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).
Division of Biochemistry, Indian Veterinary Research Institute, Izatnagar 243122, Uttar Pradesh, India.
Rao T
RT
Prasada
Division of Biochemistry, Indian Veterinary Research Institute, Izatnagar 243122, Uttar Pradesh, India.
Prasanth T
PT
Lakshmi
Division of Biochemistry, Indian Veterinary Research Institute, Izatnagar 243122, Uttar Pradesh, India.
R
R
Parvathy
Division of Biochemistry, Indian Veterinary Research Institute, Izatnagar 243122, Uttar Pradesh, India.
S
S
Murugavel
Division of Biochemistry, Indian Veterinary Research Institute, Izatnagar 243122, Uttar Pradesh, India.
Devi
D
Karuna
Division of Biochemistry, Indian Veterinary Research Institute, Izatnagar 243122, Uttar Pradesh, India.
Joshi
J
Paritosh
D020816:Amino Acid Motifs; D000818:Animals; D001665:Binding Sites; D001770:Blood Bactericidal Activity; D003186:Complement C9 / Q000276:immunology / Q000378:metabolism*; D004252:DNA Mutational Analysis; D004926:Escherichia coli / Q000276:immunology; D006041:Goats; D007155:Immunologic Factors / Q000276:immunology / Q000378:metabolism*; D016297:Mutagenesis, Site-Directed; D011485:Protein Binding; D019096:Vitronectin / Q000235:genetics / Q000276:immunology / Q000378:metabolism*
D003186:Complement C9; D007155:Immunologic Factors; D019096:Vitronectin
arginine glycine asparticacid motif; complement C9; site-directed mutagenesis; vitronectin