Download PDF
Thromb Haemost 2003; 90(02): 206-217
DOI: 10.1160/TH03-01-0034
Blood Coagulation, Fibrinolysis and Cellular Haemostasis
Schattauer GmbH

Biochemical importance of glycosylation of plasminogen activator inhibitor-1

Ann Gils   *
1   Laboratory for Pharmaceutical Biology and Phytopharmacology, Faculty of Pharmaceutical Sciences, Katholieke Universiteit Leuven, Belgium
,
Katrine E. Pedersen   *
2   Department of Molecular Biology, Aarhus University, Denmark
,
Peter Skottrup   *
2   Department of Molecular Biology, Aarhus University, Denmark
,
Anni Christensen
2   Department of Molecular Biology, Aarhus University, Denmark
,
Dominik Naessens
1   Laboratory for Pharmaceutical Biology and Phytopharmacology, Faculty of Pharmaceutical Sciences, Katholieke Universiteit Leuven, Belgium
,
Johanna Deinum
3   AstraZeneca, R&D, Mölndal, Sweden
,
Jan J. Enghild
2   Department of Molecular Biology, Aarhus University, Denmark
,
Paul J. Declerck
1   Laboratory for Pharmaceutical Biology and Phytopharmacology, Faculty of Pharmaceutical Sciences, Katholieke Universiteit Leuven, Belgium
,
Peter A. Andreasen
2   Department of Molecular Biology, Aarhus University, Denmark
› Author AffiliationsFinancial support: This work was supported by grants from the Danish Cancer Society, the Danish Natural Science Research Council, the Danish Cancer Research Foundation, and the Novo-Nordisk Foundation to PAA and by grants OT/98/37 from the Research Fund Katholieke Universiteit Leuven and G.0225.01 from the Fund for Scientific Research (FWO-Vlaanderen) to PJD. AG is a postdoctoral fellow of the Fund for Scientific Research (FWO-Vlaanderen). PS is a recipient of a scholarship from the Danish Cancer Society.
Further Information

Publication History

Received 17 January 2003

Accepted after revision 18 May 2003

Publication Date:
06 December 2017 (online)

Also available at
    Permissions and Reprints

Summary

The serpin plasminogen activator inhibitor-1 (PAI-1) is a potential target for anti-thombotic and anti-cancer therapy. PAI-1 has 3 potential sites for N-linked glycosylation. We demonstrate here that PAI-1 expressed recombinantly or naturally by human cell lines display a heterogeneous glycosylation pattern of the sites at N209 and N265, while that at N329 is not utilised. The IC50-values for inactivation of PAI-1 by 4 monoclonal antibodies differed strongly between glycosylated PAI-1 and non-glycosy-lated PAI-1 expressed in E. coli. For 3 antibodies, an overlap of the epitopes with the glycosylation sites could be excluded as explanation for the differential reactivity. The latency transition of non-glycosylated, but not of glycosylated PAI-1, was strongly accelerated by a non-ionic detergent. The different biochemical properties of glycosylated and non-glycosylated PAI-1 depended specifically on glycosylation of either one or the other of the utilised sites. The PAI-1-binding protein vitronectin reversed the changes associated with the lack of glycosylation at one of the sites. Our results stress the importance of the source of PAI-1 when studying the mechanisms of action of PAI-1-inactivating compounds of potential clinical importance.

Keywords

Thombosis - arteriosclerosis - metastasis - serpin - vitronectin

* These authors contributed equally to this study


 

