【作者】 孙明伟；

【导师】 高福；

【作者基本信息】 中国科学技术大学 ， 生物化学与分子生物学， 2014， 博士

【摘要】 主要组织相容性抗原复合体(MHC)是一种存在于脊椎动物中的由一个庞大的基因家族编码的细胞表面分子,介导白细胞,也就是常说的免疫细胞与其它白细胞或体细胞间的相互作用,在机体细胞间识别和区分自已与异己中扮演重要角色。MHC分子作为表达于个体细胞表面的抗原递呈结构,影响着不同来源抗原的T细胞反应。因而,MHC在一定程度上决定了个体对于感染物抗原的免疫应答,这其中也涉及到对疾病的易感性和自身免疫反应的发生。在人类中,MHC又叫做人类白细胞抗原(HLA)。在细胞内,经过抗原加工和蛋白酶体降解途径产生的多肽作为T细胞表位,由MHCI类分子递呈给细胞毒性T细胞(CTLs)。该过程在效应细胞免疫中发挥着重要的作用。另一方面发生了各种翻译后修饰(PTMs)的真核蛋白能够产生一系列修饰的抗原,丰富了MHC相关多肽的类型,递呈于细胞表面成为T细胞受体(TCR)识别的潜在目标。已有研究表明,一些包含翻译后修饰(如甲酰化、脱酰胺化、糖基化、乙酰化、磷酸化和半胱氨酸化)的多肽可以与各自的非修饰类似物被T细胞特异性地区分开。在某种情况下,发生于感染、炎症、细胞转化、凋亡和衰老中的翻译后修饰往往带来新的MHC相关新抗原的产生。因此,表位中的翻译后修饰以及它们在作为肿瘤和自身免疫疾病的免疫治疗疫苗候选中的应用价值受到越来越多的关注。N端乙酰化作为真核蛋白最普遍的翻译后修饰之一,能够产生一系列N端乙酰肽,并由MHC I类分子递呈于细胞表面。虽然这一修饰类型在调节蛋白质降解中扮演重要角色,然而N端乙酰肽的递呈及T细胞识别的分子基础仍然很不清楚。因此,我们解析了HLA-B39与一段天然N端乙酰化多肽的复合物高分辨率晶体结构,以及B39与非修饰多肽的复合物结构。区别于传统非修饰多肽中暴露于A口袋外部的P1残基侧链,乙酰化P1-Ser的羟基侧链则是插入抗原结合槽的A口袋中,而NH2端乙酰基团突出结合槽外参与T细胞识别。此外,N端乙酰化不仅改变了多肽的构象,还显著地造成了B39的α1-螺旋残基的构象摆动,而这可能将影响到TCR结合与免疫识别。N端乙酰肽和一系列人源和鼠源的MHC I类分子的热稳定性检测显示了其普遍降低的稳定性。以人工合成的N端乙酰化HLA-A2限制性表位免疫HLA-A2.1/Kb转基因小鼠激发的CTL应答也表明表位在N端乙酰化以后免疫原性会显著提高。这些发现首次洞察了经典的MHC I类分子对N端乙酰肽的特异性递呈模式,为基于乙酰化表位的免疫治疗和疫苗研发奠定了基础。在以上工作基础上,我们又以A型流感病毒表位为研究对象,从预测和鉴定N端乙酰化的流感病毒CTL表位出发,调查了流感病毒N端乙酰化抗原在健康人群中的T细胞免疫反应水平,进而评价流感病毒潜在N端乙酰化抗原的细胞免疫原性。研究中首次鉴定了病毒来源的N端乙酰化T细胞表位及其在健康个体中激发的特异性T细胞响应,为研究N端乙酰化表位特异性CTL免疫反应提供了试验依据,丰富了翻译后修饰的T细胞免疫表位的范畴,为理解翻译后修饰依赖性的T细胞免疫反应提供了帮助。除了N端乙酰化修饰外,磷酸化作为癌变、恶性转化和凋亡等生理活动的重要标识在肿瘤免疫治疗策略中的应用也越来越引人关注。在邻近两个位点的协同磷酸化又是磷酸化修饰组中最为常见的形式。因此,我们通过X-射线晶体学方法分别解析了一对(10肽和9肽)双磷酸化多肽与HLA-B27的pMHC复合物结构。其结果不但展示出双磷酸多肽不同于单个位点磷酸化多肽的独特递呈特征,而且在将双磷酸化多肽结构与单磷酸化多肽及未修饰多肽结构的比较中,还进一步阐明了自身抗原在经过修饰或蛋白质剪切等多重改变后作为新表位被递呈的分子基础。另外,通过圆二色谱检测,我们揭示了磷酸基团对于多肽结合力和pMHC稳定性具有磷酸化位点依赖性的影响。这些结果也为双磷酸肽这种新型翻译后修饰下的抗原加工递呈研究提供了首次结构学及热动力学依据,进一步预示了多位点磷酸化对于多肽免疫原性和T细胞识别中的潜在影响,揭示了多位点磷酸化形式的潜在T细胞表位亟待作为抗原鉴定中首要考虑和挖掘的目标。多位点磷酸肽有望成为治疗自身免疫病和癌症等的疫苗或药剂成分。除此之外,基于前人的研究结果,我们通过多肽免疫转基因小鼠的方法分别评价了7条A2限制性磷酸化表位的细胞免疫原性,客观评估了磷酸化多肽作为肿瘤疫苗的潜在应用价值。此外,研究还采用5’-RACE技术为免疫优势磷酸化特异性T细胞受体序列的获得提供了方法学依据。总之,通过免疫信息学、小鼠模型、细胞生物学和结构晶体学等方法,我们分别对MHCⅠ类分子递呈N端乙酰化和磷酸化多肽提供了免疫学及结构学调查和评价,展示了同一来源的抗原经添加基团及蛋白质剪切等修饰成为多重改变的自身抗原的面貌和机制,揭示了这些翻译后修饰多肽作为MHCⅠ类抗原的递呈机制及其在T细胞识别中的潜在影响,进而探讨了其在免疫治疗和疫苗设计中的应用价值。更多还原

【Abstract】 Major histocomaptibility complex (MHC) is a tightly linked cluster of genes in vertebrate, whose products are the cell surface molecules that plays roles in intercellular recognition and in discrimination between self and nonself. The particular set of MHC molecules as antigen-presenting structures expressed by an individual influences the repertoire of antigens to which that T cells can repond. Therefore, the MHC partly determines the immune response of an individual to antigens of infectious organisms, and it has been implicated in the susceptibility to disease and in the development of autoimmunity. In humans, the MHC is referred to as the human leukocyte antigen (HLA) complex.Polypeptides produced by antigen processing and proteasomal