title: Increased UDP-GlcNAc:Gal beta 1-3GaLNAc-R (GlcNAc to GaLNAc) beta-1, 6-N-acetylglucosaminyltransferase activity in metastatic murine tumor cell lines. Control of polylactosamine synthesis.
abstract: Malignant transformation of rodent cell lines by polyoma virus and by activated ras genes is associated with increased UDP-GlcNAc:Man alpha-R beta-1,6-N-acetylglucosaminyltransferase V (GlcNAc-transferase V) activity and it product -GlcNAc beta 1-6Man alpha 1-6Man beta 1-branched Asn-linked oligosaccharides. In this report, we have compared beta 1-6GlcNAc branching of core O- and N-linked oligosaccharides in three experimental models of malignancy, namely (a) rat2 fibroblasts and their malignant T24H-ras-transfected counterpart; (b) benign SP1 mammary carcinoma cells and two metastic sublines of SP1; and (c) the metastatic MDAY-D2 lymphoma cell line and its poorly metastatic glycosylation mutant KBL-1. In addition to the previously reported increase in GlcNAc-transferase V activity, UDP-GlcNAc:Gal beta 1-3GalNAc alpha-R (GlcNAc to GalNAc) beta-1,6-N-acetylglucosaminyltransferase (core 2 GlcNAc-transferase, EC 2.4.1.102) activity was found to be elevated by 70% in the malignant rat2 and SP1 cell lines while several other glycosyltransferase activities were not significantly different. The action of core 2 GlcNAc-transferase followed by beta 1-4Gal-transferase provides an N-acetyllactosamine antenna that can be extended with polylactosamine (i.e. repeating Gal beta 1-4GlcNAc beta 1-3) provided UDP-GlcNAc:Gal beta-R beta 1-3GlcNAc-transferase (GlcNAc-transferase) (i)) activity is present. Polylactosamine content in microsomal membrane glycoproteins was quantitated by labeling the GlcNAc termini resulting from the action of Escherichia freundii endo-beta-galactosidase with bovine galactosyltransferase/UDP-[3H] Gal. Glycopeptidase F- sensitive and -insensitive fractions were measured to assess the N- and O-linked components. In the SP1 tumor model, the metastatic sublines showed increased core 2 GlcNAc-transferase and GlcNAc-transferase V activities but no change in GlcNAc-transferase (i) activity, yet polylactosamine was increased in both O- and N-linked oligosaccharides. In rat2 cells, down-regulation of GlcNAc-transferase (i) following transformation was associated with decreased polyactosamine even though core 2 GlcNAc-transferase and GlcNAc-transferase V were elevated in the cells. Finally, a 3-fold decrease in GlcNAc-transferase V in KBL-1, the glycosylation mutant of MDAY-D2 cells, resulted in complete loss of polylactosamine in N-linked but no change in O-linked polylactosamine content. These results suggest that, provided GlcNAc-transferase (i) is not limiting, the beta 1-6-branching enzymes core 2 GlcNAc-transferase and GlcNAc-transferase V regulate the levels of polyactosamine in O- and N-linked oligosaccharides, respectively.(ABSTRACT TRUNCATED AT 400 WORDS)
authors.affiliation: Samuel Lunenfeld Research Institute, Mt. Sinai Hospital, Toronto, Ontario, Canada.
authors.forename: S
authors.initials: S
authors.lastname: Yousefi
authors.forename: E
authors.initials: E
authors.lastname: Higgins
authors.forename: Z
authors.initials: Z
authors.lastname: Daoling
authors.forename: A
authors.initials: A
authors.lastname: Pollex-Krüger
authors.forename: O
authors.initials: O
authors.lastname: Hindsgaul
authors.forename: J W
authors.initials: JW
authors.lastname: Dennis
mesh_terms: D000606:Amino Sugars / Q000096:biosynthesis*; D000818:Animals; D002240:Carbohydrate Sequence; D002471:Cell Transformation, Neoplastic / Q000378:metabolism*; D002851:Chromatography, High Pressure Liquid; D005697:Galactosides / Q000378:metabolism; D011905:Genes, ras; D005964:Glucosyltransferases / Q000378:metabolism*; D006001:Glycoconjugates / Q000737:chemistry / Q000378:metabolism*; D007700:Kinetics; D009682:Magnetic Resonance Spectroscopy; D051379:Mice; D008861:Microsomes / Q000378:metabolism; D008969:Molecular Sequence Data; D017351:N-Acetylglucosaminyltransferases; D009362:Neoplasm Metastasis; D011134:Polysaccharides / Q000096:biosynthesis*; D051381:Rats; D014407:Tumor Cells, Cultured / Q000378:metabolism*
chemical_list: D000606:Amino Sugars; D005697:Galactosides; D006001:Glycoconjugates; D011134:Polysaccharides; C066929:polylactosamine; D005964:Glucosyltransferases; D017351:N-Acetylglucosaminyltransferases; C052254:alpha-1,6-mannosylglycoprotein beta 1,6-N-acetylglucosaminyltransferase