The interface between tapasin and MHC class I: identification of amino acid residues in both proteins that influence their interaction

Prior to the binding of antigenic peptide, a complex of chaperone proteins associates with the Major Histocompatibility Complex (MHC) class I heavy chain/β₂m heterodimer. Although each dornain of the MHC class I heavy chain contains amino acid resid uses that influence chaperone binding, there are several pieces of evidence that point to an interaction between the MHC clas 1α2/α3 domains and tapasin. In egard to the site on tapasin involved in the tapasin/MHC interface, we have found that a particular region of tapasin (containing amino acid residues 334–342) is necessary for the binding of tapasin to the MHC class I heavy chain. Our results also indicate that amino acids in this region of tapasin also affect the proportion of MHC class I open forms expressed at the cell surface and MHC class I egress from the endoplasmic reticulurn. Based on these results and those obtained by other laboratories, a model for MHC class I/tapasin interaction is proposed.     

Lymphoblastoid cells express HLA-B27 homodimers both intracellularly and at the cell surface following endosomal recycling

The MHC class I allele HLA-B27 is very strongly associated with development of autoimmune spondyloarthritis, although the disease mechanism remains unknown. Class I molecules classically associate in the endoplasmic reticulum (ER) with beta2-microglobulin (beta(2)m) and antigenic peptides for cell surface expression and presentation to T cells. We have previously shown that HLA-B27 is capable of forming beta(2)m-free disulfide-bonded homodimers in vitro. Here we show that HLA-B27 forms disulfide-bonded homodimers in vivo by two distinct pathways. HLA-B27 homodimers form in the ER but appear unable to egress to the cell surface in human cells. Cell surface HLA-B27 homodimers are abundantly expressed in a variety of lymphoid cell lines. Experiments with inhibitors indicate that HLA-B27 homodimers can arise from cell-surface heterodimers via an endosome-dependent recycling pathway. HLA-B27 homodimer expression on the cell surface of 721.220 is dependent on the unpaired cysteine(67) and is inhibited by restoration of tapasin function or by incubation with peptides that bind strongly to HLA-B27 heterodimers. Cell surface expressed HLA-B27 homodimers are likely to be immunologically reactive ligands for NK family immunoreceptors and, hence, could play a pathogenic role in spondyloarthritis.     

Tapasin对HLA-E细胞表面表达的影响

目的 :探索伴侣分子Tapasin对HLA E分子细胞表面表达的影响。方法 :体外构建HLA EcDNA的逆转录病毒表达载体 ,并通过感染的方法将其转入Tapasin阴性的T2细胞系 ,利用流式细胞术检测HLA E分子在靶细胞表面的表达情况。结果 :外源HLA E基因在Tapasin阴性的T2细胞表面获得表达 (74 13% ) ,而对照细胞及经空载体转染的T2细胞表面未检测到HLA E分子的表达。结论 :HLA E分子的细胞表面表达也存在TAP非依赖的方式。     

CTP-HBcAg18-27-Tapasin融合蛋白胞内转导功能的检测

目的:观察融合蛋白CTP-HBcAg18-27-Tapasin在抗原提呈细胞内的转导功能.方法:体外分离培养近交系BALB/c小鼠髓源性DC,加入重组粒细胞-巨噬细胞集落刺激因子和白细胞介素-4培养5d,再加入脂多糖诱导DC成熟.不同剂量的CTP-HBcAg18-27-Tapasin及RP-MI-1640培养液加入细胞培养介质中,激光共聚焦显微镜下观察免疫荧光在细胞内的分布及定位,并对荧光强度进行定量分析,进一步以Western blot观察不同组细胞中Tapasin表达的差异来检测CTP-HBcAg18-27-Tapasin的转导效率.结果:成功体外诱导培养并鉴定小鼠骨髓源性树突状细胞,免疫荧光法证实CTP-HBcAg18-27-Tapasin能够穿透树突状细胞膜进入细胞质,而不能进入细胞核,且荧光强度50 μg/L CTP-HB-cAg18-27-Tapasin组依次高于10 μg/L CTP-HBcAg18-27-Tapasin组和空白组,Western blot 也证实CTP-HBcAg18-27-Tapasin能够穿透细胞膜进入树突状细胞,结果显示差异有统计学意义.结论:CTP-HBcAg18-27-Tapasin具有穿透树突状细胞膜定位于胞浆的能力.     

Tapasin Decreases Immune Responsiveness to a Model Tumor Antigen

The T-cell response against cancer is dependent on the cell surface presentation of tumor-associated or tumor-specific peptides by major histocompatibility complex (MHC) class I molecules. We found that tapasin, a chaperone protein that normally assists in the assembly of MHC class I molecules, is undetectable in an unstimulated pancreatic tumor cell line, Panc02, and only very weakly expressed after -interferon stimulation. Transfection of tapasin into the Panc02 cells did not quantitatively increase MHC class I surface expression or detectably affect MHC class I association with peptide and ₂-microglubulin (₂m). However, we found that transfected tapasin downregulated immune reactivity against a model tumor antigen, MUC1. Although tapasin has been previously shown by others to increase immune recognition of particular antigens, our results suggest that tapasin has a negative impact on the presentation of an immunodominant epitope from a specific model tumor antigen.     

