实验海洋生物学重点实验室知识库

补体系统是先天免疫的重要组成部分和免疫效应系统，在无脊椎动物免疫防御反应中发挥重要作用。目前，对甲壳动物特别是蟹类补体系统的研究尚处于起步阶段，亟待深入研究。本论文利用基因克隆、实时定量PCR、原核重组表达、RNA干扰等分子生物学技术，对三疣梭子蟹Portunus trituberculatus的C1q受体（PtgC1qR）、α2-巨球蛋白（PtA2M-1和PtA2M-2）、含硫酯蛋白（PtTEP）和甘露糖结合凝集素（PtMBL）等补体样分子进行了结构和免疫功能的研究，探讨了补体样分子之间的作用关系以及它们在三疣梭子蟹免疫系统中的作用。

补体C1q受体gC1qR是一种多功能、多配体的结合蛋白。三疣梭子蟹的PtgC1qR编码含有268个氨基酸残基的蛋白，该预测的蛋白含有1个保守的MAM33结构域，且其前56个氨基酸为线粒体靶序列。PtgC1qR基因在所有检测的组织中均有表达，且在肝胰腺中表达丰度最高。PtgC1qR在受精卵和卵裂期的表达水平显著高于囊胚期、原肠期和心跳期，暗示PtgC1qR可能是一种母源免疫基因。病原微生物刺激后，PtgC1qR在血细胞中的表达呈现时间依赖性响应模式。此外，PtgC1qR重组蛋白（rPtgC1qR）可与革兰氏阳性菌（藤黄微球菌Micrococcus luteus、金黄色葡萄球菌Staphylococcus aureus）和革兰氏阴性菌（溶藻弧菌Vibrio alginolyticus、铜绿假单胞菌Pseudomonas aeruginosa）紧密结合，并显著抑制其生长，暗示PtgC1qR在三疣梭子蟹免疫防御和免疫识别中扮演重要角色；然而rPtgC1qR对真菌（毕赤酵母菌Pichia pastoris）的结合活力较弱且未表现出抑菌活力。酚氧化酶实验结果表明，rPtgC1qR可在体外以剂量依赖方式抑制酚氧化酶活力，且在蛋白浓度为11.65 μM时几乎100%抑制酚氧化酶活力。RNAi结果表明，沉默PtgC1qR 基因后，丝氨酸蛋白酶相关基因（PtcSP1-3、PtSPH），酚氧化酶原激活相关基因（PtproPO、PtPPAF）和C3样基因（PtA2M-1 and PtTEP）的表达显著升高；然而吞噬作用相关基因（PtMyosin、PtRab5和PtArp）和PtA2M-2的表达量显著降低。

α2-巨球蛋白（alpha-2 macroglobulin, A2M）是一种广泛存在的蛋白酶抑制剂。本研究克隆获得2条A2M基因，分别命名为PtA2M-1和PtA2M-2。PtA2M-1和PtA2M-2蛋白分别含有1,541和1,516个氨基酸，均包含A2M典型的结构域。节肢动物的A2M主要在血细胞中高表达，PtA2M-1和PtA2M-2在鳃、眼柄和消化道中表达量较高，而它们在血细胞中表达量很低。在胚胎发育过程中，PtA2Ms在受精卵中表达量最高，暗示它们可能是母源基因。感染溶藻弧菌后，PtA2M-1和PtA2M-2呈相似的时间依赖性响应模式。与PtA2M-2基因相比，PtA2M-1对藤黄微球菌和毕赤酵母菌的刺激更为敏感。沉默PtA2M-1或PtA2M-2基因可显著增强酚氧化酶原激活相关基因（PtproPO和PtPPAF）和丝氨酸蛋白酶相关基因（PtcSP1-3和PtSPH）的表达，然而PtLSZ和吞噬作用相关基因（PtMyosin和PtRab5）表达显著被抑制。PtA2M-1或PtA2M-2基因沉默的三疣梭子蟹血淋巴中酚氧化酶和溶菌酶活力变化也支撑上述结果。此外，研究发现PtA2Ms可能通过Toll和NF-kB通路调控抗菌肽基因（PtALF1-3、PtCrustin1和PtCrustin3）的表达。

含硫酯蛋白（thioester-containing proteins, TEPs）是一类在进化上古老的分泌型效应蛋白。三疣梭子蟹PtTEP的开放阅读框为4,434 bp，编码一个含有1,478个氨基酸残基的多肽，该肽链具有昆虫TEP蛋白保守的序列特征。PtTEP在肝胰腺、肠、鳃等主要免疫组织中高表达，在受到细菌或真菌诱导后表达量显著升高。与受精卵时期相比，PtTEP的转录产物呈先下降后显著升高的变化趋势。利用RNA干扰技术沉默PtTEP基因后，酚氧化酶原激活相关基因（PtproPO和PtPPAF）和丝氨酸蛋白酶相关基因（PtcSP1-3和PtSPH）均显著上调，而PtLSZ和吞噬作用相关基因（PtMyosin和PtRab5）的表达显著受到抑制。PtTEP基因沉默的三疣梭子蟹血淋巴酚氧化酶和溶菌酶活力的变化也支持上述结果。此外，PtTEP基因沉默后抗菌肽基因和参与Toll和NF-κB通路激活的基因表达量均显著下降，这使得三疣梭子蟹更容易感染溶藻弧菌而死亡。

