An anti-EGF monoclonal antibody that detects intramolecular communication in factor IX.
Coagulation factor IX contains a gamma-carboxyglutamic acid (Gla) module, two epidermal growth factor-like (EGF) modules, and a serine protease region. We have characterized a mouse monoclonal antibody that binds the N-terminal EGF-like module of human factor IX with high affinity. Studies of recombinant factor IX mutants indicated that the epitope is located in the C-terminal end of the EGF-like module, which is consistent with the binding being non-Ca(2+)-dependent. The antibody bound factor IXa (K(D) = 7.6 x 10(-10) M) with about 10-fold higher affinity than factor IX (K(D) = 6.2 x 10(-9) M). Binding of the antibody to factor IXa did not affect the amidolytic activity of the protein, nor was binding affected by active site inhibition of factor IXa. These results are consistent with long-range interactions between the serine protease region and the N-terminal EGF-like module in factor IX.
Persson
K E
KE
Department of Clinical Chemistry, Lund University, University Hospital, Malmö, Malmö, S-205 02, Sweden.
Knobe
K E
KE
Stenflo
J
J
D000595:Amino Acid Sequence; D000818:Animals; D000911:Antibodies, Monoclonal / Q000737:chemistry*; D001665:Binding Sites; D002118:Calcium / Q000737:chemistry / Q000378:metabolism / Q000494:pharmacology; D004305:Dose-Response Relationship, Drug; D004591:Electrophoresis, Polyacrylamide Gel; D004797:Enzyme-Linked Immunosorbent Assay; D004815:Epidermal Growth Factor / Q000276:immunology*; D000939:Epitopes; D005164:Factor IX / Q000737:chemistry* / Q000378:metabolism*; D006801:Humans; D015151:Immunoblotting; D007700:Kinetics; D051379:Mice; D008856:Microscopy, Fluorescence; D008969:Molecular Sequence Data; D011485:Protein Binding; D011487:Protein Conformation; D017434:Protein Structure, Tertiary; D012697:Serine Endopeptidases / Q000737:chemistry; D020349:Surface Plasmon Resonance; D013997:Time Factors
D000911:Antibodies, Monoclonal; D000939:Epitopes; D004815:Epidermal Growth Factor; D005164:Factor IX; D012697:Serine Endopeptidases; D002118:Calcium