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. 1992 Oct 1;287(Pt 1):21–29. doi: 10.1042/bj2870021

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

K Lidholt 1, U Lindahl 1
PMCID: PMC1133118  PMID: 1417774

Abstract

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.

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Selected References

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  1. BITTER T., MUIR H. M. A modified uronic acid carbazole reaction. Anal Biochem. 1962 Oct;4:330–334. doi: 10.1016/0003-2697(62)90095-7. [DOI] [PubMed] [Google Scholar]
  2. Bäckström G., Hök M., Lindahl U., Feingold D. S., Malmström A., Rodén L., Jacobsson I. Biosynthesis of heparin. Assay and properties of the microsomal uronosyl C-5 epimerase. J Biol Chem. 1979 Apr 25;254(8):2975–2982. [PubMed] [Google Scholar]
  3. Dietrich C. P., Nader H. B., Buonassisi V., Colburn P. Inhibition of synthesis of heparan sulfate by selenate: possible dependence on sulfation for chain polymerization. FASEB J. 1988 Jan;2(1):56–59. doi: 10.1096/fasebj.2.1.2961646. [DOI] [PubMed] [Google Scholar]
  4. FURTH J., HAGEN P., HIRSCH E. I. Transplantable mastocytoma in the mouse containing histamine, heparin, 5-hydroxytryptamine. Proc Soc Exp Biol Med. 1957 Aug-Sep;95(4):824–828. doi: 10.3181/00379727-95-23375. [DOI] [PubMed] [Google Scholar]
  5. Forsee W. T., Rodén L. Biosynthesis of heparin. Transfer of N-acetylglucosamine to heparan sulfate oligosaccharides. J Biol Chem. 1981 Jul 25;256(14):7240–7247. [PubMed] [Google Scholar]
  6. Gallagher J. T., Lyon M., Steward W. P. Structure and function of heparan sulphate proteoglycans. Biochem J. 1986 Jun 1;236(2):313–325. doi: 10.1042/bj2360313. [DOI] [PMC free article] [PubMed] [Google Scholar]
  7. Gundlach M. W., Conrad H. E. Glycosyl transferases in chondroitin sulphate biosynthesis. Effect of acceptor structure on activity. Biochem J. 1985 Mar 15;226(3):705–714. doi: 10.1042/bj2260705. [DOI] [PMC free article] [PubMed] [Google Scholar]
  8. Helting T. Biosynthesis of heparin. Solubilization and partial purification of uridine diphosphate glucuronic acid: acceptor glucuronosyltransferase from mouse mastocytoma. J Biol Chem. 1972 Jul 10;247(13):4327–4332. [PubMed] [Google Scholar]
  9. Helting T., Lindahl U. Biosynthesis of heparin. I. Transfer of N-acetylglucosamine and glucuronic acid to low-molecular weight heparin fragments. Acta Chem Scand. 1972;26(9):3515–3523. doi: 10.3891/acta.chem.scand.26-3515. [DOI] [PubMed] [Google Scholar]
  10. Helting T., Lindahl U. Occurrence and biosynthesis of beta-glucuronidic linkages in heparin. J Biol Chem. 1971 Sep 10;246(17):5442–5447. [PubMed] [Google Scholar]
  11. Hök M., Lindahl U., Hallén A., Bäckström G. Biosynthesis of heparin. Studies on the microsomal sulfation process. J Biol Chem. 1975 Aug 10;250(15):6065–6071. [PubMed] [Google Scholar]
  12. Hök M., Riesenfeld J., Lindahl U. N-[3H]Acetyl-labeling, a convenient method for radiolabeling of glycosaminoglycans. Anal Biochem. 1982 Jan 15;119(2):236–245. doi: 10.1016/0003-2697(82)90580-2. [DOI] [PubMed] [Google Scholar]
  13. Iozzo R. V. Presence of unsulfated heparan chains on the heparan sulfate proteoglycan of human colon carcinoma cells. Implications for heparan sulfate proteoglycan biosynthesis. J Biol Chem. 1989 Feb 15;264(5):2690–2699. [PubMed] [Google Scholar]
  14. Jacobsson I., Lindahl U. Biosynthesis of heparin. Concerted action of late polymer-modification reactions. J Biol Chem. 1980 Jun 10;255(11):5094–5100. [PubMed] [Google Scholar]
  15. Jacobsson I., Lindahl U., Jensen J. W., Rodén L., Prihar H., Feingold D. S. Biosynthesis of heparin. Substrate specificity of heparosan N-sulfate D-glucuronosyl 5-epimerase. J Biol Chem. 1984 Jan 25;259(2):1056–1063. [PubMed] [Google Scholar]
  16. Jacobsson K. G., Riesenfeld J., Lindahl U. Biosynthesis of heparin. Effects of n-butyrate on cultured mast cells. J Biol Chem. 1985 Oct 5;260(22):12154–12159. [PubMed] [Google Scholar]