  • References

  • 1 Andreasen PA, Egelund R, Petersen HH. The plasminogen activation system in tumor growth, invasion, and metastasis. Cell Mol Life Sci 2000; 57: 25-40.
    CrossrefPubMedGoogle Scholar
  • 2 Booth NA. Fibrinolysis and thrombosis. Baillieres Best Pract Res Clin Haematol 1999; 12: 423-33.
    CrossrefPubMedGoogle Scholar
  • 3 Huber K, Christ G, Wojta J, Gulba D. Plasminogen activator inhibitor type-1 in cardiovascular disease. Status report 2001. Thromb Res 2001; 103 (Suppl. 01) Suppl 7-19.
    CrossrefPubMedGoogle Scholar
  • 4 Vaughan DE. Angiotensin and vascular fibrin-olytic balance. Am J Hypertension 2002; 15: 3S-8S.
    CrossrefPubMedGoogle Scholar
  • 5 Erickson LA, Fici GJ, Lund JE, Boyle TP, Polites HG, Marotti KR. Development of venous occlusions in mice transgenic for the plasminogen activator inhibitor-1 gene. Nature 1990; 346: 74-6.
    CrossrefPubMedGoogle Scholar
  • 6 Eren M, Painter CA., Atkinson JB, Declerck PJ, Vaughan DE. Age-dependent spontaneous coronary arterial thrombosis in transgenic mice that express a stable form of human plasminogen activator inhibitor-1. Circulation 2002; 106: 491-6.
    CrossrefPubMedGoogle Scholar
  • 7 Danø K, Andreasen PA, Grøndahl-Hansen J, Kristensen P, Nielsen LS, Skriver L. Plasminogen activators, tissue degradation and cancer. Adv Cancer Res 1985; 44: 139-266.
    CrossrefPubMedGoogle Scholar
  • 8 Andreasen PA, Kjøller L, Christensen L, Duffy MJ. The urokinase-type plasminogen activator system in cancer metastasis. A review. Int J Cancer 1997; 72: 1-22.
    CrossrefPubMedGoogle Scholar
  • 9 Jänicke F, Prechtl A, Thomssen C. et al. Randomized adjuvant chemotherapy trial in high-risk, lymph node-negative breast cancer patients identified by urokinase-type plasminogen activator and plasminogen activator inhibitor type 1. J Natl Cancer Inst 2001; 93: 913-20.
    CrossrefPubMedGoogle Scholar
  • 10 Look MP, Van Putten WL, Duffy MJ. et al. Pooled analysis of prognostic impact of urokinase-type plasminogen activator and its inhibitor PAI-1 in 8377 breast cancer patients. J Natl Cancer Inst 2001; 94: 116-28.
    CrossrefPubMedGoogle Scholar
  • 11 Bajou K, Noël A, Gerard RD. et al Absence of host plasminogen activator inhibitor 1 prevents cancer invasion and vascularization. Nat Med 1998; 4: 923-8.
    CrossrefPubMedGoogle Scholar
  • 12 Bajou K, Masson V, Gerard RD. et al. The plasminogen activator inhibitor PAI-1 controls in vivo tumor vascularization by interaction with proteases, not vitronectin. Implications for antiangiogenic strategies. J Cell Biol 2001; 152: 777-84.
    CrossrefPubMedGoogle Scholar
  • 13 Lambert V, Munaut C, Noël A. et al. Influence of plasminogen activator inhibitor type 1 on choroidal neovascularization. FASEB J 2001; 15: 1021-7.
    CrossrefPubMedGoogle Scholar
  • 14 McMahon GA, Petitclerc E, Stefansson S. et al. Plasminogen activator inhibitor-1 regulates tumor growth and angiogenesis. J Biol Chem 2001; 276: 33964-8.
    CrossrefPubMedGoogle Scholar
  • 15 Devy L, Blacher S, Grignet-Debrus C. et al. The pro- or antiangiogenic effect of plasmino-gen activator inhibitor 1 is dose dependent. FASEB J 2002; 16: 147-54.
    CrossrefPubMedGoogle Scholar
  • 16 Gils A, Declerck PJ. Structure-function relationships in serpins: current concepts and controversies. Thromb Haemost 1998; 80: 531-41.
    Article in Thieme ConnectPubMedGoogle Scholar
  • 17 Irving JA, Pike RN, Lesk AM, Whisstock JC. Phylogeny of the serpin superfamily: implications of patterns of amino acid conservation for structure and function. Genome Res 2000; 10: 1845-64.
    CrossrefPubMedGoogle Scholar
