el-Zaatari F A, Sams K C, Taurog J D
Harold C. Simmons Arthritis Research Center, University of Texas Southwestern Medical Center, Dallas 75235.
J Immunol. 1990 Feb 15;144(4):1512-7.
The class I MHC molecule HLA-B27 bears an unpaired Cys residue at position 67, which is predicted to face the Ag binding pocket, based on the x-ray crystallographic model of HLA-A2. To investigate the potential of this residue in the antigenic structure of HLA-B27, a panel of 11 mutant HLA-B27 genes has been created, each bearing a separate amino acid substitution at position 67. The genes were transfected into mouse L cells and the resulting cells analyzed by cytofluorography with a panel of antibodies reactive with the wild-type B27 molecule. Although previous studies had indicated that all mAb that bound the B27 molecule on human lymphocytes bound comparably to L cells transfected with the wild-type B27 gene in the absence of h beta 2-m (human beta 2-microglobulin), the first of the mutant B27 genes was found to express several mAb epitopes in the presence but not in the absence of a h beta 2-m gene. Therefore, subsequent analysis of the B27 mutant panel was conducted in L cells coexpressing the h beta 2-m gene. Under these circumstances, all of the mutants bound the monomorphic anti-class I HLA mAb W6/32 and B.9.12.1, as well as the broadly polymorphic mAb B.1.23.2. Binding to the mutant transfectants of three anti-B27 mAb that cross-react with HLA-B7, ME1, GS145.2, and GSP5.3, was directly proportional to the size of the substituted amino acid side chain. The binding of another anti-B27 mAb, B27M2, that recognizes a B27 determinant that includes the region of amino acids 77-81, was not affected by the Cys67- greater than Tyr67 substitution. Rabbit antibodies to a synthetic peptide composed of B27 amino acids 61-84 bound to both the wild-type B27 and to the Tyr67 mutant. This binding, but not the binding of ME1 or B27M2, was inhibited by the synthetic peptide. These data are interpreted as suggesting that the large amino acid substitutions at position 67 induce a limited conformational change that disrupts the epitopes of the three anti-B27, B7 mAb, that are themselves at least partially conformational. The potential implications of these findings for the role of HLA-B27 in disease pathogenesis are discussed.
I类主要组织相容性复合体（MHC）分子HLA - B27在第67位带有一个未配对的半胱氨酸残基，根据HLA - A2的X射线晶体学模型预测，该残基面向抗原结合口袋。为了研究该残基在HLA - B27抗原结构中的潜在作用，构建了一组11个突变的HLA - B27基因，每个基因在第67位带有一个单独的氨基酸替换。将这些基因转染到小鼠L细胞中，并用一组与野生型B27分子反应的抗体通过细胞荧光术分析所得细胞。尽管先前的研究表明，在不存在人β2 - 微球蛋白（hβ2 - m）的情况下，所有与人淋巴细胞上的B27分子结合的单克隆抗体（mAb）与转染野生型B27基因的L细胞的结合相当，但发现第一个突变的B27基因在存在hβ2 - m基因时表达几种mAb表位，而在不存在该基因时不表达。因此，随后在共表达hβ2 - m基因的L细胞中对B27突变体组进行了分析。在这些情况下，所有突变体都能结合单态性抗I类HLA mAb W6/32和B.9.12.1，以及广泛多态性的mAb B.1.23.2。与HLA - B7交叉反应的三种抗B27 mAb（ME1、GS145.2和GSP5.3）与突变转染子的结合与取代氨基酸侧链的大小成正比。另一种抗B27 mAb B27M2识别一个包含氨基酸77 - 81区域的B27决定簇，其结合不受Cys67→Tyr67替换的影响。针对由B27氨基酸61 - 84组成的合成肽的兔抗体与野生型B27和Tyr67突变体都结合。这种结合，但不是ME1或B27M2的结合，被合成肽抑制。这些数据被解释为表明第67位的大氨基酸替换诱导了有限的构象变化，破坏了三种抗B27、B7 mAb的表位，这些表位本身至少部分是构象性的。讨论了这些发现对HLA - B27在疾病发病机制中的作用的潜在影响。
