Tatake R J, Zeff R A
Department of Pathology, University of Connecticut Health Center, Farmington 06030, USA.
Transplantation. 1995 May 15;59(9):1343-9.
A C164Y somatic mutation in the H-2Kb class I molecule causes a disruption of the alpha 2 domain disulfide bond and results in a loss of H-2Kb cell surface expression by the 69.9.15 cell line. In vitro culture of the somatic cell variant at 30 degrees C induced weak, but reproducible, expression of the H-2Kb mutant molecule on the cell surface, which suggests that a temperature-sensitive mutation was contributing to the H-2Kb null phenotype. Based on the inherent structural instability of the mutant H-2Kb molecules synthesized by 69.9.15 cells, we sought to determine the ability of high affinity peptide-ligand to counteract the null expression of H-2Kb. Treatment of 69.9.15 cells was performed with acid-eluted cell-derived peptides, as well as synthetic H-2Kb-restricted peptides, ovalbumin (OVA) p257-264 (YSIINFEKL), and vesicular stomatitis virus-nuclear protein p52-59 (RGYVYQGL). Whereas the endogenous and vesicular stomatitis virus peptides were ineffective at inducing H-2Kb expression at either 37 degrees C or 30 degrees C, treatment with the OVA peptide at 30 degrees C gave rise to dose-dependent enhancement in H-2Kb expression, an effect that was independent of exogenous sources of bovine beta 2-microglobulin at the time of peptide treatment. By comparison, expression of H-2Kb remained unaltered when cells were treated with the OVA peptide at 37 degrees C, consistent with the temperature-sensitive expression of the mutant molecules. Decay of H-2Kb from the cell surface was similar for both 69.9.15 and RMA-S cells, an indication that binding of OVA p257-264 provided the same level of stability for class I molecules with either a cis-(69.9.15) or trans-acting (RMA-S) defect in heavy chain transport. These data provide novel evidence that transport-defective MHC class I molecules, similar in nature to those encoded by class I genes isolated from human genomic libraries, i.e., the 12.4 pseudogene with a polymorphism at amino acid position 164 (C-->F), are subject to high affinity peptide-induced stabilization which reverses the class I null phenotype.
H-2Kb I类分子中的C164Y体细胞突变导致α2结构域二硫键断裂，致使69.9.15细胞系丧失H-2Kb细胞表面表达。在30℃对该体细胞变体进行体外培养，可诱导H-2Kb突变分子在细胞表面出现微弱但可重复的表达，这表明温度敏感型突变导致了H-2Kb无效表型。基于69.9.15细胞合成的突变型H-2Kb分子固有的结构不稳定性，我们试图确定高亲和力肽配体抵消H-2Kb无效表达的能力。用酸洗脱的细胞衍生肽以及合成的H-2Kb限制性肽、卵清蛋白（OVA）p257 - 264（YSIINFEKL）和水疱性口炎病毒核蛋白p52 - 59（RGYVYQGL）处理69.9.15细胞。内源性肽和水疱性口炎病毒肽在37℃或30℃均无法有效诱导H-2Kb表达，而在30℃用OVA肽处理可使H-2Kb表达呈剂量依赖性增强，该效应在肽处理时与外源性牛β2-微球蛋白无关。相比之下，当细胞在37℃用OVA肽处理时，H-2Kb表达保持不变，这与突变分子的温度敏感型表达一致。69.9.15细胞和RMA-S细胞表面H-2Kb的降解情况相似，这表明OVA p257 - 264的结合为在重链转运中存在顺式（69.9.15）或反式作用（RMA-S）缺陷的I类分子提供了相同水平的稳定性。这些数据提供了新的证据，即运输缺陷型MHC I类分子，其性质与从人类基因组文库中分离的I类基因编码的分子相似，即氨基酸位置164（C→F）具有多态性的12.4假基因，会受到高亲和力肽诱导的稳定作用，从而逆转I类无效表型。
