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

Fingerprint

Factor IX
Monoclonal Antibodies
Alanine
Factor VIIIa
Factor VIII
Aspartic Acid
Mutagenesis
Epitopes
Catalytic Domain
Amino Acids

Keywords

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

ASJC Scopus subject areas

  • Cardiology and Cardiovascular Medicine
  • Hematology

Cite this

Discontinuous residues of factor IX constitute a surface for binding the anti-factor IX monoclonal antibody A-5. / Chang, Yu Jia; Wu, Hua Lin; Hsu, Ya Chu; Hamaguchi, Nobuko; Shi, Guey Yueh; Shen, Ming Ching; Lin, Shu Wha.

In: Thrombosis Research, Vol. 111, No. 4-5, 2003, p. 293-299.

Research output: Contribution to journalArticle

Chang, Yu Jia ; Wu, Hua Lin ; Hsu, Ya Chu ; Hamaguchi, Nobuko ; Shi, Guey Yueh ; Shen, Ming Ching ; Lin, Shu Wha. / Discontinuous residues of factor IX constitute a surface for binding the anti-factor IX monoclonal antibody A-5. In: Thrombosis Research. 2003 ; Vol. 111, No. 4-5. pp. 293-299.
@article{a2d1fa3432fa48e0854ce5bea04451a1,
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 (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.",
keywords = "Alanine-scanning mutagenesis, Conformational epitope, Factor IX, Monoclonal antibody, Recombinant proteins, Surface plasmon resonance",
author = "Chang, {Yu Jia} and Wu, {Hua Lin} and Hsu, {Ya Chu} and Nobuko Hamaguchi and Shi, {Guey Yueh} and Shen, {Ming Ching} and Lin, {Shu Wha}",
year = "2003",
doi = "10.1016/j.thromres.2003.09.025",
language = "English",
volume = "111",
pages = "293--299",
journal = "Thrombosis Research",
issn = "0049-3848",
publisher = "Elsevier Limited",
number = "4-5",

}

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

UR - http://www.scopus.com/inward/record.url?scp=0346433759&partnerID=8YFLogxK

UR - http://www.scopus.com/inward/citedby.url?scp=0346433759&partnerID=8YFLogxK

U2 - 10.1016/j.thromres.2003.09.025

DO - 10.1016/j.thromres.2003.09.025

M3 - Article

C2 - 14693178

AN - SCOPUS:0346433759

VL - 111

SP - 293

EP - 299

JO - Thrombosis Research

JF - Thrombosis Research

SN - 0049-3848

IS - 4-5

ER -