Characterization of autoantibodies against sulfatide from a V-gene phage-display library derived from patients with systemic lupus erythematosus

Abstract

Sulfatide, ceramide galactosyl-3′-sulfate, is mainly present in nervous tissue, kidney, testis, red blood cells, platelets and granulocyte. Antibodies to sulfatide are present in many patients with demyelinating peripheral neuropathy, HIV infection and systemic lupus erythematosus and may account for some of the clinical manifestations. To evaluate the effect of such antibodies, we have constructed a phage-display antibody fragment library from the lymphocytes of patients with systemic lupus erythematosus. Sulfatide-reactive phage were selected by absorption and elution on sulfatide liposomes and soluble single chain variable fragment (ScFv) were isolated from individual colonies and tested in an ELISA assay for binding to bovine brain sulfatide. Five ScFv clones that bound sulfatide were isolated. Two of the clones, PH5 and PA38, bound sulfatide but not phosphatidylserine, phosphatidylcholine, phosphatidylethanolamine, sphingomyelin or ceramide. These two clones also bound sulfatide from human red blood cells. The DNA encoding the fragments was sequenced, revealing predicted polypeptides of 19 kDa for PH5 containing only variable heavy (VH) sequences, and 31 kDa for PA38, with both VH and variable light (VL) sequences. Although they had similar antigen specificities, the VH domains of the two clones were derived from different heavy-chain families. The clustered mutational patterns in the complementarity-determining region (CDR) of the heavy chains in both clones suggest that the V-domains are the products of antigen-driven B cell clonal maturation leading to the development of sulfatide-binding specificity. These results show the presence of sulfatide-specific antibodies in lupus patients, and allow us to test the possibility that the interaction of the antibodies with sulfatide may contribute to some of the symptoms. In addition, the antibodies provide useful reagents to test the role of sulfatide in pathophysiological processes.