Identification of the predominant glycosaminoglycan-attachment site in soluble recombinant human thrombomodulin: potential regulation of functionality by glycosyltransferase competition for serine474 (original) (raw)

Abstract

Thrombomodulin (TM) is an endothelial cell thrombin receptor that converts thrombin from a procoagulant to an anticoagulant enzyme. It has previously been shown that TM is expressed in both a high-M(r) form containing chondroitin sulphate and a low-M(r) form lacking this modification. Site-directed mutagenesis of a soluble human TM derivative (TMD1) was employed to determine the attachment site(s) of this functionally important oligosaccharide on the core protein. Although there are four serine residues within the Ser/Thr-rich domain of TMD1 that might support glycosaminoglycan assembly, our analysis demonstrates that the primary site of attachment is at Ser474, and evidence is presented for low levels of attachment at Ser472. It was possible to improve the overall degree of attachment by mutating Ser472 to glutamic acid (so as to conform Ser474 to the xylosyltransferase acceptor consensus acidic-Gly-Ser-Gly-acidic); however, a significant proportion (approx. 35%) of the total TM still lacked a glycosaminoglycan moiety. Mutants that possess a substitution for Ser474 show an increased mobility of their low-M(r) form on SDS/PAGE compared with native TMD1. Isolation and sequencing of a C-terminal peptide demonstrated that this serine is modified in the low-M(r) form of native TMD1. An apparent 'acceptor consensus overlap' at Ser474 suggests that the mechanism behind the glycosaminoglycan split of TM may involve a competition for substrate between xylosyltransferase and N-acetylgalactosaminyltransferase.

131

Images in this article

Selected References

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

  1. Berg D. T., Walls J. D., Grinnell B. W. A variant enhancer/regulatory region from a cloned human prototype BK virus genome. Nucleic Acids Res. 1988 Sep 26;16(18):9057–9057. doi: 10.1093/nar/16.18.9057. [DOI] [PMC free article] [PubMed] [Google Scholar]
  2. Bourdon M. A., Krusius T., Campbell S., Schwartz N. B., Ruoslahti E. Identification and synthesis of a recognition signal for the attachment of glycosaminoglycans to proteins. Proc Natl Acad Sci U S A. 1987 May;84(10):3194–3198. doi: 10.1073/pnas.84.10.3194. [DOI] [PMC free article] [PubMed] [Google Scholar]
  3. Bourin M. C., Boffa M. C., Björk I., Lindahl U. Functional domains of rabbit thrombomodulin. Proc Natl Acad Sci U S A. 1986 Aug;83(16):5924–5928. doi: 10.1073/pnas.83.16.5924. [DOI] [PMC free article] [PubMed] [Google Scholar]
  4. Bourin M. C., Lundgren-Akerlund E., Lindahl U. Isolation and characterization of the glycosaminoglycan component of rabbit thrombomodulin proteoglycan. J Biol Chem. 1990 Sep 15;265(26):15424–15431. [PubMed] [Google Scholar]
  5. Bourin M. C., Ohlin A. K., Lane D. A., Stenflo J., Lindahl U. Relationship between anticoagulant activities and polyanionic properties of rabbit thrombomodulin. J Biol Chem. 1988 Jun 15;263(17):8044–8052. [PubMed] [Google Scholar]
  6. Cummings R. D., Kornfeld S., Schneider W. J., Hobgood K. K., Tolleshaug H., Brown M. S., Goldstein J. L. Biosynthesis of N- and O-linked oligosaccharides of the low density lipoprotein receptor. J Biol Chem. 1983 Dec 25;258(24):15261–15273. [PubMed] [Google Scholar]
