Skip to main content
PMC home page
Biochemical Journal logoLink to Biochemical Journal
. 1996 Jan 15;313(Pt 2):589–596. doi: 10.1042/bj3130589

Biosynthesis of dermatan sulphate. Defructosylated Escherichia coli K4 capsular polysaccharide as a substrate for the D-glucuronyl C-5 epimerase, and an indication of a two-base reaction mechanism.

H H Hannesson 1, A Hagner-McWhirter 1, K Tiedemann 1, U Lindahl 1, A Malmström 1
PMCID: PMC1216948  PMID: 8573097

Abstract

The capsular polysaccharide from Escherichia coli K4 consists of a chondroitin ([GlcA(beta 1-->3)GalNAc(beta 1-->4)]n) backbone, to which beta-fructofuranose units are linked to C-3 of D-glucuronic acid (GlcA) residues. Removal of the fructose units by mild acid hydrolysis provided a substrate for the GlcA C-5 epimerase, which is involved in the generation of L-iduronic acid (IdoA) units during dermatan sulphate biosynthesis. Incubation of this substrate with solubilized fibroblast microsomal enzyme in the presence of 3H2O resulted in the incorporation of tritium at C-5 of hexuronyl units. A Km of 67 x 10(-6) M hexuronic acid (equivalent to disaccharide units) was determined, which is similar to that (80 x 10(-6) M) obtained for dermatan (desulphated dermatan sulphate). Vmax was about 4 times higher with dermatan than with the K4 substrate. A defructosylated K4 polysaccharide isolated after incubation of bacteria with D-[5-3H]glucose released 3H2O on reaction with the epimerase, and thus could be used to assay the enzyme. Incubation of a K4 substrate with solubilized microsomal epimerase for 6 h in the presence of 3H2O resulted in the formation of about 5% IdoA and approximately equal amounts of 3H in GlcA and IdoA. A corresponding incubation of dermatan yielded approx. 22% GlcA, which contained virtually all the 3H label. These results are tentatively explained in terms of a two-base reaction mechanism, involving a monoprotic L-ido-specific base and a polyprotic D-gluco-specific base. Most of the IdoA residues generated by the enzyme occurred singly, although some formation of two or three consecutive IdoA-containing disaccharide units was observed.

Full Text

The Full Text of this article is available as a PDF (614.4 KB).

Selected References

These references are in PubMed. This may not be the complete list of references from this article.

