REQUIREMENT OF GLYCOSYLATION OF THE HUMAN THYROTROPIN RECEPTOR ECTODOMAIN FOR ITS REACTIVITY WITH AUTOANTIBODIES IN PATIENTS SERA

To understand the role of glycosylation on autoantibody reactivity, we expressed cDNA encoding amino acid residues 22 to 416 of the human th yrotropin receptor (TSHR), along with the baculovirus-encoded glycopro tein 67 signal sequence (ETSHR-gp) in insect cells. N-terminal sequenc e analysis revealed that the signal peptide was cleaved and confirmed the identity of ETSHR-gp protein. The molecular mass of the ETSHR-gp p rotein was 63 kDa and was higher than the expected molecular mass of 4 5 kDa, suggesting that the protein was glycosylated. Carbohydrate anal ysis showed that the protein was glycosylated and that mannose was the major oligosaccharide. A nonglycosylated recombinant ETSHR protein ex pressed earlier in our laboratory neutralized TSH-binding-inhibitory I g (TBII) activity in the sera of rabbits immunized with the protein bu t did not neutralize TBII activity in the sera of patients. In contras t, the glycosylated ETSHR-gp protein neutralized TBII activity in the sera of both experimental animals and patients with autoimmune thyroid disorders. Furthermore, only the ETSHR-gp protein completely neutrali zed the activities of stimulatory and blocking Abs in the sera of pati ents with hyperthyroidism and hypothyroidism, respectively. These data clearly show that glycosylated ETSHR-gp, but not the nonglycosylated ETSHR protein, can react with autoantibodies in patients' sera and tha t it has the epitopes required for the binding of TBII, thyroid stimul atory Abs, and thyroid stimulatory blocking Abs. Moreover, these data suggest that glycosylation might be an important determinant of autoan tigenicity of human TSHR.