Enzymatic deglycosylation of porcine thyroid peroxidase: effects on catalytic activity and immunoreactivity.
Thyroid peroxidase is a heme-containing, membrane-bound, glycoprotein enzyme that catalyzes iodination and coupling in the thyroid gland. It is also the antigen for microsomal autoantibodies that are commonly found in the serum of patients with autoimmune thyroid disease. We examined the effect of deglycosylation on the catalytic functions and the immunoreactivity of this enzyme. A highly purified, solubilized, large tryptic fragment of porcine thyroid peroxidase, retaining all of the N-linked glycosylation sites of the native enzyme and displaying full catalytic activity was used. It was deglycosylated by treatment with N-glycanase under nondenaturing conditions. The loss in relative molecular mass after treatment, determined by gel electrophoresis, was about 75% of the estimated molecular weight of the glycan portion of porcine thyroid peroxidase. Lectin blots performed with horseradish peroxidase-conjugated concanavalin A showed a similar loss in relative molecular mass but some residual carbohydrate. The intensity of the carbohydrate stain was consistent with the loss of about 75% of the glycans. Despite this loss, three different assays for catalytic activity of porcine thyroid peroxidase were not significantly decreased. Immunoreactivity measured by immunoblotting and by enzyme-linked immunosorbent assay was also unimpaired. These findings suggest that N-glycanase-sensitive glycans in porcine thyroid peroxidase do not act as antigenic determinants and play a minor role, if any, in catalytic activity and, presumably therefore, in the maintenance of protein conformation.
Moura
E G
EG
Department of Pharmacology, University of Texas Southwestern Medical Center, Dallas.
Pazos-Moura
C C
CC
Yokoyama
N
N
Dorris
M L
ML
Taurog
A
A
D000818:Animals; D004591:Electrophoresis, Polyacrylamide Gel; D004797:Enzyme-Linked Immunosorbent Assay; D006023:Glycoproteins / Q000378:metabolism; D006026:Glycoside Hydrolases / Q000494:pharmacology; D006031:Glycosylation; D015151:Immunoblotting; D007453:Iodide Peroxidase / Q000276:immunology / Q000378:metabolism*; D007455:Iodine / Q000378:metabolism; D009844:Oligosaccharides / Q000378:metabolism; D010084:Oxidation-Reduction; D013552:Swine; D013961:Thyroid Gland / Q000201:enzymology*
D006023:Glycoproteins; D009844:Oligosaccharides; D007455:Iodine; D007453:Iodide Peroxidase; D006026:Glycoside Hydrolases; C020668:glycanase