H-kininogen Binding Research Articles

Fine mapping of the H-kininogen binding site in plasma prekallikrein apple domain 2

Plasma prekallikrein (PPK), the zymogen of the contact phase protease plasma kallikrein, forms a non-covalent complex with its substrate H-kininogen (HK). HK binds to cell surface proteoglycans, indirectly anchoring this bradykinin-generating protease to endothelial cells. The heavy chain of PPK consisting of four apple domains designated A1 to A4. Previous studies indicated that a major HK binding site on PPK is within the A2 domain, with additional contributions to binding provided by the N-terminal portion of A1 and the central part of A4. To precisely map the relevant binding segments in A2, we employed a monoclonal anti-PPK antibody (PKH6) that binds to A2 and blocks HK-PPK complex formation with an apparent IC 50 of 8 nM. Using recombinant A2 C-terminal deletion mutants, we mapped the target epitope of PKH6 to the N-terminal portion of A2, residues 92–153. C-terminal deletion of A2 to residue 145 resulted in a loss of PKH6 binding, as did proteolytic cleavage of A2 at Lys140–Arg141. A comparison of HK binding to various A2 deletion mutants revealed that the major HK binding site is localized to residues 145–153 in the central portion of A2, where it overlaps with the PKH6 epitope. This sequence is conserved in the A2 domain of the related protease factor XI, explaining the unusual strong cross-reactivity of PHK6 with factor XI, as well as the similar HK-binding characteristics of PPK and factor XI.

Characterization of the H-kininogen-binding Site on Factor XI: A COMPARISON OF FACTOR XI AND PLASMA PREKALLIKREIN

Factor XI (FXI), the zymogen of the blood coagulation protease FXIa, and the structurally homologous protein plasma prekallikrein circulate in plasma in noncovalent complexes with H-kininogen (HK). HK binds to the heavy chains of FXI and of prekallikrein. Each chain contains four apple domains (F1-F4 for FXI and P1-P4 for prekallikrein). Previous studies indicated that the HK-binding site on FXI is located in F1, whereas the major HK-binding site on prekallikrein is in P2. To determine the contribution of each FXI apple domain to HK-FXI complex formation, we examined binding of recombinant single apple domain-tissue plasminogen activator fusion proteins to HK. The order of affinity from highest to lowest is F2 F4 > F1 F3. Monoclonal antibodies against F2 are superior to F4 or F1 antibodies as inhibitors of HK binding to FXI. Antibody alphaP2, raised against prekallikrein, cross-reacts with FXI F2 and inhibits FXI-HK binding with an IC(50) of 8 nm. HK binding to a platelet-specific FXI variant lacking the N-terminal half of F2 is reduced > 5-fold compared with full-length FXI. A chimeric FXI molecule in which F2 is replaced by P2 is cleaved within P2 during activation by factor XIIa, resulting in greatly reduced HK binding capacity. In contrast, wild-type FXI is not cleaved within F2, and its binding capacity for HK is unaffected by factor XIIa. Our data show that HK binding to FXI involves multiple apple domains, with F2 being most important. The findings demonstrate a similarity in mechanism for FXI and prekallikrein binding to HK.

Subcellular targeting of multiligand-binding protein gC1qR

gC1q receptor, a protein originally described as the cell surface receptor for the globular heads of complement factor C1q, has been found to bind human H-kininogen with high affinity and specificity. Therefore, gC1qR has been considered candidate kininogen docking site on the surfaces of platelets, neutrophils and endothelial cells. Recent work demonstrating that gC1qR is an intracellular protein that is tightly associated with mitochondria rather than targeted to the cell surface has challenged this view. To further probe cellular trafficking routes of gC1qR, we overexpressed human gC1qR in a mammalian cell and monitored cell surface exposure of recombinant gC1qR by virtue of its capacity to bind labeled H-kininogen. Transient transfection of COS1 cells with the full-length cDNA of human gC1qR resulted in a high level of recombinant protein that matched the pool of endogenous gC1qR present in these cells. Overexpression of gC1qR did not significantly increase the number of H-kininogen binding sites exposed by the transfected cells thus denying the possibility that alternative routing of gC1qR to the surface of COS1 cells occurs at significant levels. Hence gC1qR has the capacity to tightly bind H-kininogen, but because gC1qR is routed to mitochondria it cannot fulfill the postulated functions as a cell docking site for kininogens and complement factors.

