Convergent Synthesis of Homogeneous Glc₁Man₉GlcNAc₂-Protein and Derivatives as Ligands of Molecular Chaperones in Protein Quality Control

Abstract

A detailed understanding of the molecular mechanism of chaperone-assisted protein quality control is often hampered by the lack of well-defined homogeneous glycoprotein probes. We describe here a highly convergent chemoenzymatic synthesis of the monoglucosylated glycoforms of bovine ribonuclease (RNase) as specific ligands of lectin-like chaperones calnexin (CNX) and calreticulin (CRT) that are known to recognize the monoglucosylated high-mannose oligosaccharide component of glycoproteins in protein folding. The synthesis of a selectively modified glycoform Gal1Glc1Man9GlcNAc2-RNase was accomplished by chemical synthesis of a large N-glycan oxazoline and its subsequent enzymatic ligation to GlcNAc-RNase under the catalysis of a glycosynthase. Selective removal of the terminal galactose by a β-galactosidase gave the Glc1Man9GlcNAc2-RNase glycoform in excellent yield. CD spectroscopic analysis and RNA-hydrolyzing assay indicated that the synthetic RNase glycoforms maintained essentially the same global conformations and were fully active as the natural bovine ribonuclease B. SPR binding studies revealed that the Glc1Man9GlcNAc2-RNase had high affinity to lectin CRT, while the synthetic Man9GlcNAc2-RNase glycoform and natural RNase B did not show CRT-binding activity. These results confirmed the essential role of the glucose moiety in the chaperone molecular recognition. Interestingly, the galactose-masked glycoform Gal1Glc1Man9GlcNAc2-RNase also showed significant affinity to lectin CRT, suggesting that a galactose β-1,4-linked to the key glucose moiety does not significantly block the lectin binding. These synthetic homogeneous glycoprotein probes should be valuable for a detailed mechanistic study on how molecular chaperones work in concert to distinguish between mis-folded and folded glycoproteins in the protein quality control cycle.

Synthesis of Glc1Man9‐Glycoprotein Probes by a Misfolding/Enzymatic Glucosylation/Misfolding Sequence

“…By observing the in vitro assays using our precisely synthesized native and misfolded G1M9-IL-8 glycoprotein probes,w ea re able to suggest the role of CRTand G-II in glycoprotein QC process as follows:W ang et al reported that both native and completely denatured G1M9-RNases bind to CRTwith similar affinities, [10] and our results of10 and 12 also …”

“…[10] These elegant studies addressed the effects of oligosaccharide structures,b ut the effects of protein conformations ought to be elucidated.…”

Designer α1,6-Fucosidase Mutants Enable Direct Core Fucosylation of Intact N-Glycopeptides and N-Glycoproteins

“…Thus, The high mannose N-glycans in RNase B ( 20 ) were removed by Endo-H to afford the deglycosylated protein GlcNAc-RNase ( 22 ) in quantitative yield 72 . We found that, in contrast to the slow fucosylation of the intact RNase B ( 20 ), the core fucosylation of GlcNAc-RNase ( 22 ) was much more efficient to give the Fucα1,6GlcNAc-RNase ( 23 ) in essentially quantitative yield within 5 h under the same conditions.…”

Analysis of carbohydrates and glycoconjugates by matrix‐assisted laser desorption/ionization mass spectrometry: An update for 2011–2012

“…To investigate mechanisms of molecular chaperones (Amin et al, 2011) GalNAc 1 4(Fuc 1 3)GlcNAc-R BSA TOF/TOF Glycan array used to identify glycan as potential antigen for serodiagnosis of trichinellosis (Aranzamendi, et al, 2011) Glucose/cholesterol BSA TOF (sinapinic) Cholesterol promotes formation of AGEs (Hirasawa et al, 2011) Glyco-PEG Factor VIIa L-TOF (CHCA)…”