  • 18 Wind T, Hansen M, Jensen JK, Andreasen PA. The molecular basis for anti-proteolytic and non-proteolytic functions of plasminogen activator inhibitor-1: Roles of the reactive centre loop, the shutter region, the flexible joint region, and the small serpin fragment. Biol Chem 2002; 283: 21-36.
    PubMedGoogle Scholar
  • 19 Shore JD, Day DE, Francis-Chmura AM. et al. A fluorescent probe study of plasminogen activator inhibitor-1. Evidence for reactive center loop insertion and its role in the inhibitory mechanism. J Biol Chem 1995; 270: 5395-8.
    CrossrefPubMedGoogle Scholar
  • 20 Lawrence DA, Ginsburg D, Day DE. et al. Serpin-protease complexes are trapped as stable acyl-enzyme intermediates. J Biol Chem 1995; 270: 25309-12.
    CrossrefPubMedGoogle Scholar
  • 21 Wilczynska M, Fa M, Ohlsson PI, Ny T. The inhibition mechanism of serpins. Evidence that the mobile reactive center loop is cleaved in the native protease-inhibitor complex. J Biol Chem 1995; 270: 29652-5.
    CrossrefPubMedGoogle Scholar
  • 22 Egelund R, Rodenburg KW, Andreasen PA, Rasmussen MS, Guldberg RE, Petersen TE. An ester bond linking a fragment of a serine protease to its serpin inhibitor. Biochemistry 1998; 37: 6375-9.
    CrossrefPubMedGoogle Scholar
  • 23 Stratikos E, Gettins PG. Formation of the covalent serpin-protease complex involves translocation of the protease by more than 70 Å and full insertion of the reactive center loop into β-sheet A. Proc Natl Acad Sci USA 1999; 96: 4808-13.
    CrossrefPubMedGoogle Scholar
  • 24 Fa M, Bergström F, Hägglöf P, Wilczynska M, Johansson L, B-ÅNy T. The structure of a serpin-protease complex revealed by intra-molecular distance measurements using donor-donor energy migration and mapping of interaction sites. Structure 2000; 8: 397-405.
    CrossrefPubMedGoogle Scholar
  • 25 Huntington JA, Read RJ, Carrell RW. Structure of a serpin-protease complex shows inhibition by deformation. Nature 2000; 407: 923-6.
    CrossrefPubMedGoogle Scholar
  • 26 Egelund R, Petersen TE, Andreasen PA. A serpin-induced extensive proteolytic susceptibility of urokinase-type plasminogen activator implicates distortion of the protease substrate binding pocket and oxyanion hole in the ser-pin inhibitory mechanism. Eur J Biochem 2001; 268: 673-85.
    CrossrefPubMedGoogle Scholar
  • 27 Ye S, Cech AL, Belmares R. et al. The structure of a Michaelis serpin-protease complex. Nat Struct Biol 2001; 8: 979-83.
    CrossrefPubMedGoogle Scholar
  • 28 Mottonen J, Strand A, Symersky J. et al. Structural basis of latency in plasminogen activator inhibitor-1. Nature 1992; 355: 270-3.
    CrossrefPubMedGoogle Scholar
  • 29 Aertgeerts K, De Bondt HL, De Ranter CJ, Declerck PJ. Mechanisms contributing to the conformational and functional flexibility of plasminogen activator inhibitor-1. Nat Struct Biol 1995; 2: 891-7.
    CrossrefPubMedGoogle Scholar
  • 30 Xue Y, Björquist P, Inghardt T. et al. Interfering with the inhibitory mechanism of serpins: crystal structure of a complex formed between cleaved plasminogen activator inhibitor type 1 and a reactive-centre loop peptide. Structure 1998; 6: 627-36.
    CrossrefPubMedGoogle Scholar
  • 31 Sharp AM, Stein PE, Pannu NS. et al. The active conformation of plasminogen activator inhibitor 1, a target for drugs to control fibrinolysis and cell adhesion. Structure 1999; 7: 111-8.
    CrossrefPubMedGoogle Scholar
  • 32 Nar H, Bauer M, Stassen JM, Lang D, Gils A, Declerck PJ. Plasminogen activator inhibitor 1. Structure of the native serpin, comparison to its other conformers and implications for ser-pin inactivation. J Mol Biol 2000; 297: 683-95.
