Koeller D, Lieberman R, Miyazaki J, Appella E, Ozato K, Mann D W, Forman J
J Exp Med. 1987 Sep 1;166(3):744-60. doi: 10.1084/jem.166.3.744.
We used site-directed mutagenesis to localize serologically defined (s) and CTL (c)-defined alloantigenic determinants to discrete amino acid sequences of a murine MHC class I antigen. Based on the prediction that amino acid position 63-73 of the H-2Dd antigen forms s-allodeterminants, the H-2Ld gene was mutated in a sequential fashion to replace codons for amino acid positions 63, 65, 66, 70, and 73 with those of the H-2Dd amino acids. Epitopes of the mutant antigens expressed in L-cells were examined by the binding of a series of mAbs specific for the H-2Dd antigen. The mutant antigen M66 had substitutions at residues 63, 65, and 66, and resulted in the acquisition of a number of H-2Dd-specific s-epitopes. Mutant M70 had an additional substitution at residue 70, which led to the gain of multiple additional H-2Dd s-epitopes. Together, more than half of all the relevant H-2Dd s-epitopes were mapped into amino acid position 63-70 of the H-2Dd molecule, which was expressed in the mutant H-2Ld gene. The final mutation at residue 73 (M73) caused no new epitope gains, rather, a few Dd s-epitopes acquired by the preceding mutations were lost. All of the H-2Ld-specific s-determinants were retained in the mutant molecules, as were H-2Dd s-determinants specific for the alpha-2 or alpha-3 domains. Changes of these residues affected c-determinants defined by CTL. Anti-H-2Dd CTL cultures and an anti-H-2Dd CTL clone recognized the mutant H-2Ld molecules, M66 and M70. Some CTL clones generated against the Q10d molecule, which has an identical sequence to H-2Dd between residues 61 and 73, failed to recognize native H-2Dd or Ld but did crossreact with mutant Ld. While bulk-cultured anti-H-2Ld CTL cultures reacted strongly against M73, bulk-cultured H-2Ld restricted anti-vesicular stomatitis virus CTL did not. Finally, at the clonal level two of three anti-H-2Ld CTL clones lost reactivity with some or all of these mutant molecules. From these results we conclude that a stretch of amino acids from position 63 to 70 of the alpha-1 domain controls major s- and c-antigenic sites on the H-2Dd antigen and c-sites on H-2Ld antigen.
我们运用定点诱变技术，将血清学定义的（s）和细胞毒性T淋巴细胞（CTL）定义的同种异体抗原决定簇定位到小鼠MHC I类抗原的离散氨基酸序列上。基于H - 2Dd抗原的氨基酸位置63 - 73形成s - 同种异体决定簇的预测，H - 2Ld基因以连续方式发生突变，用H - 2Dd氨基酸的密码子替换氨基酸位置63、65、66、70和73的密码子。通过一系列对H - 2Dd抗原特异的单克隆抗体（mAb）的结合，检测在L细胞中表达的突变抗原的表位。突变抗原M66在第63、65和66位残基处有替换，导致获得了一些H - 2Dd特异的s - 表位。突变体M70在第70位残基处有额外替换，这导致又获得了多个额外的H - 2Dd s - 表位。总之，所有相关的H - 2Dd s - 表位的一半以上被定位到在突变的H - 2Ld基因中表达的H - 2Dd分子的氨基酸位置63 - 70。第73位残基的最终突变（M73）没有导致新表位的获得，相反，先前突变获得的一些Dd s - 表位丢失了。所有H - 2Ld特异的s - 决定簇都保留在突变分子中，α - 2或α - 3结构域特异的H - 2Dd s - 决定簇也保留了下来。这些残基的变化影响了CTL定义的c - 决定簇。抗H - 2Dd CTL培养物和一个抗H - 2Dd CTL克隆识别突变的H - 2Ld分子M66和M70。一些针对Q10d分子产生的CTL克隆，其在第61和73位残基之间与H - 2Dd具有相同序列，不能识别天然的H - 2Dd或Ld，但能与突变的Ld发生交叉反应。虽然大量培养的抗H - 2Ld CTL培养物对M73反应强烈，但大量培养的H - 2Ld限制的抗水疱性口炎病毒CTL则不然。最后，在克隆水平上，三个抗H - 2Ld CTL克隆中的两个与这些突变分子中的一些或全部失去了反应性。从这些结果我们得出结论，α - 1结构域中从位置63到70的一段氨基酸控制H - 2Dd抗原上的主要s - 和c - 抗原位点以及H - 2Ld抗原上的c - 位点。
