Department of Physiological Chemistry, University of Veterinary Medicine Hannover, Hannover, Germany; Department of Biochemistry and Chemistry of Nutrition, Faculty of Veterinary Medicine, Cairo University, Giza 12211, Egypt.
Department of Physiological Chemistry, University of Veterinary Medicine Hannover, Hannover, Germany.
Int J Biol Macromol. 2018 Oct 1;117:574-585. doi: 10.1016/j.ijbiomac.2018.05.196. Epub 2018 May 27.
Endoplasmin, or GRP94, is an ER-located stress inducible molecular chaperone implicated in the folding and assembly of many proteins. The Arabian one-humped camel lives in an environment of thermal stress, nevertheless is able to encounter the risk of misfolded proteins. Here, the cDNA encoding camel GRP94 was isolated by rapid amplification of cDNA ends. The isolated cDNA contained an open reading frame of 2412 bp encoding a protein of 803 amino acids with predicted molecular mass of 92.5 kDa. Nucleotide and protein BLAST analysis of cGRP94 revealed strong conservation between camel and other domestic mammals. Overexpression of cGRP94 in COS-1 cells revealed multiple isoforms including one N-glycosylated species. Immunofluorescence colocalized cGRP94 with the ER resident protein calnexin. Interestingly, none of the cGRP94 isoforms expressed in CHO cells was N-glycosylated, presumably due to folding determinants that mask the N-glycosylation sites as proposed by in silico modelling. Surprisingly, isoforms of cGRP94 were detected in the culture media of transfected cells indicating that the protein, although an ER resident, also is trafficked and secreted into the exterior milieu. The overall striking structural homologies of GRP94s among mammalian reflect their pivotal role in the ER quality control and protein homeostasis.
内质网分子伴侣(endoplasmin，或 GRP94)是一种应激诱导的内质网定位分子伴侣，参与许多蛋白质的折叠和组装。阿拉伯单峰驼生活在热应激环境中，但能够应对错误折叠蛋白质的风险。在这里，通过 cDNA 末端快速扩增技术分离出骆驼 GRP94 的 cDNA。分离出的 cDNA 含有一个 2412bp 的开放阅读框，编码一个 803 个氨基酸的蛋白质，预测分子量为 92.5kDa。cGRP94 的核苷酸和蛋白质 BLAST 分析显示，骆驼与其他家畜之间存在很强的保守性。在 COS-1 细胞中过表达 cGRP94 显示出多种同工型，包括一种 N-糖基化的物种。免疫荧光共定位显示 cGRP94 与内质网驻留蛋白 calnexin 共定位。有趣的是，CHO 细胞中表达的 cGRP94 同工型均未发生 N-糖基化，推测这是由于折叠决定因素掩盖了 N-糖基化位点，正如计算机建模所提出的那样。令人惊讶的是，在转染细胞的培养介质中检测到了 cGRP94 的同工型，这表明该蛋白虽然是内质网驻留蛋白，但也可以运输并分泌到细胞外环境中。哺乳动物 GRP94 之间惊人的结构同源性反映了它们在内质网质量控制和蛋白质平衡中的关键作用。
