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 (original) (raw)
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Biochemistry, 1996
HK31 (S565-K595) has previously been shown to encompass the binding domain for plasma prekallikrein (PK) within domain 6 of high molecular weight kininogen (HK). The complementary binding domain for HK within PK is mapped to PK56 (F56-G86), in the apple 1 domain, and to PK266 (K266-C295), in the apple 4 domain. Isothermal titration calorimetry was used to directly monitor binding among HK31, PK56, and PK266. Either PK peptide binds to HK31 in 1:1 stoichiometry, regardless of whether a binary complex is first formed between PK266 and HK31 or between PK56 and HK31. Binding of the alternate PK peptide into a ternary complex is facilitated nearly 2-fold. The ternary complex consists of 1:1:1 HK31:PK56:PK266. Furthermore, binary and ternary complex formation is entropically driven and thermodynamically favored, suggesting that the conformational changes accompany binding. Fluorescence emission spectroscopy revealed that binding of PK56 caused a limited decrease in intrinsic tryptophan fluorescence emission intensity of HK31 while binding of PK266 to HK31 or the complex of HK31/PK56 had no such effect. We conclude that the two PK peptides bind to the HK peptide at different sites. The binding between HK and PK is likely due to conformational changes which serve to juxtapose the PK binding domain within HK with the HK binding site involving two spatial proximity segments. Abstract published in AdVance ACS Abstracts, September 15, 1996. 1 Abbreviations: HK, high molecular weight kininogen; HK31, synthetic S565-K595 peptide in domain 6 of the light chain of HK; HPLC, high-performance liquid chromatography; ITC, isothermal titration calorimetry; PK, prekallikrein; PK56, synthetic F56-G86 in the apple 1 domain of plasma prekallikrein; PK266, synthetic K266-C295 in the apple 4 domain of plasma prekallikrein. Single-letter abbreviations are used to denote all amino acids.
Blood, 1998
The consequences of assembling the contact system of proteins on the surface of vascular cells has received little study. We asked whether assembly of these proteins on the surface of cultured human endothelial cells (HUVECs) results in the activation of prekallikrein (PK) and its dependent pathways. Biotinylated PK binds specifically and reversibly to HUVECs in the presence of high molecular weight kininogen (HK) (apparent Kd of 23 ± 11 nmol/L,Bmax of 1.7 ± 0.5 × 107 sites per cell [mean ± SD, n = 5 experiments]). Cell-associated PK is rapidly converted to kallikrein. Surprisingly, the activation of cell-associated HK•PK complexes is entirely independent of exogenous factor XII (Km = 30 nmol/L,Vmax = 12 ± 3 pmol/L/min in the absencevKm = 20 nmol/L,Vmax = 9.2 ± 2.1 pmol/L/min in the presence of factor XII). Rather, kallikrein formation is mediated by an endothelial cell-associated, thiol protease. Cell-associated HK is proteolyzed during the course of prekallikrein activation, relea...
Inhibition of contact activation by a kininogen peptide (HKH20) derived from domain 5
International Immunopharmacology, 2002
Contact activation can be initiated by interaction of Factor XII, prekallikrein (PK) and high molecular weight kininogen (HK) with inorganic negatively charged biologic macromolecules, or upon cell surfaces, or interaction with membrane protein derivatives such as aggregated h amyloid. The latter two examples are zinc-dependent. The interaction with cells is dependent on peptides derived from HK domains 3 and 5 termed LDC27 and HKH20, respectively. We have tested the ability of each of these peptides to inhibit HK-dependent contact activation. HKH20 inhibited activation of prekallikrein when a mixture containing HK, prekallikrein and Factor XII was incubated with dextran sulfate, gC1qR, amyloid h or endothelial cells. Comparable quantities of LDC27 had no effect. The binding of biotinylated HK or biotinylated Factor XII was inhibited in a dose response fashion by increasing concentrations of HKH20 while LDC27, again had no effect. The N-terminal region of HKH20 (amino acids 475-485) is of particular importance for binding and histidine 485 prominently enhances the reaction as assessed employing overlapping and deleted peptides. Since there is a role for HK heavy chain in binding to endothelial cells and LDC27 can be employed as an affinity ligand to isolate the binding proteins, we increased the LDC27 concentration from 10-fold to 250-fold to determine whether it is functional. Inhibition of endothelial cell-dependent prekallikrein activation required 100-fold greater concentration of LDC27 when compared to HKH20 to achieve significant inhibition. We conclude that the interactions of the light chain of HK via HKH20 is of particular importance for activation of the bradykinin forming cascade in zinc-dependent or independent reactions and is true for all ''surface'' initiators tested thus far.
