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  • Identification of further elongation and branching of dimeric type 1 chain on lactosylceramides from colonic adenocarcinoma by tandem mass spectrometry sequencing analyses.

Identification of further elongation and branching of dimeric type 1 chain on lactosylceramides from colonic adenocarcinoma by tandem mass spectrometry sequencing analyses.

The Journal of biological chemistry (2008-04-17)
Yao-Yun Fan, Shin-Yi Yu, Hiromi Ito, Akihiko Kameyama, Takashi Sato, Chi-Hung Lin, Lung-Chih Yu, Hisashi Narimatsu, Kay-Hooi Khoo
ABSTRACT

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.

MATERIALS
Product Number
Brand
Product Description

Supelco
Supelclean ENVI-Carb SPE Tube, bed wt. 250 mg, volume 3 mL, pk of 54
Supelco
Supelclean ENVI-Carb SPE Tube, bed wt. 250 mg, volume 6 mL, pk of 30