Mapping of the Discontinuous H-kininogen Binding Site of Plasma Prekallikrein: EVIDENCE FOR A CRITICAL ROLE OF APPLE DOMAIN-2

Plasma prekallikrein, a zymogen of the contact phase system, circulates in plasma as heterodimeric complex with H-kininogen. The binding is mediated by the prekallikrein heavy chain consisting of four apple domains, A1 to A4, to which H-kininogen binds with high specificity and affinity (K(D) = 1.2 x 10(-8) M). Previous work had demonstrated that a discontinuous kininogen-binding site is formed by a proximal part located in A1, a distal part exposed by A4, and other yet unidentified portion(s) of the kallikrein heavy chain. To detect relevant binding segment(s) we recombinantly expressed single apple domains and found a rank order of binding affinity for kininogen of A2 > A4 approximately A1 > A3. Removal of single apple domains in prekallikrein deletion mutants reduced kininogen binding by 21 (A1), 64 (A2), and 24% (A4), respectively, whereas deletion of A3 was without effect. Transposition of homologous A2 domain from prekallikrein to factor XI conferred high-affinity kininogen binding from the former to the latter. The principal role of A2 for H-kininogen docking to the prekallikrein heavy chain was further substantiated by the finding that cleavage of a single peptide bond in A2 drastically diminished the H-kininogen binding affinity. Furthermore, the epitope of monoclonal antibody PKH6 which blocks kallikrein-kininogen complex formation with an IC(50) of 8 nM mapped to the center portion of domain A2. Our data indicate that domain A2 and two flanking sequence segments of A1 and A4 form a discontinuous binding platform for H-kininogen on the prekallikrein heavy chain. Domain-specific antibodies directed to these critical sites efficiently interfered with contact phase-induced bradykinin release from H-kininogen.

Human H-kininogen Is a Ferritin-binding Protein

H-kininogen is a multifunctional protein: it inhibits cysteine proteases, plays a role in contact activation of the coagulation cascade, and is the precursor of the potent proinflammatory peptide bradykinin. In the experiments described here, we identify H-kininogen as a ferritin-binding protein. Ferritin is a cellular and serum protein that is elevated in acute and chronic inflammation and many cancers. Despite numerous reports of ferritin-binding protein(s) in human serum, the nature and function of these proteins remain unclear. As a first step in characterizing the interaction between ferritin and its binding protein(s), we devised a ligand blot assay and used it to guide purification of a ferritin-binding protein from human serum. Edman degradation of the purified protein determined the sequence HNLGHGHK(H)ERDQGHG, a sequence with identity to residues 421-436 of human H-kininogen. These results were confirmed by demonstrating that commercially purified H-kininogen possessed ferritin binding activity and that ferritin binding could not be detected in plasma from kininogen-deficient individuals. Ligand blot assays mapped the ferritin binding domain to the light chain of H-kininogen chain, and revealed that both H and L recombinant ferritins possess H-kininogen binding activity. The unexpected identification of H-kininogen as a ferritin-binding protein may link ferritin in the complex chain of interactions by which H-kininogen mediates its multiple effects in contact activation and inflammation.

Mapping of the Discontinuous Kininogen Binding Site of Prekallikrein: A DISTAL BINDING SEGMENT IS LOCATED IN THE HEAVY CHAIN DOMAIN A4

Prekallikrein, the precursor to the serine proteinase kallikrein, 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 nM). 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.

