Carbohydrate Moieties in Recombinant Human Thyroid Peroxidase: Role in Recognition by Antithyroid Peroxidase Antibodies in Hashimoto’s Thyroiditis

Abstract

We studied the oligosaccharide moieties of recombinant human thyroid peroxidase (hTPO) expressed in Chinese hamster ovary (CHO) cells, and the role of these glycans in hTPO antigenicity in Hashimoto's thyroiditis. To determine whether hTPO carbohydrate moieties were N-linked, O-linked, or both, and to obtain information about the characteristics of the carbohydrate component(s), we digested hTPO with deglycosylating enzymes of varying specificity. Proteins in CHO-TPO cells were labeled with [35S]methionine, and hTPO was immunoprecipitated with anti-hTPO antibodies present in Hashimoto's thyroiditis serum. Digestion with endoglycosidase (endo) F, which removes both complex and polymannose N-linked glycans, increased the electrophoretic mobility of the hTPO doublet from approximately 115 kD and 110 kD to 110 kD and 105 kD. Endo H, which acts similarly to endo F, but only on polymannose, and not complex, glycans, had a similar effect. In contrast, O-glycanase and neuraminidase, which remove O-linked glycans and terminal neuraminic acid, respectively, did not alter the mobility of radiolabeled hTPO. Radiolabeled recombinant hTPO was retained by concanavalin A, but not by wheat germ agglutinin, Ricinus communis agglutinin 1, peanut agglutinin and Ulex europaeus lectins. To determine whether or not the glycan moieties in hTPO play a role in the disease-associated epitopes in Hashimoto's thyroiditis, radiolabeled recombinant hTPO was immunoprecipitated after digestion with N-glycanase. Removal of the N-linked carbohydrate chains with endo F and endo H did not prevent antibody binding. In summary, the present data indicate that: i) hTPO expressed in CHO cells contains N-linked, but not O-linked glycan moieties; ii) the N-linked carbohydrate is primarily of the polymannose variety; and, iii) the glycan moieties do not contribute to the hTPO epitopes in Hashimoto's thyroiditis.

Human Thyroperoxidase in Its Alternatively Spliced Form (TPO2) Is Enzymatically Inactive and Exhibits Changes in Intracellular Processing and Trafficking

“…Oligosaccharide processing optionally continues in the Golgi apparatus, leading to the generation of complex-type structures. TPO1 synthetized by CHO cell lines bears only high mannose-type structures (27), but the small part of TPO1 present at the cell surface bears complex-type structures.…”

“…Indeed, as TPO2 bears only high mannose-type structures, the protein is processed through the ER and is not trafficked beyond the cis-Golgi. Incorrectly folded proteins are retained mainly in the ER (27,(32)(33)(34). This would also be true for TPO2 since the altered folding resulting from conformational changes may decrease its half-life in the cell and abolish its intracellular transport.…”

Intracellular Trafficking of Thyroid Peroxidase to the Cell Surface

“…2 and 5B). Nevertheless, in thyrocytes, TPO glycans may not be efficiently modified (43), and the extent of post-translational processing may be different in nonthyroid cell types expressing recombinant TPO (27).…”

Glycopolymers from Saccharomyces Cerevisiae BIM Y-195 with Unusual Immunochemical Properties: Isolation, Structural Identification and Prediction of Their Role in Pathogenesis/Treatment of Autoimmune Thyroid Diseases

“…Taking into account that TPO and Tg are glycoproteins, one could assume that α-1, 6-glucan competes for anti-TPO (anti-Tg) binding with carbohydrate chains of TPO (Tg). The known data suggest that the carbohydrate chains of TPO do not contribute to TPO recognition by anti-TPO (Foti and Rapoport, 1990;Giraud et al, 1992;Kiso et al, 1992;Moura et al, 1991). On the contrary, improved glycosylation of Tg weakens interaction of the thyroid antigen with anti-Tg due to distorting conformation of the polypeptide chain or masking its antigenic regions (Fenouillet, et al, 1986).…”