A 63 kDa skeletal muscle protein associated with eye muscle inflammation in Graves' disease is identified as the calcium binding protein calsequestrin

It is generally accepted that thyroid-associated ophthalmopathy (TAO) is an autoimmune disease of the eye muscle (EM) and the surrounding orbital conn ective tissue in which circulating antibodies play an important role. Antib odies against EM membrane proteins of 63-67 kDa mel. wt. seem to be the bes t markers of ophthalmopathy in patients with autoimmune thyroid disease. We purified a 63 kDa EM protein using SDS-polyacrylamide gel electrophoresis technology and TAO patients' sera as probes, digested the protein with cyan ogen bromide and sequenced immunoreactive peptides, We also screened a huma n EM library with a rabbit antiserum against 63-65 kDa proteins and affinit y purified antibodies from a TAO patient's serum that reacted with a 55 kDa EM membrane protein. From partial sequence information and from DNA sequen cing of positive cDNA clones, the protein was identified as calsequestrin, a 63 kDa calcium binding protein localized in the sarcoplasmic reticulum of the muscle fiber. As determined by Northern blotting, calsequestrin was ex pressed in EM and other skeletal muscle but not thyroid or fibroblasts, Cal sequestrin is different from the "64 kDa protein", which has been identifie d as succinate dehydrogenase flavoprotein subunit, which has a corrected me l. wt. of 67 kDa, Serum antibodies against calsequestrin were found in 40% of patients,vith clinically active TAO, but in only 4% of those with stable eye disease, and in 5% of normal subjects, by immunoblotting. Although it is possible that autoimmunity against calsequestrin plays a role in the pro gressive EM damage that characterizes ophthalmopathy it is more likely that the antibodies are secondary to a reaction against some other cell membran e protein, such as the novel thyroid and eye muscle shared protein G2s or t he TSH receptor.