实验海洋生物学重点实验室知识库

无脊椎动物通过有限基因编码的免疫受体介导了复杂的固有免疫反应网络，从而形成其赖以生存的免疫防御能力。关于无脊椎动物关键免疫受体及其功能的研究对深入认识无脊椎动物固有免疫反应的本质具有重要意义。整合素家族（Integrin family）是一类重要的免疫受体，广泛地存在于所有多细胞动物中，但目前对其在无脊椎动物固有免疫反应中的作用方式和特点还缺乏系统认知。本研究以软体动物长牡蛎为研究对象，从基因组中系统地鉴定了整合素家族所有成员，并借助比较生物学、生物信息学、分子生物学、免疫学等技术手段，分析了长牡蛎整合素家族成员的结构和进化特征，阐述了其参与配体结合和介导细胞免疫反应的方式和特点，探讨了其与配体互作并调节多种细胞免疫反应的分子基础和可能机制，具体实验结果如下：  

长牡蛎整合素家族由8个α亚基和3个β亚基组成，至少可形成8个功能配对的整合素异源二聚体，该数目多于线虫、果蝇以及海葵等无脊椎动物。在结构域组成上，相比人和果蝇的整合素，长牡蛎整合素α和β亚基都含有保守的跨膜域，其中α亚基除了拥有保守INA、Intα结构域之外，有些成员还携带了特异的FG-GAP、sulfotransfer结构域；β亚基除了拥有保守的胞外INB结构域之外，有些成员在其胞内还插入了额外的INB结构域。除此之外，长牡蛎整合素α和β亚基保守的INA和INB结构域具有高度变异性：首先，在序列长度上，长牡蛎INA和INB结构域分别由146~499个氨基酸残基和225~354个氨基酸残基构成，长度变异较大。其次，长牡蛎INA和INB结构域的序列变异性大，其中长牡蛎INA结构域之间的序列相似性和一致性分别为23.8%和0.2%，而长牡蛎INB结构域之间的序列相似性和一致性分别为69.6%和14.9%。再者，SWISS-MODEL建模分析显示，所有长牡蛎INA结构域不具备保守且典型的“大腿”和 “小腿”样结构，而大多数INB结构域除了携带保守的由6~8个α螺旋围绕4~6个β片层而形成的中心结构，还额外插入了不同数目的α螺旋或β片层结构，也表现出高度的变异性。作为重要的免疫受体，长牡蛎整合素家族成员数目发生扩张，结构多样且变异性高，可能为其行使多样化的免疫功能提供了重要的结构基础。 

在分子进化上，长牡蛎整合素家族8个α亚基中，α亚基CGI_10012356与RGD结合型α亚基聚为一支；α亚基CGI_10013155与层粘连蛋白结合型α亚基聚为一支；其余6个α亚基明显与其他进化分支分离而单独聚类，命名为牡蛎特有型α亚基分支。相比人的整合素α亚基，长牡蛎整合素家族α亚基缺乏LDV结合型α亚基，以及含αI结构域的α亚基分支。在β亚基的系统进化树中，长牡蛎的3个β亚基和昆虫纲βV进化关系相对较近且聚在一个分支，而与同属软体动物门的光滑双脐螺的β亚基发生明显分歧。推测长牡蛎整合素家族α、β亚基分别经历了独特的进化历程，可能已经高度进化为独特分支。    

长牡蛎整合素家族成员能结合RGDCP、LDVCP、 GFOGERCP和层粘连蛋白等多种配体，尤其是尽管长牡蛎整合素家族在进化上缺乏LDV结合型以及GFOGER结合型的成员，但依然能结合特异配体LDVCP和GFOGERCP，从而实现了在配体结合功能上比较完备的分工。长牡蛎整合素家族成员介导血细胞吞噬、迁移、包囊反应等多种细胞免疫反应时，也表现出了明显的功能分化，即只有特定的整合素家族成员在特定的细胞免疫反应中起主要作用。以长牡蛎整合素家族β亚基（CGI_10012179）为例，抗体封闭血细胞表面β亚基（CGI_10012179）后，血细胞吞噬、迁移和包囊反应的活性显著下降，且显著比其他3个代表性的α亚基抗体的抑制作用强，表明长牡蛎整合素家族成员介导复杂的细胞免疫反应时极大地依赖于β亚基。另外一方面，多个长牡蛎整合素家族成员在特定的细胞免疫反应中还展现出协作的特点，同时某些整合素家族成员还参与了多种细胞免疫反应，从而大大增加了长牡蛎整合素家族成员参与细胞免疫反应的多样性。       

