| Complement system is one of the effective immune effector systems inmulticellular organisms, and plays significant roles in innate immune defense.Complement related molecules exist in several categories of invertebrates, andparticipate in the host’s immune defense. However, the study about complementrelated molecules was still limited in mollusc. In the present study, through molecularbiological, bioinformatics and immunological techniques, the structural and functionalstudy of complement related molecules in Zhikong scallop Chlamys farreri and bayscallop Argopecten irradians, including C1q-domain-containing proteins(CfC1qDC-1, CfC1qDC-2and AiC1qDC-2), a thioester-containing protein (CfTEP),a C-type lectin (AiCTL-9) and a fibrinogen-related protein (CfFREP), wereperformed to investigate their function in scallop immune system.The full-length cDNA sequence of AiC1qDC-2was cloned in bay scallop byRACE approach. Its open reading frame encoded a peptide with one gC1q domain,which adopted a typical10-stranded sandwich fold with a jelly-roll topology andcontained characteristic amino acid residues of C1qDC protein family. By RT-PCR,AiC1qDC-2mRNA was mainly detected in hepatopancreas and gonad, and itsexpression level was significantly up-regulated in hemocytes when scallops werestimulated by different microorganisms. The recombinant AiC1qDC-2protein couldbind kinds of PAMPs, such as LPS, PGN, Yeast-glucan and polyI:C, and aggregatemicroorganisms including Escherichia coli, Vibro anguillarum, Bacillus subtilis andPichia Pastoris.The CfC1qDC-1mRNA expression level in hemocytes was significantlyup-regulated when Zhikong scallops were stimulated by LPS, PGN or β-glucan, andduring the embryonic development of scallop, its expression level was up-regulated from D-hinged larvae and reached the highest at eye-spot larvae. The endogenousCfC1qDC was dominantly located in the hepatopancreas, gill, kidney and gonaddetected by immunofluorescence. The recombinant protein of CfC1qDC-1could bindvarious PAMPs including LPS, PGN, β-glucan as well as polyI:C, and interact withhuman heat-aggregated IgG which could be inhibited by LPS. Furthermore,rCfC1qDC-1could enhance the phagocytic activity of scallop hemocytes towardsE.coli. Tertiary structures analysis showed that the spatial location of the cationicamino acid residues in gC1q of CfC1qDC-1was similar to that in ghB of humancomplement C1q.The full-length cDNA sequence of CfC1qDC-2was cloned from Zhikong scallopusing RACE technique. There were three gC1q domains in CfC1qDC-2, whichformed a tightly packed bell-shaped trimer as that in human complement C1q. ThemRNA transcripts of CfC1qDC-2were mainly detected in the tissues ofhepatopancreas and gonad, and the expression level was up-regulated from D-hingedlarvae and reached the highest at eye-spot larvae during the embryonic developmentof scallop. Moreover, the mRNA expression level of CfC1qDC-2in hemocytes wassignificantly up-regulated after scallops stimulated by LPS, PGN or β-glucan. Therecombinant protein of CfC1qDC-2could bind a wide variety of ligands, inludingPAMPs, microorganisms, scallop apoptotic cells and even human heat-aggregated IgGand IgM. Additionally, rCfC1qDC-2could inhibit the C1q-dependent hemolysis ofrabbit serum.The full-length cDNA sequence of AiCTL-9was cloned from bay scallop, andits amino acid sequence contained four CTLD domains and possessed the typicalstructures and characteristic motifs of C-type lectin family. The mRNA transcripts ofAiCTL-9were mainly detected in the tissues of hepatopancreas and gonad, and itsmRNA expression level in hemocytes was significantly up-regulated when thescallops were stimulated by different microorganisms. The recombinant protein ofAiCTL-9could bind kinds of PAMPs, such as LPS, β-glucan and MAN, aggregatedifferent microorganisms, such as E. coli, V. angulillarum, B. subtili and Micrococcusbuteus, and even significantly enhanced the encapsulation of scallop hemocytes. The full-length cDNA sequence of CfFREP was identified in zhikong scallopthrough homology-based cloning, and its amino acid sequence contained one FBGdomain, which have the typical structure and characteristic motifs of FREP family.The mRNA expression level of CfFREP in hemocytes was significantly up-regulatedwhen the scallops were stimulated by LPS or PGN. The endogenous CfFREP wasdominantly located in the hepatopancreas, gill, kidney and periphery of hemocytesdetected by immunofluorescence. The recombinant protein of CfFREP could bindLPS, PGN, β-glucan and polyI:C, but not aggregate any tested microorganisms. ThemRNA expression level of CfTEP in hemocytes was significantly up-regulated whenthe scallops were stimulated by LPS, PGN or β-glucan, and its expression level wasup-regulated from D-hinged larvae and reached the highest at eye-spot larvae duringthe embryonic development of scallop.The endogenous CfTEP was dominantly located in the hepatopancreas, gill,kidney, gonad and mantle of audlt scallop, and D-hinged, veliger, and eye-spot larvae.Western blotting displayed that the CfTEP protein existed as a form of multi-fragmentin scallop serum, and when TEP proteins were inactivated by injection ofmethylamine in scallop, the survival rate of scallop was significantly deduced afterthe infection of V. anguillarum.The studies about these six scallop complement related molecules indicated thatthey were similar to vertebrate complement components in both structure and function.They possessed charactirstic domains and conserved motifs of the vertebrate C1q, C3,ficolin or MBL. And they functioned as PRRs to participate in immune recognitionand as immune effectors related in the clearance of pathogens, meanwhile，couldinteract with vertebrate immunoglobulins and scallop apoptotic cells. Therefore, theymight function as the components of the ancient complment system in scallopparticipating in the innate immune defense. Meanwhile, it provided new informationfor understanding the evolution of component related molecules and the enrichmentand development of marine invertebrate immunology. |
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