The Members of the Highly Diverse Crassostrea gigas Integrin Family Cooperate for the Generation of Various Immune Responses

Abstract

Studies on invertebrate immune receptors can provide insights into characteristics specific to innate immune system. Here, eight α and three β integrins are identified from an invertebrate, the Pacific oyster Crassostrea gigas, and their possible immune functions are studied. Oyster α/β integrins exhibit a higher degree of sequence and structural variability than the members from Homo sapiens and Drosophila melanogaster. The analysis reveals that oyster RGD-and laminin-binding receptor homologs are present in the phylogenetic tree of α integrins, but the other six oyster α integrins mainly form a species-specific branch; meanwhile, oyster β integrins are clustered with insect β integrins but distinct from a member from the mollusk Biomphalaria glabrata. Although phylogenetically lacking the important α integrin branches of LDV-binding, PS3-type, and αI-containing integrins, oyster integrins can bind to most ECM ligands, including RGDCP, LDVCP, GFOGERCP, and laminin protein in a distinct binding pattern. Besides, oyster integrins are distributed in different hemocyte subpopulations, while only specific integrins are selectively involved in hemocyte phagocytosis, migration, and encapsulation, and some of them participate in more than one immune response in a sophisticated pattern. Especially, oyster β integrins are arranged in the core to mediate complex immune responses, unlike the counterparts in humans that mainly depend on αI-containing integrins to incite immune reactions. This study represents the first comprehensive attempt to reveal the structural and evolutionary features of the integrin family and their involvement in cellular immune responses in the non-model invertebrate C. gigas and sheds light on the characteristics specific to the innate immune system in the integrin family.

RGD-Labeled Hemocytes With High Migration Activity Display a Potential Immunomodulatory Role in the Pacific Oyster Crassostrea gigas

“…Confocal microscopic analysis revealed that FITC-conjugated RGDCP could label a small portion of hemocytes (RGD + hemocytes) from the oyster C. gigas with a circle of green fluorescent signals on their surfaces ( Figure 2A ). Flow cytometric analysis showed that RGD + hemocytes accounted for 8.7% of the whole hemocytes in the blank oysters (designated resting RGD + hemocytes, Figure 2B ), which was consistent with previously reported results ( 39 ). The hemocytes with negative signals accounted for the majority of oyster hemocytes representing multiple cell types and were assigned as RGD - hemocytes ( Figure 2B ).…”

“…Molecular immunology research has made great strides after the identification of most immune genes, but there are still many unknowns in the cellular immunity of this animal (42)(43)(44). The involvement of integrins in cellular immune responses and the binding of RGDCP to integrins and hemocytes in C. gigas were reported in our previous studies (38,39). In this study, RGDCP-bound positive hemocytes (RGD + hemocytes) were sorted by fluorescence-activated cell sorting (FACS), and the labeled cells were proven to have high migration activity.…”

“…The FITC-conjugated RGDCP peptide was synthesized by Sangon Biotech (Shanghai, China) and used as the probe for certain integrins to label specific populations of oyster hemocytes as previously described ( 38 , 39 ). Briefly, the collected hemocytes were resuspended in modified Leibovitz L15 medium and incubated with FITC conjugated-RGDCP (0.01 mg/ml) for 30 min.…”

“…For example, RGDCP conjugated with the fluorescent dye FITC, Cy7, or Cy5 is available for targeting integrin aVb3, which is highly expressed on the glioma cell surface, to indicate the migration activity, morphological change, and invasion processes (30)(31)(32). Considering that synthetic RGDCP has also been reported to conservatively bind hemocytes via surfacespecific integrins in many invertebrates, including D. melanogaster (33), Botryllus schlosseri (34), Pacifastacus leniusculus (35), Lymnaea stagnalis (36), Mytilus trossulus (37), and C. gigas (38)(39)(40), fluorescent RGDCP as a specific probe for invertebrate integrins may also be practical for marking a population of hemocytes and helpful for studying the main types of migrating hemocytes and their roles in the immune responses of invertebrates.…”

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“…Confocal microscopic analysis revealed that FITC-conjugated RGDCP could label a small portion of hemocytes (RGD + hemocytes) from the oyster C. gigas with a circle of green fluorescent signals on their surfaces (Figure 2A). Flow cytometric analysis showed that RGD + hemocytes accounted for 8.7% of the whole hemocytes in the blank oysters (designated resting RGD + hemocytes, Figure 2B), which was consistent with previously reported results (39). The hemocytes with negative signals accounted for the majority of oyster hemocytes representing multiple cell types and were assigned as RGDhemocytes (Figure 2B).…”