Analysis of protein S C4b-binding protein interactions by homology modeling and inhibitory antibodies.
A monoclonal antibody (mAb 6F6) directed against the beta-chain of C4b-binding protein (C4BP) was previously shown to inhibit the binding of protein S to C4BP. To localize the epitope of this antibody, 23 overlapping synthetic peptides (15-mers) covering the entire sequence (1-235) of the beta-chain of C4BP were used. When the immobilized peptides were screened for their ability to bind mAb 6F6, only peptide beta(51-65) showed high-affinity binding. The apparent affinity of mAb 6F6 for immobilized peptide beta(51-65) was somewhat similar to that for native C4BP with Kd approximately 1 nM for C4BP and approximately 9 nM for peptide beta(51-65). Peptide beta(51-65) inhibited the binding of the mAb 6F6 to immobilized C4BP with half-maximal inhibition at 30 microM peptide. Clotting assays of protein S anticoagulant cofactor activity using a factor Xa-1-stage assay with activated protein C allow measurement of free protein S in solution since only free protein S is active. Studies using such clotting assays showed that preincubation of C4BP with either mAb 6F6 or polyclonal anti-beta(31-45) antibodies inhibited the formation of the complex between C4BP and protein S. Previous studies showed that, although peptide beta(51-65) itself does not inhibit complex formation, peptide beta(31-45) does bind directly to protein S and does inhibit protein S binding to C4BP. The three-dimensional structure of the first SCR (residues 2-60) of the C4BP beta-chain was made on the basis of homology modeling.(ABSTRACT TRUNCATED AT 250 WORDS)
Fernández
J A
JA
Department of Molecular & Experimental Medicine, Scripps Research Institute, La Jolla, California 92037, USA.
Villoutreix
B O
BO
Hackeng
T M
TM
Griffin
J H
JH
Bouma
B N
BN
D000595:Amino Acid Sequence; D000818:Animals; D000911:Antibodies, Monoclonal / Q000302:isolation & purification; D000937:Antigen-Antibody Reactions; D002352:Carrier Proteins / Q000737:chemistry* / Q000276:immunology / Q000378:metabolism*; D002846:Chromatography, Affinity; D003169:Complement Inactivator Proteins; D015951:Factor Xa / Q000378:metabolism; D006023:Glycoproteins; D006801:Humans; D007700:Kinetics; D046911:Macromolecular Substances; D009682:Magnetic Resonance Spectroscopy; D051379:Mice; D008807:Mice, Inbred BALB C / Q000276:immunology; D008969:Molecular Sequence Data; D010446:Peptide Fragments / Q000138:chemical synthesis / Q000737:chemistry / Q000276:immunology; D010455:Peptides / Q000138:chemical synthesis / Q000737:chemistry / Q000276:immunology; D011485:Protein Binding; D017293:Protein S / Q000737:chemistry* / Q000378:metabolism*; D011951:Receptors, Complement / Q000737:chemistry / Q000378:metabolism; D017386:Sequence Homology, Amino Acid; D013816:Thermodynamics
D000911:Antibodies, Monoclonal; D002352:Carrier Proteins; D003169:Complement Inactivator Proteins; D006023:Glycoproteins; D046911:Macromolecular Substances; D010446:Peptide Fragments; D010455:Peptides; D017293:Protein S; D011951:Receptors, Complement; D015951:Factor Xa