  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. Casu B., Petitou M., Provasoli M., Sinaÿ P. Conformational flexibility: a new concept for explaining binding and biological properties of iduronic acid-containing glycosaminoglycans. Trends Biochem Sci. 1988 Jun;13(6):221–225. doi: 10.1016/0968-0004(88)90088-6. [DOI] [PubMed] [Google Scholar]
  4. Cheng F., Heinegård D., Malmström A., Schmidtchen A., Yoshida K., Fransson L. A. Patterns of uronosyl epimerization and 4-/6-O-sulphation in chondroitin/dermatan sulphate from decorin and biglycan of various bovine tissues. Glycobiology. 1994 Oct;4(5):685–696. doi: 10.1093/glycob/4.5.685. [DOI] [PubMed] [Google Scholar]
  5. Choi H. U., Johnson T. L., Pal S., Tang L. H., Rosenberg L., Neame P. J. Characterization of the dermatan sulfate proteoglycans, DS-PGI and DS-PGII, from bovine articular cartilage and skin isolated by octyl-sepharose chromatography. J Biol Chem. 1989 Feb 15;264(5):2876–2884. [PubMed] [Google Scholar]
  6. Cöster L., Carlstedt I., Malmström A., Särnstrand B. Biosynthesis and secretion of dermatan sulphate proteoglycans in cultures of human skin fibroblasts. Biochem J. 1984 Jun 1;220(2):575–582. doi: 10.1042/bj2200575. [DOI] [PMC free article] [PubMed] [Google Scholar]
  7. Cöster L., Fransson L. A. Isolation and characterization of dermatan sulphate proteoglycans from bovine sclera. Biochem J. 1981 Jan 1;193(1):143–153. doi: 10.1042/bj1930143. [DOI] [PMC free article] [PubMed] [Google Scholar]
  8. DISCHE Z., DEVI A. A new colorimetric method for the determination of ketohexoses in presence of aldoses, ketoheptoses and ketopentoses. Biochim Biophys Acta. 1960 Mar 25;39:140–144. doi: 10.1016/0006-3002(60)90129-3. [DOI] [PubMed] [Google Scholar]
  9. Esko J. D. Genetic analysis of proteoglycan structure, function and metabolism. Curr Opin Cell Biol. 1991 Oct;3(5):805–816. doi: 10.1016/0955-0674(91)90054-3. [DOI] [PubMed] [Google Scholar]
  10. Kjellén L., Lindahl U. Proteoglycans: structures and interactions. Annu Rev Biochem. 1991;60:443–475. doi: 10.1146/annurev.bi.60.070191.002303. [DOI] [PubMed] [Google Scholar]
  11. Maimone M. M., Tollefsen D. M. Structure of a dermatan sulfate hexasaccharide that binds to heparin cofactor II with high affinity. J Biol Chem. 1990 Oct 25;265(30):18263–18271. [PubMed] [Google Scholar]
  12. Malmström A., Fransson L. A. Biosynthesis of dermatan sulfate. I. Formation of L-iduronic acid residues. J Biol Chem. 1975 May 10;250(9):3419–3425. [PubMed] [Google Scholar]
  13. Malmström A., Fransson L. A. Structure of pig skin dermatan sulfate. 2. Demonstration of sulfated iduronic acid residues. Eur J Biochem. 1971 Feb 1;18(3):431–435. doi: 10.1111/j.1432-1033.1971.tb01260.x. [DOI] [PubMed] [Google Scholar]
  14. Norman M., Ekman G., Ulmsten U., Barchan K., Malmström A. Proteoglycan metabolism in the connective tissue of pregnant and non-pregnant human cervix. An in vitro study. Biochem J. 1991 Apr 15;275(Pt 2):515–520. doi: 10.1042/bj2750515. [DOI] [PMC free article] [PubMed] [Google Scholar]
  15. Rodriguez M. L., Jann B., Jann K. Structure and serological characteristics of the capsular K4 antigen of Escherichia coli O5:K4:H4, a fructose-containing polysaccharide with a chondroitin backbone. Eur J Biochem. 1988 Oct 15;177(1):117–124. doi: 10.1111/j.1432-1033.1988.tb14351.x. [DOI] [PubMed] [Google Scholar]
  16. Scott J. E. Proteoglycan-fibrillar collagen interactions. Biochem J. 1988 Jun 1;252(2):313–323. doi: 10.1042/bj2520313. [DOI] [PMC free article] [PubMed] [Google Scholar]
  17. 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]
  18. Westergren-Thorsson G., Persson S., Isaksson A., Onnervik P. O., Malmström A., Fransson L. A. L-iduronate-rich glycosaminoglycans inhibit growth of normal fibroblasts independently of serum or added growth factors. Exp Cell Res. 1993 May;206(1):93–99. doi: 10.1006/excr.1993.1124. [DOI] [PubMed] [Google Scholar]
  19. Westergren-Thorsson G., Schmidtchen A., Särnstrand B., Fransson L. A., Malmström A. Transforming growth factor-beta induces selective increase of proteoglycan production and changes in the copolymeric structure of dermatan sulphate in human skin fibroblasts. Eur J Biochem. 1992 Apr 1;205(1):277–286. doi: 10.1111/j.1432-1033.1992.tb16778.x. [DOI] [PubMed] [Google Scholar]
  20. Yamagata T., Saito H., Habuchi O., Suzuki S. Purification and properties of bacterial chondroitinases and chondrosulfatases. J Biol Chem. 1968 Apr 10;243(7):1523–1535. [PubMed] [Google Scholar]

Articles from Biochemical Journal are provided here courtesy of The Biochemical Society

RESOURCES