2-DIMENSIONAL GEL-ELECTROPHORESIS IDENTIFIES MINOR DIFFERENCES IN IMMUNOLOGICALLY CROSS-REACTIVE 64 KDA AUTOANTIGENS IN THE THYROID AND EYEMUSCLE

Among the candidate eye muscle autoantigens proposed as being relevant to the pathogenesis of thyroid-associated ophthalmopathy (TAO), a 64 kDa membrane autoantigen appears to be most closely associated with th e eye disorder. We have examined the tissue localization and some of t he physicochemical properties of this molecule in 3 human tissues, nam ely thyroid (THY), eye muscle (EM) and skeletal muscle (SKE), and in p ig eye muscle (PEM), by two-dimensional (2-D) [isoelectric focusing (I EF)/sodium dodecyl polyacrylamide gel electrophoresis (SDS-PAGE)] gel electrophoresis followed by Western blotting. Antibody probes used wer e whole sera from patients with TAO and antibodies affinity purified f rom TAO sera by binding to, and elution from, a sepharose-4B column co njugated with D-1, a 98 amino acid peptide fragment of a recombinant 6 4 kDa thyroid autoantigen. Soluble membrane proteins eluted from a sli ce of SDS-PAGE gel containing 60-70 kDa material was prepared from the four tissues and used as antigen for 2-D gel separation. The presence of a 64 kDa antigen in THY and EM recognized by sera from patients wi th TAO, but only rarely by those from normal individuals, was confirme d. Pretreatment of the eluted 60-70 kDa material with N-Glycosidase F to eliminate charge heterogeneity resulting from glycosylation differe nces, changed-the pl and MW of molecules recognized by TAO sera, in TH Y and EM. This suggests that the 64 kDa molecule(s) in EM and THY targ eted by sera from patients with TAO are glycoproteins and that they ar e different in the two tissues. On the other hand, the molecule recogn ized in SKE appeared to have a different MW, perhaps representing the 66 kDa protein identified in previous immunoprecipitation studies, whi le that recognized in PEM closely resembled the molecule identified in EM and THY, but with a more basic pl range.