Preliminary Study On The Phagocytosis And Migration Of Hemocytes Induced By CgC1qDC-6 In Pacific Oyster Crassostrea Gigas

C1q domain containing proteins(C1qDCs)are an ancient family of molecules that are distributed from bacteria to mammals and have formed a variety of functions in long-term evolution.The C1 qDCs refer to all proteins that contain a globular head C1 q domain(gC1q)in the C-terminus.Depending on the absence or presence of the collagen-like domain,C1 qDCs are classified as ghC1 q proteins or C1q-like proteins,respectively.ghC1 q proteins are widely present in invertebrates and vertebrates.C1 qlike proteins are mainly distributed in the chordate,a few are also found in mollusks,annelids and echinoderms.However,there is still lack of knowledge on the function of these C1q-like proteins.Previous studies had annotated 337 C1 qDC genes by analyzing the genome and transcriptome of Pacific oyster(Crassostrea gigas),and only found one C1q-like gene.In the present study,a C1 qDC with a collagen-like domain(CgC1qDC-6)was identified from C.gigas with the objectives to(1)cognize its sequence feature and expression profile at both mRNA and protein levels;(2)determine its binding activity towards various pathogen-associated molecular patterns(PAMPs)and microbes;(3)examine its activity to promote hemocyte phagocytosis and its chemotaxis activity on hemocytes.The coding sequence of CgC1qDC-6 was of 756 bp,encoding a peptide of 251 amino acids with an N-terminal signal peptide,a central collagen-like domain,and a Cterminal ghC1 q domain.CgC1qDC-6 shared high similarities from 53% to 86% with other C1q-like proteins in invertebrates,and low similarities from 32% to 37% with C1q-like proteins in vertebrates.CgC1qDC-6 was clustered with the C1 qDCs from several mollusks in the protostomia branch of the phylogenetic tree.In the present study,the tissue distribution characteristics of CgC1qDC-6 were detected from mRNA levels and protein levels,respectively.The above results showed that CgC1qDC-6 was constitutively expressed in C.gigas.Analysis of qRT-PCR results showed that the transcript of CgC1qDC-6 was expressed in all tissues tested,and the highest expression in hemocytes was 6.8-fold of that in hepatopancreas(p < 0.05).CgC1qDC-6 protein was detected by Western blot,and positive signal bands were observed in the tissues examined.The recombinant CgC1qDC-6 protein(rCgC1qDC-6)exhibited binding activity to various PAMPs including LPS,PGN,Man and Poly I:C.Microorganisms including Gram-negative bacteria(Escherichia coli and Vibrio splendidus),Gram-positive bacteria(Micrococcus luteus and Staphylococcus aureus),and fungus(Pichia pastoris).The phagocytic rates of oyster hemocytes towards V.splendidus were significantly enhanced(p < 0.05)after V.splendidus was pre-incubated with rCgC1qDC-6.The above results indicated that CgC1qDC-6 might recognize multiple pathogenic microorganisms as a pattern recognition receptor(PRR)in the innate immune response of oysters and had opsonization on hemocytes.In the chemotaxis assay,rCgC1qDC-6 could mediate the migration of oyster hemocytes in a dose-dependent manner,which exhibited a positive chemotactic effect at low concentration(< 10 nM).However,as the concentration of rCgC1qDC-6 protein increased,the chemotactic activity of oyster hemocytes was lost.Through the observation of paraffin sections,it was found that 3 hours after injection of the V.splendidus,hemocyte infiltration was occurred in the adductor muscle,it was similar to lymphocyte infiltration in mammals.Western blot assay was showed that the concentration of CgC1qDC-6 protein in untreated oysters was significantly higher than 10 nM(p < 0.05).After 3 hours of intramuscular injection with V.splendidus,the protein concentration of CgC1qDC-6 was significantly decreased(p < 0.05).The above results suggested that CgC1qDC-6 might mediate the hemocyte migration into the infected tissue,and its regulation pattern might be negatively regulated.In summary,CgC1qDC-6 belongs to C1q-like type and is constitutively expressed in different tissues of C.gigas.CgC1qDC-6 might serve as a pattern recognition receptor and mediate the hemocyte phagocytosis and migration to eliminate the invading pathogens.