Structure-activity analysis of microsomal antigen/thyroid peroxidase.
The interaction between thyroid microsomal autoantibodies and thyroid microsomal antigen/thyroid peroxidase (TPO) has been studied using both intact antigen preparations and their water-soluble trypsin fragments. In an analysis of sera from 30 patients with Graves' or Hashimoto's diseases, microsomal antibodies showed similar reactivity towards trypsin fragments (with TPO activity) and intact detergent (sodium deoxycholate, DOC)-solubilized human microsomal antigen preparations (r = 0.96). This raised the possibility that both the peroxidase-active site and the major autoantigenic site(s) of microsomal antigen were present on the same trypsin fragments. Studies with porcine TPO showed that only a few sera contained microsomal antibodies which cross-reacted strongly with the porcine preparations. Further analysis was carried out by immunoprecipitation of 125I-labelled microsomal antigen followed by SDS-PAGE and autoradiography. These studies suggest that intact human microsomal antigen (a single-chain protein with Mr = 110,000) contains an intrachain loop of amino acids formed by a disulphide bridge. Trypsin treatment cleaves the antigen close to its transmembrane section and releases a water-soluble fragment (Mr = 100,000), containing the intact disulphide-linked loop of amino acids. Further trypsin action causes cleavage of the peptide bonds within the loop in some preparations. Consequently, three major water-soluble trypsin fragments (Mr = 100,000, 73,000 and 68,000) are formed all of which contain an intact disulphide bridge and have microsomal antibody binding activities. The integrity of the disulphide bridge in intact antigen/TPO preparations and their trypsin fragments is essential for autoantibody binding activity.
Nakajima
Y
Y
Endocrine Immunology Unit, University of Wales College of Medicine, Cardiff, U.K.
Howells
R D
RD
Pegg
C
C
Jones
E D
ED
Smith
B R
BR
D000818:Animals; D001323:Autoantibodies / Q000276:immunology*; D001324:Autoantigens / Q000276:immunology*; D006111:Graves Disease / Q000201:enzymology / Q000276:immunology; D006801:Humans; D007453:Iodide Peroxidase / Q000276:immunology* / Q000302:isolation & purification / Q000378:metabolism; D007700:Kinetics; D008861:Microsomes / Q000276:immunology*; D010446:Peptide Fragments; D013552:Swine; D013961:Thyroid Gland / Q000201:enzymology / Q000276:immunology*; D013967:Thyroiditis, Autoimmune / Q000201:enzymology / Q000276:immunology; D014357:Trypsin
D001323:Autoantibodies; D001324:Autoantigens; D010446:Peptide Fragments; D007453:Iodide Peroxidase; D014357:Trypsin