Human beta2-glycoprotein I as an anticardiolipin cofactor determined using mutants expressed by a baculovirus system.
beta2-Glycoprotein I (beta2-GPI) consists of five repeats of a homologous domain. We designed a series of human beta2-GPI mutant genes, ie, three mutant genes lacking the domain(s) present in the NH2-terminal region and two of those present in the COOH-terminal region. These mutant genes were expressed in Spodoptera frugiperda insect cells (Sf9) infected with recombinant baculoviruses and the mutant proteins were secreted into the culture medium. The molecular mass of the purified mutant proteins, estimated by sodium dodecyl sulfate-polyacrylamide gel electrophoresis, was fairly consistent with the size calculated from their nucleotide sequences. Binding of beta2-GPI to solid-phase cardiolipin (CL) was diminished by the deletion of the fifth domain (domain V) from its complete structure. Thus, the phospholipid binding site of beta2-GPI is located on its domain V. Monoclonal anti-CL antibodies (aCL) derived either from NZW x BXSB (WB) F1 mice or from patients with antiphospholipid syndrome bound directly to the domain V-deleted mutant protein (DI-IV) absorbed not only on an oxygenated but also on a plain polystyrene surface. We conclude from this study that the epitope for aCL is exposed on a conformationally changed structure of beta2-GPI by interacting with negatively charged phospholipid or on the mutant protein, DI-IV.
Igarashi
M
M
Microbiology Laboratory, Yamasa Corp, Choshi, Japan.
Matsuura
E
E
Igarashi
Y
Y
Nagae
H
H
Ichikawa
K
K
Triplett
D A
DA
Koike
T
T
D000595:Amino Acid Sequence; D000818:Animals; D017153:Antibodies, Anticardiolipin / Q000378:metabolism*; D000911:Antibodies, Monoclonal / Q000276:immunology; D000937:Antigen-Antibody Reactions; D001483:Base Sequence; D001667:Binding, Competitive; D002308:Cardiolipins / Q000378:metabolism*; D002460:Cell Line; D005822:Genetic Vectors / Q000235:genetics; D006023:Glycoproteins / Q000235:genetics / Q000302:isolation & purification / Q000378:metabolism*; D006801:Humans; D051379:Mice; D008969:Molecular Sequence Data; D016296:Mutagenesis; D017924:Nucleopolyhedroviruses / Q000235:genetics; D010100:Oxygen; D011137:Polystyrenes; D011485:Protein Binding; D012091:Repetitive Sequences, Nucleic Acid; D016415:Sequence Alignment; D017384:Sequence Deletion; D018411:Spodoptera; D053482:beta 2-Glycoprotein I
D017153:Antibodies, Anticardiolipin; D000911:Antibodies, Monoclonal; D002308:Cardiolipins; D006023:Glycoproteins; D011137:Polystyrenes; D053482:beta 2-Glycoprotein I; D010100:Oxygen