Two dimensional gel electrophoresis identifies minor differences in immunologically cross-reactive 64 KDA autoantigens in the thyroid and eye muscle

Abstract

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 the eye disorder. We have examined the tissue localization and some of the physicochemical properties of this molecule in 3 human tissues, namely thyroid (THY), eye muscle (EM) and skeletal muscle (SKE), and in pig eye muscle (PEM), by two-dimensional (2-D) [isoelectric focusing (IEF)/sodium dodecyl Polyacrylamide gel electrophoresis (SDS-PAGE)] gel electrophoresis followed by Western blotting. Antibody probes used were whole sera from patients with TAO and antibodies affinity purified from TAO sera by binding to, and elution from, a sepharose-4B column conjugated with D₁, a 98 amino acid peptide fragment of a recombinant 64 kDa thyroid autoantigen. Soluble membrane proteins eluted from a slice of SDSPAGE 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 with TAO, but only rarely by those from normal individuals, was confirmed. Pretreatment of the eluted 60–70 kDa material with N-Glycosidase F to eliminate charge heterogeneity resulting from glycosylation differences, changed the pl and MW of molecules recognized by TAO sera, in THY and EM. This suggests that the 64 kDa molecule(s) in EM and THY targeted by sera from patients with TAO are glycoproteins and that they are different in the two tissues. On the other hand, the molecule recognized in SKE appeared to have a different MW, perhaps representing the 66 kDa protein identified in previous immunoprecipitation studies, while that recognized in PEM closely resembled the molecule identified in EM and THY, but with a more basic pl range.