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. 1990 Dec;87(24):9784–9788. doi: 10.1073/pnas.87.24.9784

Cell mutants defective in synthesizing a heparan sulfate proteoglycan with regions of defined monosaccharide sequence.

A L De Agostini 1, H K Lau 1, C Leone 1, H Youssoufian 1, R D Rosenberg 1
PMCID: PMC55258  PMID: 2263629

Abstract

We have demonstrated that mouse LTA cells synthesize cell-surface heparan sulfate proteoglycans (HSPGs) with regions of defined monosaccharide sequence that specifically interact with antithrombin (HSPGact). It remains unclear how HSPGact can be generated by a biosynthetic pathway with no simple template for directing the ordered assembly of monosaccharide units. To examine this issue, we treated LTA cells with ethyl methanesulfonate and then isolated seven stable mutants that synthesize only 8-27% of the wild-type HSPGact but produce normal amounts of other HSPGs. These mutants are recessive in nature and fall into at least two different complementation groups. The delineation of the molecular basis of these defects should help to elucidate the manner by which cells synthesize HSPGs with regions of defined monosaccharide sequence.

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Selected References

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

  1. Atha D. H., Lormeau J. C., Petitou M., Rosenberg R. D., Choay J. Contribution of 3-O- and 6-O-sulfated glucosamine residues in the heparin-induced conformational change in antithrombin III. Biochemistry. 1987 Oct 6;26(20):6454–6461. doi: 10.1021/bi00394a024. [DOI] [PubMed] [Google Scholar]
  2. Atha D. H., Lormeau J. C., Petitou M., Rosenberg R. D., Choay J. Contribution of monosaccharide residues in heparin binding to antithrombin III. Biochemistry. 1985 Nov 5;24(23):6723–6729. doi: 10.1021/bi00344a063. [DOI] [PubMed] [Google Scholar]
  3. Atha D. H., Stephens A. W., Rimon A., Rosenberg R. D. Sequence variation in heparin octasaccharides with high affinity for antithrombin III. Biochemistry. 1984 Nov 20;23(24):5801–5812. doi: 10.1021/bi00319a020. [DOI] [PubMed] [Google Scholar]
  4. Bame K. J., Esko J. D. Undersulfated heparan sulfate in a Chinese hamster ovary cell mutant defective in heparan sulfate N-sulfotransferase. J Biol Chem. 1989 May 15;264(14):8059–8065. [PubMed] [Google Scholar]
  5. Bienkowski M. J., Conrad H. E. Structural characterization of the oligosaccharides formed by depolymerization of heparin with nitrous acid. J Biol Chem. 1985 Jan 10;260(1):356–365. [PubMed] [Google Scholar]
  6. Brandan E., Hirschberg C. B. Differential association of rat liver heparan sulfate proteoglycans in membranes of the Golgi apparatus and the plasma membrane. J Biol Chem. 1989 Jun 25;264(18):10520–10526. [PubMed] [Google Scholar]
  7. Choay J., Petitou M., Lormeau J. C., Sinaÿ P., Casu B., Gatti G. Structure-activity relationship in heparin: a synthetic pentasaccharide with high affinity for antithrombin III and eliciting high anti-factor Xa activity. Biochem Biophys Res Commun. 1983 Oct 31;116(2):492–499. doi: 10.1016/0006-291x(83)90550-8. [DOI] [PubMed] [Google Scholar]
  8. Dmitriev B. A., Knirel Y. A., Kochetkov N. K. Selective cleavage of glycosidic linkages: studies with the O-specific polysaccharide from Shigella dysenteriae type 3. Carbohydr Res. 1975 Apr;40(02):365–372. doi: 10.1016/s0008-6215(00)82617-8. [DOI] [PubMed] [Google Scholar]
  9. Esko J. D. Detection of animal cell LDL mutants by replica plating. Methods Enzymol. 1986;129:237–253. doi: 10.1016/0076-6879(86)29073-4. [DOI] [PubMed] [Google Scholar]
  10. Esko J. D., Elgavish A., Prasthofer T., Taylor W. H., Weinke J. L. Sulfate transport-deficient mutants of Chinese hamster ovary cells. Sulfation of glycosaminoglycans dependent on cysteine. J Biol Chem. 1986 Nov 25;261(33):15725–15733. [PubMed] [Google Scholar]
  11. Esko J. D., Stewart T. E., Taylor W. H. Animal cell mutants defective in glycosaminoglycan biosynthesis. Proc Natl Acad Sci U S A. 1985 May;82(10):3197–3201. doi: 10.1073/pnas.82.10.3197. [DOI] [PMC free article] [PubMed] [Google Scholar]
  12. Fritze L. M., Reilly C. F., Rosenberg R. D. An antiproliferative heparan sulfate species produced by postconfluent smooth muscle cells. J Cell Biol. 1985 Apr;100(4):1041–1049. doi: 10.1083/jcb.100.4.1041. [DOI] [PMC free article] [PubMed] [Google Scholar]
