Identification of a region of beta 2-glycoprotein I critical for lipid binding and anti-cardiolipin antibody cofactor activity.
beta 2-Glycoprotein I (beta 2-GPI), a phospholipid-binding plasma protein, is an absolute requirement (cofactor) for the binding of autoimmune-type anti-cardiolipin (aCL) antibodies to cardiolipin (CL). The nature of this cofactor activity and the specific regions of the molecule involved have not yet been determined. We have identified a preparation of beta 2-GPI that lacks aCL antibody cofactor activity. Analysis of the structural differences between the active and inactive forms enabled identification of the region of beta 2-GPI critically important for aCL cofactor activity. The active form of beta 2-GPI bound CL and displayed cofactor activity down to 1 microgram/ml. The inactive form failed to bind CL and possessed no cofactor activity even at concentrations up to 94 micrograms/ml, indicating that the ability of beta 2-GPI to bind lipids is an absolute requirement for aCL cofactor activity. Both forms possessed identical N-terminal sequences and were recognized as essentially immunoreactively identical by polyclonal antisera to beta 2-GPI. However, the inactive form has undergone proteolytic cleavage and exists primarily as a "clipped" molecule, the polypeptide chain being cleaved between Lys-317 and Thr-318 (a potential thrombin cleavage site), with the two cleaved segments linked as a disulfide-bonded complex. This indicates that the C-terminal region is critically important for beta 2-GPI to bind lipid and for aCL cofactor activity. The clipped form of beta 2-GPI would not be suitable for use as aCL cofactor and its use may have led some investigators to conclude incorrectly that beta 2-GPI does not interact with aCL antibodies.
Hunt
J E
JE
Department of Immunology, Allergy and Infectious Disease, University of New South Wales School of Medicine, St. George Hospital, Sydney, Australia.
Simpson
R J
RJ
Krilis
S A
SA
D000595:Amino Acid Sequence; D001053:Apolipoproteins / Q000097:blood*; D002308:Cardiolipins / Q000378:metabolism*; D004591:Electrophoresis, Polyacrylamide Gel; D004797:Enzyme-Linked Immunosorbent Assay; D006023:Glycoproteins / Q000097:blood* / Q000276:immunology / Q000302:isolation & purification; D006801:Humans; D007700:Kinetics; D008969:Molecular Sequence Data; D008970:Molecular Weight; D010713:Phosphatidylcholines / Q000378:metabolism*; D011863:Radioimmunoassay; D053482:beta 2-Glycoprotein I
D001053:Apolipoproteins; D002308:Cardiolipins; D006023:Glycoproteins; D010713:Phosphatidylcholines; D053482:beta 2-Glycoprotein I