Molecular Cloning, Recombinant Expression And Functional Analysis Of A Novel Scavenger Receptor From Zhikong Scallop

Zhikong scallop Chlamys farreri is one of the most important aquaculture species in China. Scallop aquaculture has been experiencing a continual large-scale mortality, which threatens the sustainable development of the culture industry. A better understanding of defence mechanisms in scallop can help us to develop appropriate approaches for disease control, and also can provide evidence to confirm the origin and evolution of innate immunity.Scavenger receptors (SRs), encompassing diverse structural membrane proteins, maintain the stabilization of homeostasis through binding and mediating the engulfment of various non-self molecules. Among them, SRs containing scavenger receptor cysteine-rich (SRCR) domain are found almost exclusively in mammals and birds, and play important roles in host defense. In present study, a novel scavenger receptor (CfSR) was cloned and characterized from Zhikong scallop, C. farreri by using EST and RACE technology. The full-length cDNA sequence of CfSR contained a 5'terminal un-translated region (UTR) of 54 bp, 3'UTR of 169 bp with a canonical polyadenylation signal sequence AATAAA and a poly (A) tail, and an open reading frame (ORF) of 2415 bp encoding a polypeptide of 804 amino acids with a predicted molecular weight of 85.63 kDa and theoretical isoelectric point of 4.61. There was one potential O-glycosylation site (Ser/Thr), six N-glycosylation sites, a signal peptide-like hydrophobic N-terminal segment, six repeats SRCR domain (residues 27-665), an UPAR_LY6 domain (residues 671-751) and a ShK toxin domain (residues 768-804) in CfSR. All of six SRCR domains belong to classical group A domain, which containing the highly conserved six cysteine residues (Cys1-6) to form three pairs of intradomain disulfide, and the three-dimensional structure prediction suggested the high probability that the SRCR-D5 would have ligands-binding activity. There was a predicted attachment site of the sequence CTTPLCN in UPAR_LY6 domain. Besides, CfSR was detected on the outer surface of haemocytes by immunofluorescence, indicating that CfSR was anchored on the outer-membrane of cells.The mRNA transcript of CfSR in vivo was up-regulated quickly and significantly by the stimulations of lipopolysaccharide (LPS), peptidoglycan (PGN) and glucan. After treatment with LPS, the mRNA expression of CfSR increased gradually and reached the highest point at 6 h post-injection (4.15-fold compared with the control group), and then decreased gradually. Similar expression pattern of CfSR transcript was also observed in the PGN stimulated group, and its mRNA level reached maximal level (5.91-fold compared with the control group) at 12 h, and then dropped to the original level at 48 h post injection. After glucan injection, the maximal level of CfSR mRNA expression was sharply reached at 3 h (13.74-fold increased compared with the control group), and then dramatically dropped back to normal level at 6 h. After that, there was another slight ascension from 6 h to 24 h for glucan stimulation. CfSR was sensitive to the stimulation of three types of PAMPs, indicating that CfSR may play a role in the immune response against different microbes.To confirm this hypothesis, CfSR was recombined and its binding ability was detected using ELISA technique. The truncated recombined CfSR including SRCR-D5 (rtCfSR, from V456 to T804) displayed a significantly strong activity to bind acetylated LDL and dextran sulfate. This characteristic was accordant with the basic definition of SR, suggesting that CfSR was a true SR molecule. It was also detected that rtCfSR displayed a significantly strong activity to bind LPS, PGN, zymosan and mannan, but it did not bind glucan. Wherein, rtCfSR bound LPS in a Ca²⁺-depending manner. It was seldom reported that SR could recognize the cell components of fungi before, and the binding to zymosan and mannan of CfSR suggested that SR molecule of lower marine invetebrate probably had a broader binding ligand spetrum. In conclusion, CfSR is a novel SR anchored on the outer-membrane of cells. It is the first SRCR domain-containing SR identified in invertebrates. CfSR can bind not only acetylated LDL and dextran sulfate but also LPS, PGN, zymosan and mannan directly, so CfSR is a versatile PRR involved in immune response to bacterial and fungi invasion.