-Thr.