甘露糖结合凝集素（mannose-binding lectin, MBL）作为一种重要的模式识别受体，参与补体凝集素途径的激活。本研究自三疣梭子蟹中克隆获得一个新型MBL分子（PtMBL）。PtMBL的cDNA全长为994 bp，包含一个777-bp的ORF，可编码含有259个氨基酸残基的蛋白，该蛋白具有保守的糖识别结构域（carbohydrate-recognition domain, CRD），在CRD区域中含有一个QPD序列。PtMBL在肌肉、肠、胃和肝胰腺等主要免疫组织中高表达，且PtMBL的mRNAs在感染细菌或真菌的早期均显著上调。随着胚胎的发育，PtMBL的转录产物呈现显著下降趋势，并在发育至心跳期时达到最低值，结合该基因在卵巢中显著高表达，提示PtMBL是一种母源免疫因子。沉默PtMBL基因可显著提高PtTEP、酚氧化酶原激活相关基因（PtproPO和PtPPAF）和丝氨酸蛋白酶相关基因（PtcSP1-3和PtSPH）的表达，然而PtA2Ms和吞噬作用相关基因（PtArp、PtMyosin和PtRab5）的表达显著被抑制。与对照组相比，PtMBL基因沉默的三疣梭子蟹血淋巴中PO活力显著升高。此外，沉默PtMBL基因，抗菌肽基因和参与Toll和NF-κB通路激活的关键基因的表达均显著上调。

对三疣梭子蟹补体样分子的研究表明，它们与脊椎动物补体分子在结构上和免疫功能上均存在较高的相似性。研究认为，PtgC1qR和PtMBL作为模式识别受体，可通过调控下游C3样分子（PtA2M-1、PtA2M-2和PtTEP）激活原始的补体通路，三疣梭子蟹中原始的补体通路可通过调控丝氨酸蛋白酶级联反应、酚氧化酶原激活系统、吞噬作用以及Toll和NF-κB免疫通路介导的抗菌肽合成与分泌执行其免疫功能。同时，补体样分子也可以作为母源性效应分子参与对病原的免疫防御反应。综上，本研究结果表明补体样分子在三疣梭子蟹的先天免疫防御中具有重要作用，为进一步阐明无脊椎动物补体系统的进化以及系统地研究原始补体系统的功能提供了新思路和理论支撑。

Complement system, as the important component and immune effector system of innate immunity, performs a crucial role in innate immune defense of invertebrates. Until now, studies on complement system remain deficient in crustaceans, especially in brachyuran crabs. In the current study, five complement like molecules, including the receptor for C1q (PtgC1qR), alpha-2 macroglobulin (PtA2M-1 and PtA2M-2), thioester-containing protein (PtTEP) and mannose-binding lectin (PtMBL), were isolated and functional studied from Portunus trituberculatus through gene cloning, quantitative real-time PCR, prokaryotic expression system and RNA interference techniques. We further investigated the relationship and immune function of these molecules in crab immune system.

The receptor for the globular head of complement component C1q, gC1qR, is a multifunctional and multiligand binding protein. In this study, a full-length cDNA sequence of a gC1qR homolog (PtgC1qR) in P. trituberculatus was identified. PtgC1qR was a 268-amino-acid polypeptide with a conserved MAM33 domain and a mitochondrial targeting sequence in the first 56 amino acids. The transcripts of PtgC1qR were detected in all examined tissues with the highest level detected in hepatopancreas. Compared with other embryonic stages, PtgC1qR was highly expressed in the fertilized eggs and embryos at the cleavage stage, which suggests PtgC1qR may be a maternal gene. The transcripts of PtgC1qR in hemocytes exhibited time-dependent response expression pattern after challenged with bacteria (Vibrio alginolyticus, Micrococcus luteus) and fungi (Pichia pastoris). Moreover, the recombinant PtgC1qR (rPtgC1qR) exhibited strong antibacterial activity and microbial-binding activity, suggesting its crucial role in immune defense and immune recognition. Further phenoloxidase (PO) assay showed that rPtgC1qR could suppress the crab PO activity in vitro in a dose-dependent manner, and it could result in nearly 100% inhibition of PO activity under the concentration of 11.65 μM. Knockdown of PtgC1qR could significantly enhance the expression of serine protease related genes (PtcSP1-3 and PtSPH), prophenoloxidase (proPO) associated genes (PtproPO and PtPPAF) and C3-like genes (PtA2M-1 and PtTEP). However, the phagocytosis related genes (PtMyosin, PtRab5 and PtArp) and PtA2M-2 were significantly down-regulated in the PtgC1qR-silenced crabs.

