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. 2010 Jul 5;11(10):1350-5.
doi: 10.1002/cbic.201000242.

Arthrobacter endo-beta-N-acetylglucosaminidase shows transglycosylation activity on complex-type N-glycan oxazolines: one-pot conversion of ribonuclease B to sialylated ribonuclease C

Wei Huang  1 , Qiang Yang, Midori Umekawa, Kenji Yamamoto, Lai-Xi Wang
Affiliations

Arthrobacter endo-beta-N-acetylglucosaminidase shows transglycosylation activity on complex-type N-glycan oxazolines: one-pot conversion of ribonuclease B to sialylated ribonuclease C

Wei Huang et al. Chembiochem. .

Abstract

Asparagine-linked glycosylation is a major form of posttranslational modifications, which plays important roles in protein folding, intracellular signaling, and a number of other biological recognition events [1]. Glycoproteins are often characterized by their structural micro-heterogeneity where different glycoforms have the same polypeptide backbone but differ in the pendant oligosaccharides. Of particular interest are the findings that subtle difference in the attached glycans can have a significant impact on the biological functions of a given glycoprotein [2, 3]. The urgent need of pure glycoforms for functional studies and biomedical applications has stimulated a great interest in exploring new methods for making homogeneous glycoproteins [4]. Major advances include the application of native chemical ligation and expressed protein ligation for constructing full-size glycoproteins [–7], chemoselective ligation to introduce homogeneous glycans [8], and the engineering of yeast glycosylation pathways to produce single glycoforms [9]. Yet another interesting advance in the field is the endoglycosidase-catalyzed transglycosylation for glycosylation engineering and glycoprotein synthesis [–16].

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Figures

Figure 1
Figure 1
Conformational comparison of Man3GlcNAc-thiazoline (green) and CT-oxazoline (yellow) docked into substrate-binding site of Endo-A (macromolecule displayed in ribbon).
Figure 2
Figure 2
SDS-PAGE analysis of the one-pot transglycosylation reaction and the product. lane 0: marker; lane 1: RNase B; lane 2: an aliquot of the one-pot reaction mixture at 5min; lane 3: an aliquot of the one-pot reaction mixture at 3 h; lane 4: purified sialylated RNase C; lane 5: sialylated RNase C treated with Endo-M.
Figure 3
Figure 3
ESI-MS of the sialylated RNase C.
Scheme 1
Scheme 1
Semi-synthesis of sialylated N-glycan oxazoline.
Scheme 2
Scheme 2
Endo-A and EndoM-N175A act on sialylated N-glycan oxazoline for transglycosylation without product hydrolysis. Enzyme activity definition: 1 mU was defined as the amount of Endo-A or EndoM-N175A needed for transferring 1 nmol of Man3GlcNAc-oxazoline to GlcNAc-PNP in 1 min at 23 °C.
Scheme 3
Scheme 3
Endo-A and EndoM-N175A catalyzed one-pot conversion of RNase B to sialylated RNase C.
See this image and copyright information in PMC

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