PDF herunterladen
Thromb Haemost 1992; 68(05): 539-544
DOI: 10.1055/s-0038-1646314
Original Article
Schattauer GmbH Stuttgart

The Influence of Glycosylation on the Catalytic and Fibrinolytic Properties of Pro-Urokinase

Catherine Lenich
The Vascular Research Laboratory, Institute for the Prevention of Cardiovascular Disease, New England Deaconess Hospital, Harvard Medical School, Boston, MA, USA
,
Ralph Pannell
The Vascular Research Laboratory, Institute for the Prevention of Cardiovascular Disease, New England Deaconess Hospital, Harvard Medical School, Boston, MA, USA
,
Jack Henkin
1   The Abbott Laboratories, Division of Research and Development, Abbott Park, IL, USA
,
Victor Gurewich
The Vascular Research Laboratory, Institute for the Prevention of Cardiovascular Disease, New England Deaconess Hospital, Harvard Medical School, Boston, MA, USA
› Institutsangaben
Weitere Informationen

Publikationsverlauf

Received 10. April 1992

Accepted after revision 10. Juni 1992

Publikationsdatum:
04. Juli 2018 (online)

Auch verfügbar auf
  als PDF herunterladen  Lizenzen und Reprints

Summary

We previously found that human pro-UK expressed in Escherichia coli is more active in fibrinolysis than recombinant human pro-UK obtained from mammalian cell culture media. To determine whether this difference is related to the lack of glycosylation of the E. coli product, we compared the activity of E. coli-derived pro-UK [(-)pro-UK] with that of a glycosylated pro-UK [(+)pro-UK] and of a mutant of pro-UK missing the glycosylation site at Asn-302 [(-) (302) pro-UK]. The latter two pro-UKs were obtained by expression of the human gene in a mammalian cell. The nonglycosylated pro-UKs were activated by plasmin more efficiently (≈2-fold) and were more active in clot lysis (1.5-fold) than the (+)pro-UK. Similarly, the nonglycosylated two-chain derivatives (UKs) were more active against plasminogen and were more rapidly inactivated by plasma inhibitors than the (+)UK.

These findings indicate that glycosylation at Asn-302 influences the activity of pro-UK/UK and could be the major factor responsible for the enhanced activity of E. coli-derived pro-UK.

 

