Nielsen Daniel, Gyllberg Hanna, Ostlund Pernilla, Bergman Tomas, Bedecs Katarina
Department of Biochemistry and Biophysics, University of Stockholm, Stockholm, Sweden.
Biochem J. 2004 Jun 1;380(Pt 2):571-9. doi: 10.1042/BJ20040010.
We have previously shown that ScN2a cells (scrapie-infected neuroblastoma N2a cells) express 2-fold- and 4-fold-increased levels of IR (insulin receptor) and IGF-1R (insulin-like growth factor-1 receptor) respectively. In addition, the IR alpha- and beta-subunits are aberrantly processed, with apparent molecular masses of 128 and 85 kDa respectively, as compared with 136 and 95 kDa in uninfected N2a cells. Despite the 2-fold increase in IR protein, the number of (125)I-insulin-binding sites was slightly decreased in ScN2a cells [Ostlund, Lindegren, Pettersson and Bedecs (2001) Brain Res. 97, 161-170]. In order to determine the cellular localization of IR in ScN2a cells, surface biotinylation was performed, showing a correct IR trafficking and localization to the cell surface. The present study shows for the first time that neuroblastoma N2a cells express significant levels of IR-IGF-1R hybrid receptors, and in ScN2a cells the number of hybrid receptors was 2-fold higher than that found in N2a cells, potentially explaining the apparent loss of insulin-binding sites due to a lower affinity for insulin compared with the homotypic IR. Furthermore, the decreased molecular mass of IR subunits in ScN2a cells is not caused by altered phosphorylation or proteolytic processing, but rather by altered glycosylation. Enzymic deglycosylation of immunoprecipitated IR from N2a and ScN2a cells with endoglycosidase H, peptide N-glycosidase F and neuraminidase all resulted in subunits with increased electrophoretic mobility; however, the 8-10 kDa shift remained. Combined enzymic or chemical deglycosylation using anhydrous trifluoromethane sulphonic acid treatment ultimately showed that the IR alpha- and beta-subunits from ScN2a cells are aberrantly glycosylated. The increased formation of IR-IGF-1R hybrids in ScN2a cells may be part of a neuroprotective response to prion infection. The degree and functional significance of aberrantly glycosylated proteins in ScN2a cells remain to be determined.
我们之前已经表明，ScN2a细胞（羊瘙痒病感染的神经母细胞瘤N2a细胞）中胰岛素受体（IR）和胰岛素样生长因子-1受体（IGF-1R）的表达水平分别增加了2倍和4倍。此外，IR的α和β亚基加工异常，其表观分子量分别为128 kDa和85 kDa，而未感染的N2a细胞中分别为136 kDa和95 kDa。尽管IR蛋白增加了2倍，但ScN2a细胞中（125）I胰岛素结合位点的数量略有减少[奥斯特伦德、林德格伦、彼得松和贝德克斯（2001年）《脑研究》97卷，第161 - 170页]。为了确定IR在ScN2a细胞中的细胞定位，进行了表面生物素化，结果显示IR转运正确且定位于细胞表面。本研究首次表明，神经母细胞瘤N2a细胞表达显著水平的IR - IGF - 1R杂合受体，并且在ScN2a细胞中杂合受体的数量比N2a细胞中高2倍，这可能解释了胰岛素结合位点明显减少的原因，因为与同型IR相比，其对胰岛素的亲和力较低。此外，ScN2a细胞中IR亚基分子量的降低不是由磷酸化或蛋白水解加工改变引起的，而是由糖基化改变引起的。用内切糖苷酶H、肽N - 糖苷酶F和神经氨酸酶对从N2a和ScN2a细胞中免疫沉淀的IR进行酶促去糖基化，均导致亚基的电泳迁移率增加；然而，8 - 10 kDa的迁移变化仍然存在。使用无水三氟甲磺酸处理进行联合酶促或化学去糖基化最终表明，ScN2a细胞中的IRα和β亚基糖基化异常。ScN2a细胞中IR - IGF - 1R杂合体形成增加可能是对朊病毒感染的神经保护反应的一部分。ScN2a细胞中糖基化异常蛋白的程度和功能意义仍有待确定。