  7. Devereux J., Haeberli P., Smithies O. A comprehensive set of sequence analysis programs for the VAX. Nucleic Acids Res. 1984 Jan 11;12(1 Pt 1):387–395. doi: 10.1093/nar/12.1part1.387. [DOI] [PMC free article] [PubMed] [Google Scholar]
  8. Dittman W. A., Kumada T., Sadler J. E., Majerus P. W. The structure and function of mouse thrombomodulin. Phorbol myristate acetate stimulates degradation and synthesis of thrombomodulin without affecting mRNA levels in hemangioma cells. J Biol Chem. 1988 Oct 25;263(30):15815–15822. [PubMed] [Google Scholar]
  9. Dittman W. A., Majerus P. W. Structure and function of thrombomodulin: a natural anticoagulant. Blood. 1990 Jan 15;75(2):329–336. [PubMed] [Google Scholar]
  10. Doege K. J., Sasaki M., Kimura T., Yamada Y. Complete coding sequence and deduced primary structure of the human cartilage large aggregating proteoglycan, aggrecan. Human-specific repeats, and additional alternatively spliced forms. J Biol Chem. 1991 Jan 15;266(2):894–902. [PubMed] [Google Scholar]
  11. Doege K., Sasaki M., Horigan E., Hassell J. R., Yamada Y. Complete primary structure of the rat cartilage proteoglycan core protein deduced from cDNA clones. J Biol Chem. 1987 Dec 25;262(36):17757–17767. [PubMed] [Google Scholar]
  12. Eaton D., Rodriguez H., Vehar G. A. Proteolytic processing of human factor VIII. Correlation of specific cleavages by thrombin, factor Xa, and activated protein C with activation and inactivation of factor VIII coagulant activity. Biochemistry. 1986 Jan 28;25(2):505–512. doi: 10.1021/bi00350a035. [DOI] [PubMed] [Google Scholar]
  13. Esmon C. T., Esmon N. L., Harris K. W. Complex formation between thrombin and thrombomodulin inhibits both thrombin-catalyzed fibrin formation and factor V activation. J Biol Chem. 1982 Jul 25;257(14):7944–7947. [PubMed] [Google Scholar]
  14. Esmon C. T., Owen W. G. Identification of an endothelial cell cofactor for thrombin-catalyzed activation of protein C. Proc Natl Acad Sci U S A. 1981 Apr;78(4):2249–2252. doi: 10.1073/pnas.78.4.2249. [DOI] [PMC free article] [PubMed] [Google Scholar]
  15. Esmon C. T. The roles of protein C and thrombomodulin in the regulation of blood coagulation. J Biol Chem. 1989 Mar 25;264(9):4743–4746. [PubMed] [Google Scholar]
  16. Esmon N. L., Carroll R. C., Esmon C. T. Thrombomodulin blocks the ability of thrombin to activate platelets. J Biol Chem. 1983 Oct 25;258(20):12238–12242. [PubMed] [Google Scholar]
  17. Fransson L. A., Carlstedt I., Cöster L., Malmström A. Binding of transferrin to the core protein of fibroblast proteoheparan sulfate. Proc Natl Acad Sci U S A. 1984 Sep;81(18):5657–5661. doi: 10.1073/pnas.81.18.5657. [DOI] [PMC free article] [PubMed] [Google Scholar]
  18. Fulcher C. A., Gardiner J. E., Griffin J. H., Zimmerman T. S. Proteolytic inactivation of human factor VIII procoagulant protein by activated human protein C and its analogy with factor V. Blood. 1984 Feb;63(2):486–489. [PubMed] [Google Scholar]
  19. Geetha-Habib M., Campbell S. C., Schwartz N. B. Subcellular localization of the synthesis and glycosylation of chondroitin sulfate proteoglycan core protein. J Biol Chem. 1984 Jun 10;259(11):7300–7310. [PubMed] [Google Scholar]
