Calnexin and Calreticulin Binding to Human Thyroperoxidase Is Required for Its First Folding Step(s) But Is Not Sufficient to Promote Efficient Cell Surface Expression

Abstract

Human thyroperoxidase (hTPO) is a type I transmembrane-bound heme-containing glycoprotein that catalyzes the synthesis of thyroid hormones. In a previous study we stably expressed hTPO in Chinese hamster ovary cells and observed that after the synthesis, only 20% of the hTPO molecules were recognized by a monoclonal antibody (mAb 15) directed against a conformational structure, and that only 2% were able to reach the cell surface. In the present study it was proposed to determine how calnexin (CNX) and calreticulin (CRT) contribute to the folding of hTPO. Sequential immunoprecipitation was performed using anti-CNX or anti-CRT followed by anti-hTPO antibodies, and the results showed that CNX and CRT were associated with hTPO. Inhibiting the interactions between CNX or CRT and hTPO using castanospermine greatly reduced the first step(s) in the hTPO folding process. Under these conditions, the half-life of this enzyme was greatly reduced (2.5 vs. 17 h in the control experiments), and hTPO was degraded via the proteasome pathway. This reduced the rate of hTPO transport to the cell surface. Overexpression of CNX or CRT into the hTPO-CHO cells was found to enhance the first hTPO folding step(s) by 20-60%, but did not increase the level of hTPO present at the cell surface. All in all, these findings provide evidence that CNX and CRT are crucial to the first step(s) in hTPO folding, but that interactions with other molecular chaperones are required for the last folding steps to take place.

Calnexin Overexpression Increases Manganese Peroxidase Production in Aspergillus niger

“…This suggests a function for calnexin at the stage of incorporation of the prosthetic group into this hemoprotein. Furthermore, Fayadat and coworkers showed that calnexin overexpression increased the initial folding steps of the human thyroperoxidase (15) and that heme insertion was required for secretion of this hemoprotein (14).…”

Discovery and Validation of Protein Abundance Differences between Follicular Thyroid Neoplasms

“…These proteins are involved in the maturation of thyroglobulin, possibly as a part of a macromolecular process, and assist with glycosylation and folding of thyroglobulin monomers (23,24). Calreticulin, another ER protein identified in our study, plays a key role in the synthesis of glycoproteins, including thyroperoxidase (25,26). Moreover, in addition to their role in protein folding, calreticulin and HSP gp96 may trigger an anticancer immune response (27) and improve the efficiency of phagocytosis (28).…”

The ER Glycoprotein Quality Control System

“…Thyroid peroxidase deficiency (de Carvalho et al, 1994; Thyroid peroxidase + CNX/CRT Kim and Arvan, 1995;Fayadat et al, 2000) Thyroxin-binding globulin deficiency ¶ Thyroxin-binding globulin + (Miura et al, 1994;Refetoff et al, 1996) Osteogenesis imperfecta (Lamande and Bateman, 1999) Type I procollagen + Hereditary hypofibrinogenemia (Roy et al, 1996) Fibrinogen + CNX α1-Antichymotrypsin (ACT) deficiency (Callea et al, 1992) α1-Antichymotrypsin + CNX Neurophyseal diabetes insipidus (Morello et al, 2001) Vasopressin precursor protein + CNX, Prolonged Assoc. Nephrogenic diabetes insipidus (Tamarappoo et al, 1999) Aquaporin II -Charcot-Marie-Tooth disease (Thomas, 1999;Mendell, 1998) Peripheral myelin protein 22 + CNX Pelizaeus-Merzbacher disease (Yool et al, 2000;Swanton et al, 2003) Proteolipoprotein -CNX, Prolonged Assoc.…”