Hedley M L, Ozato K, Maryanski J, Tucker P W, Forman J
Graduate Program in Immunology, University of Texas Southwestern Medical Center, Dallas 75235.
J Immunol. 1989 Aug 1;143(3):1026-31.
Mutation of the 3' splice sites bordering exon 2 of the H-2Dd and H-2Kd genes generated alternatively spliced transcripts when the constructs were transfected into L cells (J. Immunol. 143:1018). The H-2Dd transcripts contained an additional 84 nucleotides derived from the first intervening sequence, whereas 60 extra bases were included in the H-2Kd mRNA. Proteins derived from these transcripts were recognized by mAb. Moreover, both Ag served as recognition elements for CTL, and the mutant H-2Kd molecule functioned as a restricting element for an Ag peptide. As a result of alternative splicing, the mutant proteins should have additional residues at their NH2 termini to increase their lengths by 28 (Dd) or 20 (Kd) amino acids. Immunoprecipitation and analysis on SDS-PAGE demonstrated that the mutant H-2Kd molecule was indeed larger than the normal H-2Kd protein, but the mutant and wild-type H-2Dd Ag were the same size. In addition, treatment of H-2Dd mutant and normal Ag with N-glycanase produced molecules of equal size, demonstrating that the mutant protein was completely glycosylated. Limited amino acid sequencing of this Ag indicated that it was normal H-2Dd. Therefore, before its transfer to the cell surface, post-translational modifications remove the additional NH2-terminal residues of the mutant Dd but not Kd protein.
当将构建体转染到L细胞中时，H-2Dd和H-2Kd基因外显子2边界处的3'剪接位点发生突变，产生了可变剪接的转录本（《免疫学杂志》143:1018）。H-2Dd转录本包含来自第一个内含序列的另外84个核苷酸，而H-2Kd mRNA中包含60个额外的碱基。这些转录本衍生的蛋白质可被单克隆抗体识别。此外，这两种抗原都作为CTL的识别元件，并且突变的H-2Kd分子作为一种抗原肽的限制元件发挥作用。由于可变剪接，突变蛋白在其NH2末端应该有额外的残基，使其长度增加28个（Dd）或20个（Kd）氨基酸。免疫沉淀和SDS-PAGE分析表明，突变的H-2Kd分子确实比正常的H-2Kd蛋白大，但突变型和野生型H-2Dd抗原大小相同。此外，用N-糖苷酶处理H-2Dd突变体和正常抗原产生大小相等的分子，表明突变蛋白已完全糖基化。对该抗原进行有限的氨基酸测序表明它是正常的H-2Dd。因此，在其转移到细胞表面之前，翻译后修饰去除了突变的Dd蛋白但不是Kd蛋白的额外NH2末端残基。