Journal of Allergy and Clinical Immunology, 2009
Background: Bradykinin formation typically requires interaction of Factor XII, prekallikrein (PK), and highmolecular-weight kininogen (HK) with negatively charged exogenous initiators or cell-surface proteins. Approximately 85% of plasma PK circulates as a complex with HK. Nonenzymatic cell-derived initiators, such as heat shock protein 90, can activate the HK-PK complex to generate kallikrein, bradykinin, and cleaved HK, even in the absence of Factor XII. Objective: We sought to determine whether PK, without activation to kallikrein, can digest HK to release bradykinin. Methods: Kallikrein was measured by using a chromogenic assay, and bradykinin levels were determined by ELISA. Cleavage of PK and HK were assessed by SDS-PAGE and Western blot analysis. Results: Cleavage of HK by PK is demonstrated without any conversion of PK to kallikrein. HK cleavage by PK is distinguished from that of kallikrein by the following: (1) stoichiometric activation of HK by PK with release of bradykinin proportional to the PK input; (2) inhibition of PK cleavage of HK by corn trypsin inhibitor, which has no effect on kallikrein; and (3) inhibition of PK cleavage of HK by a peptide derived from HK, which inhibits binding of PK to HK. The same peptide has no effect on kallikrein activation of HK. C1 inhibitor (C1INH), the major control protein of the plasma bradykinin-forming cascade, inhibits PK cleavage of HK. Conclusion: PK is an enzyme that can cleave HK to release bradykinin, and this reaction is inhibited by C1INH. This might account, in part, for circulating bradykinin levels and initiation of kinin formation in C1INH deficiency.
The involvement of proteoglycans in the human plasma prekallikrein interaction with the cell surface
PloS one, 2014
The aim of this work was to evaluate the role of human plasma prekallikrein assembly and processing in cells and to determine whether proteoglycans, along with high molecular weight kininogen (H-kininogen), influence this interaction. We used the endothelial cell line ECV304 and the epithelial cell lines CHO-K1 (wild type) and CHO-745 (deficient in proteoglycans). Prekallikrein endocytosis was studied using confocal microscopy, and prekallikrein cleavage/activation was determined by immunoblotting using an antibody directed to the prekallikrein sequence C364TTKTSTR371 and an antibody directed to the entire H-kininogen molecule. At 37°C, prekallikrein endocytosis was assessed in the absence and presence of exogenously applied H-kininogen and found to be 1,418.4±0.010 and 1,070.3±0.001 pixels/cell, respectively, for ECV304 and 1,319.1±0.003 and 631.3±0.001 pixels/cell, respectively, for CHO-K1. No prekallikrein internalization was observed in CHO-745 in either condition. Prekallikrein...
