The activated beta-integrin (Cg beta V) enhances RGD-binding and phagocytic capabilities of hemocytes in Crassostrea gigas
The activated beta-integrin (Cg beta V) enhances RGD-binding and phagocytic capabilities of hemocytes in Crassostrea gigas
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活化的β-整合素（Cg beta V）增强巨牡蛎血细胞的RGD结合和吞噬能力
DOI：
10.1016/j.fsi.2019.01.047
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发表时间：
2019
影响因子：
4.7
通讯作者：
Song Linsheng
中科院分区：
文献类型：
--
作者：
Lv Zhao;Qiu Limei;Jia Zhihao;Wang Weilin;Liu Zhaoqun;Wang Lingling;Song Linsheng
整合素是一类重要的细胞受体家族，被激活后可结合外源颗粒并促进细胞吞噬。本研究从太平洋牡蛎（Crassostreagas）中鉴定了一种新的β整合素，它具有一个胞外区、一个跨膜区和一个短的胞浆区。CgβV与吞噬相关的β整合素具有NPX[Y/F]保守基序，该基序与吞噬相关的β整合素活化有关。结果表明，Cg βV mRNA在牡蛎血细胞、性腺、闭壳肌、外套膜、鳃和肝胰腺等组织中均有表达，且在脂多糖（LPS）刺激后12 h，血细胞中Cg βV mRNA的表达量显著上调（p< 0.05），是对照组的2.29倍，CgβV蛋白主要表达于血细胞表面。牡蛎血细胞可与异硫氰酸荧光素（FITC）标记的Arg-Gly-Asp-containing peptides（RGDCPs）结合，LPS刺激后12 h RGDCPs的结合率达到37.6%，显著高于对照组的8.8%，p < 0.05，表明牡蛎血细胞表面的RGDCPs结合整合素被激活。未标记的RGDCPs和抗Cg βV抗体均能抑制血细胞对FITC标记的RGDCPs的结合能力，其中RGD阻断组（7.4%，p < 0.05）和抗Cg βV阻断组（22.1%，p < 0.05）均显著低于对照组（37.6%），表明Cg βV可能是一种与RGD结合的整合素。吞噬实验表明，LPS能增强血细胞对大肠杆菌和荧光珠的吞噬作用，其吞噬率（PR）分别为18.3%和17.4%，吞噬指数（PI）分别为5.29和37.71，均显著高于对照组（对大肠杆菌为11.0%和3.65）。大肠杆菌，荧光微球分别为9.8%和29.26%（P< 0.05）。此外，无标记的RGDCPs和抗Cg βV抗体均能显著抑制血细胞对E的吞噬作用。大肠杆菌和荧光珠。在E。大肠杆菌和荧光珠经牡蛎血清调理后，未标记的RGDCP仍能抑制血细胞对它们的吞噬作用，而抗Cg βV抗体仅能抑制血细胞对E的吞噬作用。表明只有激活的Cg βV参与了牡蛎对细菌的增强吞噬作用。此外，牡蛎血细胞整合素介导的吞噬途径中的关键组分包括GTP酶、talin蛋白、Ca ~（2+）和cAMP均受到LPS的诱导。这些结果表明，激活的Cg βV增强了血细胞的结合RGD和吞噬功能，为阐明整合素介导的软体动物吞噬功能机制提供了新的线索。
Integrins are an important family of cell receptors that can bind foreign particles and promote cell phagocytosis after they are activated. In the present study, a novel β integrin was identified from pacific oysterCrassostreagigaswith an extracellular domain, a single transmembrane segment, and a short cytoplasmic domain. It was phylogenetically clustered with phagocytosis-related insecta βV, and designated asCgβV.CgβV shared a conserved NPX[Y/F] motif related to integrin activation with other phagocytosis-related β integrins. The mRNA transcripts ofCgβV were widely detected in oyster tissues including hemocytes, gonad, adductor muscle, mantle, gill, and hepatopancreas, and the expression level in hemocytes was significantly up-regulated at 12 h after lipopolysaccharide (LPS) stimulation (p< 0.05), which was 2.29-fold higher than that in the control group.CgβV proteins were mainly observed on the hemocytes surface. The oyster hemocytes were found to bind fluorescein isothiocyanate (FITC)-labeled Arg-Gly-Asp-containing peptides (RGDCPs), and the binding capability was significantly up-regulated with the peak percentage of 37.6% at 12 h post LPS stimulation, which was higher than that in the control group (8.8%,p< 0.05), suggesting the activation of RGD-binding integrins on oyster hemocytes surface. The label-free RGDCPs and anti-CgβV antibody inhibited the binding capability of hemocytes towards FITC-labeled RGDCPs, which were significant lower in RGD blocking group (7.4%,p< 0.05) and anti-CgβV blocking group (22.1%,p< 0.05) than that in the control group (37.6%), indicating thatCgβV could be a RGD-binding integrin. Phagocytosis assay demonstrated that LPS could enhance the phagocytosis of hemocytes towardsEscherichia coliand fluorescent beads with the phagocytic rate (PR) of 18.3% and 17.4%, and phagocytic index (PI) of 5.29 and 37.71, respectively, which were significant higher than that in the control group (11.0% and 3.65 forE. coli, 9.8% and 29.26 for fluorescent beads, respectively,p< 0.05). In addition, both the label-free RGDCPs and anti-CgβV antibody significantly hindered the phagocytosis of hemocytes towardsE. coliand fluorescent beads. After theE. coliand fluorescent beads were opsonized by oyster serum, the label-free RGDCPs still inhibited the phagocytosis of hemocytes towards them, while the anti-CgβV antibody could only inhibit the phagocytosis of hemocytes towardsE. coli, suggesting that only the activatedCgβV was involved in the enhancing phagocytosis for bacteria in oyster. Moreover, the key components of conserved integrin-mediated phagocytosis pathway including GTPases, talin proteins, Ca2+and cAMP were all induced by LPS in hemocytes of oyster. All these results suggested that the activatedCgβV enhanced RGD-binding and phagocytic capabilities of hemocytes, shedding lights on the mechanisms of integrin-mediated phagocytosis in mollusks.