Identification of further elongation and branching of dimeric type 1 chain on lactosylceramides from colonic adenocarcinoma by tandem mass spectrometry sequencing analyses.
Mammalian glycan chain elongation is mostly based on extending the type 2 chain, Galbeta1-4GlcNAc, whereas the corresponding type 1 chain, Galbeta1-3GlcNAc, is not normally extended. In a broader context of developing high sensitivity mass spectrometry methodologies for glycomic identification of Le(a) versus Le(x) 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 beta3- or beta4-linked to GlcNAc at the reducing end.
Fan
Yao-Yun
YY
Institute of Biochemical Sciences, National Taiwan University, Taipei 106, Taiwan.
Yu
Shin-Yi
SY
Ito
Hiromi
H
Kameyama
Akihiko
A
Sato
Takashi
T
Lin
Chi-Hung
CH
Yu
Lung-Chih
LC
Narimatsu
Hisashi
H
Khoo
Kay-Hooi
KH
D000230:Adenocarcinoma / Q000378:metabolism*; D045744:Cell Line, Tumor; D003110:Colonic Neoplasms / Q000378:metabolism*; D019281:Dimerization; D005091:Exons; D005643:Fucose / Q000737:chemistry; D006801:Humans; D007790:Lactosylceramides / Q000737:chemistry*; D013058:Mass Spectrometry / Q000379:methods; D011134:Polysaccharides / Q000737:chemistry / Q000378:metabolism; D012333:RNA, Messenger / Q000378:metabolism; D019032:Spectrometry, Mass, Matrix-Assisted Laser Desorption-Ionization; D013347:Subcellular Fractions / Q000378:metabolism; D053719:Tandem Mass Spectrometry / Q000379:methods
D007790:Lactosylceramides; D011134:Polysaccharides; D012333:RNA, Messenger; D005643:Fucose; C037199:poly-N-acetyllactosamine