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

Yu Jia Chang, Hua Lin Wu, Ya Chu Hsu, Nobuko Hamaguchi, Guey Yueh Shi, Ming Ching Shen, Shu Wha Lin

Research output: Contribution to journalArticle

7 Citations (Scopus)

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 (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.

Original languageEnglish
Pages (from-to)293-299
Number of pages7
JournalThrombosis Research
Volume111
Issue number4-5
DOIs
Publication statusPublished - 2003
Externally publishedYes

Keywords

  • Alanine-scanning mutagenesis
  • Conformational epitope
  • Factor IX
  • Monoclonal antibody
  • Recombinant proteins
  • Surface plasmon resonance

ASJC Scopus subject areas

  • Cardiology and Cardiovascular Medicine
  • Hematology

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