Native gel electrophoresis and isoelectric focusing of a 64-kilodalton eye muscle protein shows that it is an important target for serum autoantibodies in patients with thyroid-associated ophthalmopathy and not expressed in other skeletal muscle.

Abstract

Although sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) and Western blotting are widely used to detect serum antibodies in patients with autoimmune disorders, this procedure unfolds and denatures proteins and may alter antibody-binding sites. We have used a gentle protocol for the preparation and purification of a 64-kilodalton (kDa) eye muscle (EM) membrane antigen associated with thyroid-associated ophthalmopathy (TAO) for use as antigen in immunoblotting. Pig EM membrane proteins were prepared from crude homogenates by high speed centrifugation and solubilized by hand homogenization. These native membrane proteins (NMprot) were then electrophoresed on an 8.5% polyacrylamide gel in the absence of SDS, reducing agents, or urea, and proteins from individual bands were eluted, applied to standard SDS-PAGE, and immunoblotted with selected TAO patient sera. A prominent 64-kDa protein, present in most of the bands, was recognized by autoantibodies in sera from 35% of the patients with TAO and 47% of those with Graves' hyperthyroidism without evident ophthalmopathy, but in only 4% of normal subjects. To further purify the 64-kDa protein and increase the sensitivity of immunoblotting, NMprot were separated by isoelectric focusing (IEF) in the absence of SDS, reducing agent, and urea. The 64-kDa protein appeared mainly in IEF fraction 7 and had an isoelectric point of 6.1-6.2. Similar results were found for a human EM protein of 64 kDa. Sera from groups of patients and normal subjects were tested in immunoblotting against a pig EM 64-kDa protein prepared from NMprot and purified in IEF. Tests were positive in 67% of patients with TAO, in 37.5% of those with Graves' hyperthyroidism without eye disease, in 11% of patients with Hashimoto's thyroiditis without eye disease, and in 9% of normal subjects. The 64-kDa protein was not found in other skeletal muscle. The demonstration that a native 64-kDa protein that is specifically targeted by autoantibodies in the serum of patients with TAO is expressed in EM, but not other skeletal muscle, greatly enhances its possible significance in the pathogenesis of this eye disorder.