Identification of Further Elongation and Branching of Dimeric Type 1 Chain on Lactosylceramides from Colonic Adenocarcinoma by Tandem Mass Spectrometry Sequencing Analyses
Resource
Journal of Biological Chemistry 283 (24): 16455-16468
Journal
Journal of Biological Chemistry
Pages
16455-16468
Date Issued
2008
Date
2008
Author(s)
Fan, Yao-Yun
Yu, Shin-Yi
Ito, Hiromi
Kameyama, Akihiko
Sato, Takashi
Lin, Chi-Hung
Narimatsu, Hisashi
Khoo, Kay-Hooi
Abstract
Mammalian glycan chain elongation is mostly based on extending the type 2 chain, Galβ1-4GlcNAc, whereas the corresponding type 1 chain, Galβ1-3GlcNAc, is not normally extended. In a broader context of developing high sensitivity mass spectrometry methodologies for glycomic identification of Lea versus Lex and linear versus branched poly-N-acetyllactosamine (polyLacNAc), we have now shown that the dimeric type 1 glycan chain, as carried on the lactosylceramides of a human colonic adenocarcinoma cell line, Colo205, not only can be further extended linearly but can likewise be branched at C6 of 3-linked Gal in a manner similar to polyLacNAc. A combination of chemical and enzymatic derivatization coupled with advanced mass spectrometry analyses afforded unambiguous identification of a complex mixture of type 1 and 2 hybrids as well as those fucosylated variants founded exclusively on linear and branched trimeric type 1 chain. We further showed by in vitro enzymatic synthesis that extended type 1 and the hybrid chains can be branched by all three forms of the human I branching enzymes (IGnT) currently identified but with lower efficiency and stringency with respect to branching site preference. Importantly, it was found that a better substrate is one that carries a Gal site for branching that is extended at the non-reducing end by a type 2 and not a type 1 unit, whereas the IGnTs are less discriminative with respect to whether the targeted Gal site is itself β3- or β4-linked to GlcNAc at the reducing end. © 2008 by The American Society for Biochemistry and Molecular Biology, Inc.
Other Subjects
Cell culture; Electric field effects; Mammals; Mass spectrometers; Mass spectrometry; Spectrometers; Spectrometry; Spectrum analysis; Adenocarcinoma cell lines; Branched polies; Branching enzymes; Complex mixtures; Derivatization; Enzymatic syntheses; Glycan chains; High sensitivities; Hybrid chains; In vitro; Mass spectrometry analysis; Reducing ends; Sequencing analysis; Site preferences; Tandem mass spectrometries; High performance liquid chromatography; 1,4 alpha glucan branching enzyme; aminosugar; glycan; lactosylceramide; poly n acteyllactosamine; unclassified drug; fucose; lactosylceramide; messenger RNA; poly n acetyllactosamine; poly-N-acetyllactosamine; polysaccharide; article; colon adenocarcinoma; controlled study; derivatization; enzyme synthesis; fucosylation; human; human cell; hybrid; matrix assisted laser desorption ionization time of flight mass spectrometry; outcome assessment; priority journal; sequence analysis; tandem mass spectrometry; adenocarcinoma; cell fractionation; chemistry; colon tumor; dimerization; exon; mass spectrometry; metabolism; methodology; tumor cell line; Mammalia; Adenocarcinoma; Cell Line, Tumor; Colonic Neoplasms; Dimerization; Exons; Fucose; Humans; Lactosylceramides; Mass Spectrometry; Polysaccharides; RNA, Messenger; Spectrometry, Mass, Matrix-Assisted Laser Desorption-Ionization; Subcellular Fractions; Tandem Mass Spectrometry
Type
journal article
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