Unexpected Tolerance of Glycosylation by UDP-GalNAc:Polypeptide α-N-Acetylgalactosaminyltransferase Revealed by Electron Capture Dissociation Mass Spectrometry: Carbohydrate as Potential P
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- 作者：Yayoi Yoshimura ; Takahiko Matsushita ; Naoki Fujitani ; Yasuhiro Takegawa ; Haruhiko Fujihira ; Kentarou Naruchi ; Xiao-Dong Gao ; Naomi Manri ; Takeshi Sakamoto ; Kentaro Kato ; Hiroshi Hinou ; Shin-Ichiro Nish
- 刊名：Biochemistry
- 出版年：2010
- 出版时间：July 20, 2010
- 年：2010
- 卷：49
- 期：28
- 页码：5929-5941
- 全文大小：1273K
- 年卷期：v.49,no.28(July 20, 2010)
- ISSN：1520-4995
文摘
UDP-GalNAc:polypeptide α-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 α-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 α-GalNAc residue at potential glycosylation sites by ppGalNAcT2 proceeds smoothly in various unnatural glycopeptides having α-Man, α-Fuc, and β-Gal residues as well as α-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 α-, β-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 α-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.