  20. Hanover J. A., Elting J., Mintz G. R., Lennarz W. J. Temporal aspects of the N- and O-glycosylation of human chorionic gonadotropin. J Biol Chem. 1982 Sep 10;257(17):10172–10177. [PubMed] [Google Scholar]
  21. Hanover J. A., Lennarz W. J., Young J. D. Synthesis of N- and O-linked glycopeptides in oviduct membrane preparations. J Biol Chem. 1980 Jul 25;255(14):6713–6716. [PubMed] [Google Scholar]
  22. Hayashi T., Zushi M., Yamamoto S., Suzuki K. Further localization of binding sites for thrombin and protein C in human thrombomodulin. J Biol Chem. 1990 Nov 25;265(33):20156–20159. [PubMed] [Google Scholar]
  23. Hofsteenge J., Stone S. R. The effect of thrombomodulin on the cleavage of fibrinogen and fibrinogen fragments by thrombin. Eur J Biochem. 1987 Oct 1;168(1):49–56. doi: 10.1111/j.1432-1033.1987.tb13385.x. [DOI] [PubMed] [Google Scholar]
  24. Hofsteenge J., Taguchi H., Stone S. R. Effect of thrombomodulin on the kinetics of the interaction of thrombin with substrates and inhibitors. Biochem J. 1986 Jul 1;237(1):243–251. doi: 10.1042/bj2370243. [DOI] [PMC free article] [PubMed] [Google Scholar]
  25. Jackman R. W., Beeler D. L., VanDeWater L., Rosenberg R. D. Characterization of a thrombomodulin cDNA reveals structural similarity to the low density lipoprotein receptor. Proc Natl Acad Sci U S A. 1986 Dec;83(23):8834–8838. doi: 10.1073/pnas.83.23.8834. [DOI] [PMC free article] [PubMed] [Google Scholar]
  26. Kaji T., Akai T., Hayashi T., Sakuragawa N. Effect of thrombin on the production of glycosaminoglycans by cultured endothelial cells. Thromb Res. 1991 Jun 1;62(5):509–517. doi: 10.1016/0049-3848(91)90024-q. [DOI] [PubMed] [Google Scholar]
  27. Kaji T., Akai T., Hayashi T., Sakuragawa N. Thrombin decreases glycosaminoglycans content of endothelial cells in culture. Thromb Res. 1991 Feb 15;61(4):375–384. doi: 10.1016/0049-3848(91)90651-c. [DOI] [PubMed] [Google Scholar]
  28. Kisiel W., Canfield W. M., Ericsson L. H., Davie E. W. Anticoagulant properties of bovine plasma protein C following activation by thrombin. Biochemistry. 1977 Dec 27;16(26):5824–5831. doi: 10.1021/bi00645a029. [DOI] [PubMed] [Google Scholar]
  29. 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]
  30. Koyama T., Parkinson J. F., Aoki N., Bang N. U., Müller-Berghaus G., Preissner K. T. Relationship between post-translational glycosylation and anticoagulant function of secretable recombinant mutants of human thrombomodulin. Br J Haematol. 1991 Aug;78(4):515–522. doi: 10.1111/j.1365-2141.1991.tb04481.x. [DOI] [PubMed] [Google Scholar]
  31. Krueger R. C., Jr, Fields T. A., Hildreth J., 4th, Schwartz N. B. Chick cartilage chondroitin sulfate proteoglycan core protein. I. Generation and characterization of peptides and specificity for glycosaminoglycan attachment. J Biol Chem. 1990 Jul 15;265(20):12075–12087. [PubMed] [Google Scholar]
  32. Krueger R. C., Jr, Fields T. A., Mensch J. R., Jr, Schwartz N. B. Chick cartilage chondroitin sulfate proteoglycan core protein. II. Nucleotide sequence of cDNA clone and localization of the S103L epitope. J Biol Chem. 1990 Jul 15;265(20):12088–12097. [PubMed] [Google Scholar]