  • REFERENCES

  • 1 Kasai S, Arimura H, Nishida M, Suyama T. Primary structure of single-chain pro-urokinase. J Biol Chem 1985; 260: 12382-12389
    PubMedGoogle Scholar
  • 2 Kruithof EKO, Tran-Thang C, Ransijn A, Bachmann F. Demonstration of fast-acting inhibitor of plasminogen activators in human plasma. Blood 1984; 64: 907-913
    PubMedGoogle Scholar
  • 3 Holmes WE, Pennica DM, Blaber M, Rey MW, Günzler WA, Steffens GJ, Heyneker HL. Cloning and expression of the gene for pro-urokinase in Escherichia coli. Bio-technology. 1985; 3: 923-929
    PubMedGoogle Scholar
  • 4 Steffens GJ, Günzler WA, Otting F, Frankus E, Flohe L. The complete amino acid sequence of low molecular mass urokinase from human urine. Hoppe-Seyler's Z Physiol Chem 1982; 363: 1043-1058
    PubMedGoogle Scholar
  • 5 McLellan WL, Vetterlein D, Roblin R. The glycoprotein nature of human plasminogen activators. FEBS Lett 1980; 115: 181-184
    CrossrefPubMedGoogle Scholar
  • 6 Buko AM, Kentzer EJ, Petros A, Menon G. and others Characterization of a posttranslational fucosylation in the growth factor domain of urinary plasminogen activator. Proc Natl Acad Sci USA 1991; 88: 3992-3996
    CrossrefPubMedGoogle Scholar
  • 7 Gurewich V, Pannell R, Louie S, Kelley P, Suddith RL, Greenlee R. Effective and fibrin-specific clot lysis by a zymogen precursor form of urokinase (pro-urokinase). J Clin Invest 1984; 73: 1731-1739
    CrossrefPubMedGoogle Scholar
  • 8 The PRIMI trial study group Randomized double-blind trial of recombinant pro-urokinase against streptokinase in acute myocardial infarction. Lancet 1989; 1: 863-868
    PubMedGoogle Scholar
  • 9 Loscalzo J, Wharton TP, Kirshenbaum JM, Levine HJ, Flaherty JT, Topol EJ, Ramaswamy K, Kosowsky BD, Salem DN, Ganz P, Brinker JA, Gurewich V, Muller JE. Pro-urokinase for Myocardial Infarction study group. Clot-selective coronary thrombolysis with pro-urokinase. Circulation 1989; 79: 776-782
    CrossrefPubMedGoogle Scholar
  • 10 Lijnen HR, Van Hoef B, Collen D. Comparative kinetic analysis of the activation of human plasminogen by natural and recombinant single-chain urokinase-type plasminogen activator. Biochim Biophys Acta 1986; 884: 402-408
    CrossrefPubMedGoogle Scholar
  • 11 Lenich C, Pannell R, Gurewich V. A comparison of some of the enzymatic properties of recombinant glycosylated and non-glycosy-lated pro-urokinase (pro-UK). Thromb Haemostas 1991; 6 Abs 2221
    PubMedGoogle Scholar
  • 12 Orsini G, Brandazza A, Sarmientos P, Molinari A, Lansen J, Cauet G. Efficient renaturation and fibrinolytic properties of prourokinase and a deletion mutant expressed in Escherichia coli as inclusion bodies. Eur J Biochem. 1991; 195: 691-697
    PubMedGoogle Scholar
  • 13 Pain R. Protein folding for pleasure and for profit. Trends Biochem Sci 1987; 12: 309-312
    CrossrefPubMedGoogle Scholar
  • 14 Pannell R, Gurewich V. The activation of plasminogen by single-chain urokinase or by two-chain urokinase: a demonstration that single-chain urokinase has a low catalytic activity (pro-urokinase). Blood 1987; 69: 22-26
    PubMedGoogle Scholar
  • 15 Lenich C, Pannell R, Gurewich V. The effect of the carboxy-terminal lysine of urokinase on the catalysis of plasminogen activation. Thromb Res 1991; 64: 69-80
    CrossrefPubMedGoogle Scholar
  • 16 Castellino FJ, Powell JR. Human plasminogen. Methods Enzymol 1981; 80: 365-378
    PubMedGoogle Scholar
  • 17 Panyim S, Chalkley P. High resolution acrylamide gel electrophoresis of histones. Arch Biochem Biophys 1969; 130: 337-346
    CrossrefPubMedGoogle Scholar
  • 18 Barlow GH, Summaria L, Robbins KC. Molecular weight studies on human plasminogen and plasmin. J Biol Chem 1969; 244: 1138-1141
    PubMedGoogle Scholar
  • 19 Barlow GH. Urinary and kidney cell plasminogen activator (urokinase). Methods Enzymol 1976; 45: 239-244
    PubMedGoogle Scholar
  • 20 Chibber BAK, Morris JP, Castellino FJ. Effects of human fibrinogen and its cleavage products on activation of human plasminogen by streptokinase. Biochemistry 1985; 24: 3429-3434
    CrossrefPubMedGoogle Scholar
  • 21 Liu J, Gurewich V. A comparative study of the promotion of tissue plasminogen activator and pro-urokinase-induced plasminogen activation by fragments D and E-2 of fibrin. J Clin Invest 1991; 88: 2012-2017
    CrossrefPubMedGoogle Scholar
  • 22 Rampling MW, Gaffney PJ. The sulphite precipitation method for fibrinogen measurement; its use on small samples in the presence of fibrinogen degradation products. Clin Chim Acta 1976; 67: 43-52
    CrossrefPubMedGoogle Scholar
  • 23 Eriksson E, Ranby M, Gyzander E, Risberg B. Determination of plasminogen activator inhibitor in plasma using t-PA and a chromogenic single-point poly-D-lysine stimulated assay. Thromb Res 1988; 50: 091-101
    CrossrefPubMedGoogle Scholar
  • 24 Scully MF, Ellis V, Watahiki Y, Kakkar VV. Activation of pro-urokinase by plasmin: non-Michaelian kinetics indicates a mechanism of negative cooperativity. Arch Biochem Biophys 1989; 268 (2): 438-446
    PubMedGoogle Scholar
  • 25 Longstaff C, Clough AM, Gaffney PJ. Kinetics of plasmin activation of single chain urinary-type plasminogen activator (scu-PA) and demonstration of a high affinity interaction between scu-PA and plasminogen. J Biol Chem 1992; 267 (1): 173-179
    PubMedGoogle Scholar
  • 26 Danø K, Reich E. Plasminogen activator from cells transformed by an oncogenic virus. Inhibitors of the activation reaction. Biochim Biophys Acta 1979; 566: 138-151
    CrossrefPubMedGoogle Scholar
  • 27 Lemaire G, Drapier J-C, Petit J-F. Importance, localization and functional properties of the cell-associated form of plasminogen activator in mouse peritoneal macrophages. Biochim Biophys Acta 1983; 755: 332-343
    CrossrefPubMedGoogle Scholar
  • 28 Urden G, Hamsten A, Wiman B. Comparison of plasminogen activator inhibitor activity and antigen in plasma samples. Clin Chim Acta 1987; 169: 189-196
    CrossrefPubMedGoogle Scholar
  • 29 Wittwer AJ, Howard S. Glycosylation at Asn-184 inhibits the conversion of single-chain to two-chain tissue-type plasminogen activator by plasmin. Biochemistry 1990; 29: 4175-4180
    CrossrefPubMedGoogle Scholar
  • 30 Wittwer AJ, Howard SC, Carr LS, Harakas NK, Feder J, Parekh RB, Rudd PM, Dwek RA, Rademacher TW. Effects of N-glycosylation on in vitro activity of Bowes Melanoma and human colon fibroblast derived tissue plasminogen activator. Biochemistry 1989; 28: 7662-7669
    CrossrefPubMedGoogle Scholar
  • 31 Gonzalez-Gronow M, Grenett HE, Fuller GM, Pizzo SV. The role of carbohydrate in the function of human plasminogen: comparison of the protein obtained from molecular cloning and expression in Escherichia coli and COS cells. Biochim Biophys Acta. 1990; 1039: 269-276
    PubMedGoogle Scholar
  • 32 Marquart M, Walter J, Deisenhofer J, Bode W, Huber R. The geometry of the reactive site and of the peptide groups in trypsin, trypsinogen and its complexes with inhibitors. Acta Crystallogr 1983; B39: 480-490
    PubMedGoogle Scholar
  • 33 Cool DE, Edgell C-JS, Louie GV, Zoller MJ, Brayer GD, MacGilliv-ray RTA. Characterization of human blood coagulation factor XII cDNA. Prediction of the primary structure of factor XII and the tertiary structure of β -factor XIIa. J Biol Chem 1985; 260: 13666-13676
    PubMedGoogle Scholar