title: Glycosynthases enable a highly efficient chemoenzymatic synthesis of N-glycoproteins carrying intact natural N-glycans.
abstract: Homogeneous N-glycoproteins carrying defined natural N-glycans are essential for detailed structural and functional studies. The transglycosylation activity of the endo-beta-N-acetylglucosaminidases from Arthrobacter protophormiae (Endo-A) and Mucor hiemalis (Endo-M) holds great potential for glycoprotein synthesis, but the wild-type enzymes are not practical for making glycoproteins carrying native N-glycans because of their predominant activity for product hydrolysis. In this article, we report studies of two endoglycosidase-based glycosynthases, EndoM-N175A and EndoA-N171A, and their usefulness in constructing homogeneous N-glycoproteins carrying natural N-glycans. The oligosaccharide oxazoline corresponding to the biantennary complex-type N-glycan was synthesized and tested with the two glycosynthases. The EndoM-N175A mutant was able to efficiently transfer the complex-type glycan oxazoline to a GlcNAc peptide and GlcNAc-containing ribonuclease to form the corresponding homogeneous glycopeptide/glycoprotein. The EndoA-N171A mutant did not recognize the complex-type N-glycan oxazoline but could efficiently use the high-mannose-type glycan oxazoline for transglycosylation. These mutants possess the transglycosylation activity but lack the hydrolytic activity toward the product. Kinetic studies revealed that the dramatically enhanced synthetic efficiency of the EndoA-N171A mutant was due to the significantly reduced hydrolytic activity toward both the Man(9)GlcNAc oxazoline and the product as well as to its enhanced activity for transglycosylation. Thus, the two mutants described here represent the first endoglycosidase-based glycosynthases enabling a highly efficient synthesis of homogeneous natural N-glycoproteins.
authors.affiliation: Institute of Human Virology and Department of Biochemistry & Molecular Biology, University of Maryland School of Medicine, Baltimore, Maryland 21201, USA.
authors.forename: Wei
authors.initials: W
authors.lastname: Huang
authors.forename: Cishan
authors.initials: C
authors.lastname: Li
authors.forename: Bing
authors.initials: B
authors.lastname: Li
authors.forename: Midori
authors.initials: M
authors.lastname: Umekawa
authors.forename: Kenji
authors.initials: K
authors.lastname: Yamamoto
authors.forename: Xinyu
authors.initials: X
authors.lastname: Zhang
authors.forename: Lai-Xi
authors.initials: LX
authors.lastname: Wang
mesh_terms: D001173:Arthrobacter / Q000201:enzymology; D002240:Carbohydrate Sequence; D006023:Glycoproteins / Q000138:chemical synthesis* / Q000378:metabolism; D006026:Glycoside Hydrolases / Q000737:chemistry* / Q000378:metabolism; D006031:Glycosylation; D008958:Models, Molecular; D008969:Molecular Sequence Data; D009089:Mucor / Q000201:enzymology; D009844:Oligosaccharides / Q000138:chemical synthesis / Q000378:metabolism; D010080:Oxazoles / Q000737:chemistry / Q000378:metabolism; D011134:Polysaccharides / Q000138:chemical synthesis* / Q000378:metabolism
chemical_list: D006023:Glycoproteins; D009844:Oligosaccharides; D010080:Oxazoles; D011134:Polysaccharides; D006026:Glycoside Hydrolases