ISOLATION AND CHARACTERIZATION OF A VITRONECTIN-BINDING SURFACE PROTEIN FROM STAPHYLOCOCCUS-AUREUS

In a previous study we demonstrated that cells of Staphylococcus aureu s strain V8 bind I-125-labelled vitronectin in a receptor-ligand type of interaction, and a protein having a molecular mass of 60 kDa was id entified as a putative high-affinity staphylococcal vitronectin-bindin g protein (Liang, O.D. et al. (1993) Biochim. Biophys. Acta 1225, 57-6 3). In the present communication we report on the isolation and prelim inary characterisation of the 60 kDa vitronectin-binding protein. The bacterial cell surface proteins were released by stirring bacteria wit h 1 M LiCl at 37 degrees C for 2 h and separated on an FPLC Mono-Q col umn with a gradient of 0-0.5 M NaCl in 20 mM Tris buffer at pH 9.0. Fr actions containing vitronectin-binding activity, assayed on microtiter plates with immobilised human vitronectin, were collected and SDS-PAG E analysis showed the content to be a single protein band at the 60 kD a position. In Western blot experiments the protein transblotted onto nitrocellulose membranes could bind soluble vitronectin. Its amino-ter minal 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), suggest ing that they are identical molecules. This was supported by ligand bl otting experiments where both vitronectin and heparan sulfate were sho wn to bind to the same protein band in parallel strips.