Quality control mechanism for secretory proteins in intracellular protein sorting pathway

在内质网（ER）腔中，许多分泌性蛋白质获得N-连接的寡糖侧链，这可能有助于蛋白质的折叠、组装和分选。本研究通过在细胞中表达N-糖基化缺陷的组织蛋白酶E（Cathepsin E，CE）突变体或用N-糖基化抑制剂处理细胞，研究了N-糖基化对CE折叠、稳定性和定位的作用，并试图了解这些糖基化缺陷的CE在细胞中是如何受到质量控制的。当用衣霉素处理表达外源大鼠CE基因的正常大鼠肾（NRK）细胞时，观察到CE在细胞内的快速降解。布雷菲德菌素A，巴弗洛霉素铝和氯化铵的降解不受抑制，这表明降解发生在前高尔基室。蛋白酶体抑制剂（如lactacystin或ALLN）对CE的降解没有影响，CE的去稳定化并不完全是由于酶的N-糖基化被消除，因为发现非糖基化的CE突变体在NRK细胞中稳定保留。在相同类型的衣霉素处理，没有变化，观察到在细胞内的稳定性的BiP（GRP 78），ER居民分子伴侣。这些结果表明，衣霉素诱导一些未知的蛋白酶体非依赖性的降解系统内的ER，以消除蛋白质的缺陷，在适当的N-糖基化。

In the endoplasmic reticulum (ER) lumen, many of secretory proteins acquire N-linked oligosaccharide side chains which may help folding assembly and sorting of the proteins. In the present study, we examined roles of the N-glycosylation on cathepsin E (CE) in its folding, stability and localization by expressing N-glycosylation-deficient mutant CE in cells or by treatment of the cells with a N-glycosylation inhibitor, and tried to know how these glycosylation-deficient CEs suffered the quality control in the cells. When normal rat kidney(NRK) cells expressing exogenous rat CE gene were treated with tunicamycin, rapid degradation of CE within the cells was observed. The degradation was not inhibited by brefeldin A,bafilolmycin Al and NH4Cl, suggesting that the degradation occurs in pre-Golgi compartments. Moreover, the degradation was not affected by inhibitors for proteasome, such as lactacystin or ALLN.The de-stabilization of CE was not solely due to the elimination of N-glycosylation of the enzyme, because non-glycosylation mutant of CE was found to be stably retained in the NRK cells. In the same type of tunicamycin treatment, no change was observed in the intracellular stability of BiP (GRP78), an ER resident molecular chaperone. These results suggest that tunicamycin induces some unknown proteasome-independent degradation system within the ER to eliminate proteins defect in properN-glycosylation.