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Monitoring of Glycoprotein Quality Control System with a Series of Chemically Synthesized Homogeneous Native and Misfolded Glycoproteins

Author:
Kiuchi, Tatsuto, Izumi, Masayuki, Mukogawa, Yuki, Shimada, Arisa, Okamoto, Ryo, Seko, Akira, Sakono, Masafumi, Takeda, Yoichi, Ito, Yukishige, Kajihara, Yasuhiro
Source:
Journal of the American Chemical Society 2018 v.140 no.50 pp. 17499-17507
ISSN:
1520-5126
Subject:
calnexin, calreticulin, disulfide bonds, endoplasmic reticulum, erythropoietin, glucosidases, glucosylation, high performance liquid chromatography, interferon-beta, interleukin-8, isomerases, liver, monitoring, oligosaccharides, quality control, rats, spectrometers, transferases
Abstract:
The glycoprotein quality control (GQC) system in the endoplasmic reticulum (ER) effectively uses chaperone-type enzymes and lectins such as UDP-glucose:glycoprotein glucosyltransferase (UGGT), calnexin (CNX), calreticulin (CRT), protein disulfide bond isomerases (ERp57 or PDIs), and glucosidases to generate native-folded glycoproteins from nascent glycopolypeptides. However, the individual processes of the GQC system at the molecular level are still unclear. We chemically synthesized a series of several homogeneous glycoproteins bearing M9-high-mannose type oligosaccharides (M9-glycan), such as erythropoietin (EPO), interferon-β (IFN-β), and interleukin 8 (IL8) and their misfolded counterparts, and used these glycoprotein probes to better understand the GQC process. The analyses by high performance liquid chromatography and mass spectrometer clearly showed refolding processes from synthetic misfolded glycoproteins to native form through folding intermediates, allowing for the relationship between the amount of glucosylation and the refolding of the glycoprotein to be estimated. The experiment using these probes demonstrated that GQC system isolated from rat liver acts in a catalytic cycle regulated by the fast crosstalk of glucosylation/deglucosylation in order to accelerate refolding of misfolded glycoproteins.
Agid:
6263059