Chang Yu-Jia, Wu Hua-Lin, Hsu Ya-Chu, Hamaguchi Nobuko, Shi Guey-Yueh, Shen Ming-Ching, Lin Shu-Wha
Institute of Basic Medical Sciences, College of Medicine, National Cheng Kung University, Tainan, Taiwan.
Thromb Res. 2003;111(4-5):293-9. doi: 10.1016/j.thromres.2003.09.025.
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.
抗人凝血因子IX单克隆抗体A-5（Mab A-5）已广泛用于凝血因子IX的结构-功能研究。Mab A-5识别人类凝血因子IX（FIX）的催化结构域。先前已确定Mab A-5结合的重要区域位于凝血因子IX重链的氨基末端，涵盖氨基酸残基181-310[《血液》（74）971]。我们在该区域选择了20个位置进行丙氨酸扫描诱变。我们发现，Mab A-5不能与在第228位和第252位发生取代的重组凝血因子IX突变体发生反应。Mab A-5与FIXD276A（在第276位用丙氨酸取代天冬氨酸的凝血因子IX）和FIXK201A/D203A（在第201位和第203位的双丙氨酸取代）的反应程度也较低。野生型FIX、FIXK201A/D203A和FIXD276A与Mab A-5结合的表观解离速率常数（K(D)）分别为6.0×10^(-9)、1.4×10^(-8)和2.0×10^(-8)M。这两种FIX突变体的K(D)值增加主要是由于解离速率增加。这些受影响的残基构成了与凝血因子VIIIa结合表面相对的一个表面。我们得出结论，Mab A-5的表位位于由残基228、252、276以及201或203组成的一个表面上。这个表面可能对凝血因子VIII结合不重要，但在凝血因子IX上包含一个强抗原区域。
