title: Assessment of glycosaminoglycan-protein linkage tetrasaccharides as acceptors for GalNAc- and GlcNAc-transferases from mouse mastocytoma.
abstract: Two glycosaminoglycan-protein linkage tetrasaccharide-serine compounds, GlcAbeta1-3Galbeta1-3Galbeta1-4Xylbeta1-O-Ser and GlcAbeta1-3Gal(4-O-sulfate)beta1-3Galbeta1-4Xylbeta1-O -Ser, were tested as hexosamine acceptors, using UDP-[3H]GlcNAc and UDP-[3H]GalNAc as sugar donors, and solubilized mouse mastocytoma microsomes as enzyme source. The nonsulfated Ser-tetrasaccharide was found to function as an acceptor for a GalNAc residue, whereas the Ser-tetrasaccharide containing a sulfated galactose unit was inactive. Characterization of the radio-labelled product by digestion with alpha-N-acetylgalactosaminidase and beta-N-acetylhexosaminidase revealed that the [3H]GalNAc unit was alpha-linked, as in the product previously synthesized using serum enzymes, and not beta-linked as found in the chondroitin sulfate polymer. Heparan sulfate/heparin biosynthesis could not be primed by either of the two linkage Ser-tetrasaccharides, since no transfer of [3H]GlcNAc from UDP-[3H]GlcNAc could be detected. By contrast, transfer of a [3H]GlcNAc unit to a [GlcAbeta1-4GlcNAcalpha1-4]2-GlcAbeta1-4-aMan hexasaccharide acceptor used to assay the GlcNAc transferase involved in chain elongation, was readily detected. These results are in agreement with the recent proposal that two different N-acetylglucosaminyl transferases catalyse the biosynthesis of heparan sulfate. Although the mastocytoma system contains both the heparan sulfate/heparin and chondroitin sulfate biosynthetic enzymes the Ser-tetrasaccharides do not seem to fulfil the requirements to serve as acceptors for the first HexNAc transfer reactions involved in the formation of these polysaccharides.
authors.affiliation: Department of Medical and Physiological Chemistry, University of Uppsala, The Biomedical Center, Sweden. Kerstin.Lidholt@medkem.uu.se
authors.forename: K
authors.initials: K
authors.lastname: Lidholt
authors.forename: M
authors.initials: M
authors.lastname: Fjelstad
authors.forename: U
authors.initials: U
authors.lastname: Lindahl
authors.forename: F
authors.initials: F
authors.lastname: Goto
authors.forename: T
authors.initials: T
authors.lastname: Ogawa
authors.forename: H
authors.initials: H
authors.lastname: Kitagawa
authors.forename: K
authors.initials: K
authors.lastname: Sugahara
mesh_terms: D000818:Animals; D006025:Glycosaminoglycans / Q000378:metabolism*; D012515:Mast-Cell Sarcoma / Q000201:enzymology*; D051379:Mice; D017350:N-Acetylgalactosaminyltransferases / Q000378:metabolism*; D017351:N-Acetylglucosaminyltransferases / Q000378:metabolism*; D009844:Oligosaccharides / Q000378:metabolism*; D011506:Proteins / Q000378:metabolism*; D013379:Substrate Specificity
chemical_list: D006025:Glycosaminoglycans; D009844:Oligosaccharides; D011506:Proteins; D017350:N-Acetylgalactosaminyltransferases; D017351:N-Acetylglucosaminyltransferases