  33. Krusius T., Ruoslahti E. Primary structure of an extracellular matrix proteoglycan core protein deduced from cloned cDNA. Proc Natl Acad Sci U S A. 1986 Oct;83(20):7683–7687. doi: 10.1073/pnas.83.20.7683. [DOI] [PMC free article] [PubMed] [Google Scholar]
  34. Kunkel T. A. Rapid and efficient site-specific mutagenesis without phenotypic selection. Proc Natl Acad Sci U S A. 1985 Jan;82(2):488–492. doi: 10.1073/pnas.82.2.488. [DOI] [PMC free article] [PubMed] [Google Scholar]
  35. Kurosawa S., Galvin J. B., Esmon N. L., Esmon C. T. Proteolytic formation and properties of functional domains of thrombomodulin. J Biol Chem. 1987 Feb 15;262(5):2206–2212. [PubMed] [Google Scholar]
  36. Kurosawa S., Stearns D. J., Jackson K. W., Esmon C. T. A 10-kDa cyanogen bromide fragment from the epidermal growth factor homology domain of rabbit thrombomodulin contains the primary thrombin binding site. J Biol Chem. 1988 May 5;263(13):5993–5996. [PubMed] [Google Scholar]
  37. Laemmli U. K. Cleavage of structural proteins during the assembly of the head of bacteriophage T4. Nature. 1970 Aug 15;227(5259):680–685. doi: 10.1038/227680a0. [DOI] [PubMed] [Google Scholar]
  38. Lentz S. R., Sadler J. E. Inhibition of thrombomodulin surface expression and protein C activation by the thrombogenic agent homocysteine. J Clin Invest. 1991 Dec;88(6):1906–1914. doi: 10.1172/JCI115514. [DOI] [PMC free article] [PubMed] [Google Scholar]
  39. Lu R. L., Esmon N. L., Esmon C. T., Johnson A. E. The active site of the thrombin-thrombomodulin complex. A fluorescence energy transfer measurement of its distance above the membrane surface. J Biol Chem. 1989 Aug 5;264(22):12956–12962. [PubMed] [Google Scholar]
  40. López-Casillas F., Cheifetz S., Doody J., Andres J. L., Lane W. S., Massagué J. Structure and expression of the membrane proteoglycan betaglycan, a component of the TGF-beta receptor system. Cell. 1991 Nov 15;67(4):785–795. doi: 10.1016/0092-8674(91)90073-8. [DOI] [PubMed] [Google Scholar]
  41. Marlar R. A., Kleiss A. J., Griffin J. H. Mechanism of action of human activated protein C, a thrombin-dependent anticoagulant enzyme. Blood. 1982 May;59(5):1067–1072. [PubMed] [Google Scholar]
  42. McClelland A., Kühn L. C., Ruddle F. H. The human transferrin receptor gene: genomic organization, and the complete primary structure of the receptor deduced from a cDNA sequence. Cell. 1984 Dec;39(2 Pt 1):267–274. doi: 10.1016/0092-8674(84)90004-7. [DOI] [PubMed] [Google Scholar]
  43. McCormick D., van der Rest M., Goodship J., Lozano G., Ninomiya Y., Olsen B. R. Structure of the glycosaminoglycan domain in the type IX collagen-proteoglycan. Proc Natl Acad Sci U S A. 1987 Jun;84(12):4044–4048. doi: 10.1073/pnas.84.12.4044. [DOI] [PMC free article] [PubMed] [Google Scholar]
  44. Moore K. L., Andreoli S. P., Esmon N. L., Esmon C. T., Bang N. U. Endotoxin enhances tissue factor and suppresses thrombomodulin expression of human vascular endothelium in vitro. J Clin Invest. 1987 Jan;79(1):124–130. doi: 10.1172/JCI112772. [DOI] [PMC free article] [PubMed] [Google Scholar]
  45. Nawa K., Sakano K., Fujiwara H., Sato Y., Sugiyama N., Teruuchi T., Iwamoto M., Marumoto Y. Presence and function of chondroitin-4-sulfate on recombinant human soluble thrombomodulin. Biochem Biophys Res Commun. 1990 Sep 14;171(2):729–737. doi: 10.1016/0006-291x(90)91207-9. [DOI] [PubMed] [Google Scholar]
