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

Abstract

The glycoprotein quality control (GQC) system in the endoplasmic reticulum (ER) effectively uses chaperone-type enzymes and lectins such as UDPglucose: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 M9high-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.

Clamping, bending, and twisting inter-domain motions in the misfold-recognising portion of UDP-glucose:glycoprotein glucosyl-transferase

“…Having concluded that UGGT's promiscuity is not dependent on the flexible linker between the catalytic domain and the N-terminal misfold sensing region, but is likely underpinned by the motions uncovered by the MD simulations, the question remains regarding UGGT's reported ability to survey not only folding of small-and medium-size glycoprotein monomers, but also quaternary structure of glycoprotein oligomers and larger multi-glycoprotein complexes [23] [24] [25]. The Indeed, the MD conformational landscape observed in this work reaches extremely compact conformations, which may explain the activity of the enzyme against synthetic glycopeptides [16][17][18][19][20]. Importantly, monomeric UGGT can recognize and re-glucosylate a misfolded glycoprotein only if it can bridge the distance between a folding defect and at least one of the glycoprotein's N-linked glycosylation sites.…”

“…Since the discovery of UGGT back in 1989 [11,12], activity studies have used a range of glycoprotein substrates, such as urea-misfolded bovine thyroglobulin [11], mutants of exo-(1,3)-β-glucanase [13], RNase BS [14,15], small size synthetic compounds bearing high-mannose glycans attached to fluorescent aglycon moieties such as 'TAMRA' and 'BODIPY' [16,17] and chemically synthesized misfolded glycoproteins [18][19][20], to mention only a few. Although a comprehensive list of physiological UGGT substrate glycoproteins has not been compiled, and the molecular detail on UGGT:substrate interactions remains uncharacterized, it is apparent that the enzyme is highly promiscuous.…”

Clamping, bending, and twisting inter-domain motions in the misfold-recognizing portion of UDP-glucose: Glycoprotein glucosyltransferase

“…Since the discovery of UGGT in 1989 (Parodi, 2007;Trombetta et al, 1989), UGGT activity studies have used a range of glycoprotein substrates (Trombetta et al, 1989;Taylor et al, 2004;Ritter and Helenius, 2000;Ritter et al, 2005), small-size glycosylated synthetic compounds (Totani et al, 2006(Totani et al, , 2009, and chemically synthesized misfolded glycoproteins (Izumi et al, 2016a(Izumi et al, , 2016bKiuchi et al, 2018). In addition to glycoprotein monomers, UGGT also surveys the quaternary structure of glycoprotein oligomers and larger multi-glycoprotein complexes (Keith et al, 2005;Zhang et al, 2011;Gardner and Kearse, 1999).…”

Clamping, Bending, and Twisting Inter-Domain Motions in the Misfold-Recognising Portion of UDP-Glucose: Glycoprotein Glucosyl-Transferase

“…Since the discovery of UGGT in 1989 (Parodi, 2007;Trombetta et al, 1989), UGGT activity studies have used a range of glycoprotein substrates (Trombetta et al, 1989;Taylor et al, 2004;Ritter and Helenius, 2000;Ritter et al, 2005), small-size glycosylated synthetic compounds (Totani et al, 2006(Totani et al, , 2009, and chemically synthesized misfolded glycoproteins (Izumi et al, 2016a(Izumi et al, , 2016bKiuchi et al, 2018). In addition to glycoprotein monomers, UGGT also surveys the quaternary structure of glycoprotein oligomers and larger multi-glycoprotein complexes (Keith et al, 2005;Zhang et al, 2011;Gardner and Kearse, 1999).…”