Isolation and Characterization of the Kininogen-binding Protein p33 from Endothelial Cells: IDENTITY WITH THE gC1q RECEPTOR

Kininogens, the precursor proteins of the vasoactive kinins, bind specifically, reversibly, and saturably to platelets, neutrophils, and endothelial cells. Two domains of the kininogens expose major cell binding sites: domain D3 that is shared by H- and L-kininogen and domain D5H that is exclusively present in H-kininogen. Previously we have mapped the kininogen cell binding sites to 27 residues of D3 ("LDC27") and 20 residues of D5H ("HKH20"", respectively (Herwald, H., Hasan, A. A. K., Godovac-Zimmermann, J., Schmaier, A. H., and Müller-Esterl, W. (1995) J. Biol. Chem. 270, 14634-14642; Hasan, A. A. K., Cines, D. B., Herwald, H., Schmaier, A. H., and Müller-Esterl, W. (1995) J. Biol. Chem. 270, 19256-19261). The corresponding kininogen acceptor site(s) exposed by the cell surfaces are still poorly defined. Using a non-ionic detergent, Nonidet P-40, we have been able to solubilize kininogen binding sites from an endothelial cell line, EA.hy926, in their functionally active form. Affinity chromatography of the solubilized kininogen binding sites on HKH20, a synthetic peptide representing the D5H cell binding site, allowed us to isolate a 33-kDa protein ("p33") that binds specifically and reversibly to H-kininogen with a KD (apparent dissociation constant) of 9 +/- 2 nM. Preparative SDS electrophoresis followed by NH2-terminal amino acid sequence analysis identified the kininogen-binding protein p33 as the gC1q receptor ("gC1qR"), an extrinsic membrane protein that interacts with the globular domains of the complement component C1q. The purified p33 binds C1q with moderate affinity, KD = 240 +/- 10 nM. Recombinant expression of the corresponding cDNA in Escherichia coli demonstrated that p33 binds H-kininogen, but not L-kininogen. Peptide HKH20 but not peptide LDC27 inhibited binding of H-kininogen to the recombinant p33 in a concentration-dependent manner, indicating that H-kininogen binds to p33 via domain D5H. Recombinant p33 efficiently inhibited the binding of H-kininogen to EA.hy926 cells. Factor XII, but not prekallikrein, competed with H-kininogen binding to p33. These findings suggest that an endothelial binding protein mediates the assembly of critical components of the kinin-generating pathway on the surface of endothelial cells, thereby linking the early events of kinin formation and complement activation.

Parallel mechanisms of high molecular weight kininogen action as a cofactor in kallikrein inactivation and prekallikrein activation reactions.

The mechanism by which high molecular weight kininogen (H-kininogen) potentiates the heparin-accelerated inhibition of plasma kallikrein by antithrombin [Olson, S. T., Sheffer, R., & Francis, A. M. (1993) Biochemistry (preceding paper in this issue)] was investigated at I = 0.15, pH 7.4, 25 degrees C. Single-chain, two-chain, and light-chain, but not heavy-chain, forms of H-kininogen were similarly effective in potentiating the heparin-accelerated antithrombin-kallikrein reaction, indicating that the light-chain region of the protein was responsible for promoting kallikrein inactivation and that cleavage of H-kininogen did not significantly affect this promoting activity. H-kininogen potentiation increased in a saturable manner with increasing kininogen concentration, reflecting a KD (23 +/- 8 nM) similar to that previously measured for H-kininogen binding to kallikrein by equilibrium methods. Both H-kininogen-stimulated and unstimulated heparin rate enhancements initially increased with increasing heparin concentration in a manner corresponding to the saturation of antithrombin with heparin (KD = 10-30 nM). However, at higher heparin concentrations, the stimulated but not the unstimulated heparin rate enhancement decreased in parallel with the saturation of a protein-heparin interaction with a KD (0.4 +/- 0.2 microM) comparable to that directly measured for the H-kininogen-heparin interaction (2.0 +/- 0.2 microM). These results implied that H-kininogen stimulation required the formation of a quaternary complex in which antithrombin and H-kininogen-kallikrein complex were bound to the same heparin chain. In keeping with this interpretation, a synthetic heparin pentasaccharide representing the antithrombin binding sequence accelerated the antithrombin-kallikrein reaction to an extent similar to that of full-length heparin chains containing this sequence, but the pentasaccharide acceleration was not stimulated by H-kininogen. The importance of H-kininogen-kallikrein complex binding to heparin for kininogen stimulation was further indicated from the marked salt dependence of the second-order rate constant for the association of H-kininogen-kallikrein complex but not free kallikrein with antithrombin-heparin complex, under conditions where saturation of the two binary complexes was maintained. Kinetic analyses of antithrombin-kallikrein reactions as a function of the inhibitor concentration indicated that the KD for an initial antithrombin-kallikrein encounter complex was decreased 20-fold by heparin binding to antithrombin and an additional 200-fold by H-kininogen also binding to kallikrein. By contrast, rate constants for the conversion of the encounter complex to a stable complex were comparable for all reactions.(ABSTRACT TRUNCATED AT 400 WORDS)