  46. Nuwayhid N., Glaser J. H., Johnson J. C., Conrad H. E., Hauser S. C., Hirschberg C. B. Xylosylation and glucuronosylation reactions in rat liver Golgi apparatus and endoplasmic reticulum. J Biol Chem. 1986 Oct 5;261(28):12936–12941. [PubMed] [Google Scholar]
  47. Owen W. G., Esmon C. T. Functional properties of an endothelial cell cofactor for thrombin-catalyzed activation of protein C. J Biol Chem. 1981 Jun 10;256(11):5532–5535. [PubMed] [Google Scholar]
  48. Parkinson J. F., Garcia J. G., Bang N. U. Decreased thrombin affinity of cell-surface thrombomodulin following treatment of cultured endothelial cells with beta-D-xyloside. Biochem Biophys Res Commun. 1990 May 31;169(1):177–183. doi: 10.1016/0006-291x(90)91451-w. [DOI] [PubMed] [Google Scholar]
  49. Parkinson J. F., Grinnell B. W., Moore R. E., Hoskins J., Vlahos C. J., Bang N. U. Stable expression of a secretable deletion mutant of recombinant human thrombomodulin in mammalian cells. J Biol Chem. 1990 Jul 25;265(21):12602–12610. [PubMed] [Google Scholar]
  50. Parkinson J. F., Vlahos C. J., Yan S. C., Bang N. U. Recombinant human thrombomodulin. Regulation of cofactor activity and anticoagulant function by a glycosaminoglycan side chain. Biochem J. 1992 Apr 1;283(Pt 1):151–157. doi: 10.1042/bj2830151. [DOI] [PMC free article] [PubMed] [Google Scholar]
  51. Preissner K. T., Delvos U., Müller-Berghaus G. Binding of thrombin to thrombomodulin accelerates inhibition of the enzyme by antithrombin III. Evidence for a heparin-independent mechanism. Biochemistry. 1987 May 5;26(9):2521–2528. doi: 10.1021/bi00383a018. [DOI] [PubMed] [Google Scholar]
  52. Preissner K. T., Koyama T., Müller D., Tschopp J., Müller-Berghaus G. Domain structure of the endothelial cell receptor thrombomodulin as deduced from modulation of its anticoagulant functions. Evidence for a glycosaminoglycan-dependent secondary binding site for thrombin. J Biol Chem. 1990 Mar 25;265(9):4915–4922. [PubMed] [Google Scholar]
  53. RAPAPORT S. I., SCHIFFMAN S., PATCH M. J., AMES S. B. The importance of activation of antihemophilic globulin and proaccelerin by traces of thrombin in the generation of intrinsic prothrombinase activity. Blood. 1963 Feb;21:221–236. [PubMed] [Google Scholar]
  54. Roberts K. P., Griswold M. D. Characterization of rat transferrin receptor cDNA: the regulation of transferrin receptor mRNA in testes and in Sertoli cells in culture. Mol Endocrinol. 1990 Apr;4(4):531–542. doi: 10.1210/mend-4-4-531. [DOI] [PubMed] [Google Scholar]
  55. Roth J. Cytochemical localization of terminal N-acetyl-D-galactosamine residues in cellular compartments of intestinal goblet cells: implications for the topology of O-glycosylation. J Cell Biol. 1984 Feb;98(2):399–406. doi: 10.1083/jcb.98.2.399. [DOI] [PMC free article] [PubMed] [Google Scholar]
  56. Suzuki K., Kusumoto H., Deyashiki Y., Nishioka J., Maruyama I., Zushi M., Kawahara S., Honda G., Yamamoto S., Horiguchi S. Structure and expression of human thrombomodulin, a thrombin receptor on endothelium acting as a cofactor for protein C activation. EMBO J. 1987 Jul;6(7):1891–1897. doi: 10.1002/j.1460-2075.1987.tb02448.x. [DOI] [PMC free article] [PubMed] [Google Scholar]
