Systematic and Targeted Glycomic Analysis to Map the Expression of Specific Terminal Glycotopes in Relation to Over-expression of β1,3-Galactosyltransferase V
Date Issued
2008
Date
2008
Author(s)
Chen, Yen-Ying
Abstract
Glycosylation plays essential roles in cell-cell recognition, communication and adhesion by means of specific glycotopes. Among the important glycobiology issues to be addressed is the specific elongation of core structures, leading to terminal decoration with bioactive glycotopes. Type II chain (Galβ1-4GlcNAc), N-acetyllactosamine (LacNAc), is a ubiquitous disaccharide building block of mammalian glycoconjugates, which may be further elongated with additional LacNAc units into the so-called polylactosaminoglycans. In contrast, anther basic unit which differs only in linkage, namely the type I chain, or Galβ1-3GlcNAc, is generally not extended. Among the rare exceptions are the multi-fucosylated extended type I chains carried on the lactosylceramides of a colorectal cancer cell line, Colo205. β1,3-galactosyltransferase V (β3Gal-T5), a crucial enzyme for type I chain formation, was suggested to be involved with in vivo biosynthesis of extended type I chains. In pursuit of this, β3Gal-T5 was over-expressed in another colorectal cell line, DLD-1, which did not synthesize extended type I chain naturally. A concerted strategy combing antibody-dependent detection and tandem mass spectrometry (MS), which would distinguish the subtle linkage variation between type I and II chains, was developed and implemented to delineate the induced glycomic alteration. We showed that DLD-1 transfected with β3Gal-T5 (DLD3GT5-1) strongly expressed type I glycotopes whereas type II glycotopes were significantly decreased. A complementary low and high energy CID MALDI-MS/MS analysis validated the presence of extended type I chains and associated fucosylated glycotopes on glycolipids and N-glycans of DLD3GT5-1. Furthermore, the comprehensive survey through product ion-dependent MS3 in LC-MS/MS analysis performed on LTQ-Orbitrap showed that type I-derived Lea was extensively biosynthesized on N-glycans upon β3Gal-T5 over-expression, in contrast with predominant type II-derived Lex in mock transfectant. Finally, semi-quantitative analysis based on MALDI-MS profiles revealed a significantly suppressed expression of sialylation and polylactosaminoglycans but elevated fucosylation in N-glycans and O-glycans, thus suggesting the determining roles of β3Gal-T5 in glycome shaping in colorectal cancer cell lines.
Subjects
glycomics
mass spectrometry
glycosylation
glycosyltransferase
SDGs
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