title: Overexpression of N-acetylglucosaminyltransferases III and V in human melanoma cells. Implications for MCAM N-glycosylation.
abstract: An important role in cancer pathogenesis is attributed to N-glycans with "bisecting" N-acetylglucosamine and beta1-6 branches but the exact mechanisms still remain to be elucidated. Two structures are formed by Golgi beta-1,4-mannosyl-glycoprotein 4-beta-N-acetylglucosaminyltransferase (EC = 2.4.1.144, GnT-III) and alpha-1,6-mannosylglycoprotein 6-beta-N-acetylglucosaminyltransferase A (EC = 2.4.1.155, GnT-V) respectively. The enzymes are encoded by MGAT3 and MGAT5 genes. The aim of this study was to establish two human melanoma cell lines with induced overexpression of GnT-III or GnT-V and to perform a preliminary functional characterization. WM-266-4 cells were stably transfected with human MGAT3 or MGAT5 cDNAs. GnT-III and GnT-V activities were assayed with a novel HPLC method based on labeling of N-glycan acceptor with 2-aminobenzamide (adapted from Taniguchi et al., 1989). Higher expression and activities of glycosyltransferases were detected. Increased amounts of "bisected" and beta1-6 branched N-glycans were present on melanoma cell adhesion molecule (known as MCAM/MUC18). However, cells did not display significant differences in viability and capabilities to migrate through an endothelial layer. To the best of our knowledge, the result of our study is the first to demonstrate that "bisected" N-glycans can be carried by MCAM. Moreover, increased modification of this protein by the two glycosyltransferases in WM-266-4-GnT-III cells was the consequence of the overexpression of only one enzyme. The obtained model can be useful for studying mechanisms of N-glycans branching and better understanding of their role in cancer progression. The proposed modification of the glycosyltransferase activity assay has shown to be a good alternative for 2-aminopyridine based HPLC systems.
authors.affiliation: Department of Glycoconjugate Biochemistry, Institute of Zoology, Jagiellonian University, Gronostajowa 9, 30-387 Kraków, Poland. Electronic address: monika.bubka@uj.edu.pl.
authors.forename: Monika
authors.initials: M
authors.lastname: Bubka
authors.affiliation: Department of Glycoconjugate Biochemistry, Institute of Zoology, Jagiellonian University, Gronostajowa 9, 30-387 Kraków, Poland.
authors.forename: Paweł
authors.initials: P
authors.lastname: Link-Lenczowski
authors.affiliation: Department of Glycoconjugate Biochemistry, Institute of Zoology, Jagiellonian University, Gronostajowa 9, 30-387 Kraków, Poland.
authors.forename: Marcelina
authors.initials: M
authors.lastname: Janik
authors.affiliation: Department of Glycoconjugate Biochemistry, Institute of Zoology, Jagiellonian University, Gronostajowa 9, 30-387 Kraków, Poland.
authors.forename: Ewa
authors.initials: E
authors.lastname: Pocheć
authors.affiliation: Department of Glycoconjugate Biochemistry, Institute of Zoology, Jagiellonian University, Gronostajowa 9, 30-387 Kraków, Poland.
authors.forename: Anna
authors.initials: A
authors.lastname: Lityńska
mesh_terms: D000217:Acyltransferases / Q000235:genetics* / Q000378:metabolism*; D051929:CD146 Antigen / Q000378:metabolism; D045744:Cell Line, Tumor; D002465:Cell Movement; D002470:Cell Survival; D042783:Endothelial Cells / Q000473:pathology; D015870:Gene Expression; D006031:Glycosylation; D006801:Humans; D008545:Melanoma / Q000473:pathology*; D017351:N-Acetylglucosaminyltransferases / Q000235:genetics* / Q000378:metabolism*; D011134:Polysaccharides / Q000378:metabolism; D014162:Transfection; D015854:Up-Regulation
chemical_list: D051929:CD146 Antigen; C497902:MCAM protein, human; D011134:Polysaccharides; D000217:Acyltransferases; C021802:2-acylglycerol O-acyltransferase; D017351:N-Acetylglucosaminyltransferases; C052254:alpha-1,6-mannosylglycoprotein beta 1,6-N-acetylglucosaminyltransferase
keywords: Activity assay; Glycosyltransferases; MCAM; Melanoma; N-glycosylation; Transendothelial migration