Ly-6D of Japanese flounder (Paralichthys olivaceus) functions as a complement regulator and promotes host clearance of pathogen

The Lymphocyte antigen-6 (Ly-6) superfamily has been considered to play an important role in the innate immunity of mammals. The functions of Ly-6 proteins are diverse since their low sequence homology. Currently, the function of Ly-6D, a member of Ly-6 family proteins, is completely unknown in teleost. In the present study, we identified and characterized a Ly-6D homologue (named PoLy-6D) from the teleost fish Paralichthys olivaceus and examined its immune function. PoLy-6D possesses a hydrophobic signal peptide, a LU domain including a conserved "LXCXXC" motif in N-terminus and a "CCXXXXCN" motif in C-terminus. Under normal physiological condition, PoLy-6D expression distributes in all the examined tissues, the highest three tissues are successively spleen, head kidney, and blood. When infected by extracellular and intracellular bacterial pathogens and viral pathogen, PoLy-6D expression was induced and the patterns vary with different types of microbial pathogens infection and different immune tissues. In vitro experiment showed recombinant PoLy-6D (rPoLy-6D) inhibited the lysis of rabbit red blood cells by serum and selectively improved bacterial survival in serum. After serum were treated by antibody of rPoLy-6D, bacteriostatic effect of serum was obviously enhanced. These results indicate the importance of PoLy-6D as a complement regulator. rPoLy-6D possessed the binding activity to multiple bacteria but did not exhibit antimicrobial activities. The interaction between rPoLy-6D and bacteria suggests that PoLy-6D is involved in host clearance of pathogens probably by serving as a receptor for pathogens. Overexpression of PoLy-6D in vivo promoted the host defense against invading E. piscicida. These findings add new insights into the regulation mechanism of the complement system in teleost and emphasize the importance of Ly6D products for the control of pathogen infection.