REEVALUATION OF THE PREVALENCES OF SERUM AUTOANTIBODIES REACTIVE WITH64-KD EYE MUSCLE PROTEINS IN PATIENTS WITH THYROID-ASSOCIATED OPHTHALMOPATHY

Serum autoantibodies reactive with eye muscle proteins of ''64 kilodal tons (kd)'' are frequently found in patients with Graves' hyperthyroid ism and thyroid-associated ophthalmopathy (TAO). Earlier, we cloned a 64-kd protein that was identified as calsequestrin, a calcium-binding protein localized in the sarcoplasmic reticulum of striated muscle and extensively studied another cloned 64-kd protein, called 1D, which is expressed in thyroid and eye muscle, and some other tissues. Using a monoclonal antibody against calsequestrin, a polyclonal antibody again st 1D and a TAO patient serum reactive with the ''64-kd protein,'' as probes, we performed Western blots of porcine eye muscle membrane. We identified three different proteins in the 63 to 67 kd molecular weigh t range that were targeted by antibodies in sera from patients with TA O. It was not possible to differentiate antibodies reactive with calse questrin and 1D because these two proteins have very similar molecular weights-63 to 64 kd-and band appearance in Western blotting. A 67-kd protein was most frequently recognized by TAO patients' sera. Serum an tibodies reactive with the 67-kd protein were detected in 73% of patie nts with active TAO of 1 year duration or less, in 37% of patients wit h TAO of more than 3 years' duration, in 35% with Graves' hyperthyroid ism without evident ophthalmopathy, in 30% of patients with Hashimoto' s thyroiditis, and in 16% of normal subjects. Serum antibodies reactiv e with calsequestrin/1D were detected in 47% of patients with active T AO of less than 1 year, in 22% of patients with TAO longer than 3 year s, 17% with Graves' hyperthyroidism without evident ophthalmopathy, in 10% of patients with Hashimoto's thyroiditis, and in 21% of normal su bjects. The prevalence of anti-67-kd protein antibodies in TAO patient s corresponded to those reactive with the so called ''64-kd protein'' that we have reported previously. In conclusion, we were able to impro ve the accuracy of the Western blots by comparing the molecular weight of positive bands using specific antibodies reactive with eye muscle antigens as probes. The previously recognized, and extensively studied , ''64-kd protein'' is now shown to have a molecular weight of 67 kd, The role of the various eye muscle antibodies in the diagnosis and man agement of the ophthalmopathy associated with Graves' hyperthyroidism needs to be addressed in prospective studies using purified or recombi nant full-length proteins.