Proteomic identification of specific glycosyltransferases functionally implicated for the biosynthesis of a targeted glyco-epitope

Chi Hung Lin, Chia Wei Lin, Kay Hooi Khoo*

*Corresponding author for this work

    Research output: Contribution to journalArticlepeer-review

    6 Citations (Scopus)


    Functional glycomic and glycoproteomic analyses often entail correlating the mapped glycosylation pattern of a cell against the activities of specific glycosyltransferases it expresses. While the mRNA transcripts can be readily mapped, the expression of a functional glycosyltransferase at protein level has defied most current proteomic approaches. To enable identification of these low abundant Golgi residing membrane bound proteins, we have developed a novel semigel-based shotgun proteomic workflow incorporating subcellular fractionation and one-step affinity enrichment of the detergent solubilized Golgi preparation on resins derivatized with nucleotide diphosphates. Applying the strategy to a colonic adenocarcinoma, Colo205, which is known to aberrantly synthesize abundant fucosylated extended type 1 chain, we first validated that β3-galactosyltransferase 5 (β3GalT5) is indeed the overexpressed β3GalT. This and β4GalT1 are the two galactosyltrasferases which were positively identified by proteomic analysis of the eluted fractions from uridine diphosphate (UDP)-affinity column. Substituting UDP with a guanidine diphosphate (GDP)-affinity column and monitoring the eluted fractions for enriched α3/4-fucosyltransferase (FucT) activities, we then identified FucT3 and FucT6 as the two major α3/4FucTs expressed in Colo205 at the protein level. Our proteomic analysis demonstrated that not all GDP-utilizing glycosyltransferases bind and are retained similarly by the GDP-affinity column and that specific activity assay along with optimization of binding and elution conditions is critical for successful identification of a particular subset of the targeted glycosyltransferases. Only OFUT1, a protein O-FucT, was additionally identified to coelute with the α3/4FucT activity, and not other GDP-fucose utilizing FucTs.

    Original languageEnglish
    Pages (from-to)475-483
    Number of pages9
    Issue number3
    Publication statusPublished - Feb 2008


    • Affinity capture
    • Glycosyltransferases
    • Subcellular fractionation


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