  57. Suzuki K., Stenflo J., Dahlbäck B., Teodorsson B. Inactivation of human coagulation factor V by activated protein C. J Biol Chem. 1983 Feb 10;258(3):1914–1920. [PubMed] [Google Scholar]
  58. Takahashi N., Takahashi Y., Putnam F. W. Structure of human hemopexin: O-glycosyl and N-glycosyl sites and unusual clustering of tryptophan residues. Proc Natl Acad Sci U S A. 1984 Apr;81(7):2021–2025. doi: 10.1073/pnas.81.7.2021. [DOI] [PMC free article] [PubMed] [Google Scholar]
  59. Thonar E. J., Lohmander L. S., Kimura J. H., Fellini S. A., Yanagishita M., Hascall V. C. Biosynthesis of O-linked oligosaccharides on proteoglycans by chondrocytes from the swarm rat chondrosarcoma. J Biol Chem. 1983 Oct 10;258(19):11564–11570. [PubMed] [Google Scholar]
  60. Tsiang M., Lentz S. R., Sadler J. E. Functional domains of membrane-bound human thrombomodulin. EGF-like domains four to six and the serine/threonine-rich domain are required for cofactor activity. J Biol Chem. 1992 Mar 25;267(9):6164–6170. [PubMed] [Google Scholar]
  61. Vehar G. A., Davie E. W. Preparation and properties of bovine factor VIII (antihemophilic factor). Biochemistry. 1980 Feb 5;19(3):401–410. doi: 10.1021/bi00544a001. [DOI] [PubMed] [Google Scholar]
  62. Walker F. J., Sexton P. W., Esmon C. T. The inhibition of blood coagulation by activated Protein C through the selective inactivation of activated Factor V. Biochim Biophys Acta. 1979 Dec 7;571(2):333–342. doi: 10.1016/0005-2744(79)90103-7. [DOI] [PubMed] [Google Scholar]
  63. Wang X. F., Lin H. Y., Ng-Eaton E., Downward J., Lodish H. F., Weinberg R. A. Expression cloning and characterization of the TGF-beta type III receptor. Cell. 1991 Nov 15;67(4):797–805. doi: 10.1016/0092-8674(91)90074-9. [DOI] [PubMed] [Google Scholar]
  64. Wen D. Z., Dittman W. A., Ye R. D., Deaven L. L., Majerus P. W., Sadler J. E. Human thrombomodulin: complete cDNA sequence and chromosome localization of the gene. Biochemistry. 1987 Jul 14;26(14):4350–4357. doi: 10.1021/bi00388a025. [DOI] [PubMed] [Google Scholar]
  65. Wigler M., Pellicer A., Silverstein S., Axel R., Urlaub G., Chasin L. DNA-mediated transfer of the adenine phosphoribosyltransferase locus into mammalian cells. Proc Natl Acad Sci U S A. 1979 Mar;76(3):1373–1376. doi: 10.1073/pnas.76.3.1373. [DOI] [PMC free article] [PubMed] [Google Scholar]
  66. Wilson I. B., Gavel Y., von Heijne G. Amino acid distributions around O-linked glycosylation sites. Biochem J. 1991 Apr 15;275(Pt 2):529–534. doi: 10.1042/bj2750529. [DOI] [PMC free article] [PubMed] [Google Scholar]
  67. Ye J., Esmon C. T., Johnson A. E. The chondroitin sulfate moiety of thrombomodulin binds a second molecule of thrombin. J Biol Chem. 1993 Feb 5;268(4):2373–2379. [PubMed] [Google Scholar]
  68. Zimmermann D. R., Ruoslahti E. Multiple domains of the large fibroblast proteoglycan, versican. EMBO J. 1989 Oct;8(10):2975–2981. doi: 10.1002/j.1460-2075.1989.tb08447.x. [DOI] [PMC free article] [PubMed] [Google Scholar]
  69. Zushi M., Gomi K., Yamamoto S., Maruyama I., Hayashi T., Suzuki K. The last three consecutive epidermal growth factor-like structures of human thrombomodulin comprise the minimum functional domain for protein C-activating cofactor activity and anticoagulant activity. J Biol Chem. 1989 Jun 25;264(18):10351–10353. [PubMed] [Google Scholar]