Biochemical Journal, 2004
The kininogenase activities of mouse (mK1), rat (rK1) and human (hK1) tissue kallikreins were assayed with the bradykinin-containing synthetic peptides Abz-MTEMARRPPGFSPFRSVTVQ-NH 2 (where Abz stands for o-aminobenzoyl) and Abz-MTS-VIRRPPGFSPFRAPRV-NH 2 , which correspond to fragments Met 374 -Gln 393 and Met 375 -Val 393 of mouse and rat LMWKs (lowmolecular-mass kininogens) with the addition of Abz. Bradykinin was released from these peptides by the mK1-and rK1-mediated hydrolysis of Arg-Arg and Arg-Ser (or Arg-Ala) peptide bonds. However, owing to preferential hydrolysis of Phe-Arg compared with the Arg-Ala bond in the peptide derived from rat LMWK, hK1 released bradykinin only from the mouse LMWK fragment and preferentially released des-[Arg 9 ]bradykinin from the rat LMWK fragment (Abz-MTSVIRRPPGFSPFRAPRV-NH 2 ). The formation of these hydrolysis products was examined in more detail by determining the kinetic parameters for the hydrolysis of synthetic, internally quenched fluorescent peptides containing six N-or C-terminal amino acids of bradykinin added to the five downstream or upstream residues of mouse and rat kininogens respectively. One of these peptides, Abz-GFSPFRAPRVQ-EDDnp (where EDDnp stands for ethylenediamine 2,4-dinitrophenyl), was preferentially hydrolysed at the Phe-Arg bond, confirming the potential des-[Arg 9 ]bradykinin-releasing activity of hK1 on rat kininogen. The proline residue that is two residues upstream of bradykinin in rat kininogen is, in part, responsible for this pattern of hydrolysis, since the peptide Abz-GFSPFRASRVQ-EDDnp was preferentially cleaved at the Arg-Ala bond by hK1. Since this peptidase accepts the arginine or phenylalanine residue at its S 1 subsite, this preference seems to be determined by the prime site of the substrates. These findings also suggested that the effects observed in rats overexpressing hK1 should consider the activation of B1 receptors by des-[Arg 9 ]bradykinin. For further comparison, two short internally quenched fluorescent peptides that bind to hK1 with affinity in the nM range and some inhibitors described previously for hK1 were also assayed with mK1 and rK1.
Comparison of human and porcine tissue kallikrein substrate specificities
Immunopharmacology, 1999
Little is known about the species specificity of tissue kallikrein-kininogen interaction since the kinetic parameters for Lys-bradykinin release from kininogen by tissue kallikreins from different animal species have not been reported. We have now determined the kinetic parameters for hydrolysis by human and porcine tissue kallikrein, hK1 and pK1, respectively Ž. Berg et al., 1992 of two series of intramolecularly quenched fluorogenic peptides having the sequences that flank the scissile Arg-Ser or Met-Lys bond in human and bovine kininogen. Results have shown that peptides having sequences from human kininogen are better substrates for hK1 and peptides derived from bovine kininogen are better substrates for pK1. Ž. X X Kinetic data for hydrolysis of the Arg-Ser bond showed that differences in the interaction of residue s in positions P-P 2 10 contribute to the efficiency of the cleavage and may be responsible for differences in their susceptibilities to the two kallikreins. Significant variations in the kinetic data were observed for the hydrolysis of the Met-Lys bond in substrates with w 370 381 x an N-terminal extension at sites P-P. The highest k rK value in the hydrolysis of Abz-Gln-Gln-bkng-EDDnp 3 9 cat m by pk1 demonstrates an important interaction of subsites S-S with Gln and Thr residues in the bovine kininogen segment. 5 4 A Gln 370-Gln 391 bovine kininogen fragment used to study the cleavage of both Met-Lys and Arg-Ser bonds in the same molecule confirmed the importance of an extended interaction site for species specificity among tissue kallikreins.
Journal of Hepatology, 1992
We partially purified the glycoproteins prokallikrein and kallikrein from rat plasma. The purification of rat plasma kallikrein may result in two forms: an intact form (a, Mr 84-87 kDa) and a partially degraded form (fl, Mr 46-51 kDa). The a-form is composed of a heavy chain (Mr 50 kDa) and a light chain (Mr 34-37 kDa) linked by a disulfide bond. The catalytic site is found on the light chain. The fl-form has a partially degraded heavy chain (Mr 28 kDa). Using a preparation of exsanguinated and perfused rat liver, we verified that rat plasma prokallikrein is not activated by the liver and that neither the proenzyme nor the light chain is removed by the organ. Both forms (a and fl) of the active enzyme are similarly removed from the perfusate. We also observed that the clearance of plasma kallikrein is temperature-dependent, and not affected by substances that inhibit binding to galactosyl-, mannosyl-, fucosyl-or phosphomannosyl-specific lectins, but inhibited by fl-galactosides. We suggest that: (a) the binding site to hepatocytes is latent on prokallikrein and is located on its heavy chain, more specifically on the 28-kDa fragment still present in the fl form of the active enzyme and (b) plasma kallikrein is recognized by an S-type lectin.