Endoproteolytic cleavage of human thyroperoxidase: role of the propeptide in the protein folding process.
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.
Le Fourn
Valérie
V
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.
Ferrand
Mireille
M
Franc
Jean-Louis
JL
D000595:Amino Acid Sequence; D000818:Animals; D000911:Antibodies, Monoclonal / Q000737:chemistry; D001120:Arginine / Q000737:chemistry; D001324:Autoantigens / Q000737:chemistry* / Q000378:metabolism*; D019921:Biotinylation; D020126:Brefeldin A / Q000737:chemistry; D016466:CHO Cells; D006224:Cricetinae; D003545:Cysteine / Q000737:chemistry; D003593:Cytoplasm / Q000378:metabolism; D018076:DNA, Complementary / Q000378:metabolism; D004591:Electrophoresis, Polyacrylamide Gel; D004721:Endoplasmic Reticulum / Q000378:metabolism; D045683:Furin / Q000737:chemistry; D017353:Gene Deletion; D006031:Glycosylation; D006418:Heme / Q000737:chemistry; D006801:Humans; D047468:Immunoprecipitation; D007453:Iodide Peroxidase / Q000737:chemistry* / Q000378:metabolism*; D033862:Iron-Binding Proteins / Q000737:chemistry* / Q000378:metabolism*; D008239:Lysine / Q000737:chemistry; D017038:Mannosyl-Glycoprotein Endo-beta-N-Acetylglucosaminidase / Q000737:chemistry; D008957:Models, Genetic; D018832:Molecular Chaperones / Q000737:chemistry; D008969:Molecular Sequence Data; D008985:Monensin / Q000737:chemistry; D016296:Mutagenesis; D016297:Mutagenesis, Site-Directed; D043524:Peptide-N4-(N-acetyl-beta-glucosaminyl) Asparagine Amidase / Q000737:chemistry; D010455:Peptides / Q000737:chemistry; D017510:Protein Folding; D011499:Protein Processing, Post-Translational; D017434:Protein Structure, Tertiary; D017386:Sequence Homology, Amino Acid; D013961:Thyroid Gland / Q000378:metabolism; D013997:Time Factors; D014162:Transfection
D000911:Antibodies, Monoclonal; D001324:Autoantigens; D018076:DNA, Complementary; D033862:Iron-Binding Proteins; D018832:Molecular Chaperones; D010455:Peptides; D020126:Brefeldin A; D006418:Heme; D008985:Monensin; D001120:Arginine; C056658:TPO protein, human; D007453:Iodide Peroxidase; D017038:Mannosyl-Glycoprotein Endo-beta-N-Acetylglucosaminidase; D045683:Furin; D043524:Peptide-N4-(N-acetyl-beta-glucosaminyl) Asparagine Amidase; D008239:Lysine; D003545:Cysteine