Mapping of Epitopes on Human Tissue-Type Plasminogen Activator with Recombinant Deletion Mutant Proteins

Anton-Jan van Zonneveld; Harry Veerman; Just P J Brakenhoff; Lucien A Aarden; Jean-Francois Cajot; Hans Pannekoek

doi:10.1055/s-0038-1651067

Thrombosis and Haemostasis, Table of Contents

Thromb Haemost 1987; 57(01): 082-086
DOI: 10.1055/s-0038-1651067

Original Articles

Schattauer GmbH Stuttgart

Mapping of Epitopes on Human Tissue-Type Plasminogen Activator with Recombinant Deletion Mutant Proteins

Anton-Jan van Zonneveld

The Central Laboratory of the Netherlands Red Cross Blood Transfusion Service, Depts. of Molecular Biology and Autoimmune Diseases, Amsterdam, The Netherlands

,

Harry Veerman

The Central Laboratory of the Netherlands Red Cross Blood Transfusion Service, Depts. of Molecular Biology and Autoimmune Diseases, Amsterdam, The Netherlands

,

Just P J Brakenhoff

The Central Laboratory of the Netherlands Red Cross Blood Transfusion Service, Depts. of Molecular Biology and Autoimmune Diseases, Amsterdam, The Netherlands

,

Lucien A Aarden

The Central Laboratory of the Netherlands Red Cross Blood Transfusion Service, Depts. of Molecular Biology and Autoimmune Diseases, Amsterdam, The Netherlands

,

Jean-Francois Cajot

^*The Swiss Institute for Experimental Cancer Research, Epalinges, Switzerland

,

Hans Pannekoek

The Central Laboratory of the Netherlands Red Cross Blood Transfusion Service, Depts. of Molecular Biology and Autoimmune Diseases, Amsterdam, The Netherlands

› Author Affiliations

Abstract

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Summary

An antigen assay based on a monoclonal antibody directed against the light chain of tissue-type plasminogen activator (t-PA) was developed to quantify seven recombinant (r) t-PA deletion mutant proteins. These recombinant proteins were then employed to map different epitopes on t-PA which interact with a panel of twenty-three monoclonal anti-t-PA antibodies. Twenty were directed against domains on the heavy chain, two against the “finger” domain, three against the “epidermal growth factor-like” domain, five against the kringle 1 domain, and ten against the kringle 2 domain. Only three monoclonal anti-t-PA antibodies interact with the light chain. The finding that the epitopes of each of the monoclonals could be determined with the deletion mutant proteins supports the hypothesis of autonomous folding of structural domains and emphasizes the validity of the use of the recombinant t-PA-deletion mutant proteins for structure-function studies.

Keywords

Recombinant tissue-type plasminogen activator - Monoclonal antibodies - Epitopes - Domain structure

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References

References
1 Binder BR, Spragg J, Austen KF. Purification and characterization of human vascular plasminogen activator derived from blood vessel perfusates. J Biol Chem 1979; 254: 1998-2003
2 Wallen P, Riinby M, Bergsdorf N, Kok P. Purification and characterization of tissue plasminogen activator; on the occurrence of two different forms and their enzymatic properties. In: Progress in Fibrinolysis. Davidson JF, Nilsson IM, Asted B. (eds.), vol. 5 pp. 229-232 Edinburgh: Churchill Livingstone; 1981
3 Pennica D, Holmes WE, Kohr WJ, Harkins RN, Vehar GA, Ward CA, Bennet WF, Yelverton E, Seeburg PH, Heyneker HL, Goeddel DV. Cloning and expression of human tissue-type plasminogen activator cDNA in E. coli . Nature 1983; 301: 214-222
4 Banyai L, Varadi A, Patthy L. Common evolutionary origin of fibrinbinding structures of fibronectin and tissue-type plasminogen activator. FEBS Lett 1983; 163: 37-41
5 Verstraete H, Bory M, Collen D, Erbel R, Lennane RJ, Mathey D, Michels HR, Schartl M, Uebis R, Bernard R, Brower RW, De Bono DP, Huhmann W, Lubsen J, Meyer J, Rutsch W, Schmidt W, von Essen R. Randomnised trial of intravenous recombinant tissue-type plasminogen activator versus intravenous streptokinase in acute myocardial infarction. The Lancet 1985; 1: 842-847
6 Williams DO, Borer J, Braunwald E, Chesebro JH, Cohen LS, Dalen J, Dodge HT, Francis CK, Knatterud G, Ludbrook P, Markis JE, Mueller H, Desvigne-Nickens P, Passamani ER, Powers ER, Rao AK, Roberts R, Ross A, Ryan TJ, Sobel BE, Winniford M, Zaret B. Intravenous recombinant tissue-type plasminogen activator in patients with acute myocardial infarction: a report from the NHLBI thrombolysis in myocardial infarction trial. Circulation 1986; 73: 338-346
7 Thorsen S, Glas-Greenwalt P, Astrup T. Differences in binding to fibrin of urokinase and tissue-type plasminogen activator. Thromb Diath Haemorrh 1972; 28: 65-74
8 Hoylaerts M, Rijken DC, Lijnen HR, Collen D. Kinetics of the activation of plasminogen by human tissue plasminogen activator. J Biol Chem 1982; 257: 338-346
9 MacDonald ME, Van Zonneveld A-J, Pannekoek H. Functional analysis of the human tissue-type plasminogen activator protein: the light chain. Gene 1986; 42: 59-67
10 Van Zonneveld A-J, Veerman H, Pannekoek H. Autonomous functions of structural domains of human tissue-type plasminogen activator. Proc Natl Acad Sci USA 1986; 83: 4670-4674
11 Kagitani H, Tagawa M, Hatanaka K, Ikari T, Saito A, Bando H, Okada K, Matsuo O. Expression in E. coli of finger-domain lacking tissue-type plasminogen activator with high fibrin affinity. FEBS Lett 1985; 189: 145-149
12 Mattler L, Marks CA, Bang NU. Factors influencing tissue-type plasminogen activator binding to fibrin and fibrin-dependent plasminogen activation. Thrombos Haemostas 1985; 54: 266 (Abstr)
13 MacGregor IR, Micklem LR, James K, Pepper DS. Characterization of epitopes on human tissue plasminogen activator recognized by a group of monoclonal antibodies. Thromb Haemostas 1985; 53: 45-50
14 Wallen P, Bergsdorf N, Pohl G, Stigbrand T. Differentiation between one-chain and two-chain tissue type plasminogen activator by monoclonal antibodies. Haemostasis 1984; 14: 42
15 Kohler G, Milstein C. Continuous cultures of fused cells secreting antibody of predefined specificity. Nature 1975; 256: 495-497
16 Aarden L, Lansdorp P, De Groot E. A growth factor for hybridoma cells produced by human monocytes. Lymphokines 1985; 10: 175-185
17 Lopata MA, Cleveland DW, Sollner-Webb B. High level transient expression of a chloroamphenicol acetyl transferase gene by DEAE mediated DNA transfection coupled with a dimethyl sulfoxide or glycerol shock treatment. Nucleic Acids Res 1984; 12: 5707-5717
18 Hunter WM, Greenwood FC. Preparation of ¹³¹lodine-labeled human growth hormone of high specific activity. Nature 1962; 194: 495-496
19 Patthy L. Evolution of the proteases of blood coagulation and fibrinolysis by assembly from modules. Cell 1985; 41: 657-663
20 Gilbert W. Genes-in-pieces revisited. Science 1985; 228: 823-824