Identification Of Integrin Family Members And Their Interacting Ligands And The Mechanisms Underlying Their Mediation Of Cellular Immunity In Oyster Crassostrea Gigas

Invertebrates successfully cope with diverse environmental immune challenges via complex innate immune response networks mediated by limited immune receptors encoded by germline genes,and studies on the key immune receptors and their functions in invertebrates are very helpful in the understanding of the nature of innate immune responses in invertebrates.The integrin family is a group of newly identified immune receptors widely present in all multicellular animals.However,the comprehensive understanding of their role and characteristics in innate immune responses remain unclear in invertebrates.In the present study,all members of the integrin gene family were systematically identified in the genome of an invertebrate pacific oyster Crassostrea gigas,and their structural and evolutionary characteristics were further analyzed.With the comparative biology,bioinformatic,molecular biology,and immunologic methods,the ways and patterns of their involvements in the ligand binding and cellular immune responses,and the molecular and cellular mechanisms underlying the immune processes mediated by integrins were also explored in oyster C.gigas.The C.gigas integrin family consisted of 8?subunits and 3?subunits,forming at least 8 functionally paired integrin heterodimers.The C.gigas integrin family member was more than that from other invertebrates such as Caenorhabditis elegans,Drosophila melanogaster and Nematostella vectensis.In terms of domain architecture,oyster 8?subunits and 3?subunits contained the conserved transmembrane domains.Compared to the integrins from humans Homo sapiens and flies D.melanogaster,oyster 8?subunits owned the conserved INA and Int?domains,and also carried the specific FG-GAP and sulfotransfer domains,while an oyster?subunit harbored an extra intracellular INB domain besides the conserved extracellular INB domain.In addition,the conserved INA and INB domains of oyster integrins were highly variable.First,the oyster INA and INB domains were composed of 146-499 amino acid residues and 225-354 amino acid residues,respectively,and thus the sequence length of oyster INA and INB domains displayed high variability.Secondly,the sequence similarity and identity among the INA domains of oyster integrins were 23.8%and0.2%,respectively,and the sequence similarity and identity among the INB domains of oyster integrins were 69.6%and 14.9%,respectively,contributing to the high sequence variability.Furthermore,SWISS-MODEL modeling analysis showed that the tridimensional structure of the oyster INA and INB domains were also highly variable and diverse.As important immune receptors,integrin gene family expanded,and displayed high variability and diversity in structures,which might provide the structural basis for integrins-mediating diverse immune functions in oyster C.gigas.Evolutionarily,the oyster integrin family underwent a unique process.Among the 8?subunits,the?subunit CGI₁0012356 was clustered into the clade of RGD-binding?subunits,the?subunit CGI₁0013155 was clustered into the clade of laminin-binding?subunits,and the remaining 6?subunits were obviously segregated from others and assigned into the oyster-specific?branch.The oyster?s indeed lacked the LDV receptors and?I-domain receptors compared to that in humans.In the phylogenetic tree of the?subunits,the three oyster?subunits of were closely related to the insecta?Vs,but significantly distantly related to the?subunits from the same phylum species Biomphalaria glabrata.These results collectively indicated that oyster integrin family composed of species-specific?s and?s could be highly evolved into a distinct evolutionary branch.Oyster integrins could bind to ligands such as RGDCP,laminin,LDVCP and GFOGERCP although oyster evolutionarily lacked of LDV-binding and GFOGER-binding integrin family members.So oyster integrins could display multiple ligand-binding activities as human integrins,but with different functional division compared to human integrins.Oyster integrins could also participate in various cellular immune responses such as hemocytes phagocytosis,migration,and encapsulation with distinct functional division.Specifically,only specific integrin family members played the major roles in specific cellular immune responses.In addition,some oyster integrins exhibited synergistic characteristics in specific cellular immune responses,and some integrin family members were also involved in a variety of cellular immune responses,thereby greatly increasing the diversity and complexity of integrins-mediating cellular immune responses.It was worth noting that the?subunit?CGI₁0012179?was selected as a representative of oyster?subunits.In the present study,the representative?CGI₁0012179 appeared to be involved in all the tested cellular immune responses of phagocytosis,migration and encapsulation in blockage assays,and the antibodies of?CGI₁0012179 could inhibit these three cellular immune responses with the most inhibition effects compared to that of the other three representative?subunits,indicating that oyster integrins seemed to be able to mediate multiple cellular immune responses,and largely depended on?subunits.According to the serum pull-down assay and mass spectrometry analysis,a ligand of oyster integrin?subunits,CgPEPCK,was identified from oyster serum.It was initially considered as a conserved phosphoenolpyruvate carboxykinase with GTP-binding activity in cytoplasm,playing an important role in the process of gluconeogenesis.In the present study,it was unexpectedly found that CgPEPCK could be secreted into oyster serum from hemocytes,serving as a secreted PRR with immune recognition to LPS,PGN and various microorganisms.In addition,several ligands of oyster integrin?subunits were also