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 thyrotropin receptor (TSHR), along with the baculovirus-encoded glycoprotein 67 signal sequence (ETSHR-gp) in insect cells. N-terminal sequence analysis revealed that the signal peptide was cleaved and confirmed the identity of ETSHR-gp protein. The molecular mass of the ETSHR-gp protein was 63 kDa and was higher than the expected molecular mass of 45 kDa, suggesting that the protein was glycosylated. Carbohydrate analysis showed that the protein was glycosylated and that mannose was the major oligosaccharide. A nonglycosylated recombinant ETSHR protein expressed earlier in our laboratory neutralized TSH-binding-inhibitory Ig (TBII) activity in the sera of rabbits immunized with the protein but did not neutralize TBII activity in the sera of patients. In contrast, 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 neutralized the activities of stimulatory and blocking Abs in the sera of patients 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 that it has the epitopes required for the binding of TBII, thyroid stimulatory Abs, and thyroid stimulatory blocking Abs. Moreover, these data suggest that glycosylation might be an important determinant of autoantigenicity of human TSHR.
Seetharamaiah
G S
GS
Department of Microbiology and Immunology, University of Texas Medical Branch, Galveston 77555, USA.
Dallas
J S
JS
Patibandla
S A
SA
Thotakura
N R
NR
Prabhakar
B S
BS
D000818:Animals; D019138:Antibodies, Blocking / Q000378:metabolism; D000937:Antigen-Antibody Reactions; D001323:Autoantibodies / Q000097:blood* / Q000378:metabolism; D016367:Baculoviridae / Q000276:immunology; D016466:CHO Cells; D006224:Cricetinae; D006031:Glycosylation; D006801:Humans; D017434:Protein Structure, Tertiary; D011989:Receptors, Thyrotropin / Q000096:biosynthesis / Q000737:chemistry / Q000276:immunology* / Q000378:metabolism*; D014760:Viral Fusion Proteins / Q000276:immunology
D019138:Antibodies, Blocking; D001323:Autoantibodies; D011989:Receptors, Thyrotropin; D014760:Viral Fusion Proteins