Identification of second arginine‐glycine‐aspartic acid motif of ovine vitronectin as the complement C9 binding site and its implication in bacterial infection

Vitronectin (Vn), a multifunctional protein of blood and extracellular matrix, interacts with complement C9. This interaction may modulate innate immunity. Details of Vn–C9 interactions are limited. Vn–C9 interactions were assessed by employing a goat homologous system and observing Vn binding to C9 in three different assays. Using recombinant fragments, C9 binding was mapped to the N‐terminus of Vn. Site directed mutagenesis was performed to alter the second arginine glycine aspartic acid (RGD) sequence (RGD‐2) of Vn. Changing R to G or D to A in RGD‐2 caused significant decrease in Vn binding to C9 whereas changing of R to G in the first RGD motif (RGD‐1) had no effect on Vn binding to C9. These results imply that the RGD‐2 of goat Vn is involved in C9 binding. In a competitive binding assay, the presence of soluble RGD peptide inhibited Vn binding to C9 whereas heparin had no effect. Vn binding to C9 was also evaluated in terms of bacterial pathogenesis. Serum dependent inhibition of Escherichia coli growth was significantly reverted when Vn or its N‐fragment were included in the assay. The C‐fragment, which did not support C9 binding, also partly nullified serum‐dependent inhibition of bacterial growth, probably through other serum component(s).