树鼩MHC I类基因的特征及人剪切异构体HLA-A11ΔE4的功能研究
文献类型：学位论文
| 作者 | 张喜鹤 |
| 学位类别 | 博士 |
| 答辩日期 | 2015-07 |
| 授予单位 | 中国科学院研究生院 |
| 授予地点 | 北京 |
| 导师 | 郑永唐 |
| 关键词 | 主要组织相容性复合体 树鼩 选择性剪接 剪接异构体 二聚体 NK杀伤 |
| 其他题名 | Gene and Molecular characterization of the tree shrew (Tupaia belangeri) MHC class I gene and The function study of splicing variants of HLA-A11ΔE4 |
| 中文摘要 | 主要组织相容性复合体（MHC）I类分子在天然免疫和获得性免疫中都发挥着重要作用，不但可以通过识别自然杀伤细胞（Killer cell Ig-like receptor，NK）表面的杀伤细胞免疫球蛋白样受体（KIR）等抑制NK的活化而保护正常的细胞免受杀伤，还可以通过递呈抗原至相应的T淋巴细胞而启动下游免疫反应清除靶细胞。MHC I类分子是由具有高度多态性的重链（Haven chain：HC）和保守的轻链β2m（β2微球蛋白：β2-microglobulin）所组成，这类分子几乎表达在所有有核细胞的表面。成熟的MHC I 类分子的α1和α2结构域所组成的口袋可以识别一个8-13个氨基酸长度的抗原肽；α3结构域和β2微球蛋白起着支撑α1和α2结构域的作用。 鉴于在免疫系统中特殊的作用，MHC是在各种疾病进程的研究以及动物模型的建立中都不可忽视的重要的分子。目前对HLA的研究已经很透彻了，对非人灵长类动物MHC的研究也较多，包括恒河猴，食蟹猴以及平顶猴等常用的模型动物。树鼩作为新兴的动物模型，在免疫、视觉、神经和听觉系统的研究中应用越来越广，而目前对树鼩MHC I 类基因的研究非常少。有鉴于此，我们首次从树鼩体内分离出了全长的MHC I 类基因并通过分析揭示了树鼩MHC I类基因的基本结构和分子特征。我们发现与灵长类相比，树鼩MHC分子的CD8+ T细胞结合位点、N-糖基化位点和4个形成链内二硫键的半胱氨酸位点都很保守，说明这些分子可能具有与经典的MHC一样的空间结构，暗示这些分子很可能具有经典MHC分子的传统功能。通过分析我们发现树鼩MHC基因也表现出以下特性：1）高度的多态性；2）单元型遗传；3）连锁不平衡。值得注意的是，树鼩MHC分子与人和恒河猴相比相似度虽然很高，但是少了5个氨基酸：分别位于外显子2、5和6。基因组序列分析表明树鼩MHC分子也是由8个外显子和7个内含子组成，其中外显子长度与灵长类相似而内含子的长度有较大的差异。核酸序列分析还发现外显子2和3表现出了较高的多态性，其它位点则相对比较保守。我们还发现树鼩MHC I类分子存在各种形式的剪接异构体，其中所有的选择性剪接都发生在外显子2到4之间。将通过分析树鼩MHC分子与其他物种特别是人的同源性，揭示树鼩在进化上的地位，为更好地开发其做为动物模型奠定基础。 蛋白作为生命活动的执行者是由基因通过转录并经过翻译而产生的，通常情况下在由DNA向RNA转录时，由于剪接信号的存在，外显子会被选择性的保留下来而内含子会被选择性的切除，最后产生由基因组内各个外显子组成的RNA。而在非正常情况下会产生外显子被切掉或者内含子被保留的情况，称为选择性剪切（Alternative Spliced）。选择性剪切通常的定义是：前体RNA（pre-mRNAs）的外显子被选择性的切掉或者内含子被保留下来而产生了新的在结构和功能上不同于全长mRNA和蛋白的异构体。选择性剪切极大的增加了基因转录产物的数量并丰富了蛋白的多样性。 选择性剪切普遍存在于几乎所有的基因中：研究表明在人的基因组中大约90%的基因都存在不同形式的剪接异构体（Splicing Variants），在免疫系统和神经系统中，选择性剪切更是普遍存在。这些剪切异构体影响了几乎所有系统的功能。越来越多的证据表明选择性剪切与很多疾病的发生和进程有关联，尤其是自身免疫疾病。HLA作为人体内多态性最高的基因之一也存在多种形式的剪切异构体，但是到目前为止对HLA剪切异构体的研究主要集中在非经典的HLA-G上，而对经典的HLA分子的剪切异构体功能的研究还是空白。有鉴于此，我们分离得到了HLA I类分子的剪切异构体HLA-A11ΔE4。这个剪切异构体的α3结构域被选择性切掉，而α3结构域的缺失并没有影响开放阅读框(ORF)，其它外显子都可以正常编码蛋白。我们发现这个异构体定位在细胞膜上且不能结合β2m。进一步的研究还验证了这个异构体不但可以形成同源二聚体也可以与HLA-A11结合形成异源二聚体。HLA-A11ΔE4在表达HLA-A11的个体中普遍存在，其表达量不但在不同的刺激条件下差异明显，在PBMC中不同亚群中的表达量也有较大差别。重要的是HLA-A11ΔE4能抑制NK细胞对靶细胞的杀伤，而HLA-A11ΔE4与全长分子HLA-A11同时存在时能更有效地抑制NK的杀伤。最后我们还发现与HLA-A11一样，HLA-A11ΔE4抑制NK的杀伤是通过结合细胞表面的KIR3DL2分子，而其在胞内的信号传导也同HLA-A11一样通过MAPK和Akt信号通路。 |
| 英文摘要 | The major histocompatibility complex (MHC) class I molecules play a pivotal role in the adaptive immunity and innate immunity, which can not only regulate the activity of natural killer cell (NK) by intereacting with Killer cell Ig-like receptor (KIR), but also can present antigenic peptides to cytotoxic T lymphocytes (CTL) to clear the target cell. MHC I molecules are expressed on the surface of almost all nucleated cells, which made of a highly polymorphic heavy chain (HC) and a nonpolymorphic light chain β2-microglobulin (β2m). The groove formed by the α1 and α2 domains of the matured MHC class Imolecules can binds a peptide which as long as 8-13 amino acids. The function of α3 domain in extracellular region is associte with β2m and support theα1 and α2 domains. For the important functions in immune systerm, the genomic structure and molecular characterization of MHC class I genes have been thoroughly investigated in numerous mammals, for example, human and non-human primates: Macaca mulatta, crab-eating macaque and Macaca leonina. As a new modeled animals, tree shrews have increasingly been employed in a variety of ?elds, e.g. neurology, psychosocial stress, myopia, acoustic, and in particular immunology. To date, however, little is known about MHC class I genes in tree shrew. To address this de?ciency, we