分子伴侣Tapasin在介导特异性细胞毒性T淋巴细胞反应中的作用

病毒及肿瘤细胞的清除很大程度上依赖于特异性CD8＋细胞毒性T淋巴细胞（CTL）,CTL通过T细胞受体（TCR）特异性识别病毒肽段并通过MHC-I类分子提呈引起感染细胞融解,经典MHC-I类分子在内质网内的装配要求有抗原肽配体和β2微球蛋白（β2m）的存在,而这一过程需要伴侣分子（chaperones）如Tapasin的参与。Tapasin是抗原递呈相关转运体（TAP）相关蛋白的基因产物,与MHC-I类分子同为免疫球蛋白超家族成员,在介导特异性CTL反应中有着重要作用,本文简述了Tapasin分子结构特征、在介导特异性CTL反应中的作用及与疾病的联系。     

分子伴侣Tapasin在介导免疫反应中的作用

分子伴侣Tapasin是抗原提呈相关转运蛋白的基因产物,和MHC-Ⅰ同为免疫球蛋白超家族的成员,与MHC分子和多肽装配密切相关,是MHC-Ⅰ类分子与TAP之间的桥梁.Tapasin在抗原提呈过程中有着重要作用,因而了解其生物学特征、在介导免疫反应中的作用及与疾病的相关性具有重要意义.     

The role of tapasin in MHC class I protein trafficking in embryos and T cells

Preimplantation mouse embryos express both classical (class Ia) and nonclassical (class Ib) MHC class I proteins, and yet are not rejected by the maternal immune system. Although the function of the embryonic MHC class Ia proteins is unknown, one MHC class Ib protein, Qa-2, the product of the preimplantation embryo development (Ped) gene, actually enhances reproductive success. Similar in structure to MHC class Ia proteins, Qa-2 protein is a trimer of the alpha (heavy) chain, β₂ microglobulin and a bound peptide. Studies on the folding, assembly and trafficking of MHC class Ia molecules to the cell surface have revealed this process to be dependent on multiple protein chaperone molecules, but information on the role of chaperone molecules in Qa-2 expression is incomplete. Here, we report the detection of mRNA for four chaperone molecules (TAP1, TAP2, calnexin and tapasin) in preimplantation embryos. We then focused on the role of the MHC-dedicated chaperone, tapasin, on Qa-2 protein expression. First, we demonstrated that tapasin protein is expressed by preimplantation embryos. Then, we used tapasin knockout mice to evaluate the role of tapasin in Qa-2 protein expression on both T cells and preimplantation embryos. We report here that optimal cell surface expression of Qa-2 is dependent on tapasin in both T cells and preimplantation embryos. Identification of the molecules involved in regulation of MHC class I protein expression in early embryos is an important first step in gaining insight into mechanisms of escape of embryos from destruction by the maternal immune system.     

胞质转导肽-HBcAg18-27-Tapasin体外诱导小鼠髓源性树突状细胞成熟及在T淋巴细胞增殖中的作用

目的 观察融合蛋白胞质转导肽(CTP) -HBcAg18-27 -Tapasin诱导体外培养的小鼠髓源性树突状细胞(DC)成熟和对T淋巴细胞增殖的作用.方法 体外分离、培养近交系BALB/c小鼠髓源性DC,加入重组粒细胞-巨噬细胞集落刺激因子和IL-4培养5d,再加入脂多糖诱导成熟.10 μg/L CTP HBcAg18-27-Tapasin、50 μg/L CTP-HBcAg18-27-Tapasin、10 μg/L CTP-HBcAg18-27及RPMI-1640培养液加入培养介质.流式细胞术测定DC表面分子表达,ELISA法测定DC培养上清液中的IL-12p70的水平,细胞计数试剂盒检测T淋巴细胞增殖反应,流式细胞术检测增殖的T淋巴细胞内的细胞因子.多个样本均数间的比较采用单因素方差分析,组间两两比较采用LSD法.结果 成功体外诱导小鼠髓源性DC; CTP-HBcAg18-27Tapasin能明显上调DC表面分子CD80、CD86及主要组织相容性复合体Ⅰ分子的表达;50 μg/L CTP-HBcAg18-27-Tapasin组诱导DC分泌的IL 12p70水平为(61.12±10.25)pg/mL,依次高于10μg/LCTP-HBcAg18-27 -Tapasin组的(50.43±10.42) pg/mL、10μg/L CTD HBcAg18-27组的(40.17±8.54) pg/mL和空白组的(30.51±8.03) pg/mL(F=15.85,P=0.030和P=0.037);CTP HBcAg18-27-Tapasin诱导DC刺激T淋巴细胞增值能力明显高于对照组;流式细胞仪检测融合蛋白诱导的CTL水平50 μg/L CTP-HBcAg18-27-Tapasin组为(2.05±0.41)％、10 μg/L CTP-HBcAg18-27-Tapasin组为(1.06±0.10)％,高于10 μg/L CTP-HBcAg18-27组的(0.45±0.11)％和空白组的(0.09±0.02)％(F- 60.22,P=0.003).结论 CTP- HBcAg18-27-Tapasin可以促进DC的分化、成熟,增强DC刺激T淋巴细胞增殖能力并能增加CTL的表达.