SITE-DIRECTED MUTAGENESIS OF CLASS-I HLA GENES - ROLE OF GLYCOSYLATION IN SURFACE EXPRESSION AND FUNCTIONAL RECOGNITION
SITE-DIRECTED MUTAGENESIS OF CLASS-I HLA GENES - ROLE OF GLYCOSYLATION IN SURFACE EXPRESSION AND FUNCTIONAL RECOGNITION
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DOI：
10.1084/jem.166.5.1329
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发表时间：
1987-11-01
影响因子：
15.3
通讯作者：
BIRO, PA
中科院分区：
文献类型：
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作者：
BARBOSA, JA;SANTOSAGUADO, J;BIRO, PA
我们利用寡核苷酸定向突变和基因转移技术研究了人类白细胞抗原-B7分子上的糖基在mAb和CTL识别中的作用。在人类白细胞抗原-B7的第86位氨基酸中，天冬酰胺被保守地替换为谷氨酰胺，从而取消了所有人类白细胞抗原分子上存在的独特的糖基化位点。第二个突变的B7分子是用天冬氨酸-天冬氨酸-苏氨酸取代176-178位氨基酸残基上的赖氨酸-天冬氨酸/赖氨酸三肽，从而在176位氨基酸产生N-连接的糖链，该糖链存在于所有已知的小鼠H-2 I类抗原上。在将基因转移到小鼠和人类细胞受体后，突变的和正常的人类白细胞抗原B7M176+表达了相同水平的表面蛋白。然而，两个被认为识别不同的HLA-B7分子表位的单抗(MB40.2和MB40.3)的结合被完全消除。相反，经间接免疫荧光和流式细胞术分析12株与单态和多态决定簇反应的人类白细胞抗原-B7单抗后，突变体没有表面表达(小鼠L细胞)或最低表面表达(人RD细胞或小鼠L细胞共表达人β2微球蛋白[β2M])。免疫沉淀分析表明细胞内存在变性突变蛋白。衣霉素处理不能挽救HLAB7M86抗原在细胞表面的表达，而干扰素能诱导更高水平的表面表达。衣霉素处理不允许单抗MB40.2或MB40.3与HLAB7M176+突变抗原结合，提示糖链本身并不直接参与这些单抗表位的识别或构象。进一步突变B7M86-分子以在第176位产生糖链部分(B7M86-/176+)并不能挽救正常的表面表达水平。最后，两个突变都没有影响12个同种特异性CTL克隆的识别。在人类细胞转染体表面的低表达足以实现裂解，尽管效率降低，而裂解可以通过干扰素诱导更高水平的表达而增加。因此，在已有的mAb和同种异体特异性CTL克隆识别中，HLA抗原上的碳水化合物部分起到的作用很小，甚至根本不存在。
We have investigated the role of the carbohydrate moiety on the HLA-B7 molecule in mAb and CTL recognition using oligonucleotide-directed mutagenesis and gene transfer techniques. A conservative substitution of asparagine to glutamine at amino acid 86 in HLA-B7 was created to abolish the unique glycosylation site present on all HLA molecules. A second mutant B7 molecule was made by substituting asparagine-aspartic acid-threonine for the resident lysine-aspartic acid/lysine tripeptide at amino acids 176-178, thus creating an N-linked glycan at amino acid 176 which is additionally present on all known murine H-2 class I antigens. Upon gene transfer into mouse and human cell recipients, the HLA-B7M176+ mutant and normal HLA-B7 expressed identical levels of surface protein. However, the binding of two mAbs (MB40.2 and MB40.3) thought to recognize different epitopes of the HLA-B7 molecule was completely eliminated. In contrast, the HLA-B7M86- mutant displayed no surface expression (mouse L cells) or minimal surface expression (human RD cells or mouse L cells coexpressing human .beta.2 microglobulin [.beta.2m]) after indirect immunofluorescence (IIF) and flow cytometric analysis with a panel of 12 HLA-B7 mAb reactive with monomorphic and polymorphic determinants. Immunoprecipitation analysis demonstrated that intracellular denatured mutant protein was present. Tunicamycin treatment did not rescue the expression of HLA-B7M86- antigens to the cell surface; while interferon did induce higher levels of surface expression. Tunicamycin treatment did not allow binding of the mAbs MB40.2 or MB40.3 to HLA-B7M176+ mutant antigens, suggesting that the carbohydrate moiety itself was not directly involved in the recognition or conformation of these mAb epitopes. Further mutation of the B7M86- molecule to create a glycan moiety at amino acid position 176 (B7M86-/176+) did not rescue normal levels of surface expression. Finally, neither mutation was seen to affect recognition by a panel of 12 allospecific CTL clones. The low expression of HLA-B7M86- on the surface of human cell transfectants was sufficient to achieve lysis, albeit at a reduced efficiency, and lysis could be increased by interferon induction of higher levels of expression. Thus, the carbohydrate moiety on HLA antigens plays a minimal or nonexistent role in recognition by available mAb and allospecific CTL clones.