Differential reactivities of recombinant glycosylated ectodomains of mouse and human thyrotropin receptors with patient autoantibodies.
We expressed the extracellular domain of the mouse TSH receptor (mET-gp) using the baculovirus expression system. The recombinant protein was identified as mET-gp by immunoblotting and N-terminal amino acid sequencing. Carbohydrate analysis of the recombinant protein showed that the protein is glycosylated. Experimental antibodies raised against the extracellular domain of the human TSHr (ETSHr) were assayed for reactivity against mET-gp and glycosylated human ETSHr (ETSHr-gp) in an ELISA and found to be comparable. Similarly, both mET-gp and ETSHr-gp proteins neutralized the TSH binding inhibitory immunoglobulin (TBII) activity of rabbit anti-ETSHr antibodies in a RRA. However, when these proteins were compared for their ability to neutralize TBII and blocking activities (TSBAb) of IgG from patients with thyroid autoimmune disorders, only ETSHr-gp was able to neutralize these activities. In contrast, mET-gp partially neutralized, whereas ETSHr-gp completely neutralized the stimulatory (TSAb) activities of IgG from patients. Analyses of reactivities of these two proteins against a panel of antipeptide and monoclonal antibodies and their protein sequences showed differences in some specific epitopes. These data showed that in spite of significant homology between the two proteins, they exhibit specific epitope differences that are sufficient to cause divergence in their ability to react with patient autoantibodies to TSHr. This suggests that the two proteins might differ in their three-dimensional structure.
Patibandla
S A
SA
Department of Microbiology, The University of Texas Medical Branch, Galveston 77555, USA.
Seetharamaiah
G S
GS
Dallas
J S
JS
Thotakura
N R
NR
Peake
R L
RL
Prabhakar
B S
BS
D000595:Amino Acid Sequence; D000818:Animals; D000937:Antigen-Antibody Reactions; D001323:Autoantibodies / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D016367:Baculoviridae; D002240:Carbohydrate Sequence; D018076:DNA, Complementary / Q000737:chemistry; D004797:Enzyme-Linked Immunosorbent Assay; D005822:Genetic Vectors; D006031:Glycosylation; D006801:Humans; D018828:Immunoglobulins, Thyroid-Stimulating; D051379:Mice; D008969:Molecular Sequence Data; D011817:Rabbits; D011989:Receptors, Thyrotropin / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D018411:Spodoptera
D001323:Autoantibodies; D018076:DNA, Complementary; D018828:Immunoglobulins, Thyroid-Stimulating; D011989:Receptors, Thyrotropin; C094372:thyrotropin-binding inhibitory immunoglobulin