Henle K J, Wang S Y, Nagle W A, Lumpkin C K
Department of Medicine, University of Arkansas for Medical Sciences, Arkansas Children's Hospital Research Institute, Little Rock.
Exp Cell Res. 1994 Feb;210(2):185-91. doi: 10.1006/excr.1994.1028.
High intracellular levels of heat shock proteins and enhanced protein glycosylation are two phenomena closely associated with the cellular stress response. GP50 is the major heat-induced glycoprotein in Chinese hamster ovary (CHO) cells; however, GP50 is not well characterized, and its function is unknown. J6 is a gene originally identified in F9 murine teratocarcinoma cells after exposure to retinoic acid. In this study we show that J6 is heat-inducible and codes for a protein that shares characteristics with GP50. Western blotting of CHO cell homogenates, using a J6 polyclonal antibody, showed a single band with a molecular weight identical to that of GP50. Thermotolerant cells showed increased levels of the J6/GP50 protein. Heat-shocked CHO cells also accumulated transiently high levels of J6 mRNA between 2 and 7 h following 10 min at 45 degrees C. These induction kinetics are similar to those for GP50 labeling with D-[3H]mannose and to the activation of major heat shock genes, e.g., hsp70. Hybrid selection of J6 mRNA from CHO cells, followed by in vitro translation, produced a single band on SDS-PAGE with a molecular weight identical to that of deglycosylated GP50. Neither cellular proliferation (exponential growth versus plateau phase) nor the specific heat shock temperature (41.5 degrees C versus 45 degrees C) had significant effects on J6 induction by heat stress. Stress conditions other than hyperthermia, including ethanol, arsenite, and hypoxia, increased J6 mRNA levels. Conversely, J6 mRNA was reduced by quercetin, brefeldin A, okadaic acid, uv, and hydrogen peroxide. Our data support the hypothesis that J6 is a heat shock gene with a gene product identical to the polypeptide moiety of GP50.
细胞内热休克蛋白水平升高和蛋白质糖基化增强是与细胞应激反应密切相关的两种现象。GP50是中国仓鼠卵巢（CHO）细胞中主要的热诱导糖蛋白；然而，GP50的特征尚未明确，其功能也未知。J6是最初在F9小鼠畸胎瘤细胞经视黄酸处理后鉴定出的一个基因。在本研究中，我们表明J6是热诱导型的，其编码的蛋白质与GP50具有共同特征。用J6多克隆抗体对CHO细胞匀浆进行蛋白质印迹分析，显示出一条分子量与GP50相同的单条带。耐热细胞中J6/GP50蛋白水平升高。在45℃处理10分钟后，热休克的CHO细胞在2至7小时内也短暂积累了高水平的J6 mRNA。这些诱导动力学与用D-[3H]甘露糖标记GP50的动力学以及主要热休克基因（如hsp70）的激活动力学相似。从CHO细胞中杂交选择J6 mRNA，然后进行体外翻译，在SDS-PAGE上产生了一条分子量与去糖基化的GP50相同的单条带。细胞增殖（指数生长期与平台期）和特定热休克温度（41.5℃与45℃）对热应激诱导J6均无显著影响。除热疗外的应激条件，包括乙醇、亚砷酸盐和缺氧，均可增加J6 mRNA水平。相反，槲皮素、布雷菲德菌素A、冈田酸、紫外线和过氧化氢可降低J6 mRNA水平。我们的数据支持这样的假设，即J6是一个热休克基因，其基因产物与GP50的多肽部分相同。