  13. Gum J. R., Jr, Raetz C. R. Biochemical and immunological characterization of mutant L-M cells with altered levels of dibutyryl cyclic AMP-inducible alkaline phosphatase. Mol Cell Biol. 1985 May;5(5):1184–1187. doi: 10.1128/mcb.5.5.1184. [DOI] [PMC free article] [PubMed] [Google Scholar]
  14. Gum J. R., Jr, Raetz C. R. Dibutyryl cAMP-inducible alkaline phosphatase in animal cell plasma membranes: fluorescence detection of mutant clones on polyester cloth. Proc Natl Acad Sci U S A. 1983 Jul;80(13):3918–3922. doi: 10.1073/pnas.80.13.3918. [DOI] [PMC free article] [PubMed] [Google Scholar]
  15. Ishihara M., Fedarko N. S., Conrad H. E. Involvement of phosphatidylinositol and insulin in the coordinate regulation of proteoheparan sulfate metabolism and hepatocyte growth. J Biol Chem. 1987 Apr 5;262(10):4708–4716. [PubMed] [Google Scholar]
  16. Jordan R. E., Favreau L. V., Braswell E. H., Rosenberg R. D. Heparin with two binding sites for antithrombin or platelet factor 4. J Biol Chem. 1982 Jan 10;257(1):400–406. [PubMed] [Google Scholar]
  17. KIT S., DUBBS D. R., PIEKARSKI L. J., HSU T. C. DELETION OF THYMIDINE KINASE ACTIVITY FROM L CELLS RESISTANT TO BROMODEOXYURIDINE. Exp Cell Res. 1963 Aug;31:297–312. doi: 10.1016/0014-4827(63)90007-7. [DOI] [PubMed] [Google Scholar]
  18. 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]
  19. Lam L. H., Silbert J. E., Rosenberg R. D. The separation of active and inactive forms of heparin. Biochem Biophys Res Commun. 1976 Mar 22;69(2):570–577. doi: 10.1016/0006-291x(76)90558-1. [DOI] [PubMed] [Google Scholar]
  20. Lindahl U., Bäckström G., Thunberg L., Leder I. G. Evidence for a 3-O-sulfated D-glucosamine residue in the antithrombin-binding sequence of heparin. Proc Natl Acad Sci U S A. 1980 Nov;77(11):6551–6555. doi: 10.1073/pnas.77.11.6551. [DOI] [PMC free article] [PubMed] [Google Scholar]
  21. Lindahl U., Bäckström G., Thunberg L. The antithrombin-binding sequence in heparin. Identification of an essential 6-O-sulfate group. J Biol Chem. 1983 Aug 25;258(16):9826–9830. [PubMed] [Google Scholar]
  22. Marcum J. A., Atha D. H., Fritze L. M., Nawroth P., Stern D., Rosenberg R. D. Cloned bovine aortic endothelial cells synthesize anticoagulantly active heparan sulfate proteoglycan. J Biol Chem. 1986 Jun 5;261(16):7507–7517. [PubMed] [Google Scholar]
  23. Marcum J. A., McKenney J. B., Rosenberg R. D. Acceleration of thrombin-antithrombin complex formation in rat hindquarters via heparinlike molecules bound to the endothelium. J Clin Invest. 1984 Aug;74(2):341–350. doi: 10.1172/JCI111429. [DOI] [PMC free article] [PubMed] [Google Scholar]
  24. Marcum J. A., Rosenberg R. D. Heparinlike molecules with anticoagulant activity are synthesized by cultured endothelial cells. Biochem Biophys Res Commun. 1985 Jan 16;126(1):365–372. doi: 10.1016/0006-291x(85)90615-1. [DOI] [PubMed] [Google Scholar]
  25. Oosta G. M., Gardner W. T., Beeler D. L., Rosenberg R. D. Multiple functional domains of the heparin molecule. Proc Natl Acad Sci U S A. 1981 Feb;78(2):829–833. doi: 10.1073/pnas.78.2.829. [DOI] [PMC free article] [PubMed] [Google Scholar]
  26. Raetz C. R., Wermuth M. M., McIntyre T. M., Esko J. D., Wing D. C. Somatic cell cloning in polyester stacks. Proc Natl Acad Sci U S A. 1982 May;79(10):3223–3227. doi: 10.1073/pnas.79.10.3223. [DOI] [PMC free article] [PubMed] [Google Scholar]
  27. Rosenberg R. D., Armand G., Lam L. Structure-function relationships of heparin species. Proc Natl Acad Sci U S A. 1978 Jul;75(7):3065–3069. doi: 10.1073/pnas.75.7.3065. [DOI] [PMC free article] [PubMed] [Google Scholar]
  28. Rosenberg R. D., Damus P. S. The purification and mechanism of action of human antithrombin-heparin cofactor. J Biol Chem. 1973 Sep 25;248(18):6490–6505. [PubMed] [Google Scholar]
  29. Rosenberg R. D., Lam L. Correlation between structure and function of heparin. Proc Natl Acad Sci U S A. 1979 Mar;76(3):1218–1222. doi: 10.1073/pnas.76.3.1218. [DOI] [PMC free article] [PubMed] [Google Scholar]
  30. Shively J. E., Conrad H. E. Stoichiometry of the nitrous acid deaminative cleavage of model amino sugar glycosides and glycosaminoglycuronans. Biochemistry. 1970 Jan 6;9(1):33–43. doi: 10.1021/bi00803a005. [DOI] [PubMed] [Google Scholar]
  31. Stone A. L., Beeler D., Oosta G., Rosenberg R. D. Circular dichroism spectroscopy of heparin-antithrombin interactions. Proc Natl Acad Sci U S A. 1982 Dec;79(23):7190–7194. doi: 10.1073/pnas.79.23.7190. [DOI] [PMC free article] [PubMed] [Google Scholar]

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