    CrossrefPubMedGoogle Scholar
  • 33 Stout TJ, Graham H, Buckley DI, Matthews DJ. Structures of active and latent PAI-1: a possible stabilizing role for chloride ions. Biochemistry 2000; 39: 8460-9.
    CrossrefPubMedGoogle Scholar
  • 34 Egelund R, Einholm AP, Pedersen KE. et al. A regulatory hydrophobic area in the flexible joint region of plasminogen activator inhibitor-1, defined with fluorescent activity-neutralizing ligands. Ligand-induced serpin polymerization. J Biol Chem 2001; 276: 13077-86.
    CrossrefPubMedGoogle Scholar
  • 35 Debrock S, Declerck PJ. Neutralization of plasminogen activator inhibitor-1 inhibitory properties: identification of two distinct mechanisms. Biochim Biophys Acta 1997; 1337: 257-66.
    CrossrefPubMedGoogle Scholar
  • 36 Gils A, Declerck PJ. Modulation of plasmino-gen activator inhibitor 1 by Triton X-100 – Identification of two consecutive conformational transitions. Thromb Haemost 1998; 80: 286-91.
    Article in Thieme ConnectPubMedGoogle Scholar
  • 37 Huber R, Carrell RW. Implications of the three-dimensional structure of alpha 1-anti-trypsin for structure and function of serpins. Biochemistry 1989; 28: 8951-66.
    CrossrefPubMedGoogle Scholar
  • 38 Tucker HM, Mottonen J, Goldsmith EJ, Gerard RD. Engineering of plasminogen activator inhibitor-1 to reduce the rate of latency transition. Nat Struct Biol 1995; 6: 442-5.
    CrossrefPubMedGoogle Scholar
  • 39 Gils A, Knockaert I, Brouwers E, Declerck PJ. Glycosylation-dependent conformational transitions in plasminogen activator inhibitor-1: Evidence for the presence of two active conformations. Fibrinolysis Proteolysis 2000; 14: 58-64.
    CrossrefPubMedGoogle Scholar
  • 40 Andreasen PA, Egelund R, Jensen S, Rodenburg KW. Solvent effects on activity and conformation of plasminogen activator inhibitor-1. Thromb Haemost 1999; 81: 407-14.
    Article in Thieme ConnectPubMedGoogle Scholar
  • 41 Hansen M, Busse MN, Andreasen PA. Importance of the amino-acid composition of the shutter region of plasminogen activator inhibitor-1 for its transitions to latent and substrate forms. Eur J Biochem 2001; 268: 6274-83.
    CrossrefPubMedGoogle Scholar
  • 42 Munch M, Heegaard CW, Andreasen PA. Interconversions between active, inert and substrate forms of denatured/refolded type-1 plasminogen activator inhibitor. Biochim Biophys Acta 1993; 1202: 29-37.
    CrossrefPubMedGoogle Scholar
  • 43 Strömqvist M, Andersson JO, Bostrom S. et al. Separation of active and inactive forms of recombinant human plasminogen activator inhibitor type 1 (PAI-1) expressed in Chinese Hamster Ovary cells: Comparison with native human PAI-1. Protein Expr Purif 1994; 5: 309-16.
    CrossrefPubMedGoogle Scholar
  • 44 Gils A, Knockaert I, Declerck PJ. Substrate behavior of plasminogen activator inhibitor-1 is not associated with a lack of insertion of the reactive site loop. Biochemistry 1996; 35: 7474-81.
    CrossrefPubMedGoogle Scholar
  • 45 Petersen HH, Hansen M, Schousboe SL, Andreasen PA. Localisation of epitopes for monoclonal antibodies to urokinase-type plasminogen activator. Relationship between epitope localization and effects of antibodies on molecular interactions of the enzyme. Eur J Biochem 2001; 268: 4430-9.
    CrossrefPubMedGoogle Scholar
  • 46 Guex N, Peitsch MC. SWISS-MODEL and the Swiss-PdbViewer: an environment for comparative protein modeling. Electrophoresis 1997; 18: 714-2723.
    CrossrefPubMedGoogle Scholar
  • 47 Verhamme I, Kvassman J-O, Day D. et al. Accelerated conversion of human plasmino-gen activator inhibitor-1 to its latent form by antibody binding. J Biol Chem 1999; 274: 17511-7.
