A NEW FUNCTION OF HUMAN KININOGENS: THE AMINO-TERMINAL REGION OF DOMAIN 1 INVOLVES AN EF HAND-LIKE STRACTURE FOR METAL BINDING

Abstract

Two types of kininogens in mammalian plasma, high molecular weight (HMW) and low molecular weight (LMW) kininogens, are the precursors of kinin. Especially, HMW kininogen circulates in the plasma as a complex with prekallikrein and factor XI, and functions as a cofactor in the initial phase reactions of intrinsic blood coagulation cascade. Recently, it has been found that the kininogens have inhibitory activity toward cysteine proteinases. The heavy chain portion, which is identical for HMW and LMW kininogens, is composed of three domains, domain 1, 2 and 3. Each the domain 2 and 3 has a reactive site as a cysteine proteinase inhibitor. However, physiological function of domain 1 remains still unknown. By using the antibody recognizing the interaction between HMW kininogen and Ca2+ (anti-HMW kininogen-Ca2+ antibody) as a probe, we newly found the Ca2+ binding site in the domain 1.Anti-HMW kininogen-Ca2+ antibody was isolated from anti-HMW kininogen antiserum as an antibody which bound to a HMW kininogen-Sepharose column equilibrated with 40 mM Tris-HCl buffer, pH 7.5, containing 1.0 M NaCl and 1 mM CaCl2, and was eluted with 3 mM EDTA. Resulting from the characterization by ELISA, this antibody specifically recognized the CB-1 region (CNBr-cleavage fragment 1: 1-160 amino acid sequence) of the heavy chain of kininogen molecules in the presence of Ca2+ or Mg2+. Furthermore, circular dichroism (CD) experiments showed that the conformational changes of HMW kininogen and heavy chain were induced by the addition of metal ions such as Ca2+ or Mg2+, and that this change was due to the conformational change of the CB-1 region. The dissociation constant (Kd) for heavy chain measured by Ca2+ titration analysis by CD at 214 nm was found to be 0.33 ± 0.09 mM. The number of Ca2+ binding sites of heavy chain calculated from Hill plot was 1.15 ± 0.04. The EF handlike structure found in the amino-terminal portion of the heavy chain of kininogen molecules strongly supported the above data. This indicates a possibility that kininogens play an important role as a Ca2+ binding protein.