借助pull-down以及质谱分析技术，鉴定了长牡蛎整合素α亚基的一个血清配体——CgPEPCK，它是一个保守的烯醇式丙酮酸激酶，具有保守的GTP结合活性，在胞内糖异生过程当中发挥重要作用。除此之外，CgPEPCK可以被分泌至血清，兼具识别LPS、PGN以及多种病原微生物的免疫功能。另外，发现β亚基的一类血清配体可能是众多结构和功能相似的C1qDC蛋白，其中CgC1qDC-5可以作为分泌型PRR结合LPS、Lipid A以及多种病原细菌，并对细菌具有调理促吞噬的作用。抗体封闭的实验证明，血细胞膜上的β整合素CgIntegrin可以通过结合LPS以识别细菌，继而作为吞噬受体直接介导血细胞对细菌的吞噬过程，同时还能作为血清CgC1qDC-5的受体，介导CgC1qDC-5依赖的促吞噬过程。

LPS刺激后，长牡蛎血细胞中β整合素CgβV转录本的表达水平显著上升，整合素激活保守机制上游分子GTP酶和talin的表达水平，以及激活保守机制下游分子Ca²⁺和cAMP的表达水平均被诱导大量表达，这些分子与哺乳动物巨噬细胞表面整合素被LPS激活时的变化趋势一致，表明长牡蛎整合素可能被外源LPS保守地激活。整合素激活后，长牡蛎血细胞结合FITC-RGDCP的比例显著升高。进一步用RGDCP和CgβV抗体封闭血细胞后，发现血细胞结合FITC-RGDCP的比例都显著下降，且CgβV抗体的抑制作用显著弱于RGDCP，表明CgβV作为长牡蛎RGD结合型整合素之一被LPS激活。LPS刺激后，血细胞对大肠杆菌的吞噬水平也显著增强。进一步用CgβV抗体封闭血细胞后，发现血细胞对未被血清调理的大肠杆菌的吞噬水平显著下降，表明整合素CgβV激活后促进了血细胞对细菌的吞噬作用。另一方面，大肠杆菌经血清调理后，血细胞对其吞噬水平显著上升，表明血清具有调理促吞噬的作用。而进一步用CgβV抗体封闭血细胞后，发现血细胞对被血清调理的大肠杆菌的吞噬水平显著下降，表明整合素CgβV激活后可能还促进了血清的促吞噬作用。   

借助FITC-RGDCP靶定长牡蛎血细胞膜上的RGD结合型整合素以标记出RGD⁺血细胞，发现RGD⁺血细胞数大约占全血细胞的8.7%。RGD⁺血细胞直径为5-10 μm，细胞核质比较大，胞质中大颗粒较少，且有较高的MPO、ROS和Ca²⁺水平。灿烂弧菌刺激24小时后，RGD⁺血细胞比例显著升高至18.1%。借助FACS技术分选出灿烂弧菌刺激前后的RGD⁺血细胞，并进行转录组测序分析。转录组结果显示，RGD⁺血细胞主要高表达整合素-ECM互作相关基因、细胞骨架相关的F-actin、PI3K、Rho、ROCK、MLCK等一些与迁移通路相关的基因，具有较高迁移活性的潜力。灿烂弧菌刺激后，RGD⁺血细胞表面的受体整合素激活，促进迁移的相关基因表达上调，且抑制迁移的相关基因表达下调，使得其迁移活性进一步增强。神经内分泌因子也可能参与调节RGD⁺血细胞的迁移活性，因为用抑制剂（SCH 23390）阻断兴奋性神经递质多巴胺的受体后，显著抑制了RGD⁺血细胞的迁移活性。测定长牡蛎血细胞的迁移活性后发现，灿烂弧菌刺激后，长牡蛎全血细胞的迁移活性显著升高；RGD⁺血细胞的迁移活性也显著升高，而RGD^-血细胞的迁移活性没有发生显著变化，表明只有RGD⁺血细胞主要以增强迁移的方式响应灿烂弧菌刺激。此外，灿烂弧菌刺激后，RGD⁺血细胞中免疫调节因子IL-17及其受体基因整体呈上调的表达趋势，同时长牡蛎全血细胞中Cg-BigDefensins、Cg-Defensins以及Cg-BPI等众多抗菌肽基因也表达上调，从而可能增强长牡蛎血细胞的抗菌免疫反应。      

