Mapping of a putative surface-binding site of human coagulation factor XII.
We have localized the binding epitope(s) of two murine monoclonal antibodies (B7C9 and P5-2-1) that were shown previously to inhibit the activation of human coagulation factor XII by negatively charged surfaces. A factor XII cDNA expression library in lambda gt11 was screened with antibody B7C9, and 16 immunoreactive bacteriophage were isolated. Fusion proteins from each of the recombinant phage were reactive with both monoclonal antibodies. Two of the phage cDNA inserts were found to code for amino acid residues -6-+31 and +1-+47 of factor XII, respectively, thereby defining the limits of the antigenic peptide to amino acids +1-+31. Each of the remaining 14 recombinant phage contained longer factor XII cDNA inserts that included sequences coding for the amino-terminal 31 amino acid residues. These results were confirmed by direct binding of antibody B7C9 to synthetic peptides containing amino acids 1-14 and 1-28 of factor XII. Further experiments with a set of nested peptides also indicated that amino acid residues 1-4 were essential but not sufficient for binding of B7C9 to the peptides. Hydrophobicity analysis of the amino-terminal region of plasma factor XII revealed a highly hydrophilic region between amino acid residues 5 and 15 that contained positively charged lysine residues at positions 8, 11, and 13. We conclude that a major epitope(s) recognized by monoclonal antibodies B7C9 and P5-2-1 is present in the amino-terminal 28 amino acids of factor XII. It is proposed that binding of these antibodies to factor XII blocks interaction of the positively charged region between residues 5 and 15 with negatively charged surfaces, thereby inhibiting activation.
Clarke
B J
BJ
Department of Pathology, McMaster University, Hamilton, Ontario, Canada.
Côté
H C
HC
Cool
D E
DE
Clark-Lewis
I
I
Saito
H
H
Pixley
R A
RA
Colman
R W
RW
MacGillivray
R T
RT
D000911:Antibodies, Monoclonal / Q000276:immunology*; D010582:Bacteriophage lambda / Q000235:genetics; D001666:Binding Sites, Antibody; D001667:Binding, Competitive; D003001:Cloning, Molecular; D004247:DNA / Q000235:genetics; D004797:Enzyme-Linked Immunosorbent Assay; D000939:Epitopes / Q000276:immunology*; D004926:Escherichia coli / Q000235:genetics; D005174:Factor XII / Q000235:genetics / Q000276:immunology* / Q000378:metabolism; D006801:Humans; D015151:Immunoblotting; D007124:Immunoenzyme Techniques; D010446:Peptide Fragments / Q000276:immunology*; D011993:Recombinant Fusion Proteins / Q000096:biosynthesis
D000911:Antibodies, Monoclonal; D000939:Epitopes; D010446:Peptide Fragments; D011993:Recombinant Fusion Proteins; D005174:Factor XII; D004247:DNA