Ayu (Plecoglossus altivelis) CD46 isoforms protect the cells from autologous complement attack

CD46 is an important immune regulatory receptor with multiple functions. However, studies on the function of teleost CD46, especially the different CD46 isoforms are limited. In this study, we identified three membrane cofactor protein (MCP, CD46) gene isoforms from ayu (Plecoglossus altivelis) and tentatively named as PaCD46 isoforms. PaCD46 isoforms were generated by alternative splicing and all consisted of four conserved short consensus repeats (SCRs), and the variable serine-threonine-proline-rich domain, transmembrane hydrophobic domain, and cytoplasmic tail. Phylogenetic analysis showed that the isoforms clustered together with other fish CD46 and then with higher animal CD46. Western blotting analysis of peripheral blood mononuclear cells (PBMC) revealed three bands, all of which had much larger molecular weights than the theoretical values of the three PaCD46 isoforms. Moreover, three PaCD46 isoforms were individually expressed on HEK293 cells, and Western blotting showed the similar band profile to that of PBMC. The recombinant extracellular domain of the PaCD46 isoforms, obtained by expression in Pichia pastoris, significantly reduced hemolysis activity of ayu sera. Furthermore, each of the three PaCD46 isoforms respectively protected the HEK293 cells expressing the isoform. The isoforms were also identified for their protection of autologous PBMC from complement activation. These results provided the first evidence that PaCD46 isoforms may be complement regulatory proteins to prevent complement-induced damage to self-tissue.