A novel approach for N-glycosylation studies using detergent extracted microsomes.
Recently, it has become apparent that asparagine-linked (N-linked) oligosaccharide at an early stage of processing can play an important role in quality control of the secretory pathway. Here, we have developed a system for better understanding of the N-glycosylation machinery and its involvement in quality control in the endoplasmic reticulum (ER). Rough microsomes (RM) treated with 0.18% Tx-100 (TxRM) preserved translocation activities to a similar extent detected in RM. TxRM were depleted of many soluble proteins including glucosidase II, BiP and Erp72, but maintained approximately 80% of calnexin, a membrane protein. More importantly, TxRM revealed insufficient glycosylation of T cell receptor-alpha (TCR-alpha), suggesting that a factor or factors extracted with 0.18% Tx-100 is responsible for facilitating the transfer of oligosaccharides to the protein. In addition, the top band of TCR-alpha translated in TxRM migrated slower than that in RM, but faster than that in RM treated with castanospermine (CST), an inhibitor of glucosidase I/II. This suggests that the trimming of the inner two glucose sugars is impaired by the loss of glucosidase II. Furthermore, we demonstrated that TCR-alpha coprecipitated with calnexin migrated between unglucosylated and diglucosylated forms on SDS-PAGE. Thus, the treatment of RM with low concentration of detergent is a very powerful method for elucidating not only N-glycosylation processes but also other biological functions such as quality control in the ER.
Yuki
Hideo
H
Department of Anatomy, Biology and Medicine, Faculty of Medicine, Oita University, 1-1 Idaigaoka, Hasama-machi, Oita, Japan.
Hamanaka
Ryoji
R
Shinohara
Tetsuji
T
Sakai
Kumiko
K
Watanabe
Makoto
M
D000818:Animals; D037281:Calnexin / Q000378:metabolism; D004285:Dogs; D019673:Genes, T-Cell Receptor alpha / Q000187:drug effects; D006031:Glycosylation; D050958:HSP47 Heat-Shock Proteins / Q000378:metabolism; D066298:In Vitro Techniques; D008861:Microsomes / Q000187:drug effects / Q000378:metabolism*; D017830:Octoxynol / Q000378:metabolism / Q000494:pharmacology*; D009844:Oligosaccharides / Q000378:metabolism; D010179:Pancreas / Q000166:cytology / Q000378:metabolism; D000520:alpha-Glucosidases / Q000378:metabolism
D050958:HSP47 Heat-Shock Proteins; D009844:Oligosaccharides; D037281:Calnexin; D017830:Octoxynol; C034251:4-nitrophenyl-alpha-glucosidase; D000520:alpha-Glucosidases