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J. Biol. Chem., Vol. 280, Issue 6, 4568-4577, February 11, 2005

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Endoproteolytic Cleavage of Human Thyroperoxidase

ROLE OF THE PROPEPTIDE IN THE PROTEIN FOLDING PROCESS^*

Valérie Le Fourn, Mireille Ferrand, and Jean-Louis Franc

From the Laboratoire de Recherche Interactions Cellulaires Neuroendocriniennes, Unité Mixte de Recherche 6544, CNRS-Université de la Méditerranée, Faculté de Médecine, Institut Fédératif de Recherche Jean-Roche, 13916 Marseille, France and U555 INSERM, Faculté de Médecine, 13385 Marseille, France

Human thyroperoxidase (hTPO), the key enzyme involved in thyroid hormone synthesis, is synthesized in the form of a 933-amino acid polypeptide that subsequently undergoes posttranslational modifications such as N- and O-glycosylation and heme fixation. In the present study, it was established that the N-terminal part of hTPO is cleaved during the maturation of the enzyme. In the first set of experiments performed in this study, Chines hamster ovary (CHO) cells transfected with hTPO cDNA generated four different species after deglycosylation, namely a 98-kDa species, which corresponds to the full-length deglycosylated hTPO, and two 94-kDa and one 92-kDa species, which were truncated in the N-terminal parts. The three latter forms were detected only at the cell surface. A proprotein convertase inhibitor prevented these cleavages, and experiments using monensin and brefeldin A showed that they occurred in a post-endoplasmic reticulum compartment. Site-directed mutagenesis studies were performed in which Arg⁶⁵ was identified as one of the cleavage sites. In the second part of the study, hTPO from human thyroid glands was purified using a monoclonal antibody recognizing the folded form of hTPO. Amino acid determination showed that the N-terminal part of this protein begins at Thr¹⁰⁹. This cleavage process differs from that observed in CHO cells. The fact that this hTPO was endoglucosaminidase H-sensitive indicated that the cleavage of the propeptide occurs in the endoplasmic reticulum. To analyze the role of the hTPO prosequence, cDNAs with and without prosequence (Cys¹⁵–Lys¹⁰⁸) were transfected into CHO cells. hTPO propeptide deletion drastically decreased the proportion of the folded hTPO form, and under these conditions the cell surface activity disappeared completely. These results strongly suggest that the prosequence plays a crucial role as an intramolecular chaperone, facilitating the folding of hTPO.

Received for publication, July 16, 2004 , and in revised form, November 29, 2004.

^* This work was supported in part by CNRS, Université de la Méditerranée (UMR6544), INSERM (U555), and the Ligue Nationale contre le Cancer (to J. -L. F.). The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

Supported during this work by the Association pour le Développement des Recherches Médicales and by the Fondation pour la Recherche Médicale

To whom correspondence should be addressed: ICNE, UMR 6544 CNRS-Université de la Méditerranée, Faculté de Médecine, Bd P. Dramard,13916 Marseille Cedex 20, France. Tel.: 33-4-91-69-8715; Fax: 33-4-91-69-8920; E-mail: franc.jl{at}jean-roche.univ-mrs.fr.

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