Jethmalani S M, Henle K J
Department of Medicine, University of Arkansas for Medical Sciences, Little Rock 72205.
Biochem Biophys Res Commun. 1994 Nov 30;205(1):780-7. doi: 10.1006/bbrc.1994.2733.
Selective glycosylation of "prompt" stress glycoproteins (P-SG), mainly P-SG67 and P-SG64 (M(r) of 64,000, pI = 5.1), occurs immediately during acute heat-stress. In the present study, P-SG64 was purified by sequential gel filtration, anion-exchange, affinity chromatography, and two-dimensional isoelectric focusing/SDS-PAGE. Purified P-SG64 was further characterized by microsequencing of a peptide fragment, PT-61, which showed a 100% sequence homology with calreticulin, suggesting that P-SG64 is identical to calreticulin. PT-61 also showed 55%, 58% and 63% sequence homologies with calnexin, HIV-1 gp120 and HIV-2 envelope polyprotein, respectively. 45Ca2+ overlay studies confirmed Ca(2+)-binding of P-SG64. P-SG67 was also recently identified as calreticulin (8), which suggests that CHO cells either have two isoforms of calreticulin or express variable states of calreticulin glycosylation during acute heat stress. The role of intracellular Ca2+ ([Ca2+]i) during heat-induced "prompt" glycosylation was also examined and indicated an 8-fold increase in [Ca2+]i. Chelation of this increased cytoplasmic Ca2+ by BAPTA reduced glycosylation of P-SG67/P-SG64/calreticulin only by approximately 20%. This observation suggests that altered [Ca2+]i homeostasis is not directly linked to calreticulin glycosylation, instead, heat-induced calreticulin glycosylation is a Ca(2+)-independent effect.
“快速”应激糖蛋白（P-SG），主要是P-SG67和P-SG64（分子量64,000，pI = 5.1）的选择性糖基化在急性热应激期间立即发生。在本研究中，通过连续凝胶过滤、阴离子交换、亲和色谱和二维等电聚焦/SDS-PAGE对P-SG64进行了纯化。通过对肽片段PT-61进行微测序对纯化的P-SG64进行了进一步表征，结果显示其与钙网蛋白有100%的序列同源性，表明P-SG64与钙网蛋白相同。PT-61与钙连蛋白、HIV-1 gp120和HIV-2包膜多蛋白的序列同源性分别为55%、58%和63%。45Ca2+覆盖研究证实了P-SG64与Ca2+的结合。最近还发现P-SG67也是钙网蛋白（8），这表明CHO细胞要么有两种钙网蛋白异构体，要么在急性热应激期间表达钙网蛋白糖基化的可变状态。还研究了细胞内Ca2+（[Ca2+]i）在热诱导的“快速”糖基化过程中的作用，结果表明[Ca2+]i增加了8倍。用BAPTA螯合这种增加的细胞质Ca2+仅使P-SG67/P-SG64/钙网蛋白的糖基化降低了约20%。这一观察结果表明，[Ca2+]i稳态的改变与钙网蛋白糖基化没有直接联系，相反，热诱导的钙网蛋白糖基化是一种不依赖Ca2+的效应。
