Two classes of germline genes both derived from the V(H)1 family direct the formation of human antibodies that recognize distinct antigenic sites in the C2 domain of factor VIII.
Most plasmas from patients with inhibitors contain antibodies that are reactive with the C2 domain of factor VIII. Previously, we have shown that the variable heavy chain (V(H)) regions of antibodies to the C2 domain are encoded by the closely related germline gene segments DP-10, DP-14, and DP-88, which all belong to the V(H)1 gene family. Here, we report on the isolation and characterization of additional anti-C2 antibodies that are derived from V(H) gene segments DP-88 and DP-5. Competition experiments using murine monoclonal antibodies CLB-CAg 117 and ESH4 demonstrated that antibodies derived from DP-5 and DP-88 bound to different sites within the C2 domain. Epitope mapping studies using a series of factor VIII/factor V hybrids revealed that residues 2223 to 2332 of factor VIII are required for binding of the DP-10-, DP-14-, and DP-88-encoded antibodies. In contrast, binding of the DP-5-encoded antibodies required residues in both the amino- and carboxy-terminus of the C2 domain. Inspection of the reactivity of the antibodies with a series of human/porcine hybrids yielded similar data. Binding of antibodies derived from germline gene segments DP-10, DP-14, and DP-88 was unaffected by replacement of residues 2181 to 2243 of human factor VIII for the porcine sequence, whereas binding of the DP-5-encoded antibodies was abrogated by this replacement. Together these data indicate that antibodies assembled from V(H) gene segments DP-5 and the closely related germline gene segments DP-10, DP-14, and DP-88 target 2 distinct antigenic sites in the C2 domain of factor VIII.
van den Brink
Edward N
EN
Department of Plasma Proteins, CLB, Amsterdam, the Netherlands.
Bril
Wendy S
WS
Turenhout
Ellen A M
EA
Zuurveld
Marleen
M
Bovenschen
Niels
N
Peters
Marjolein
M
Yee
Thynn Thynn
TT
Mertens
Koen
K
Lewis
Deborah A
DA
Ortel
Thomas L
TL
Lollar
Pete
P
Scandella
Dorothea
D
Voorberg
Jan
J
D000595:Amino Acid Sequence; D000818:Animals; D000918:Antibody Specificity; D018604:Epitope Mapping; D000939:Epitopes / Q000737:chemistry / Q000378:metabolism; D019143:Evolution, Molecular; D005169:Factor VIII / Q000737:chemistry / Q000276:immunology*; D005803:Genes, Immunoglobulin / Q000276:immunology*; D006467:Hemophilia A / Q000276:immunology; D006801:Humans; D007143:Immunoglobulin Heavy Chains / Q000235:genetics*; D007135:Immunoglobulin Variable Region / Q000235:genetics*; D007518:Isoantibodies / Q000096:biosynthesis / Q000276:immunology* / Q000302:isolation & purification; D008969:Molecular Sequence Data; D005810:Multigene Family; D017434:Protein Structure, Tertiary
D000939:Epitopes; D007143:Immunoglobulin Heavy Chains; D007135:Immunoglobulin Variable Region; D007518:Isoantibodies; D005169:Factor VIII