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.
Herwald
H
H
Institute for Physiological Chemistry and Pathobiochemistry, University of Mainz, Germany.
Jahnen-Dechent
W
W
Alla
S A
SA
Hock
J
J
Bouma
B N
BN
Müller-Esterl
W
W
D000595:Amino Acid Sequence; D001345:Autoradiography; D001665:Binding Sites; D001667:Binding, Competitive; D004591:Electrophoresis, Polyacrylamide Gel; D004797:Enzyme-Linked Immunosorbent Assay; D000939:Epitopes / Q000378:metabolism*; D006801:Humans; D007457:Iodine Radioisotopes; D007700:Kinetics; D007704:Kininogens / Q000302:isolation & purification / Q000378:metabolism*; D046911:Macromolecular Substances; D008961:Models, Structural; D008969:Molecular Sequence Data; D008970:Molecular Weight; D010446:Peptide Fragments / Q000737:chemistry / Q000302:isolation & purification; D010455:Peptides / Q000138:chemical synthesis / Q000378:metabolism; D011288:Prekallikrein / Q000378:metabolism*
D000939:Epitopes; D007457:Iodine Radioisotopes; D007704:Kininogens; D046911:Macromolecular Substances; D010446:Peptide Fragments; D010455:Peptides; D011288:Prekallikrein