CgGalectin-9 Is Involved In Regulating Haemocyte Autophagy Induced By Vibrio Splendidus In Pacific Oyster Crassostrea Gigas

The recognition of PAMPs by PRRs triggers the first induced defence against invasive pathogens.Galectin,a lectin with high affinity forβ-galactosides,is an important PRR,occupying an important position in the innate immune system.The Pacific oyster Crassostrea gigas is a mollusk that inhabits the intertidal zone,due to its special living environment,it has evolved a complex innate immune system,which can recognize and eliminate various invasive microbes through a series of complex and delicate immune responses.In the present study,a novel tandem-repeat galectin（CgGal-9）was characterized from C.gigas by using the technical means of bioinformatics,molecular biology,cell biology,and immunology to understand its molecular and expression characteristics,the function of binding and agglutinating microbes,and its regulation on autophagy of haemocytes.The main results are as follows.1.Molecular characteristics and expression characteristics of CgGal-9 in C.gigasThe c DNA sequence of CgGal-9 gene was cloned from C.gigas.The open reading frame of CgGal-9 was 981 bp,and encoded a polypeptide composed of 326 amino acids.The theoretical molecular weight of CgGal-9 was 36.12 k Da and the isoelectric point was 8.77.CgGal-9 contained two carbohydrate recognition domains（CRDs）and two highly conservedβ-galactosid-binding motifs（Hx NPR and WGx EE）within CRDs connected by 50 amino acid residues in the CRDs.Multiple sequence alignment revealed that CgGal-9 was similar to Galectin-9s in different species,both of which had two conserved CRDs,belonging to tandem repeating galactolectins.In the phylogenetic tree,Galectin-9s in vertebrates and invertebrates were divided into two distinct clades,CgGal-9 was first clustered with Ca Gal-9 from Crassostrea angulate and then grouped with invertebrate galectin-9s.Transcriptome analysis showed that the expression level of CgGal-9 increased from the early stage of type D larvae（ED1）and reached the highest level in the juvenile stage.The expression level of CgGal-9 in hemolymph of C.gigas was higher than that of other tested tissues,and the expression level in granulocytes was higher than that in agranulocytes and semigranulocytes.The m RNA expression level of CgGal-9 was significantly increased after stimulation by LPS or Vibrio splendidus,and CgGal-9 protein was mainly distributed in the cytoplasm of haemocytes with a small amount in the cell membrane of some granulocytes.2.Recognition and agglutination function of CgGal-9 to pathogenic microbesCgGal-9 could co-locate with V.splendidus in haemocytes of C.gigas after co-incubation with the FITC-labeled V.splendidus.The recombinant protein of CgGal-9（r CgGal-9）has the activity of binding lipopolysaccharide（LPS）,peptidoglycans（PGN）and mannose（MAN）,among which the binding activity to MAN is strong,and the binding activity of r CgGal-9 to the above PAMPs is concentration-dependent.The r CgGal-9 showed broad-spectrum binding activity against Gram-negative bacteria（V.splendidus,Escherichia coli）,Gram-positive bacteria（Micrococcus luteus,Staphylococcus aureus,Bacillus subtilis）and fungi（Pichia pastoris）in the absence of Ca²⁺,and showed agglutination activity to V.splendidus,E.coli,M.luteus,S.aureus and P.pastoris in the presence of Ca²⁺,among them,it has strong binding and agglutination activity to P.pastoris.3.CgGal-9 is involved in the regulation of haemocytes autophagy induced by V.splendidusAfter CgGal-9 was blocked by Anti-CgGal-9 antibody,the autophagy levels in haemocytes were detected by flow cytometry after V.splendidus stimulated for 3 h.The results showed that compared with negative serum control group,the autophagy levels of haemocytes were significantly decreased in CgGal-9 antibody-blocking group after V.splendidus stimulation for 3h,which was 0.76-fold（p<0.05）of that in the negative serum control group.After CgGal-9 was blocked by Anti-CgGal-9 antibody,the m RNA expression levels of Cg Beclin1,Cg ATG5,Cg ATG16,Cg P62 and Cg LC3 were significantly lower than that in the control group,and the m RNA expression level of Cgm TOR was significantly higher than that in the control group.After si RNA was used to interfere the expression of CgGal-9,the m RNA expression levels of Cg Beclin1,Cg ATG5,Cg P62,and Cg LC3 were significantly lower than the negative control group（NC）,and Cgm TOR was significantly higher than negative control group.In summary,a new tandem-repeat galectin（designated CgGal-9）was identified from C.gigas,and its expression level was high in granulocytes.CgGal-9 showed a broad-spectrum of binding activity against a variety of PAMPs and microbes,and has agglutination activity to a variety of microbes,among which the binding and agglutination activity to P.pastoris was strong.CgGal-9was involved in regulating the expression of autophagy-related genes in haemocytes of C.gigas.The results provide a good reference for further exploring the structure and function of shellfish lectin.