title: Preparation of a sialic acid-binding protein from Streptococcus mitis KS32AR.
abstract: A recent report has identified a lectin on the surfaces of several strains of Streptococcus mitis and Streptococcus sanguis with specificity for an N-acetylneuraminic acid alpha 2,3-galactose-beta 1,3-N-acetylgalactosamine sequence (P.A. Murray, M.J. Levine, L.A. Tabak, and M.S. Reddy, Biochem. Biophys. Res. Commun. 106:390-396, 1982). In the present study, purification and characterization of this sialic acid-binding protein (SABP) was begun. A clinical isolate of S. mitis was grown to mid stationary phase in synthetic FMC medium and then extracted with lithium 3,5-diiodosalicylate. Lyophilized extract was subjected to gel filtration on a Sephadex G-200 column, giving four protein peaks (A to D). Peak B, shown by hemagglutination assay to contain SABP, was next subjected to affinity chromatography on a Sepharose-4B matrix coupled to fetuin glycopeptides. After an extensive washing, peak B materials bound to the affinity matrix were eluted with buffered N-acetylneuraminic acid. Sodium dodecyl sulfate-polyacrylamide gel electrophoresis with 2-mercaptoethanol on 7.5% gels of affinity-purified materials revealed components of 96, 70, and 65 kilodaltons (kDa). Without reducing agent, only the 65-kDa band and materials which did not penetrate the gel were visualized, suggesting that the 96- and 70-kDa components were disulfide linked. The chemical cross-linking agent, disuccinimidyl suberate, was used to demonstrate specific interactions between the SABP preparation and [14C]fetuin glycopeptides. After cross-linking, sodium dodecyl sulfate-polyacrylamide gel electrophoresis and fluorography revealed the 96- and 70-kDa components, indicating that the SABP is at least bivalent. These findings support our previous suggestion that human salivary glycoproteins facilitate clearance of selected oral streptococci via specific interactions between sialic acid-containing oligosaccharides and a carbohydrate-binding protein on the bacterial cell surface.
mesh_terms: D000268:Adhesiveness; D002250:Carbon Radioisotopes; D002850:Chromatography, Gel; D004591:Electrophoresis, Polyacrylamide Gel; D006020:Glycopeptides / Q000378:metabolism; D037102:Lectins / Q000032:analysis* / Q000302:isolation & purification; D009439:Neuraminidase / Q000032:analysis; D011487:Protein Conformation; D012463:Saliva / Q000382:microbiology; D063265:Sialic Acid Binding Immunoglobulin-like Lectins; D013291:Streptococcus / Q000032:analysis* / Q000254:growth & development
chemical_list: D002250:Carbon Radioisotopes; D006020:Glycopeptides; D037102:Lectins; D063265:Sialic Acid Binding Immunoglobulin-like Lectins; D009439:Neuraminidase