degradation of intracellular proteins serve as cytotoxic T lymphocyte (CTL) epitopes presented by MHC class I molecules, which play a critical role in effective cellular immunity. Eukaryotic proteins bearing various post-translational modifications (PTMs) can generate a class of modified antigens, which contribute to a special repertoire of MHC-associated peptides presented at the cell surface as potential targets for T-cell receptor (TCR)-mediated recognition. Evidence suggests that peptides containing PTMs, including formylation, deimination, glycosylation, acetylation, phosphrylation, cysteinylation, contribute to the pool of MHC-bound peptides presented at the cell surface and represent potential targets for T cell recognition. Indeed, most naturally occurring peptides bearing PTMs can be discriminated from their unmodified homologs specifically by T cells. In some cases, quantitative and qualitative changes in PTM that occur during infection, inflammation, cellular transformation, death and aging result in the display of new MHC-associated neoantigens. The significance of PTMs upon epitopes and their use as vaccine candidates for the immunotherapy of cancer and autoimmune diseases have been increasingly appreciated.As one of the most common PTMs of eukaryotic proteins, Nα-terminal acetylation generates a class of Nα-acetylpeptides that are presented by MHC class I at the cell surface. Although such PTMs play a pivotal role in adjust the proteolysis, the molecular basis for the presentation and T-cell recognition of Na-acetylpeptides retains largely unknown. Herein, we determined a high-resolution crystallographic structure of HLA-B39complexed with an Nα-acetylpeptide derived from natural cellular processing, and also the complexes of B39with unmodified peptides. Unlike the NH2-free PI residues of unmodified peptides, the hydroxyl side chain of the acetylated Pl-Ser inserts into pocket A of the antigen-binding groove, and the Na-linked acetyl protrudes out of the groove for the T-cell recognition. Moreover, the Nt-acetylation not only alters the conformation of the peptide, but also notably switches the residues in the α1-helix of B39, which may impact the T-cell engagement. The thermostability measurements of complexes between Nα-acetylpeptides and a series of MHC class I molecules derived from human and murine revealed universal diminished stabilities. The CTL responses stimulated by synthetic Nt-acetylated HLA-A2restricted epitopes in HLA-A2.1/Kb transgenic mice suggested immunogenic enhancement after the Nt-acetylation of epitope. These findings provided the first insight into the mode of Nα-acetylpeptide-specific presentation by classical MHC class I molecules and shed light on the potential of acetylepitope-based immune intervene and vaccine development.With above understanding, to