Loo T W, Clarke D M
Medical Research Council Group in Membrane Biology, Department of Medicine and Department of Biochemistry, University of Toronto, Ontario M5S 1A8, Canada.
J Biol Chem. 1998 Dec 4;273(49):32373-6. doi: 10.1074/jbc.273.49.32373.
Human P-glycoprotein is synthesized in HEK 293 cells as two major products: the 150-kDa core-glycosylated intermediate and the 170-kDa mature proteins. The 150- and 170-kDa proteins were not detected in mutants such as G341C. The major protein in this mutant was a 130-kDa proteolytic degradation product. This result suggested that the mutant protein was misfolded and sensitive to proteolytic digestion during or immediately after synthesis. We found that mutation of Arg113, located in the first extracellular loop of P-glycoprotein and near the consensus glycosylation sites, to Ala, Lys, Glu, Met, or Cys blocked formation of the 130-kDa product. Introduction of R113A into mutant G341C resulted in the synthesis of a mature (170 kDa) and functional transporter. Similarly, when R113A was introduced into misprocessed mutants, there was increased synthesis of the 150-kDa core-glycosylated intermediate. Maturation of the core-glycosylated intermediate into the mature enzyme, however, was not observed. These results suggest that polytopic proteins are accessible to proteases in the lumen of the endoplasmic reticulum during biosynthesis and that proteases are important contributors to the quality control mechanism involved in protein folding. It is also shown that unstable proteins can be made more stable by removal of hypersensitive proteolytic sites.
人P-糖蛋白在HEK 293细胞中作为两种主要产物合成：150 kDa的核心糖基化中间体和170 kDa的成熟蛋白。在诸如G341C等突变体中未检测到150 kDa和170 kDa的蛋白。该突变体中的主要蛋白是130 kDa的蛋白水解降解产物。这一结果表明，突变蛋白在合成过程中或合成后立即发生错误折叠并对蛋白水解消化敏感。我们发现，位于P-糖蛋白第一个细胞外环且靠近共有糖基化位点的精氨酸113突变为丙氨酸、赖氨酸、谷氨酸、甲硫氨酸或半胱氨酸会阻止130 kDa产物的形成。将R113A引入突变体G341C导致合成成熟的（170 kDa）功能性转运蛋白。同样，当将R113A引入加工错误的突变体时，150 kDa的核心糖基化中间体的合成增加。然而，未观察到核心糖基化中间体成熟为成熟酶。这些结果表明，多结构域蛋白在生物合成过程中在内质网腔中可被蛋白酶作用，并且蛋白酶是参与蛋白质折叠的质量控制机制的重要因素。还表明，通过去除超敏蛋白水解位点可以使不稳定蛋白变得更稳定。
