Molecular Identification And Functional Characterization Of The Scavenger Receptor Cysteine-rich(SRCR) Proteins And Macrophage Migration Inhibitory(MIF)in Red Drum Sciaenops Pcellatus

This article is focused on the gene identification, induced expression pattern and biological function of a scavenger receptor cysteine-rich (SRCR) proteins and MIF in red drum Sciaenops ocellatus.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. Macrophage migration inhibitory factor (MIF) is a multi-functional cytokine involved in immunoregulation and inflammation.We identified and characterized a teleost SRCR protein from red drum Sciaenops ocellatus. The protein was named S. ocellatus SRCR1(SoSRCRPI). SoSRCRPI is410-residue in length and was predicted to be a transmembrane protein. SoSRCRPI expression was detected mainly in immune-relevant tissues and upregulated by bacterial and viral infection. The expressed SoSRCRPI was localized on cell surface. Recombinant SoSRCRPI (rSoSRCRPl) 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 SoSRCRPI, the mutant proteins were created. Compared to rSoSRCRP1, all mutants were significantly reduced in the ability of bacterial interaction. Taken together, these results indicate that SoSRCRPI 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. In the study of MIF, SoMIF, from red drum Sciaenops ocellatus. SoMIF is composed of115residues and shares85%-99%overall sequence identities with the MIF of a number of teleost. SoMIF expression was detected in a wide range of tissues and upregulated by bacterial and viral infection in a time-dependent manner. The expressed SoMIF was secreted into the extracellular milieu. Recombinant SoMIF (rSoMIF) purified from Escherichia coli inhibited the migration of both HK monocytes and lymphocytes, and this inhibitory effect was abolished by the presence of anti-rSoMIF antibodies. When rSoMIF was administered into red drum, it stimulated the production of reactive oxygen species in HK monocytes both in the presence and absence of pathogen infection. In vivo infection study showed that compared to untreated fish, fish pre-treated with rSoMIF before bacterial infection exhibited significantly lower bacterial loads in blood, kidney, spleen, and liver. Taken together, these results indicate that SoMIF is a secreted protein that regulates immune cell trafficking and is involved in pathogen-induced immune response.