Identification Of Complement-related Molecules CgMASPL-2 And CggC1qR And Its Immune Function In Crassostrea Gigas

The complement system is an important component of innate immunity,which plays an important role in the identification and elimination of pathogens and other immune responses.Although the complement molecules such as mannose-bindinglectin serine protease（MASP）and complement Receptor of globular heads of C1q（gC1q R）have been found in invertebrates,there is lack of in-depth study on its role in the complement system.In the present study,CgMASPL-2 and CggC1qR were cloned and identified from Crassostrea gigas,and their functions in the anti-pathogenic infection were further analyzed.The results were as follows:The open readingframe of CgMASPL-2 is 2757 bp,encoding918 amino acids,which contains three（Domain first found in C1r,C1s,u EGF,and bone morphogenetic protein.CUB）domains,one（Epidermal growth factor-like domain,EGF）domain,one（Immunoglobulin,IG）domain,one（Immunoglobulin C-2 Type,IGc2）domain,and one（Trypsin-like serine protease,Tryp-SPC）domain.The results of domain alignment showed that the domain composition of CgMASPL-2 was different from those of MASPs from Homo sapiens,Mus musculus,Xenopus laevis,Branchiostoma belcheri,Lampetra japonicum,Ciona intestinalis and Exaiptasia diaphana,but the same as those of MASPL from Littorina littorea and Mytilus californianus.The similarity between CgMASPL-2 and other MASPs in H.sapiens,M.musculus,B.belcheri,X.laevis,C.intestinalis,E.diaphana,L.japonicum,L.littorea and M.californianus was 26%-98%.Phylogenetic tree analysis showed that CgMASPL-2 was grouped with one branch with M.californianus MCASP-2-like.The evolutionary relationships of CgMASPL-2 and L.littorea Ll MRe M1 were closer with those from vertebrates.The open readingframe of CggC1qR is 771 bp,encoding256 amino acids containingone（This mitochondrial matrix protein family contains members of the MAM33 family,MAM33）domain.The similarity between CggC1qR and other gC1q Rs from H.sapiens,Danio rerio,M.musculus,Drosophila melanogaster,Penaeus chinensis,P.monodon,P.leniusculus,C.virginica and Musca domestica was 75%-100%.Phylogenetic tree analysis showed that CggC1qR was firstly clustered with C.virginica CvgC1q R and then fell into the cluster of invertebrates.The tissue distribution and expression pattern of CgMASPL-2 and CggC1qR in haemocytes after Vibrio splendidus stimulation were examined by q RT-PCR.The m RNA transcripts of CgMASPL-2 were expressed in mantle,labial palp,gonad,hepatopancreas,gills,adductor muscle and haemolymph,with the highest expression in hemolymph,followed by adductor muscle.The m RNA expressions of CgMASPL-2in haemocytes increased significantly at 48 h,72 h and 96 h after the stimulation with V.splendidus.The m RNA transcripts of CggC1qR were expressed in mantle,labial palp,gonad,hepatopancreas,gills,adductor muscle and haemolymph,with the highest expression in gonad,followed by adductor muscle.There were no significant difference about the m RNA expressions of CggC1qR in haemocytes after V.splendidus stimulation.The recombinant protein of the 3×CUB-EGF domain of CgMASPL-2 had thebindingactivity to microbes and pathogen associated molecular patterns（PAMPs）.The recombinant protein of the 3×CUB-EGF domain of CgMASPL-2 could bind Gram-positive bacteria（Staphylococcus aureus and Micrococcus luteus）,Gram-negative bacteria（Escherichia coli,Vibrio anguillarum and V.splendidus）and fungi（Pichia pastoris）and exhibited relatively higher bindingactivity to V.anguillarum,S.luteus,P.pastoris,and V.splendidus.r CgMASPL-2-3×CUB-EGF could also bind _CggC1q^lipopoly _R^sa _had^ccha _bind^{ride（LPS）}_{ingact i vity}^{and pep}_{to S}.^t _aureus^idoglyc,_V^an.^（PGN）_anguill._{a rum}^The,_{Bacillus sub}^{recombinant prot}_{tilis and}^ein _V^of.splendidus.Besides,r CggC1qR could inhibit the growth of S.aureus,E.coli and V.anguillarum in vitro.In anti-CgMASPL-2 treated oysters,the m RNA expression levels of CgIL17-1（0.05-fold that in the negative serum group,respectively,p<0.001）and CgIL17-2（0.01-fold that in the negative serum group,respectively,p<0.05）in haemocytes were significantly decreased after V.splendidus stimulation.In r CggC1qR-treated oysters,The m RNA expression levels of CgIL17-1（37.77-fold that in the negative serum group,respectively,p<0.05）,CgIL17-5（6.25-fold that in the negative serum group,respectively,p<0.05）and CgIL17-6（4.24-fold that in the negative serum group,respectively,p<0.05）in haemocytes were all significantly increased after V.splendidus stimulation.In anti-CgMASPL-2 treated oysters,the m RNA expression levels of CgATG-5（0.31-fold that in the negative serum group,respectively,p<0.05）and CgLC3（0.14-fold that in the negative serum group,respectively,p<0.0001）in haemocytes were significantly decreased after V.splendidus stimulation.The above results indicated that CgMASPL-2 and CggC1qR had the function of recognizingmicrobes and PAMPs.CgMASPL-2 could regulate the m RNA expressions of CgIL17-1,CgIL17-2,CgATG-5 and CgLC3 after V.splendidus stimulation.CggC1qR could regulate the m RNA expressions of CgIL17-1,CgIL17-5 and CgIL17-6 after V.splendidus stimulation.This study revealed that CgMASPL-2 and CggC1qR were involved in the immune response process induced by pathogens,which provided an important theoretical support for exploringthe immunologic function of the complement system.