Effects of deglycosylation of human thyroperoxidase on its enzymatic activity and immunoreactivity.
Thyroid peroxidase (TPO) is a glycoprotein enzyme which catalyses the iodination of thyroglobulin and the coupling of iodinated tyrosines. Human TPO (hTPO) is the microsomal antigen recognized by the autoantibodies in the serum of patients with autoimmune thyroid disease. An active detergent-solubilized immunoaffinity-purified hTPO was deglycosylated, either by peptide N-glycosidase F (PNGase F) or by endo-beta-N-acetylglucosaminidase H (endo H), and the enzymatic activity and immunoreactivity of the native and deglycosylated forms were compared. Electrophoretic controls and affinoblotting with concanavalin A showed that deglycosylation was not total and that it was more pronounced with endo H than with PNGase F. The enzymatic activity of hTPO was inhibited by endo H deglycosylation, but not by PNGase F deglycosylation; this inhibition was not due to aggregation and/or insolubilization of the molecule subsequent to deglycosylation. Immunoreactivity was monitored by enzyme-linked immunosorbent assay (ELISA) with 13 mouse monoclonal antibodies, rabbit polyclonal antibodies and antibodies from serum of patients with Hashimoto's thyroiditis. In contrast with enzymatic activity, immunoreactivity was not modified or was slightly enhanced (with four monoclonal antibodies) by deglycosylation. The results indicate that strong, if not total, deglycosylation induces a modification of the tertiary structure of hTPO, which affects the enzymatic site but does not modify markedly the epitopes implicated in the recognition of the molecule by the antibodies tested.
Giraud
A
A
INSERM U 38, Faculté de Médecine, Marseille, France.
Franc
J L
JL
Long
Y
Y
Ruf
J
J
D000581:Amidohydrolases / Q000378:metabolism; D000818:Animals; D000911:Antibodies, Monoclonal / Q000276:immunology; D002499:Centrifugation, Density Gradient; D004591:Electrophoresis, Polyacrylamide Gel; D004797:Enzyme-Linked Immunosorbent Assay; D000939:Epitopes; D006031:Glycosylation; D006801:Humans; D007453:Iodide Peroxidase / Q000737:chemistry* / Q000276:immunology; D017038:Mannosyl-Glycoprotein Endo-beta-N-Acetylglucosaminidase / Q000378:metabolism; D051379:Mice; D043524:Peptide-N4-(N-acetyl-beta-glucosaminyl) Asparagine Amidase; D011817:Rabbits; D013967:Thyroiditis, Autoimmune / Q000276:immunology
D000911:Antibodies, Monoclonal; D000939:Epitopes; D007453:Iodide Peroxidase; D017038:Mannosyl-Glycoprotein Endo-beta-N-Acetylglucosaminidase; D000581:Amidohydrolases; D043524:Peptide-N4-(N-acetyl-beta-glucosaminyl) Asparagine Amidase