analyzed the structure and characteristic of the tree shrew MHC class I genes (Tube-MHC I) and performed a comparative gene analysis of the tree shrew and other mammal species. We found that among these peptides, the cysteines, CD8+ interaction and N-glycosylation sites are all well conserved, which implied that these moecules can perform the function of classic MHC class I molecules: present antigents. We also found three characters oin tree shrew MHC class I genes: highty polymorphism, Haplotype heredity and balancing selection. Otherwise, we found that all MHC class I genes showed 5 amino acid residues shorter than those in HLA-A or Mafa-A: one in exon 2, three in exon 5 and one in exon 6. Furthermore, the genomic sequence of the tree shrew MHC class I gene was identi?ed to be 3180 bp in length and had a similar exon–intron organization and exon length to humans, but the length of introns makes a great difference. The nucleic acid sequence analysis showed that the exon 2 and exon 3 are highly polymorphism and other sites are conserved. Besides, in tree shrews, we found that exon 2 and exon 4 manifested a high frequency of alternative splicing, with only parts of the region truncated instead of the entire exon. These ?ndings provide valuable contributions in furthering our understanding of the structure, molecular polymorphism, and function of the MHC class I genes in tree shrews, further improving their utility as an animal model in biomedical research. Alternative splicing (AS) is an important mechanism to increase protein diversity, which is ubiquitous in many of the genes, especially those dealing with the immune system. It have reported that Alternative splicing affects more than 90% of the genes in the human genome, which imply that AS are critical for normal functioning of virtually every system. Many reports have demonstrated that AS associte with various diseases, expecrally that autoimmune diseases. As the most polymorphism gene, HLA I transcripts have been reported to undergo alternative splicing by numerous investigators. However, the study of AS on HLA are focus on HLA-G, and little is known about the AS on classic HLA I gene. Here, we show that HLA-A11 can be alternatively spliced, generating a novel HLA-A11 isoform HLA-A11ΔE4 which devoid of α3 domain. For the entire α3 were deleted and α2 and transmembrane domain are complete, the deletion did not alter the open reading frame(ORF) which indicated that the transmembrane domain and the cytoplasmic domain are encoded and functional. We also demonstrated that HLA-A11ΔE4 reached the cell surface without β2-microglobulin (β2m). Furthernore, we demonstrated that HLA-A11ΔE4 proteins are more stable than HLA-A11 and not noly homodimerize but also forms heterodimer with HLA-A and HLA-B. The expression analysis showed that HLA-A11ΔE4 are high expression in monocytes, CD4+ and CD8+ cells, and in activated immune cells the expression level are much higher. In function study we prove that the splicing variants can inhibite NK Cytolysis to protects target cells from NK lysis by intraact with KIR3DL2. We further proved that HLA-A11ΔE4 inhibite degranulation and cytokines expression of NK cells. The transfer of inhibition signal of both HLA-A11ΔE4 and HLA-A11 are controled by MAPK and Akt Signaling pathway. |
| 语种 | 中文 |
| 源URL | [http://159.226.149.26:8080/handle/152453/10139]  |
| 专题 | 昆明动物研究所_分子免疫药理学 |
| 推荐引用方式 GB/T 7714 | 张喜鹤. 树鼩MHC I类基因的特征及人剪切异构体HLA-A11ΔE4的功能研究[D]. 北京. 中国科学院研究生院. 2015. |
入库方式： OAI收割
来源：昆明动物研究所
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