Mapping of the high molecular weight kininogen binding site of prekallikrein. Evidence for a discontinuous epitope formed by distinct segments of the prekallikrein heavy chain

Prekallikrein, a glycoprotein involved in contact phase activation, circulates in plasma in the form of a binary complex with high molecular weight kininogen (H-kininogen). The binding to H-kininogen is mediated by the prekallikrein heavy chain consisting of four repetitive domains, A1-A4. To define more precisely the region(s) involved in kininogen binding, we have employed an affinity cross-linking strategy with a synthetic peptide of 31 residues which mimics the prekallikrein binding site of H-kininogen. Cross-linking of the radiolabeled peptide to (pre)kallikrein revealed a binding segment in the NH2-terminal portion of the prekallikrein heavy chain; another binding segment was located in the COOH-terminal part of the heavy chain. The latter binding segment is harbored by a previously identified fragment of the kallikrein heavy chain involved in H-kininogen binding (Page, J.D., and Colman, R.W. (1991) J. Biol. Chem. 266, 8143-8148). Chemical cleavage of the heavy chain cross-linked with the radiolabeled peptide mapped the NH2-terminal binding segment to 60 residues (positions 53-112) of A1. Synthesis of a peptide (positions 56-86) and development of specific antibodies to this peptide narrowed down the kininogen binding segment to 31 residues of the center portion of A1. This NH2-terminal segment is equivalent to a kininogen binding site previously identified in factor XI (Baglia, F.A., Jameson, B.A., and Walsh, P.N. (1992) J. Biol. Chem. 267, 4247-4252). We conclude that prekallikrein exposes at least two segments on its heavy chain portion which form a continuous surface thereby facilitating the intimate binding of the zymogen to its nonenzymatic cofactor, H-kininogen.

Mapping of the H-kininogen binding site exposed by the prekallikrein heavy chain.

Mapping of the H-kininogen binding site exposed by the prekallikrein heavy chain.

High molecular weight kininogen-binding site of prekallikrein probed by monoclonal antibodies.

A panel of monoclonal antibodies against human prekallikrein was raised in mice and characterized with respect to the major antigenic epitopes. Of 18 antibodies, nine were directed against the light chain portion performing the proteolytic function of activated kallikrein, and nine recognized the heavy chain mediating the binding of prekallikrein to high molecular weight (H-)kininogen. Among the anti-heavy chain antibodies, one (PK6) interfered with the procoagulant activity of prekallikrein, and prolonged in a concentration-dependent manner the activated partial thromboplastin time of reconstituted prekallikrein-deficient plasma (Fletcher type). Antibody PK6 was subtyped IgG1,k and had an apparent Kass of 6.8 +/- 0.44.10(8) M-1 for prekallikrein. Functional analyses revealed that PK6 does not interfere with prekallikrein activation by activated Hageman factor (beta-F XIIa), and has no effect on the kininogenase function of activated kallikrein. Monoclonal antibody PK6 but none of the other anti-heavy chain antibodies completely prevented complex formation of prekallikrein with H-kininogen, and readily dissociated preformed complexes of prekallikrein and H-kininogen. Likewise, Fab' and F(ab')2 fragments of PK6 blocked H-kininogen binding to prekallikrein. A synthetic peptide of 31 amino acid residues encompassing the entire prekallikrein binding region of H-kininogen effectively competed with PK6 for prekallikrein binding indicating that the target epitope of PK6 is juxtaposed to, if not incorporated in the H-kininogen-binding site of prekallikrein. Extensive cross-reactivity of PK6 with another H-kininogen-binding protein of human plasma, i.e. factor XI, suggested that the structure of the target epitope of PK6 is well conserved among prekallikrein and factor XI, as would be expected for the kininogen-binding site shared by the two proteins. It is anticipated that monoclonal antibody PK6 will be an important tool for the precise mapping of the hitherto unknown kininogen-binding site of prekallikrein.