Arginine Residues of Streptococcus gordonii Hsa Essential for Binding Activity

Objectives: Hsa is a large serine rich protein of Streptococcus gordonii DL1 and mediates binding to α2-3-linked sialic acid termini of platelet glycoprotein Ibα, and erythrocyte membrane protein glycophorin A and band 3. This interaction appears to be mediated by an NR2 region of Hsa. The binding interaction appears to be mediated by an NR2 region of Hsa. However, the molecular details of the interaction between Hsa NR2 region and these glycoproteins are not well understood. In the present study, we analyzed the amino-acid residues of Hsa NR2 region involved in sialic-acid recognition.
Methods: We prepared seven mutants of Hsa NR2 fused with GST, each containing a point mutation of an arginine residue. Using these GST fusion proteins, we performed hemagglutination assay, platelet aggregation assay, and sugar binding assay. Furthermore, we established S. gordonii DL1 derivatives that encoded the corresponding Hsa mutant protein, and determined the ability of these derivatives to adhere to human blood cells.
Results: Two of these mutants R340N and R365N showed significant reduction in hemagglutination, in platelet aggregation, and in adhesion to human leukocytes. Sugar binding assay showed that these mutants completely eliminated the binding of both Neu5Acα(2-3)Galβ(1-3)GalNAc and Neu5Acα(2-3)Galβ(1-4)Glc.
Conclusions: These results indicate that the Arg340 and Arg365 residues of Hsa play an important role in the binding of Hsa to host cell receptors.