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长牡蛎免疫球蛋白超家族成员结构和功能的分析
其他题名The structural and functional study of the immunoglobulin superfamily members in Crassostrea gigas
刘聪辉
学位类型博士
导师宋林生
2016-05-13
学位授予单位中国科学院大学
学位授予地点北京
学位专业海洋生物学
关键词长牡蛎 免疫球蛋白超家族 “选择适应性免疫” 细胞黏附分子 Siglec
摘要免疫球蛋白超家族(IgSF)是所有含有免球蛋白样三维拓扑结构的蛋白分子的群体集合。脊椎动物中含有基于免疫球蛋白的适应性免疫,无脊椎动物虽然缺乏高度可变的免疫球蛋白,但已有研究表明无脊椎动物中具有类似适应性免疫的“选择适应性免疫”(alternative adaptive immunity),无脊椎动物中的免疫球蛋白超家族(IgSF)成员分子可能在无脊椎动物的“选择适应性免疫”应答中发挥重要作用。目前对于软体动物IgSF成员结构和免疫功能的了解非常有限。本文采用生物信息学、分子生物学、免疫学等手段,鉴定并分析了长牡蛎IgSF分子的结构和进化特征,探讨了IgSF分子在长牡蛎个体发育和免疫微生物(河口弧菌、溶藻弧菌、塔氏弧菌、灿烂弧菌和鳗弧菌)和PAMPs(LPS、PGN、GLU和poly(I:C))刺激的表达模式,查明了几种IgSF成员分子(CgJAM-A-L、CgSiglec-1和CgNCAM)的结构与免疫学功能。
通过结构域分析,从长牡蛎中共鉴定得到了268个IgSF成员分子, 分别含有1-36个Ig 结构域,其中162个基因由Ig结构域串联重复而成,而其余106个基因(39.6%)则由Ig结构域与其它结构域重组而成。注释分析提示长牡蛎IgSF主要具有识别和粘附的功能,其中共有217个IgSF成员分子被预测具有识别和结合的功能。HMM-3D功能聚类的分子结果对GO注释的结果做了进一步的补充,将部分在Blast2GO软件中未能成功注释基因与其他明确注释基因聚类到了一起。根据已报道海洋动物基因组中IgSF的进化分析发现,IgSF家族体现出以海绵动物、棘皮动物和半索动物为节点的阶段性扩张趋势。
转录组分析发现PAMPs和弧菌刺激后表达量显著升高的IgSF分子数量分别达到116和166个,提示长牡蛎IgSF可能在免疫应答反应中发挥重要功能。发现大量的IgSF在免疫响应中具有高度的特异性,且含较少Ig结构域的IgSF分子功能多样性更为丰富,表明长牡蛎IgSF在进化扩张过程中存在结构和功能的分化。在灿烂弧菌刺激的转录组分析中筛选获得大量的IgSF单碱基突变和可变剪接,并且有大量突变位点具备对灿烂弧菌二次刺激的特异性,暗示长牡蛎IgSF可能通过点突变和可变剪接产生多样性,且点突变形成的新个体变异型可能参与 “选择适应性免疫”应答。另外通过分析长牡蛎不同时期的转录组数据发现,部分长牡蛎IgSF能够在特定发育时期上调表达,可能参与母源免疫和卵裂期、胚胎期、幼虫期和稚贝型的发育调控。
非跨膜型的CgJAM-A-L是在长牡蛎中发现的一个JAM家族分子同源基因,含有三个串联的I-set型Ig 结构域。它能够识别并结合多种PAMPs(LPS、PGN、MAN、LTA、poly(I:C)和GLU)和微生物(灿烂弧菌、鳗弧菌、大肠杆菌、金黄色葡萄球菌、藤黄微球菌、耶罗维亚酵母和毕赤酵母),并在长牡蛎的固有免疫应答中发挥调理作用。同时CgJAM-A-L的结构进化信息表明了JAM家族分子与抗体分子可能具有一个多功能的“祖先基因”,并在进化中实现了功能的特异性分化,从而分别形成JAM和抗体分子。跨膜型的CgSiglec-1基因包含两个Ig结构域和两个胞内的受体酪氨酸抑制基序(ITIM),并在长牡蛎的免疫识别和诱导下游级联反应中发挥连接作用。它在各组织中广泛分布,并且血淋巴细胞中的转录本能够在灿烂弧菌刺激后显著地上调。CgSiglec-1能够结合多种PAMPs(pSIAS、LPS和PGN),并且通过抑制下游信号通路,在血淋巴细胞的凋亡、吞噬和细胞因子的释放中发挥调节作用。跨膜型的CgNCAM包含五个Ig结构域、两个FnIII结构域和一个跨膜区,并可能在长牡蛎的固有免疫中发挥模式识别作用。它与一种植物的凝集素具有较高的相似性,并且在微生物刺激后,血淋巴细胞中的mRNA能够显著上调。重组的rCgNCAM蛋白具有对多种PAMPs(LPS、GLU和MAN)和微生物(灿烂弧菌、鳗弧菌、大肠杆菌、藤黄微球菌、耶罗维亚酵母和毕赤酵母)的广谱识别能力。同时CgNCAM还可能在长牡蛎Extracellular traps的形成中发挥重要的作用。三中不同类型的IgSF共同说明长牡蛎IgSF的主要功能是识别和粘附,游离型的IgSF可能发挥调理分子的作用,而跨膜型IgSF能够作为抗原分子的表面受体并调节下游的免疫通路。
综上所述,长牡蛎中的IgSF数目众多,种类丰富,并能够在免疫应答中发挥重要作用。长牡蛎IgSF可能通过点突变和可变剪接产生基因的多样性,且新形成的个体变异型可能参与“选择适应性免疫”应答。IgSF成员分子CgJAM-A-L、CgSiglec-1和CgNCAM能识别结合多种外源微生物,并分别发挥调理分子、免疫调节分子和表面受体的作用。该结果探索了无脊椎动物中IgSF分子与高等动物的异质性,初步揭示无脊椎动物IgSF分子在“选择适应性免疫”中的作用,为进一步研究IgSF分子的进化提供了分子生物学证据,丰富和发展了海洋无脊椎动物免疫学内容。
其他摘要
Immunoglobulin superfamily (IgSF) is a multiple collection of proteins containing one or more immunoglobulin (Ig) domains, which performs a wide range of functions in various fundamental processes, such as immune response, neural cell development, anti-tumoral action, and simply in cell adhesion. The vertebrate possessed adaptive immunity based on immunoglobulin. There is no immunoglobulin found in invertebrate, but the IgSF members in invertebrate can potentially generate tens of thousands of protein isoforms, suggesting their function as an alternative adaptive immunity mechanism. Although alternative adaptive immunity of oysters was evidenced in previous reports, the knowledge of structure and function of invertebrate IgSF is still limited. In the present study, the structure characteristics, expression profile and phylogeny relationship of IgSFs, as well as their roles in alternative adaptive immune response and developmental stages in oysters were investigated using bioinformatics methods. Several members of IgSF (CgJAM-A-L, CgSiglec-1 and CgNCAM) were further characterized in the immune response of oyster using immunological methods.
