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

Although sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SD S-PAGE) and Western blotting are widely used to detect serum antibodie s in patients with autoimmune disorders, this procedure unfolds and de natures proteins and may alter antibody-binding sites. We have used a gentle protocol for the preparation and purification of a 64-kilodalto n (kDa) eye muscle (EM) membrane antigen associated with thyroid-assoc iated ophthalmopathy (TAO) for use as antigen in immunoblotting. Pig E M membrane proteins were prepared from crude homogenates by high speed centrifugation and solubilized by hand homogenization. These native m embrane proteins (NM(prot)) were then electrophoresed on an 8.5% polya crylamide gel in the absence of SDS, reducing agents, or urea, and pro teins 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 autoantibodie s in sera from 35% of the patients with TAO and 47% of those with Grav es' hyperthyroidism without evident ophthalmopathy, but in only 4% of normal subjects. To further purify the 64-kDa protein and increase the sensitivity of immunoblotting, NM(prot) were separated by isoelectric focusing (IEF) in the absence of SDS, reducing agent, and urea. The 6 4-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 o f 64 kDa. Sera from groups of patients and normal subjects were tested in immunoblotting against a pig EM 64-kDa protein prepared from NM(pr ot) and purified in IEF. Tests were positive in 67% of patients with T AO, 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 oth er skeletal muscle. The demonstration that a native 64-kDa protein tha t is specifically targeted by autoantibodies in the serum of patients with TAO is expressed in EM, but not other skeletal muscle, greatly en hances its possible significance in the pathogenesis of this eye disor der.