Mapping of the discontinuous kininogen binding site of prekallikrein: A distal binding segment is located in the heavy chain domain A4

Research output: Contribution to journalArticle

Abstract

Prekallikrein, the precursor to the serine proteinase kailikrein, circulates in plasma in an equimolar complex with H-kininogen. The binding to H-kininogen is mediated by the kallikrein heavy chain consisting of four "apple" domains, A1-A4, which attaches to H-kininogen with high specificity and affinity (KD = 83 UM). At least two distinct portions of the kallikrein heavy chain form this H-kininogen binding site: a proximal segment located in the NH2-terminal fragment of the heavy chain encompassing A1, and distal segment(s) located in COOH-terminal fragment spanning domains A2-A4. The proximal binding segment has been located to amino acid positions 56-86 of A1. To precisely map the distal binding segment, we have identified monoclonal antibodies directed to the COOH-terminal fragment which interfere with the H-kininogen-prekallikrein complex formation. Monoclonal antibody 13G11 binds to recombinant apple domain A4 but not to domain A3 of the prekallikrein heavy chain. Deletion mutagenesis of domain A4 narrowed down the target epitope of 13G11 to the center portion of domain A4, positions 284-331. Direct binding studies of H-kininogen to various domain A4 constructs revealed that the distal H-kininogen binding portion is located on a segment of 48 residues, which overlaps the 13G11 epitope. Hence the tight interaction of H-kininogen and prekallikrein is mediated by at least two separate sequence segments located in domains A1 and A4, respectively, of the prekallikrein heavy chain. The isolated distal binding segment significantly prolongs the partial thromboplastin time of reconstituted Williams plasma thus stressing the critical role of the prekallikrein-H-kininogen complex formation in the initiation of the endogenous blood coagulation cascade.

Details

Authors
  • Heiko Herwald
  • Thomas Renné
  • Joost C.M. Meijers
  • Dominic W. Chung
  • Jimmy D. Page
  • Robert W. Colman
  • Werner Müller-Esterl
External organisations
  • University of Mainz
  • University Medical Center Utrecht
  • University of Washington
  • Temple University
  • Pfizer Inc.
Research areas and keywords

Subject classification (UKÄ) – MANDATORY

  • Medicinal Chemistry
Original languageEnglish
Pages (from-to)13061-13067
JournalJournal of Biological Chemistry
Volume271
Issue number22
Publication statusPublished - 1996 May 31
Publication categoryResearch
Peer-reviewedYes
Externally publishedYes