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 Al 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 IC50 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.
Renné
Thomas
T
Institute of Clinical Biochemistry and Pathobiochemistry, Julius-Maximilians University at Würzburg, Josef-Schneider-Strasse 2, D-97080 Würzburg, Germany. thomas@renne.net
Sugiyama
Akiko
A
Gailani
David
D
Jahnen-Dechent
Willi
W
Walter
Ulrich
U
Müller-Esterl
Werner
W
D000911:Antibodies, Monoclonal / Q000276:immunology; D001665:Binding Sites; D015153:Blotting, Western; D004591:Electrophoresis, Polyacrylamide Gel; D018604:Epitope Mapping; D000939:Epitopes / Q000276:immunology / Q000378:metabolism*; D006801:Humans; D007118:Immunoassay; D019679:Kininogen, High-Molecular-Weight / Q000378:metabolism*; D020842:Plasma Kallikrein / Q000235:genetics / Q000276:immunology / Q000378:metabolism*; D011288:Prekallikrein / Q000235:genetics / Q000276:immunology / Q000378:metabolism*; D011993:Recombinant Fusion Proteins / Q000235:genetics / Q000276:immunology / Q000378:metabolism; D017384:Sequence Deletion
D000911:Antibodies, Monoclonal; D000939:Epitopes; D019679:Kininogen, High-Molecular-Weight; D011993:Recombinant Fusion Proteins; D011288:Prekallikrein; D020842:Plasma Kallikrein