title: Discontinuous residues of factor IX constitute a surface for binding the anti-factor IX monoclonal antibody A-5.
abstract: Anti-human factor IX monoclonal antibody, A-5 (Mab A-5), has been widely used in structure-function studies for factor IX. Mab A-5 recognizes the catalytic domain of human factor IX (FIX). Regions important for Mab A-5 binding have previously been localized to the amino terminus of the heavy chain of factor IX, encompassing amino acid residues 181-310 [Blood (74) 971]. We have selected 20 positions in this region for alanine-scanning mutagenesis. We found that Mab A-5 failed to react with the recombinant factor IX mutants with substitutions at positions 228 and 252. Mab A-5 also reacted to a lesser extent to FIXD276A (factor IX with alanine substitution for aspartic acid at residue 276) and FIXK201A/D203A (double alanine substitutions at residues 201 and 203). The apparent dissociation rate constants (K(D)) in binding Mab A-5 were 6.0 x 10(-9), 1.4 x 10(-8) and 2.0 x 10(-8) M, for wild-type FIX, FIXK201A/D203A and FIXD276A, respectively. The increased K(D) values of the two FIX mutants are mainly owing to the increased dissociation rates. These affected residues constitute a surface opposite from the factor VIIIa binding surface. We conclude that the epitope for Mab A-5 is on a surface composed of residues 228, 252, 276, and 201 or 203. This surface, which may not be important for factor VIII binding, contains a strong antigenic region on factor IX.
authors.affiliation: Institute of Basic Medical Sciences, College of Medicine, National Cheng Kung University, Tainan, Taiwan.
authors.forename: Yu-Jia
authors.initials: YJ
authors.lastname: Chang
authors.forename: Hua-Lin
authors.initials: HL
authors.lastname: Wu
authors.forename: Ya-Chu
authors.initials: YC
authors.lastname: Hsu
authors.forename: Nobuko
authors.initials: N
authors.lastname: Hamaguchi
authors.forename: Guey-Yueh
authors.initials: GY
authors.lastname: Shi
authors.forename: Ming-Ching
authors.initials: MC
authors.lastname: Shen
authors.forename: Shu-Wha
authors.initials: SW
authors.lastname: Lin
mesh_terms: D019943:Amino Acid Substitution; D000911:Antibodies, Monoclonal / Q000737:chemistry* / Q000276:immunology*; D000936:Antigen-Antibody Complex / Q000737:chemistry* / Q000276:immunology*; D001665:Binding Sites; D018604:Epitope Mapping / Q000379:methods*; D000939:Epitopes / Q000737:chemistry / Q000276:immunology; D005164:Factor IX / Q000737:chemistry* / Q000276:immunology*; D006801:Humans; D007700:Kinetics; D016297:Mutagenesis, Site-Directed; D011485:Protein Binding; D011487:Protein Conformation; D011994:Recombinant Proteins / Q000737:chemistry / Q000276:immunology; D013329:Structure-Activity Relationship; D013918:Thrombin Time / Q000379:methods
chemical_list: D000911:Antibodies, Monoclonal; D000936:Antigen-Antibody Complex; D000939:Epitopes; D011994:Recombinant Proteins; D005164:Factor IX