    CrossrefPubMedGoogle Scholar
  • 48 Wind T, Jensen MA, Andreasen PA. Epitope mapping for four monoclonal antibodies against human plasminogen activator inhibitor type-1. Implications for antibody-mediated PAI-1 neutralisation and vitronectin binding. Eur J Biochem 2001; 268: 1095-106.
    CrossrefPubMedGoogle Scholar
  • 49 Declerck PJ, Verstreken M, Collen D. Immunoassay of murine t-PA, uPA and PAI-1 using monoclonal antibodies raised in gene-inactivated mice. Thromb Haemost 1995; 74: 1305-9.
    Article in Thieme ConnectPubMedGoogle Scholar
  • 50 Naessens D, Gils A, Compernolle G, Declerck PJ. Elucidation of the epitope of a latency. inducing antibody: Identification of new molecular target for PAI-1 inhibition. Thromb Haemost 2003; 90: 52-58.
    Article in Thieme ConnectPubMedGoogle Scholar
  • 51 Gorlatova NV, Elokdah H, Fan K, Crandall DL, Lawrence DA. Mapping of a conformational epitope on plasminogen activator inhibitor-1 by random mutagenesis. implications for serpin function. J Biol Chem 2003; 278: 16329-35.
    CrossrefPubMedGoogle Scholar
  • 52 Keijer J, Linders M, van Zonneveld AJ, Ehrlich HJ, de Boer JP, Pannekoek H. The interaction of plasminogen activator inhibitor 1 with plasminogen activators (tissue-type and urokinase-type) and fibrin: Localization of interaction sites and physiologic relevance. Blood 1991; 78: 401-9.
    CrossrefPubMedGoogle Scholar
  • 53 Schousboe SL, Egelund R, Kirkegaard T, Preissner KT, Rodenburg KW, Andreasen PA. Vitronectin and substitution of β-strand 5a residue Lys325 potentiate activity-neutralization of plasminogen activator inhibitor-1 by monoclonal antibodies against the α-helix F region. Thromb Haemost 2000; 83: 742-51.
    Article in Thieme ConnectPubMedGoogle Scholar
  • 54 Ngo T-H, Hoylaerts MF, Knockaert I, Brouwers E, Declerck PJ. Identification of a target site in plasminogen activator inhibitor-1 that allows neutralisation of its inhibitory properties concomitantly with an allosteric up-regulation of its anti-adhesive properties. J Biol Chem 2001; 276: 26243-8.
    CrossrefPubMedGoogle Scholar
  • 55 Imperiali B, O’Connor SE. Effect of N-linked glycosylation on glycopeptide and glycoprotein structure. Curr Opin Chem Biol 1999; 3: 643-9.
    CrossrefPubMedGoogle Scholar
  • 56 Worwald MR, Dwek RA. Glycoproteins: Glycan presentation and protein folding. Structure 1999; 7: R155-R160.
    CrossrefPubMedGoogle Scholar
  • 57 Sui G-C, Wiman B. Stability of plasminogen activator inhibitor-1: Role of Tyrosine 221. FEBS Lett 1998; 423: 319-23.
    CrossrefPubMedGoogle Scholar
  • 58 Jensen JK, Wind T, Andreasen PA. The vitro-nectin binding area of plasminogen activator inhibitor-1, mapped by mutagenesis and protection against an inactivating organochemical ligand. FEBS Lett 2002; 521: 91-4.
    CrossrefPubMedGoogle Scholar
  • 59 Jankova L, Harrop SJ, Saunders DN. et al. Crystal structure of the complex of plasmino-gen activator inhibitor 2 with a peptide mimicking the reactive center loop. J Biol Chem 2001; 276: 43374-82.
    CrossrefPubMedGoogle Scholar
  • 60 Björquist P, Ehnebom J, Inghardt T, Deinum J. Epitopes on plasminogen activator inhibitor type-1 important for binding to tissue plasminogen activator. Biochim Biophys Acta 1997; 1341: 87-98.
    CrossrefPubMedGoogle Scholar
  • 61 Björquist P, Ehnebom J, Deinum J. Protein movement during complex-formation between tissue plasminogen activator and plasminogen activator inhibitor-1. Biochim Biophys Acta 1999; 1431: 24-9.
    CrossrefPubMedGoogle Scholar