Karp D R, Teletski C L, Jaraquemada D, Maloy W L, Coligan J E, Long E O
Laboratory of Immunogenetics, National Institute of Allergy and Infectious Diseases, National Institutes of Health, Bethesda, Maryland 20892.
J Exp Med. 1990 Mar 1;171(3):615-28. doi: 10.1084/jem.171.3.615.
To test for the assembly of human MHC class II molecules having an alpha chain from one isotype (HLA-DR, -DQ, or -DP) and the beta chain of another (mixed isotypic pairs), murine fibroblasts were transfected with expressible cDNAs encoding the different class II alpha and beta chains. A rapid and efficient transient transfection system was developed using a polyoma virus-based vector. Typically, 30-50% of cells transfected using this system expressed high levels of class II molecules on their surface, but only with matched isotypic pairs. Biochemical analysis of cells transfected with matched or mixed isotypic pairs of the DR and DP molecules revealed that only matched chains could pair efficiently inside the cell. Thus, the lack of expression of the two mixed isotypic pairs is due to inefficient primary assembly of the class II molecule and not to a processing or transport defect. To define what region of the beta chains controlled their assembly with alpha chains, a series of chimeric cDNA molecules containing both DR and DP beta chain sequences were constructed. Expression of these chimeric beta chains with DR and DP alpha chains was determined by cytofluorimetry and biochemical analysis. Both alpha chains paired with beta chains in which only the beta 1 domain was isotypically matched. In contrast, the pattern of expression of chimeras made at other points within the beta 1 domain was different for DR and DP. These data show that different areas of primary sequence are important for the assembly of different human class II isotypes, and suggest that HLA-DR and -DP molecules have different secondary or tertiary structures in their NH2-terminal domains.
为了检测具有来自一种同种型（HLA-DR、-DQ或-DP）的α链和另一种同种型的β链（混合同种型对）的人MHC II类分子的组装情况，将编码不同II类α链和β链的可表达cDNA转染至鼠成纤维细胞。使用基于多瘤病毒的载体开发了一种快速高效的瞬时转染系统。通常，使用该系统转染的细胞中有30%-50%在其表面高水平表达II类分子，但仅在匹配的同种型对内如此。对用DR和DP分子的匹配或混合同种型对转染的细胞进行生化分析发现，只有匹配的链才能在细胞内有效配对。因此，两种混合同种型对缺乏表达是由于II类分子的初级组装效率低下，而不是由于加工或运输缺陷。为了确定β链的哪个区域控制其与α链的组装，构建了一系列包含DR和DPβ链序列的嵌合cDNA分子。通过细胞荧光测定法和生化分析确定这些嵌合β链与DR和DPα链的表达情况。两条α链均与仅β1结构域同种型匹配的β链配对。相比之下，在β1结构域内其他位点构建的嵌合体的表达模式对于DR和DP是不同的。这些数据表明，一级序列的不同区域对于不同人II类同种型的组装很重要，并表明HLA-DR和-DP分子在其NH2末端结构域具有不同的二级或三级结构。
