Biosynthesis of heparin. The D-glucuronosyl- and N-acetyl-D-glucosaminyltransferase reactions and their relation to polymer modification.

摘要: Oligosaccharides with the general structure [GlcA-GlcNAc]n-GlcA-aMan (aMan is 2,5-anhydro-D-mannose), derived from Escherichia coli K5 capsular polysaccharide, were found to serve as monosaccharide acceptors for a GlcNAc-transferase, solubilized mouse mastocytoma microsomal fraction and implicated in heparin biosynthesis. Digestion of these oligosaccharides beta-D-glucuronidase yielded GlcA-transferase that acts concert GlcNAc-transferase. Assays based on oligosaccharide showed broad pH optima two enzymes, centred around 6.5 GlcNAc-transferase 7.0 GlcA-transferase. The an absolute requirement Mn2+, whereas was stimulated by Ca2+ Mg2+ but not Mn2+. GlcNAc acceptor ability increased increasing chain length, reflected apparent Km, which 60 microM hexasaccharide 6 hexadecasaccharide. By contrast, Km [GlcNAc-GlcA]n-aMan reaction higher, approximately 0.5 mM, unaffected size. After chemical modification obtain mixed N-substituents (N-unsubstituted, N-acetylated or N-sulphated GlcN residues), transfer be virtually independent N-substituent pattern sequence. GlcA-transferase, other hand, marked preference non-reducing-terminal GlcNAc-GlcA-GlcNSO3- sequence, would thus have lower enzyme than corresponding fully structure. These results, along our previous finding elongation system strongly promoted concomitant N-sulphation nascent [Lidholt, Kjellen & Lindahl (1989) Biochem. J. 261, 999-1007], raise possibility glycosyltransferases N-deacetylase/N-sulphotransferase act during elongation, assembled into complex.