Selective binding of thyrotropin receptor autoantibodies to recombinant extracellular domain of thyrotropin/lutropin-chorionic gonadotropin receptor chimeric proteins

Gattadahalli Seetharamaiah, Jun Zhuang, Juan Huang, Sai A. Patibandla, Shashi Kaithamana, Kazuo Tahara, Leonard D. Kohn, Bellur S. Prabhakar

Research output: Contribution to journalArticle

3 Citations (Scopus)

Abstract

The extracellular domain of the glycosylated human thyrotropin receptor (ET-gp) contains epitopes that can adsorb pathogenic antibodies from sera of patients with Graves' disease (GD). In an attempt to define the regions within the ETSHR with which autoantibodies interact, we expressed extracellular domains of eight thyrotropin receptor/chorionic gonadotropin receptor (TSHR/LH-CGR) chimeric proteins in insect cells. The levels of expression were high and chimeric proteins were glycosylated. Chimeric proteins designated as EMc2+4 and EMc2+3+4, in which amino acids (aa) 90-165 and 261-370, and aa 90-370, respectively, of TSHR were replaced with corresponding aa of LH-CGR, partially reversed the thyrotropin binding inhibitory immunoglobulin (TBII) activity of experimental anti-TSHR antisera (anti-ET-gp). The other six chimeras almost completely reversed the TBn activity of these anti-ET-GP antisera. Next, we tested the ability of these chimeric proteins to reverse the TBII activity of GD patients' sera. Similar to our earlier study, ET-gp protein reversed the TBII activity of all eight GD patients' sera tested. Chimera EMc2, in which aa 90-165 of TSHR has been replaced with corresponding aa of LH-CGR, and EMc2+4 partially reversed the TBII activity of only three of the eight GD patients' sera. However, the other six chimeric proteins failed to neutralize the TBII activity of any of GD patients' sera. These data showed the following: (1) There is considerable heterogeneity amongst autoantibodies in GD patients' sera, (2) The TBII activity of some, but not others, is dependent on aa 90-165 and 261-370, and (3) Most Graves' sera, with TBII activity, failed to react with chimeric proteins in which either N-terminal or C-terminal regions of the extra cellular domain of the TSHR were replaced with corresponding regions of LH- CGR. These results suggest that the TBII activity of GD patients' sera is dependent on conformational epitopes and replacement of certain regions of TSHR with homologous regions of LH-CGR results in sufficient alteration in the conformation of the protein leading to loss of reactivity.

Original languageEnglish (US)
Pages (from-to)879-886
Number of pages8
JournalThyroid
Volume9
Issue number9
StatePublished - 1999
Externally publishedYes

Fingerprint

LH Receptors
Thyrotropin Receptors
Thyrotropin
Luteinizing Hormone
Graves Disease
Autoantibodies
Serum
Amino Acids
Proteins
Immune Sera
Epitopes
Insect Proteins
Protein Conformation
thyrotropin-binding inhibitory immunoglobulin
Antibodies

ASJC Scopus subject areas

  • Endocrinology

Cite this

Selective binding of thyrotropin receptor autoantibodies to recombinant extracellular domain of thyrotropin/lutropin-chorionic gonadotropin receptor chimeric proteins. / Seetharamaiah, Gattadahalli; Zhuang, Jun; Huang, Juan; Patibandla, Sai A.; Kaithamana, Shashi; Tahara, Kazuo; Kohn, Leonard D.; Prabhakar, Bellur S.

In: Thyroid, Vol. 9, No. 9, 1999, p. 879-886.

