Shrimal Shiteshu, Gilmore Reid
Department of Biochemistry and Molecular Pharmacology, University of Massachusetts Medical School, Worcester, MA 01605, USA.
Department of Biochemistry and Molecular Pharmacology, University of Massachusetts Medical School, Worcester, MA 01605, USA
Glycobiology. 2015 Jul;25(7):774-83. doi: 10.1093/glycob/cwv018. Epub 2015 Mar 19.
A defect in the assembly of the oligosaccharide donor (Dol-PP-GlcNAc(2)Man(9)Glc(3)) for N-linked glycosylation causes hypoglycosylation of proteins by the oligosaccharyltransferase (OST). Mammalian cells express two OST complexes that have different catalytic subunits (STT3A or STT3B). We monitored glycosylation of proteins in asparagine-linked glycosylation 6 (ALG6) deficient cell lines that assemble Dol-PP-GlcNAc(2)Man(9) as the largest oligosaccharide donor. Based upon pulse labeling experiments, 30-40% of STT3A-dependent glycosylation sites and 20% of STT3B-dependent sites are skipped in ALG6-congenital disorders of glycosylation fibroblasts supporting previous evidence that the STT3B complex has a relaxed preference for the fully assembled oligosaccharide donor. Glycosylation of STT3B-dependent sites was more severely reduced in the ALG6 deficient MI8-5 cell line. Protein immunoblot analysis and RT-PCR revealed that MI8-5 cells express 2-fold lower levels of STT3B than the parental Chinese hamster ovary cells. The combination of reduced expression of STT3B and the lack of the optimal Dol-PP-GlcNAc(2)Man(9)Glc(3) donor synergize to cause very severe hypoglycosylation of proteins in MI8-5 cells. Thus, differences in OST subunit expression can modify the severity of hypoglycosylation displayed by cells with a primary defect in the dolichol oligosaccharide assembly pathway.
用于N-连接糖基化的寡糖供体（Dol-PP-GlcNAc(2)Man(9)Glc(3)）组装缺陷会导致寡糖基转移酶（OST）对蛋白质进行低糖基化修饰。哺乳动物细胞表达两种具有不同催化亚基（STT3A或STT3B）的OST复合物。我们监测了在组装Dol-PP-GlcNAc(2)Man(9)作为最大寡糖供体的天冬酰胺连接糖基化6（ALG6）缺陷细胞系中蛋白质的糖基化情况。基于脉冲标记实验，在支持先前证据（即STT3B复合物对完全组装的寡糖供体偏好较宽松）的ALG6先天性糖基化障碍成纤维细胞中，30 - 40%的STT3A依赖性糖基化位点和20%的STT3B依赖性位点被跳过。在ALG6缺陷的MI8-5细胞系中，STT3B依赖性位点的糖基化被更严重地降低。蛋白质免疫印迹分析和RT-PCR显示，MI8-5细胞中STT3B的表达水平比亲本中国仓鼠卵巢细胞低2倍。STT3B表达降低与缺乏最佳Dol-PP-GlcNAc(2)Man(9)Glc(3)供体相结合，协同导致MI8-5细胞中蛋白质非常严重的低糖基化。因此，OST亚基表达的差异可以改变在多萜醇寡糖组装途径存在原发性缺陷的细胞中所表现出的低糖基化的严重程度。
