Localization of the binding site for a factor VIII activity neutralizing antibody to amino acid residues Asp1663-Ser1669.
We have identified the Factor VIII amino acid sequence Asp-Tyr-Asp-Asp-Thr-Ile-Ser (1663-1669) as the binding site of a Factor VIII activity neutralizing antibody (28 Bethesda units/mg). The binding site of another neutralizing antibody (10 Bethesda units/mg) overlapped only at Asp1663 and Tyr1664, whereas an antibody with minimal neutralizing activity (0.2 Bethesda units/mg) bound only at Asp1665-Ser1669. Residues comprising antibody binding sites were determined by blocking Factor VIII neutralization and/or binding to insolubilized Factor VIII with overlapping peptides, or with variant peptides in which a single amino acid was deleted or replaced with glycine. Eight additional antibodies to flanking sequences, and with similar affinities for Factor VIII, had little or no neutralizing activity (0-3.0 Bethesda units/mg). These studies suggest that Asp1663 and Tyr1664 may be structural features important to Factor VIII function.
Shima
M
M
Research Institute of Scripps Clinic, Scripps Clinic and Research Foundation, La Jolla, California 92037.
Fulcher
C A
CA
de Graaf Mahoney
S
S
Houghten
R A
RA
Zimmerman
T S
TS
D000595:Amino Acid Sequence; D000906:Antibodies; D000936:Antigen-Antibody Complex; D000939:Epitopes / Q000032:analysis*; D005169:Factor VIII / Q000276:immunology* / Q000302:isolation & purification; D006801:Humans; D007700:Kinetics; D009500:Neutralization Tests; D010455:Peptides / Q000138:chemical synthesis
D000906:Antibodies; D000936:Antigen-Antibody Complex; D000939:Epitopes; D010455:Peptides; D005169:Factor VIII