Research output: Contribution to journalArticle

Seetharamaiah, G, Zhuang, J, Huang, J, Patibandla, SA, Kaithamana, S, Tahara, K, Kohn, LD & Prabhakar, BS 1999, 'Selective binding of thyrotropin receptor autoantibodies to recombinant extracellular domain of thyrotropin/lutropin-chorionic gonadotropin receptor chimeric proteins', Thyroid, vol. 9, no. 9, pp. 879-886.
Seetharamaiah, Gattadahalli ; Zhuang, Jun ; Huang, Juan ; Patibandla, Sai A. ; Kaithamana, Shashi ; Tahara, Kazuo ; Kohn, Leonard D. ; Prabhakar, Bellur S. / Selective binding of thyrotropin receptor autoantibodies to recombinant extracellular domain of thyrotropin/lutropin-chorionic gonadotropin receptor chimeric proteins. In: Thyroid. 1999 ; Vol. 9, No. 9. pp. 879-886.
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abstract = "The extracellular domain of the glycosylated human thyrotropin receptor (ET-gp) contains epitopes that can adsorb pathogenic antibodies from sera of patients with Graves' disease (GD). In an attempt to define the regions within the ETSHR with which autoantibodies interact, we expressed extracellular domains of eight thyrotropin receptor/chorionic gonadotropin receptor (TSHR/LH-CGR) chimeric proteins in insect cells. The levels of expression were high and chimeric proteins were glycosylated. Chimeric proteins designated as EMc2+4 and EMc2+3+4, in which amino acids (aa) 90-165 and 261-370, and aa 90-370, respectively, of TSHR were replaced with corresponding aa of LH-CGR, partially reversed the thyrotropin binding inhibitory immunoglobulin (TBII) activity of experimental anti-TSHR antisera (anti-ET-gp). The other six chimeras almost completely reversed the TBn activity of these anti-ET-GP antisera. Next, we tested the ability of these chimeric proteins to reverse the TBII activity of GD patients' sera. Similar to our earlier study, ET-gp protein reversed the TBII activity of all eight GD patients' sera tested. Chimera EMc2, in which aa 90-165 of TSHR has been replaced with corresponding aa of LH-CGR, and EMc2+4 partially reversed the TBII activity of only three of the eight GD patients' sera. However, the other six chimeric proteins failed to neutralize the TBII activity of any of GD patients' sera. These data showed the following: (1) There is considerable heterogeneity amongst autoantibodies in GD patients' sera, (2) The TBII activity of some, but not others, is dependent on aa 90-165 and 261-370, and (3) Most Graves' sera, with TBII activity, failed to react with chimeric proteins in which either N-terminal or C-terminal regions of the extra cellular domain of the TSHR were replaced with corresponding regions of LH- CGR. These results suggest that the TBII activity of GD patients' sera is dependent on conformational epitopes and replacement of certain regions of TSHR with homologous regions of LH-CGR results in sufficient alteration in the conformation of the protein leading to loss of reactivity.",
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AU - Seetharamaiah, Gattadahalli

AU - Zhuang, Jun

AU - Huang, Juan

AU - Patibandla, Sai A.

AU - Kaithamana, Shashi

AU - Tahara, Kazuo

AU - Kohn, Leonard D.

AU - Prabhakar, Bellur S.

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N2 - The extracellular domain of the glycosylated human thyrotropin receptor (ET-gp) contains epitopes that can adsorb pathogenic antibodies from sera of patients with Graves' disease (GD). In an attempt to define the regions within the ETSHR with which autoantibodies interact, we expressed extracellular domains of eight thyrotropin receptor/chorionic gonadotropin receptor (TSHR/LH-CGR) chimeric proteins in insect cells. The levels of expression were high and chimeric proteins were glycosylated. Chimeric proteins designated as EMc2+4 and EMc2+3+4, in which amino acids (aa) 90-165 and 261-370, and aa 90-370, respectively, of TSHR were replaced with corresponding aa of LH-CGR, partially reversed the thyrotropin binding inhibitory immunoglobulin (TBII) activity of experimental anti-TSHR antisera (anti-ET-gp). The other six chimeras almost completely reversed the TBn activity of these anti-ET-GP antisera. Next, we tested the ability of these chimeric proteins to reverse the TBII activity of GD patients' sera. Similar to our earlier study, ET-gp protein reversed the TBII activity of all eight GD patients' sera tested. Chimera EMc2, in which aa 90-165 of TSHR has been replaced with corresponding aa of LH-CGR, and EMc2+4 partially reversed the TBII activity of only three of the eight GD patients' sera. However, the other six chimeric proteins failed to neutralize the TBII activity of any of GD patients' sera. These data showed the following: (1) There is considerable heterogeneity amongst autoantibodies in GD patients' sera, (2) The TBII activity of some, but not others, is dependent on aa 90-165 and 261-370, and (3) Most Graves' sera, with TBII activity, failed to react with chimeric proteins in which either N-terminal or C-terminal regions of the extra cellular domain of the TSHR were replaced with corresponding regions of LH- CGR. These results suggest that the TBII activity of GD patients' sera is dependent on conformational epitopes and replacement of certain regions of TSHR with homologous regions of LH-CGR results in sufficient alteration in the conformation of the protein leading to loss of reactivity.

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