Discontinuous residues of factor IX constitute a surface for binding the anti-factor IX monoclonal antibody A-5

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• 作者：Chang ; Yu-Jia ; Wu ; Hua-Lin ; Hsu ; Ya-Chu ; Hamaguchi ; Nobuko ; Shi ; Guey-Yueh ; Shen ; Ming-Ching ; et. al.
• 关键词：Factor IX ; Alanine-scanning mutagenesis ; Recombinant proteins ; Monoclonal antibody ; Conformational epitope ; Surface plasmon resonance ; FVIIa-TF ; factor VIIa-tissue factor ; FVIIIa ; factor VIIIa ; FIX ; factor IX ; FIXa ; factor IXa ; FX ; factor X ; FXIa ; factor
• 刊名：Thrombosis Research
• 出版年：2003
• 出版时间：2003
• 年：2003
• 卷：111
• 期：4-5
• 页码：293-299
• 全文大小：380 K

文摘

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 6.0×10⁻⁹, 1.4×10⁻⁸ and 2.0×10⁻⁸ 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.