Alpha-2 macroglobulin (A2M) is a ubiquitous protease inhibitor. Herein, two distinct A2M genes (designated as PtA2M-1 and PtA2M-2, respectively) were isolated from P. trituberculatus. PtA2M-1 and PtA2M-2 encoded proteins with 1,541 or 1,516 amino acids, respectively, containing the typical functional domains of A2M. Unlike highly expressed in hemocytes of most arthropods, PtA2M-1 and PtA2M-2 were predominantly detected in gill, eyestalk and digestive tracks. During the embryonic stages, PtA2Ms were found to be expressed most highly in fertilized eggs, suggesting their maternal origin. After challenged with V. alginolyticus, the transcripts of PtA2Ms showed similar time-dependent response expression pattern, while PtA2M-1 was more sensitive to M. luteus and P. pastoris infection than PtA2M-2. Knockdown of PtA2M-1 or PtA2M-2 could significantly enhance the expression of proPO-associated genes (PtproPO and PtPPAF) and serine protease related genes (PtcSP1-3 and PtSPH), however, PtLSZ and the phagocytosis-related genes (PtMyosin and PtRab5) were effectively inhibited. These results were further supported by the PO and lysozyme activities in hemolymph of the PtA2M-1- or PtA2M-2-silenced crabs. In addition, PtA2M-1 or PtA2M-2 could regulate the expression of antimicrobial peptide (AMP) genes (PtALF1-3, PtCrustin1 and PtCrustin3) through the Toll and NF-κB pathways.

Thioester-containing proteins (TEPs) are an ancient superfamily of secreted effector proteins. In the present study, a TEP gene, designated as PtTEP, was identified from P. trituberculatus. The open reading frame (ORF) of PtTEP was 4,434 bp in length, encoded a polypeptide with 1,478 amino acids containing the conserved sequence features of insect TEP. PtTEP was highly expressed in most immune-related tissues, such as intestine, gill and hepatopancreas, and the expression of PtTEP was significantly upregulated in hemocytes after bacterial and fungal challenges. Compared to that in fertilized eggs, the transcripts of PtTEP showed up-regulated in the blastula, gastrula and heart beating stages. RNA interference-mediated suppression of PtTEP could significantly increase the expression of proPO-associated genes (PtproPO and PtPPAF) and serine protease related genes (PtcSP1-3 and PtSPH) but lead to a lower expression of PtLSZ and phagocytosis-related genes (PtMyosin and PtRab5). These results were further supported by the PO and lysozyme activities in hemolymph of the PtTEP-silenced crabs. Moreover, silencing of PtTEP reduced the expression of AMP genes and genes involved in activation of the Toll and NF-κB pathways and additionally increased the mortality of V. alginolyticus infected crabs.

Mannose-binding lectin (MBL) is an essential pattern recognition receptor involved in activation of the complement lectin pathway. In this study, a novel MBL gene (PtMBL) was cloned from P. trituberculatus. The complete cDNA of PtMBL gene was 994 bp in length with an ORF of 777 bp. It encoded a protein with 259 amino acids containing the conserved carbohydrate-recognition domain (CRD) domain with QPD motif. PtMBL was highly expressed in most immune-related tissues, such as muscle, intestine, stomach and hepatopancreas, and the expression of PtMBL was significantly upregulated at early time after being challenged with bacteria and fungi. With the embryonic development, the transcripts of PtMBL were decreased significantly and reached to the lowest level at the heart-beating stage. Combined with high expression in ovary, it is suggested that PtMBL may be a maternal immune factor. Knockdown of PtMBL could significantly enhance the expression of PtTEP, proPO-associated genes (PtproPO and PtPPAF) and serine protease related genes (PtcSP1-3 and PtSPH), while the expression of PtA2Ms and phagocytosis-related genes (PtArp, PtMyosin and PtRab5) was significantly suppressed. The PO activities were increased significantly in hemolymph of the PtMBL-silenced crabs. Furthermore, silencing of PtMBL gene enhanced the expression of AMP genes and genes involved in activation of the Toll and NF-κB pathways.

The studies on complement-like molecules from the swimming crab indicated that they were as similar with vertebrate complement molecules in both structure and immune function. Research showed that PtgC1qR and PtMBL as PRRs were participated in activation of the primitive complement system through regulating C3-like molecules (PtA2M-1, PtA2M-2 and PtTEP), and the primitive complement system in P. trituberculatus may perform its immune function via the serine protease cascades, proPO-activating system, phagocytosis, as well as the synthesis and secretion of AMP mediated by Toll and NF-κB immune pathways. Moreover, complement-like molecules may be maternally-derived immune factors to participate in the immune defense response to pathogens. Taken together, our results indicated that the complement-like molecules played an important role in the innate immune defense of the swimming crab. Meanwhile, it provided new clues and theoretical support for further understanding the evolution of complement system in invertebrates and systematically investigating the function of primitive complement system.