Arthrobacter Endo-β-N-Acetylglucosaminidase Shows Transglycosylation Activity on Complex-Type N-Glycan Oxazolines: One-Pot Conversion of Ribonuclease B to Sialylated Ribonuclease C
✍ Scribed by Wei Huang; Qiang Yang; Midori Umekawa; Kenji Yamamoto; Lai-Xi Wang
- Publisher
- John Wiley and Sons
- Year
- 2010
- Tongue
- English
- Weight
- 415 KB
- Volume
- 11
- Category
- Article
- ISSN
- 1439-4227
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✦ Synopsis
Asparagine-linked glycosylation is a major form of post-translational modification and 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, whereby different glycoforms have the same polypeptide backbone but differ in the pendant oligosaccharides. Of particular interest is the finding that subtle differences in the attached glycans can have a significant impact on the biological functions of a given glycoprotein. [2, 3] The urgent need for 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 to constructing full-size glycoproteins, [5][6][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 endoglycosidase-catalyzed transglycosylation for glycosylation engineering and glycoprotein synthesis. [10][11][12][13][14][15][16] Endo-b-N-acetylglucosaminidases (ENGases) of the glycosyl hydrolase family 85 (GH85) are endoglycosidases that release N-glycans from glycoproteins by hydrolyzing the glycosidic bond in the chitobiose (GlcNAcb1-4GlcNAc) core. Two ENGases, Endo-A from Arthrobacter protophormiae and Endo-M from Mucor hiemalis, have been used for glycopeptide and glycoprotein synthesis because of their potent transglycosylation activity. [10][11][12][13][14][15][16] The two enzymes have distinct substrate specificity. Endo-M can hydrolyze both high-mannose-type and complextype N-glycans, but Endo-A has been shown to act only on high-mannose-type N-glycans.
An interesting development in the field is the exploration of synthetic sugar oxazolines, mimics of the oxazolinium ion intermediate generated in a substrate-assisted mechanism, as activated donor substrates for enzymatic transglycosylation. [11][12][13][14] To address the problem of product hydrolysis, glycosynthase mutants of Endo-M and Endo-A were generated that lack the [a] Dr.