Molecular and functional characterization of bovine C5a receptor

Abstract

Anaphylatoxin C5a is an important chemotactic factor for bovine neutrophils, and is the earliest inflammatory agent formed during bovine mastitis. Bovine neutrophils respond to C5a and its truncated form C5a des arg with similar affinities unlike human neutrophils. Therefore, to test the hypothesis that the bovine C5a receptor structure and signaling differ from that of the human C5a receptor, we cloned and analyzed the bovine C5a receptor. In the present investigation, the bovine C5a receptor encoding cDNA from bovine bone marrow was cloned and the recombinant C5a receptor protein was expressed in CHO-K1 cells. Analysis of predicted C5a receptor amino acid sequence demonstrated 69.1%, 71.3%, 59.4%, 61.6%, and 33.2% identity with that of human, dog, mouse, rat, and trout respectively. The bovine C5a receptor mediated signaling was via pertussis toxin insensitive Gá-protein, and mediated L-selectin shedding on the activated neutrophils. The homologous-, and heterologous desensitization experiments provided further evidence that C5a and C5a des arg could desensitize C5a receptor signaling as well as IL-8 receptor mediated signaling. We further analyzed the expression profiles of the C5a receptor on bovine peripheral blood leukocytes with the receptor-specific antibody and FITC labeled C5a des arg. The monoclonal antibody as well as C5a des arg FITC failed to detect C5a receptor on peripheral blood mononuclear cells. Furthermore, we probed for the expression of C5a receptor gene and protein expression in Mac-T cell line--an immortalized bovine mammary epithelial cell line--, to test the hypothesis that bovine epithelial cells express C5a receptor protein. The results of RT-PCR analysis and FACS analysis for the expression of C5a receptor demonstrated the presence of the C5a receptor in these cells. This investigation led to the elucidation of the structure and function of bovine C5a receptor in neutrophils. The analysis of the C5a receptor expression in peripheral blood leukocytes demonstrated that it was constitutively expressed in neutrophils only. Mac-T, a mammary epithelial cell line expresses C5a receptor. Future studies understanding the function of C5a receptor in these cells will contribute to the knowledge of bovine inflammation.