E. L. E. F.
CARING AND SHARING, Newsletter 4
Anticardiolipin Antibody and ß₂ -Glycoprotein I

Takao Koike, M.D., and Eiji Matsuura, Ph.D.*

Second Department of Internal Medicine, Hokkaido University School of Medicine
Sapporo JAPAN

* Immunology Laboratory, Yamasa Co., Inc.
Chosih JAPAN

The appearance of anticardiolipin antibodies (aCL) is know to be associated with thromboembolic manifestations, intra uterine fetal loss and thrombocytopenia in patients with systemic lupus erythematosus (SLE), and related other autoimmune disorders. The titel, antiphospholipid syndrome, has been used to define the patients characterized be these pathologic features. The aCL react with cardiolipin (CL) in the presence of a 50-kDa serum cofactor. The cofactor, which was identified to be ß₂ -glycoprotein I ( ß₂ -GPI) by sequencing the N-terminal amino acids, not only enhances PL binding by antibodies in SLE but also depresses it by antibodies associated with syphilis. We isolated a complete cDNA clone from a human hepatoma cell line, HepG2, and analyzed the nucleotide and deduced amino acid sequence of human ß₂ -GPI. Further, we developed the baculovirus/Sf9 cell expression system and obtained the recombinant ß₂ -GPI protein. The recombinant ß₂ -GPI expressed in the insect cells was a biologically active aCL cofactor.

Introduction

Autoantibodies against negatively charged phospholipid have been frequently detected in sera from patients with systemic lupus erythematosus (SLE) and other connective tissue disorders. Much evidence has been reported that such antibodies related with thromboembolic manifestations such as cerebral or myocardial infarctions, pulmonary embolism, deep venous thrombosis, intrauterine fetal death due to placental infarction, neurological defects and thrombocytopenia (1-7). The titel, antiphospholipid syndrome, was defined to the patients characterized by these pathological features (8).

Recent studies showed that anticardiolipin cofactor mß₂ -glycoprotein I (ß₂-GPI) or apolipoprotein Hn was required for binding of anticardiolipin antibodies (aCL) raised in the patients with SLE and related other autoimmune disorders (9-11). In this report we describe the biochemical and molecular characteristics of human aCL-cofactor, ß₂-GPI, and the biological significance of this protein in thrombus formation is discussed.

Cofactor-dependency of a CL from SLE

We purified aCL-cofactor by CL-polyacrylamide gel column, DEAE-Cellulose column and affinity chromatography using columns conjugated with monoclonal anti-human IgG.

The cofactor migrated with an apparent molecular mass of 50 kD (Fig.1) (11).

The typical binding patterns of sequential dilutions of two types of aCL (one from SLE patient and the other from a syphilitic patient) to CL were determined by using the purified cofactor in the aCL-enzyme immunoassay (EIA) (Fig.2). Effects of cofactor obtained from bovine serum albumin (BSA) used as a supplement were negligible. The aCL was not detected in normal sera (Panel A). The purified cofactor (151g/ml) was continuously present with the binding of aCL in SLE sera to the solid-phase CL (Panel B), and the binding curve was similar to those obtained by conventional aCL-EIA using fetal bovine serum (FBS) (5). Even with a 1/40-fold dilution, the endogenous cofactor activity was present in the serum sample even in absence of the cofactor. There was virtually no binding of antibody without the additional cofactor once the serum sample had been diluted to more than 1/200-fold, corresponding to the cofactor level of approximately 11g/ml or less. The binding of aCL in the syphilitic sera to the solid phase CL was lowered in the present of exogenous cofactor (Panel C).

We further examined aCL-positive sera from SLE patients and from syphilitics, respectively. We confirmed that the addition of human cofactor enhanced the titer for SLE and depressed it for syphilis (Fig. 3). The findings indicate that the complex of CL and the human cofactor can be recognized only by the antibodies raised in the sera of SLE patients and that aCL could be classified at least into two groups, according to the differences in cofactor dependency (11).

To corroborate the cofactor dependency of aCL binding, we carried out competitive inhibition experiments with an electrophoretically pure preparation of the serum cofactor and CL micelles in our EIA system. Human cofactor enhanced aCL titer from patients with SLE. When cofactor was mixed with CL micelles (1001g/ml) before aCL assay, antibody binding to CL was lost, confirming the requirement of cofactor for accurate measurement of aCL (Fig. 4). To see wether aCL binds to both plasma cofactor and CL, serum from patients with SLE containing a high titer of aCL (diluted 1 in 400 with BSA buffer to avoid the influence of cofactor in the patient or bovine serum) was preincubated with purified cofactor. The mixture was then placed in wells coated with CL in the presence of absence of 101g/ml of human cofactor. There was little inhibitory effect of cofactor on CL binding when cofactor was present in wells. However, in the absence of cofactor in wells, serum CL binding activity was increased in proportion to the increment of cofactor preincubated with serum (Fig. 5).

We confirmed that aCL in SLE serum does not recognize cofactor or CL alone, but recognizes either the complex of CL and cofactor or novel epitope on the cofactor that appear after binding to CL and phospholipids (12).

References

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