Toyofuku K, Wada I, Hirosaki K, Park J S, Hori Y, Jimbow K
Division of Dermatology & Cutaneous Sciences, Faculty of Medicine, University of Alberta, T6G 2S2, Canada.
J Biochem. 1999 Jan;125(1):82-9. doi: 10.1093/oxfordjournals.jbchem.a022272.
To understand the process of expression of tyrosinase, a key enzyme of melanogenesis, we examined its maturation in the endoplasmic reticulum (ER) by using a heterogeneous expression system. When human tyrosinase cDNA was introduced into COS 7 cells, tyrosinase activity was minimally detected. Immunofluorescence study revealed that tyrosinase was immunolocalized in the nuclear rim, the reticular network, and the punctuated structures. Because a cytoplasmic tail of tyrosinase-gene family protein functions as a lysosomal targeting signal in non-melanocytic cells, and immature and/or misfolded molecules are selectively retained in the ER, the observed localization suggested the inefficient maturation in the COS 7 cells. We thus examined if supplementation of calnexin, a membrane-bound chaperone with affinity for oligosaccharide-processing intermediates containing monoglucose, could improve the process. As expected, the activity was enhanced approximately 2-fold by co-transfection of cDNA encoding calnexin. In contrast, co-transfection of the cytosolic tail-free calnexin, which inhibits calnexin function by allowing premature egress of its ligands from the ER, suppressed expression of this enhanced tyrosinase activity. When alpha-glucosidase activity, which is required for calnexin function, was inhibited by castanospermine (CST) treatment, expression of tyrosinase activity was completely abolished. To confirm the direct involvement of calnexin in tyrosinase maturation, the interaction of calnexin with tyrosinase was examined. Immunoprecipitation of calnexin from extracts of [35S]methionine labeled cells with anti-calnexin antibody revealed that the association is highest immediately after the pulse and that nascent tyrosinase is gradually dissociated upon chase. The association was completely inhibited when CST was included in the medium. Hence, we suggest that the proper folding of tyrosinase is largely dependent on its direct interaction with calnexin for the determined duration in the ER.
为了解黑素生成的关键酶酪氨酸酶的表达过程，我们通过使用异源表达系统研究了其在内质网（ER）中的成熟情况。将人酪氨酸酶cDNA导入COS 7细胞时，仅能检测到极低水平的酪氨酸酶活性。免疫荧光研究表明，酪氨酸酶定位于核边缘、网状网络和点状结构中。由于酪氨酸酶基因家族蛋白的胞质尾部在非黑素细胞中作为溶酶体靶向信号起作用，并且未成熟和/或错误折叠的分子会被选择性地保留在内质网中，因此观察到的定位表明COS 7细胞中成熟效率低下。因此，我们研究了补充钙连蛋白（一种对含单葡萄糖的寡糖加工中间体具有亲和力的膜结合伴侣蛋白）是否可以改善这一过程。正如预期的那样，通过共转染编码钙连蛋白的cDNA，活性提高了约2倍。相反，共转染无胞质尾部的钙连蛋白（通过使其配体过早地从内质网中释放来抑制钙连蛋白功能）会抑制这种增强的酪氨酸酶活性的表达。当通过栗精胺（CST）处理抑制钙连蛋白功能所需的α-葡萄糖苷酶活性时，酪氨酸酶活性的表达完全被消除。为了证实钙连蛋白直接参与酪氨酸酶的成熟，我们研究了钙连蛋白与酪氨酸酶的相互作用。用抗钙连蛋白抗体从[35S]甲硫氨酸标记的细胞提取物中免疫沉淀钙连蛋白表明，脉冲后立即结合率最高，并且新生酪氨酸酶在追踪过程中逐渐解离。当培养基中加入CST时，这种结合完全被抑制。因此，我们认为酪氨酸酶的正确折叠在很大程度上取决于其在内质网中与钙连蛋白在确定的时间段内的直接相互作用。