以上研究结果表明，长牡蛎整合素家族成员数目发生扩张，结构多样且变异性高，已高度进化为单独分支，这些成员具有完备的配体结合功能，可直接识别病原介导多种细胞免疫反应，也可与血清中多种配体协作共同调节细胞免疫反应。当免疫激活整合素及其信号通路号后，整合素单独介导的吞噬作用及其与血清配体共同介导的调理促吞噬作用进一步增强。长牡蛎RGD结合型整合素介导免疫反应时依赖的RGD⁺血细胞具有较高的迁移活性，该活性不单单被迁移通路相关的分子调节，还被神经内分泌因子调节。病原菌灿烂弧菌刺激后，RGD⁺血细胞可能主要作为调节性细胞，通过分泌细胞因子IL-17以促进血细胞抗菌肽的表达，从而增强抗菌免疫反应。以上研究结果首次探明了软体动物长牡蛎整合素家族成员的结构与进化特征，并初步揭示了其在固有免疫反应中的作用方式与特点以及分子与细胞基础，丰富了整合素家族进化的理论，同时为软体动物乃至所有无脊椎动物中依赖众多免疫受体介导的固有免疫反应复杂网络增添了新的分支。

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 α subunits and 3 β 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 α subunits and 3 β subunits contained the conserved transmembrane domains. Compared to the integrins from humans Homo sapiens and flies D. melanogaster, oyster 8 α subunits owned the conserved INA and Intα domains, and also carried the specific FG-GAP and sulfotransfer domains, while an oyster β 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% and 0.2%, 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 α subunits, the α subunit CGI_10012356 was clustered into the clade of RGD-binding α subunits, the α subunit CGI_10013155 was clustered into the clade of laminin-binding α subunits, and the remaining 6 α subunits were obviously segregated from others and assigned into the oyster-specific α branch. The oyster αs indeed lacked the LDV receptors and αI-domain receptors compared to that in humans. In the phylogenetic tree of the β subunits, the three oyster β subunits of were closely related to the insecta βVs, but significantly distantly related to the β subunits from the same phylum species Biomphalaria glabrata. These results collectively indicated that oyster integrin family composed of species-specific αs and βs 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 β subunit (CGI_10012179) was selected as a representative of oyster β subunits. In the present study, the representative β CGI_10012179 appeared to be involved in all the tested cellular immune responses of phagocytosis, migration and encapsulation in blockage assays, and the antibodies of β CGI_10012179 could inhibit these three cellular immune responses with the most inhibition effects compared to that of the other three representative α subunits, indicating that oyster integrins seemed to be able to mediate multiple cellular immune responses, and largely depended on β subunits.

According to the serum pull-down assay and mass spectrometry analysis, a ligand of oyster integrin α 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 β 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 β integrin CgIntegrin 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 β integrin CgβV 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 CgβV antibodies revealed that the percentage of hemocytes binding to FITC-RGDCP were significantly decreased, and the inhibition of CgβV antibody was significantly weaker than that of RGDCP, indicating that CgβV 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 CgβV 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 CgβV promoted the hemocytic phagocytosis of bacteria mediated by CgβV. 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 CgβV antibody, the phagocytosis level of hemocytes to E. coli opsonized by oyster serum was significantly decreased, indicating that the activation of integrin CgβV 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 μm 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 β-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 β-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.