The Structural And Functional Study Of The Immunoglobulin Superfamily Members In Crassostrea Gigas

Immunoglobulin superfamily(IgSF) is a multiple collection of proteins containing one or more immunoglobulin(Ig) domains, which performs a wide range of functions in various fundamental processes, such as immune response, neural cell development, anti-tumoral action, and simply in cell adhesion. The vertebrate possessed adaptive immunity based on immunoglobulin. There is no immunoglobulin found in invertebrate, but the IgSF members in invertebrate can potentially generate tens of thousands of protein isoforms, suggesting their function as an alternative adaptive immunity mechanism. Although alternative adaptive immunity of oysters was evidenced in previous reports, the knowledge of structure and function of invertebrate IgSF is still limited. In the present study, the structure characteristics, expression profile and phylogeny relationship of IgSFs, as well as their roles in alternative adaptive immune response and developmental stages in oysters were investigated using bioinformatics methods. Several members of IgSF(CgJAM-A-L, CgSiglec-1 and CgNCAM) were further characterized in the immune response of oyster using immunological methods.A total of 268 IgSF members containing 1-36 Ig domains were identified from the oyster genome by domain analysis, and 162 members were composed by adjacent Ig domains while the others were mixer with multiple kinds of domain. The Gene Ontology annotation implies that the primary functions of IgSF in oyster were recognition and adhesion under the circumstance that 217 members were predicted with a recognition and binding role. A functional cluster based hidden Markov model algorithm was employed to classify the Ig SF and to further predict genes miss-annotated in Gene Ontology. Additionally, IgSF was characterized of a phase-wisely expansive pattern at the evolutionary status of sponges, echinoderm and hemichordate.The IgSF members play important roles in the immune response of oyster. The expression level of 116 and 166 members were significantly up-regulated post the PAMPs and microorganisms challenge, respectively. A functional divergence was supposed during the evolution of oyster IgSF, since plenty of members displayed highly specify to various challenge. Meanwhile more varied functional roles were found within the members containing less Ig domains. Several members can undergo alternative splicing and single nucleotide mutation, potentially generating tens of thousands of protein isoforms after the challenge of Vibrio splendidus. Thereinto, abundant specific single nucleotide mutation was verified upon the secondary challenge of V. splendidus, suggesting a potential mechanism of the alternative adaptive immunity. Additionally, the IgSF members were up-regulated in specific developmental stages, suggesting they may also function in the development and maternal immunity of oyster.CgJAM-A-L, containing three I-set Ig domains, was identified as an evolutionary primitive structural homolog of JAM-A from oyster,endowed with the binding and recognizing capability to a variety of PAMPs(LPS、PGN、MAN、LTA、poly(I:C) and GLU) and microorganisms(Micrococcus luteus, Staphylococcus aureus, Escherichia coli, V. anguillarum, V. splendidus, Pastoris pastoris and Yarrowia lipolytica) as well as opsonic function. It is involuntarily to suspect that there would be a lineage-specific evolution pattern from one comprehensive and versatile precursor to several specific and efficient ramified molecules of JAMs and immunoglobulins. CgSiglec-1, containing two I-set Ig domains and cytosolic immunoreceptor tyrosine-based inhibitory motifs(ITIMs), could act as a bridge molecule between invader recognition and immune response activation. It displayed a ubiquitous expression pattern in all the selected tissues and was up-regulated by the challenge of V. splendidus in hemocytes. Purified rCgSiglec-1 exhibited a wide binding spectrum to PAMPs(pSIAS, LPS and PGN) and could modulate the immune response including apoptosis, phagocytosis and the release of cytokines via the inhibition of signal transduction. CgNCAM could act as a pattern recognition receptor(PRR) involved in the innate immunity of oyster. It shared a close evolutionary relationship with a plant lectin and its mRNA transcripts significantly increased after microbe stimulation. The purified rCgNCAM protein exhibited a wide binding spectrum to PAMPs(LPS 、 GLU and MAN) and microorganisms(M. luteus, E. coli, V. anguillarum, V. splendidus, P. pastoris and Y. lipolytica). And it was also involved in the formation of Extracellular traps(ETs) in oyster hemocytes. The functional analysis of three IgSF members collectively indicated that recognition and binding might be the main function of IgSF in oyster. The dissociative IgSF might function as opsonin, while the transmembrane IgSF could take role as pathogen recognition receptor and activator of the downstream immune response.All the results suggested that oysters possess an abundant and diverse collection of IgSF members which act crucial roles in the immune response. Several IgSF members potentially function in alternative adaptive immunity upon the secondary challenge of V. splendidus. Three IgSF members including CgJAM-A-L, CgSiglec-1 and CgNCAM were involved in oposonization, immunoregulation and immunoagglutination of oysters, respectively. The results contribute to exploring the heterogeneity of invertebrate IgSF. Meanwhile, it provides new information for understanding the role of invertebrate IgSF in alternative adaptive immunity as well as the enrichment and development of marine invertebrate immunology.