Loo T W, Clarke D M
Medical Research Council Group in Membrane Biology, Department of Medicine and Department of Biochemistry, University of Toronto, Toronto, Ontario M5S 1A8, Canada.
J Biol Chem. 1998 Jun 12;273(24):14671-4. doi: 10.1074/jbc.273.24.14671.
Misprocessed mutants of human P-glycoprotein accumulate as core-glycosylated intermediates in the endoplasmic reticulum and are rapidly degraded. Trypsin digestion was used to test for structural differences between mature and core-glycosylated forms of P-glycoprotein. We found that the core-glycosylated wild-type and mutant P-glycoproteins were both 100-fold more sensitive to trypsin compared with the mature form of the wild-type enzyme. This result suggested that the core-glycosylated forms of both wild-type and mutant P-glycoproteins have similar unfolded structures, whereas the mature enzyme is folded into a more compact structure. The core-glycosylated mutant P-glycoproteins could be converted to the mature trypsin-resistant form by synthesis in the presence of drug substrate. Addition of proteasome inhibitor MG-132 to stabilize the core-glycosylated intermediate resulted in the accumulation but not maturation of the mutant protein. Further analysis showed that the second transmembrane domain TMD2 also became more resistant to trypsin digestion only after coexpression with TMD1 in the presence of substrate. Taken together, these results suggest that simply stabilizing the core-glycosylated intermediate is not sufficient to promote maturation of the processing mutants and that drug substrates induce maturation by promoting superfolding of the transmembrane domains.
人P-糖蛋白的错误加工突变体以内质网中的核心糖基化中间体形式积累，并迅速降解。使用胰蛋白酶消化来测试P-糖蛋白成熟形式和核心糖基化形式之间的结构差异。我们发现，与野生型酶的成熟形式相比，核心糖基化的野生型和突变型P-糖蛋白对胰蛋白酶的敏感性均高100倍。该结果表明，野生型和突变型P-糖蛋白的核心糖基化形式具有相似的未折叠结构，而成熟酶折叠成更紧密的结构。核心糖基化的突变型P-糖蛋白可以通过在药物底物存在下合成而转化为成熟的抗胰蛋白酶形式。添加蛋白酶体抑制剂MG-132以稳定核心糖基化中间体导致突变蛋白的积累但未成熟。进一步分析表明，仅在底物存在下与TMD1共表达后，第二个跨膜结构域TMD2对胰蛋白酶消化也变得更具抗性。综上所述，这些结果表明，简单地稳定核心糖基化中间体不足以促进加工突变体的成熟，并且药物底物通过促进跨膜结构域的超折叠来诱导成熟。
