TY - JOUR
T1 - Discontinuous residues of factor IX constitute a surface for binding the anti-factor IX monoclonal antibody A-5
AU - Chang, Yu Jia
AU - Wu, Hua Lin
AU - Hsu, Ya Chu
AU - Hamaguchi, Nobuko
AU - Shi, Guey Yueh
AU - Shen, Ming Ching
AU - Lin, Shu Wha
PY - 2003
Y1 - 2003
N2 - 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 (KD) 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 KD 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.
AB - 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 (KD) 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 KD 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.
KW - Alanine-scanning mutagenesis
KW - Conformational epitope
KW - Factor IX
KW - Monoclonal antibody
KW - Recombinant proteins
KW - Surface plasmon resonance
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U2 - 10.1016/j.thromres.2003.09.025
DO - 10.1016/j.thromres.2003.09.025
M3 - Article
C2 - 14693178
AN - SCOPUS:0346433759
SN - 0049-3848
VL - 111
SP - 293
EP - 299
JO - Thrombosis Research
JF - Thrombosis Research
IS - 4-5
ER -