  17. Kusche M., Bäckström G., Riesenfeld J., Petitou M., Choay J., Lindahl U. Biosynthesis of heparin. O-sulfation of the antithrombin-binding region. J Biol Chem. 1988 Oct 25;263(30):15474–15484. [PubMed] [Google Scholar]
  18. Kusche M., Hannesson H. H., Lindahl U. Biosynthesis of heparin. Use of Escherichia coli K5 capsular polysaccharide as a model substrate in enzymic polymer-modification reactions. Biochem J. 1991 Apr 1;275(Pt 1):151–158. doi: 10.1042/bj2750151. [DOI] [PMC free article] [PubMed] [Google Scholar]
  19. LEVY L., PETRACEK F. J. Chemical and pharmacological studies on N-resulfated heparin. Proc Soc Exp Biol Med. 1962 Apr;109:901–905. doi: 10.3181/00379727-109-27372. [DOI] [PubMed] [Google Scholar]
  20. LOWRY O. H., ROSEBROUGH N. J., FARR A. L., RANDALL R. J. Protein measurement with the Folin phenol reagent. J Biol Chem. 1951 Nov;193(1):265–275. [PubMed] [Google Scholar]
  21. Lidholt K., Kjellén L., Lindahl U. Biosynthesis of heparin. Relationship between the polymerization and sulphation processes. Biochem J. 1989 Aug 1;261(3):999–1007. doi: 10.1042/bj2610999. [DOI] [PMC free article] [PubMed] [Google Scholar]
  22. Lidholt K., Riesenfeld J., Jacobsson K. G., Feingold D. S., Lindahl U. Biosynthesis of heparin. Modulation of polysaccharide chain length in a cell-free system. Biochem J. 1988 Sep 1;254(2):571–578. doi: 10.1042/bj2540571. [DOI] [PMC free article] [PubMed] [Google Scholar]
  23. Lidholt K., Weinke J. L., Kiser C. S., Lugemwa F. N., Bame K. J., Cheifetz S., Massagué J., Lindahl U., Esko J. D. A single mutation affects both N-acetylglucosaminyltransferase and glucuronosyltransferase activities in a Chinese hamster ovary cell mutant defective in heparan sulfate biosynthesis. Proc Natl Acad Sci U S A. 1992 Mar 15;89(6):2267–2271. doi: 10.1073/pnas.89.6.2267. [DOI] [PMC free article] [PubMed] [Google Scholar]
  24. Lindahl U., Bäckström G., Jansson L., Hallén A. Biosynthesis of heparin. II. Formation of sulfamino groups. J Biol Chem. 1973 Oct 25;248(20):7234–7241. [PubMed] [Google Scholar]
  25. Pejler G., Bäckström G., Lindahl U., Paulsson M., Dziadek M., Fujiwara S., Timpl R. Structure and affinity for antithrombin of heparan sulfate chains derived from basement membrane proteoglycans. J Biol Chem. 1987 Apr 15;262(11):5036–5043. [PubMed] [Google Scholar]
  26. Pettersson I., Kusche M., Unger E., Wlad H., Nylund L., Lindahl U., Kjellén L. Biosynthesis of heparin. Purification of a 110-kDa mouse mastocytoma protein required for both glucosaminyl N-deacetylation and N-sulfation. J Biol Chem. 1991 May 5;266(13):8044–8049. [PubMed] [Google Scholar]
  27. Riesenfeld J., Hök M., Lindahl U. Biosynthesis of heparin. Concerted action of early polymer-modification reactions. J Biol Chem. 1982 Jan 10;257(1):421–425. [PubMed] [Google Scholar]
  28. SILBERT J. E. INCORPORATION OF 14C AND 3H FROM NUCLEOTIDE SUGARS INTO A POLYSACCHARIDE IN THE PRESENCE OF A CELL-FREE PREPARATION FROM MOUSE MAST CELL TUMORS. J Biol Chem. 1963 Nov;238:3542–3546. [PubMed] [Google Scholar]
  29. Shaklee P. N., Conrad H. E. Hydrazinolysis of heparin and other glycosaminoglycans. Biochem J. 1984 Jan 1;217(1):187–197. doi: 10.1042/bj2170187. [DOI] [PMC free article] [PubMed] [Google Scholar]
  30. Shively J. E., Conrad H. E. Formation of anhydrosugars in the chemical depolymerization of heparin. Biochemistry. 1976 Sep 7;15(18):3932–3942. doi: 10.1021/bi00663a005. [DOI] [PubMed] [Google Scholar]
  31. Sugumaran G., Silbert J. E. Relationship of sulfation to ongoing chondroitin polymerization during biosynthesis of chondroitin 4-sulfate by microsomal preparations from cultured mouse mastocytoma cells. J Biol Chem. 1990 Oct 25;265(30):18284–18288. [PubMed] [Google Scholar]
  32. Vann W. F., Schmidt M. A., Jann B., Jann K. The structure of the capsular polysaccharide (K5 antigen) of urinary-tract-infective Escherichia coli 010:K5:H4. A polymer similar to desulfo-heparin. Eur J Biochem. 1981 May 15;116(2):359–364. doi: 10.1111/j.1432-1033.1981.tb05343.x. [DOI] [PubMed] [Google Scholar]

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