Elliott T J, Eisen H N
Center for Cancer Research, Massachusetts Institute of Technology, Cambridge 02139.
Proc Natl Acad Sci U S A. 1990 Jul;87(13):5213-7. doi: 10.1073/pnas.87.13.5213.
Recent findings suggest that peptide fragments of newly synthesized proteins may associate intracellularly with nascent chains of class I histocompatibility antigens (termed MHC-I proteins because they are encoded by genes of the major histocompatibility complex) and that these peptide adducts may be required for the folding or stability and perhaps even the transport of these proteins to the cell surface. To determine whether these proteins can be reconstituted from their separated subunits into ostensibly native molecules in the absence of added peptides, we denatured a purified human MHC-I protein (HLA-A2) with 4 M NaSCN, separated its heavy (alpha) and light (beta 2-microglobulin) chains by gel filtration, and then mixed them in the presence of a 3-fold molar excess of beta 2-microglobulin and absence of added peptides. The reconstituted protein, recovered in 10% yield, was indistinguishable from native A2 in its reactivity with a monoclonal antibody (BB7.7) and its ability to specifically activate A2-specific CD8+ T cells. Inasmuch as the reconstituted A2 contained no detectable peptide adducts (we estimate less than 1 per 100 on a molar basis, assuming peptides of 2-5 kDa), the results suggest that peptide-free A2 can be recognized by CD8+ T cells and that peptide adducts are not essential for the MHC-I protein to maintain an ostensibly native structure.
最近的研究结果表明，新合成蛋白质的肽片段可能在细胞内与I类组织相容性抗原的新生链（因其由主要组织相容性复合体的基因编码，故称为MHC-I蛋白）结合，并且这些肽加合物可能是这些蛋白质折叠、稳定乃至运输到细胞表面所必需的。为了确定在不添加肽的情况下，这些蛋白质能否从其分离的亚基重新组装成表面上的天然分子，我们用4M硫氰酸钠使纯化的人MHC-I蛋白（HLA-A2）变性，通过凝胶过滤分离其重链（α链）和轻链（β2-微球蛋白），然后在β2-微球蛋白摩尔过量3倍且不添加肽的情况下将它们混合。以10%的产率回收的重组蛋白，在与单克隆抗体（BB7.7）的反应性及其特异性激活A2特异性CD8+T细胞的能力方面，与天然A2无法区分。由于重组的A2不含可检测到的肽加合物（假设肽的分子量为2-5kDa，我们估计每100个分子中肽加合物少于1个），结果表明无肽的A2可被CD8+T细胞识别，并且肽加合物对于MHC-I蛋白维持表面上的天然结构并非必不可少。
