title: Unexpected tolerance of glycosylation by UDP-GalNAc:polypeptide alpha-N-acetylgalactosaminyltransferase revealed by electron capture dissociation mass spectrometry: carbohydrate as potential protective groups.
abstract: UDP-GalNAc:polypeptide alpha-N-acetylgalactosaminyltransferases (ppGalNAcTs, EC 2.4.1.41), a family of key enzymes that initiate posttranslational modification with O-glycans in mucin synthesis by introduction of alpha-GalNAc residues, are structurally composed of a catalytic domain and a lectin domain. It has been known that multiple Ser/Thr residues are assigned in common mucin glycoproteins as potential O-glycosylation sites and more than 20 distinct isoforms of this enzyme family contribute to produce densely O-glycosylated mucin glycoproteins. However, it seems that the functional role of the lectin domain of ppGalNAcTs remains unclear. We considered that electron capture dissociation mass spectrometry (ECD-MS), a promising method for highly selective fragmentation at peptide linkages of glycopeptides to generate unique c and z series of ions, should allow for precise structural characterization to uncover the mechanism in O-glycosylation of mucin peptides by ppGalNAcTs. In the present study, it was demonstrated that a system composed of an electrospray source, a linear RFQ ion trap that isolates precursor ions, the ECD device, and a TOF mass spectrometer is a nice tool to identify the preferential O-glycosylation sites without any decomposition of the carbohydrate moiety. It should be noted that electrons used for ECD are accelerated within a range from 1.75 to 9.75 eV depending on the structures of glycopeptides of interest. We revealed for the first time that additional installation of a alpha-GalNAc residue at potential glycosylation sites by ppGalNAcT2 proceeds smoothly in various unnatural glycopeptides having alpha-Man, alpha-Fuc, and beta-Gal residues as well as alpha-GalNAc residues. The results may suggest that ppGalNAcT2 did not differentiate totally presubstituted sugar residues in terms of configuration of functional groups, d-, l-configuration, and even alpha-, beta-stereochemistry at an anomeric carbon atom when relatively short synthetic peptides were employed for the acceptor substrates. Unexpected characteristics of ppGalNAcT2 motivated us to challenge site-directed installation of alpha-GalNAc residues at desired position(s) by protecting some hydroxyl groups of Thr/Ser residues with selectively removable sugars, notably a novel concept as "carbohydrate as protective groups", toward a goal of the systematic chemical and enzymatic synthesis of biologically important mucin glycopeptides.
authors.affiliation: Graduate School of Life Science and Research Center for Post-Genome Science and Technology, Hokkaido University, Sapporo 001-0021, Japan.
authors.forename: Yayoi
authors.initials: Y
authors.lastname: Yoshimura
authors.forename: Takahiko
authors.initials: T
authors.lastname: Matsushita
authors.forename: Naoki
authors.initials: N
authors.lastname: Fujitani
authors.forename: Yasuhiro
authors.initials: Y
authors.lastname: Takegawa
authors.forename: Haruhiko
authors.initials: H
authors.lastname: Fujihira
authors.forename: Kentarou
authors.initials: K
authors.lastname: Naruchi
authors.forename: Xiao-Dong
authors.initials: XD
authors.lastname: Gao
authors.forename: Naomi
authors.initials: N
authors.lastname: Manri
authors.forename: Takeshi
authors.initials: T
authors.lastname: Sakamoto
authors.forename: Kentaro
authors.initials: K
authors.lastname: Kato
authors.forename: Hiroshi
authors.initials: H
authors.lastname: Hinou
authors.forename: Shin-Ichiro
authors.initials: S
authors.lastname: Nishimura
mesh_terms: D002241:Carbohydrates; D020134:Catalytic Domain; D004583:Electrons; D006020:Glycopeptides / Q000737:chemistry* / Q000378:metabolism*; D006031:Glycosylation; D006801:Humans; D037102:Lectins / Q000737:chemistry; D013058:Mass Spectrometry; D009077:Mucins / Q000737:chemistry / Q000378:metabolism; D017350:N-Acetylgalactosaminyltransferases / Q000737:chemistry* / Q000378:metabolism*; D010455:Peptides; D011134:Polysaccharides / Q000737:chemistry; D013912:Threonine; D014530:Uridine Diphosphate; D000097763:Polypeptide N-acetylgalactosaminyltransferase
chemical_list: D002241:Carbohydrates; D006020:Glycopeptides; D037102:Lectins; D009077:Mucins; D017350:N-Acetylgalactosaminyltransferases; D010455:Peptides; D011134:Polysaccharides; D013912:Threonine; D014530:Uridine Diphosphate; D000097763:Polypeptide N-acetylgalactosaminyltransferase