investigate the CTL-based cellular immunity against Nt-acetylated antigen of influenza virus, we used influenza A virus as the object of study, started with the prediction and identification of Nt-acetylated CTL epitopes derived from influenza virus, thereby evaluated their immunogenicities. In this study, we firstly identified an Nt-acetylated viral CTL epitope which stimulates specific T-cell responses in HLA-A24+healthy donors. This result contributed to the category of MHC-bound CTL epitopes, and provided support to understand the PTM-dependent T celluar immunity.Besides Nt-acetylation, phosphorylation as a signature of cancer, malignant transformation, and apoptosis has been regarded increasingly. And phosphorylation at two proximal sites is a common format in phosphoproteome. Thus, we solved the structures of a couple of diphosphorylated peptides (a10mer and a9mer) complexed with HLA-B27, respectively. These data clarified not only the particular features of diphosphopeptides other than peptides containing a single phosphate displayed by MHC class I, but also the molecular basis of the presentation of self antigens which serve as neoepitopes after the multiple altering of PTMs and protein slicing. In addition, we showed the phosphosite-dependent effect of phosphates on peptide binding affinity by using circular dichroism spectroscopy. Our work provided the first-ever structural and thermodynamic insights into the presentation of diphosphopeptides by MHC class I, and highlighted a potential influence of such special modification event on antigenic identity and TCR engagement. Multisite phosphopeptides represent a library of new potential drug and vaccine candidates against autoimmune disease and tumor require further exploration.Moreover, based on previous reports, we surveyed the celluar immunogenicity of seven HLA-A2restricted phosphopeptides via inoculation of HLA-A2transgenic mice with peptides, and objectively evaluated the value of phosphopeptides which be applied as potential tumor vaccines. Furthermore, we also used5’-RACE system to obtain the sequences of phosphopeptide-specific TCR repertoires, which provided methodological approaches for further study in this area.In summary, in this study we made structural and immunological investigations on Nt-acetylated and phosphorylated peptides presented by MHC class I molecules using the method combination of immunoinformatics, mouse-model, cell biology, and structural crystallography. Our results revealed the aspect and mechanism of antigens that function as’multi-altered self’ through the PTMs of adding groups and protein slicing, and suggested the potential influence of the PTMs on antigen presentation and T-cell recognition. The application values of such antigens containing PTMs in immunotherapy and vaccine design are discussed.更多还原