Epitope mapping of the anti-urokinase monoclonal antibody 5B4 by isolated domains of urokinase

A. Corti, E. Sarubbi, A. Soffientini, M. L. Nolli, A. Zanni, M. Galimberti, F. Parenti, G. Cassani

Research output: Contribution to journalArticlepeer-review

Abstract

The amino terminal fragment (ATF) of urokinase-type plasminogen activator (uPA) is a degradation product comprising the entire growth factor-like and kringle domains. It has been previously shown that ATF is able to bind to the u-PA receptor through the growth factor-like domain and that the anti u-PA monoclonal antibody 5B4 (Mab 5B4) binds to ATF preventing u-PA receptor binding. To localize more precisely the epitope recognized by Mab 5B4, ATF was subfragmented by controlled enzymatic proteolysis with V8 protease. Three subfragments of 4,000 M(r) (F-4k), 11,000 M(r) (F-11k) and 12,000 M(r) (F-12k) were purified from the reaction mixture and characterized. SDS-PAGE under reducing and non-reducing conditions, N-terminal aminoacid sequence analysis and C-terminal aminoacid analysis of each fragment indicate that F-4k and F-11k correspond to intact growth factor-like domain and kringle domain (residues 4-43 and 44-135 respectively) while F-12k corresponds to the kringle domain cleaved in the first loop at the glu52-gly53 bond. By Western blot and competitive binding experiments we show that Mab 5B4 recognizes an epitope located on the kringle domain of u-PA and that the binding is strongly reduced when the kringle contains an additional cleavage in its first loop. Since the receptor binding site of u-PA has been previously shown to be located on the growth factor-like domain, Mab 5B4 inhibits the binding of uPA to its cellular receptor likely by steric hindrance. Besides the proven utility in epitope localization of anti u-PA monoclonal antibodies, these u-PA fragments may represent powerful tools for studies of structure-function relationship of u-PA.

Original languageEnglish
Pages (from-to)934-939
Number of pages6
JournalThrombosis and Haemostasis
Volume62
Issue number3
Publication statusPublished - 1989

Keywords

  • Growth factor-like domain
  • Kringle
  • monoclonal antibody
  • Plasminogen Activator
  • Urokinase-type
  • V8 protease

ASJC Scopus subject areas

  • Hematology

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