A total of 268 IgSF members containing 1-36 Ig domains were identified from the oyster genome by domain analysis, and 162 members were composed by adjacent Ig domains while the others were mixer with multiple kinds of domain. The Gene Ontology annotation implies that the primary functions of IgSF in oyster were recognition and adhesion under the circumstance that 217 members were predicted with a recognition and binding role. A functional cluster based hidden Markov model algorithm was employed to classify the IgSF and to further predict genes miss-annotated in Gene Ontology. Additionally, IgSF was characterized of a phase-wisely expansive pattern at the evolutionary status of sponges, echinoderm and hemichordate.
The IgSF members play important roles in the immune response of oyster. The expression level of 116 and 166 members were significantly up-regulated post the PAMPs and microorganisms challenge, respectively. A functional divergence was supposed during the evolution of oyster IgSF, since plenty of members displayed highly specify to various challenge. Meanwhile more varied functional roles were found within the members containing less Ig domains. Several members can undergo alternative splicing and single nucleotide mutation, potentially generating tens of thousands of protein isoforms after the challenge of Vibrio splendidus. Thereinto, abundant specific single nucleotide mutation was verified upon the secondary challenge of V. splendidus, suggesting a potential mechanism of the alternative adaptive immunity. Additionally, the IgSF members were up-regulated in specific developmental stages, suggesting they may also function in the development and maternal immunity of oyster.
CgJAM-A-L, containing three I-set Ig domains, was identified as an evolutionary primitive structural homolog of JAM-A from oyster,endowed with the binding and recognizing capability to a variety of PAMPs (LPS、PGN、MAN、LTA、poly(I:C) and GLU) and microorganisms (Micrococcus luteus, Staphylococcus aureus, Escherichia coli, V. anguillarum, V. splendidus, Pastoris pastoris and Yarrowia lipolytica) as well as opsonic function. It is involuntarily to suspect that there would be a lineage-specific evolution pattern from one comprehensive and versatile precursor to several specific and efficient ramified molecules of JAMs and immunoglobulins. CgSiglec-1, containing two I-set Ig domains and cytosolic immunoreceptor tyrosine-based inhibitory motifs (ITIMs), could act as a bridge molecule between invader recognition and immune response activation. It displayed a ubiquitous expression pattern in all the selected tissues and was up-regulated by the challenge of V. splendidus in hemocytes. Purified rCgSiglec-1 exhibited a wide binding spectrum to PAMPs (pSIAS, LPS and PGN) and could modulate the immune response including apoptosis, phagocytosis and the release of cytokines via the inhibition of signal transduction. CgNCAM could act as a pattern recognition receptor (PRR) involved in the innate immunity of oyster. It shared a close evolutionary relationship with a plant lectin and its mRNA transcripts significantly increased after microbe stimulation. The purified rCgNCAM protein exhibited a wide binding spectrum to PAMPs (LPS、GLU and MAN) and microorganisms (M. luteus, E. coli, V. anguillarum, V. splendidus, P. pastoris and Y. lipolytica). And it was also involved in the formation of Extracellular traps (ETs) in oyster hemocytes. The functional analysis of three IgSF members collectively indicated that recognition and binding might be the main function of IgSF in oyster. The dissociative IgSF might function as opsonin, while the transmembrane IgSF could take role as pathogen recognition receptor and activator of the downstream immune response.
 
All the results suggested that oysters possess an abundant and diverse collection of IgSF members which act crucial roles in the immune response. Several IgSF members potentially function in alternative adaptive immunity upon the secondary challenge of V. splendidus. Three IgSF members including CgJAM-A-L, CgSiglec-1 and CgNCAM were involved in oposonization, immunoregulation and immunoagglutination of oysters, respectively. The results contribute to exploring the heterogeneity of
学科领域生物学
语种中文
文献类型学位论文
条目标识符http://ir.qdio.ac.cn/handle/337002/116992
专题实验海洋生物学重点实验室
作者单位中国科学院海洋研究所
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刘聪辉. 长牡蛎免疫球蛋白超家族成员结构和功能的分析[D]. 北京. 中国科学院大学,2016.
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