Biosynthesis of heparin. The D-glucuronosyl- and N-acetyl-D-glucosaminyltransferase reactions and their relation to polymer modification.
Oligosaccharides with the general structure [GlcA-GlcNAc]n-GlcA-aMan (aMan is 2,5-anhydro-D-mannose), derived from the Escherichia coli K5 capsular polysaccharide, were found to serve as monosaccharide acceptors for a GlcNAc-transferase, solubilized from a mouse mastocytoma microsomal fraction and implicated in heparin biosynthesis. Digestion of these oligosaccharides with beta-D-glucuronidase yielded acceptors for the GlcA-transferase that acts in concert with the GlcNAc-transferase. Assays based on the oligosaccharide acceptors showed broad pH optima for the two enzymes, centred around pH 6.5 for the GlcNAc-transferase and around pH 7.0 for the GlcA-transferase. The GlcNAc-transferase showed an absolute requirement for Mn2+, whereas the GlcA-transferase was stimulated by Ca2+ and Mg2+ but not by Mn2+. The GlcNAc acceptor ability of the [GlcA-GlcNAc]n-GlcA-aMan oligosaccharides increased with increasing chain length, as reflected by the apparent Km, which was 60 microM for a hexasaccharide but 6 microM for a hexadecasaccharide. By contrast, the Km for [GlcNAc-GlcA]n-aMan oligosaccharides in the GlcA-transferase reaction was higher, approximately 0.5 mM, and unaffected by acceptor size. After chemical modification of the oligosaccharides to obtain mixed N-substituents (N-unsubstituted, N-acetylated or N-sulphated GlcN residues), GlcNAc transfer was found to be virtually independent of the N-substituent pattern of the acceptor sequence. The GlcA-transferase, on the other hand, showed marked preference for an acceptor with a non-reducing-terminal GlcNAc-GlcA-GlcNSO3- sequence, which would thus have a lower Km for the enzyme than the corresponding fully N-acetylated structure. These results, along with our previous finding that chain elongation in a mastocytoma microsomal system is strongly promoted by concomitant N-sulphation of the nascent chain [Lidholt, Kjellén & Lindahl (1989) Biochem. J. 261, 999-1007], raise the possibility that the glycosyltransferases and the N-deacetylase/N-sulphotransferase act in concert during chain elongation, assembled into an enzyme complex.
Lidholt
K
K
Department of Veterinary Medical Chemistry, Swedish University of Agricultural Sciences, Uppsala.
Lindahl
U
U
D000818:Animals; D002240:Carbohydrate Sequence; D014453:Glucuronosyltransferase / Q000378:metabolism*; D006493:Heparin / Q000096:biosynthesis*; D006863:Hydrogen-Ion Concentration; D007700:Kinetics; D008407:Mast Cells / Q000378:metabolism; D008670:Metals / Q000494:pharmacology; D051379:Mice; D008969:Molecular Sequence Data; D017351:N-Acetylglucosaminyltransferases / Q000378:metabolism*; D011135:Polysaccharides, Bacterial / Q000378:metabolism; D013329:Structure-Activity Relationship; D013379:Substrate Specificity; D014407:Tumor Cells, Cultured
D008670:Metals; D011135:Polysaccharides, Bacterial; D006493:Heparin; D017351:N-Acetylglucosaminyltransferases; C037649:N-acetyllactosaminide beta-1,6-N-acetylglucosaminyltransferase; D014453:Glucuronosyltransferase