identified from oyster serum.They could be a plurality of C1qDC proteins with similar structures and functions.Among them,CgC1qDC-5 could function as a secreted PRR to recognize LPS,Lipid A and various bacteria,and could promote hemocytic phagocytosis as an opsonin.The antibody blocking assay proved that a?integrin Cg Integrin acted as a receptor for CgC1qDC-5 and participated in CgC1qDC-5-mediated pro-phagocytosis,and it could also recognize bacteria by binding to LPS,and directly mediate the hemocytic phagocytosis of bacteria as a phagocytic receptor.Oyster integrins could be activated by exogenous LPS.After LPS stimulation,the expression level of?integrin Cg?V transcript in oyster hemocytes increased significantly.In addition,the expression levels of the conserved upstream and downstream molecules in activation pathway of integrins including GTPase and talin,and Ca²⁺and cAMP were all induced in oyster hemocytes by LPS.After integrins were activated by exogenous LPS,the percentage of hemocytes binding to FITC-RGDCP was significantly increased.Further blocking of hemocytes with RGDCP and Cg?V antibodies revealed that the percentage of hemocytes binding to FITC-RGDCP were significantly decreased,and the inhibition of Cg?V antibody was significantly weaker than that of RGDCP,indicating that Cg?V was activated by LPS as one of oyster RGD-binding integrins.After LPS stimulation,the phagocytic level of hemocytes to Escherichia coli was also significantly enhanced.The blocking of hemoyctes with Cg?V antibody revealed that hemocytes showed a significant decrease in the phagocytic level of E.coli which was not opsonized by oyster serum,indicating that the activation of integrin Cg?V promoted the hemocytic phagocytosis of bacteria mediated by Cg?V.After E.coli was opsonized by oyster serum,the hemocytic phagocytosis of bacteria increased significantly,indicating that oyster serum has the effect of oposonization to promote phagocytosis.After blocking hemocytes with Cg?V antibody,the phagocytosis level of hemocytes to E.coli opsonized by oyster serum was significantly decreased,indicating that the activation of integrin Cg?V also enhanced the effect of oposonization of oyster serum.The RGD-binding integrins on oyster hemocytes membrane were targeted by the specific probes of FITC-RGDCP to label RGD⁺hemocytes,and the percentage of RGD⁺hemocytes was found to be about 8.7%of the whole oyster hemocytes.RGD⁺hemocytes were 5-10?m in diameter,with relatively large Nuclear-Cytoplasmic ratio,with fewer large particles in cytoplasm,and higher levels of MPO,ROS and Ca²⁺.After Vibrio splendidus stimulation at 24 h,the percentage of RGD⁺hemocytes increased significantly to 18.1%.Afterwards,RGD⁺hemocytes were sorted by FACS before and after stimulation with V.splendidus,and the RNA-seq was further conducted.The results of transcriptional analysis showed that RGD⁺hemocytes mainly expressed some genes related to the migration pathway before V.splendidus stimulation.After V.splendidus stimulation,the integrins on the surface of RGD⁺hemocytes was activated,the expression of genes involved in promoting migration was up-regulated,and the expression of genes involved in inhibiting migration was down-regulated.Further detection of the migration activity of hemocytes revealed that the migration activity of the whole hemocytes of oyster was significantly increased after V.splendidus stimulation,the migration activity of RGD⁺hemocytes was also significantly increased,while the migration activity of RGD^-hemocytes did not change significantly,indicating only RGD⁺hemocytes responded to V.splendidus stimulation with higher migration activity.According to the hints of transcriptional analysis,the dopamine receptors were blocked by the inhibitor?SCH 23390?,and the migration activity of RGD⁺hemocytes was significantly decreased,indicting neuroendocrine factors participated in the regulation of the migration activity of RGD⁺hemocytes after V.splendidus stimulation.In addition,transcriptional results also hinted that the expressions of IL-17s and their receptor genes were up-regulated in RGD⁺hemocytes,and many antibacterial peptide genes were also up-regulated after V.splendidus stimulation,which might enhance the antibacterial immune responses in oyster C.gigas.In summary,oyster integrin gene family expanded,and had diverse and high variable structures,which might have been highly evolved into a distinct branch.Oyster integrins had the functional broad spectrum in multiple ligand-binding and cellular immune responses with large?-dependence,exhibiting highly and complexly functional division and cooperation.Oyster integrins could directly initiate cellular immune responses by recognizing pathogens,and multiple ligands present in the oyster serum promoted cellular immune responses via their receptor integrins.Integrin-mediated immune responses were further enhanced after the oyster?-integrins were activated by exogenous stimulus.The RGD⁺hemocytes for oyster RGD-binding integrin mediating the immune response had high migration activity,which was not only regulated by migration-related pathway molecules,but also by neuroendocrine factors.After V.splendidus stimulation,RGD⁺hemocytes might act mainly as immune regulatory cells,secreting the cytokine IL-17 to promote the expression of antimicrobial peptides,thereby enhancing the antibacterial immune response of oyster.The above results firstly formulated the structural and evolutionary characteristics of integrin family in an invertebrate oyster C.gigas,and revealed the functional roles and characteristics as well as the molecular and cellular basis of this integrin family in the innate immune responses in oyster,which enriched the evolution theory of integrin family and added a new branch to the complex network of innate immune responses mediated by numerous immune receptors in mollusks and even all invertebrates.