Identification and characterization of a cell surface scavenger receptor cysteine-rich protein of Sciaenops ocellatus: Bacterial interaction and its dependence on the conserved structural features of the SRCR domain

Abstract

The scavenger receptor cysteine-rich (SRCR) proteins are secreted or membrane-bound receptors with one or multiple SRCR domains. Members of the SRCR superfamily are known to have diverse functions that include pathogen recognition and immunoregulation. In teleost, although protein sequences with SRCR structure have been identified in some species, very little functional investigation has been carried out. In this study, we identified and characterized a teleost SRCR protein from red drum Sciaenops ocellatus. The protein was named S. ocellatus SRCR1 (SoSRCRP1). SoSRCRP1 is 410-residue in length and was predicted to be a transmembrane protein, with the extracellular region containing a collagen triple helix repeat and a SRCR domain. The SRCR domain has six conserved cysteines, of which, C338 and C399, C351 and C409, and C379 and C389 were predicted to form three disulfide bonds. SoSRCRP1 expression was detected mainly in immune-relevant tissues and upregulated by bacterial and viral infection. In head kidney leukocytes, bacterial infection stimulated the expression of SoSRCRP1, and the expressed SoSRCRP1 was localized on cell surface. Recombinant SoSRCRP1 (rSoSRCRP1) corresponding to the SRCR domain was purified from Escherichia coli and found to be able to bind Gram-negative and Gram-positive bacteria. To examine the structure–function relationship of SoSRCRP1, the mutant proteins SoSRCRP1M1, SoSRCRP1M2, SoSRCRP1M3, and SoSRCRP1M4 were created, which bear C351S and C409S, C338S, C379S, and R325A mutations respectively. Compared to rSoSRCRP1, all mutants were significantly reduced in the ability of bacterial interaction, with the highest reduction observed with SoSRCRP1M4. Taken together, these results indicate that SoSRCRP1 is a cell surface-localized SRCR protein that binds bacterial ligands in a manner that depends on the conserved structural features of the SRCR domain.