Proteomic identification of specific glycosyltransferases functionally implicated for the biosynthesis of a targeted glyco-epitope.
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 beta3-galactosyltransferase 5 (beta3GalT5) is indeed the overexpressed beta3GalT. This and beta4GalT1 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 alpha3/4-fucosyltransferase (FucT) activities, we then identified FucT3 and FucT6 as the two major alpha3/4FucTs 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 alpha3/4FucT activity, and not other GDP-fucose utilizing FucTs.
Lin
Chi-Hung
CH
Chemical Biology and Molecular Biophysics Program, Taiwan International Graduate Program, Academia Sinica, Taipei, Taiwan.
Lin
Chia-Wei
CW
Khoo
Kay-Hooi
KH
D000595:Amino Acid Sequence; D045744:Cell Line, Tumor; D000939:Epitopes / Q000096:biosynthesis*; D006031:Glycosylation; D016695:Glycosyltransferases / Q000378:metabolism*; D006056:Golgi Apparatus / Q000378:metabolism*; D006801:Humans; D008969:Molecular Sequence Data; D020543:Proteome / Q000378:metabolism*; D053719:Tandem Mass Spectrometry / Q000379:methods
D000939:Epitopes; D020543:Proteome; D016695:Glycosyltransferases