Role of Heme in Intracellular Trafficking of Thyroperoxidase and Involvement of H2O2 Generated at the Apical Surface of Thyroid Cells in Autocatalytic Covalent Heme Binding

Abstract

Thyroperoxidase (TPO) is a glycosylated hemoprotein that plays a key role in thyroid hormone synthesis. We previously showed that in CHO cells expressing human TPO (hTPO) only 2% of synthesized hTPO reaches the cell surface. Herein, we investigated the role of heme moiety insertion in the exit of hTPO from the endoplasmic reticulum. Peroxidase activity at the cell surface and cell surface expression of hTPO were decreased by ∼30 and ∼80%, respectively, with succinyl acetone, an inhibitor of heme biosynthesis, and were increased by 20% with holotransferrin and aminolevulinic acid, precursors of heme biosynthesis. Results were similar with holotransferrin plus aminolevulinic acid or hemin, but hemin increased cell surface activity more efficiently (+120%) relative to the control. It had been suggested (DePillis, G., Ozaki, S., Kuo, J. M., Maltby, D. A., and Ortiz de Montellano, P. R. (1997)J. Biol. Chem. 272, 8857–8960) that covalent attachment of heme to mammalian peroxidases could be an H2O2-dependent autocatalytic processing. In our study, heme associated intracellularly with hTPO, and we hypothesized that there was insufficient exposure to H2O2 in Chinese hamster ovary cells before hTPO reached the cell surface. After a 10-min incubation, 10 μm H2O2 led to a 65% increase in cell surface activity. In contrast, in thyroid cells, H2O2 was synthesized at the apical cell surface and allowed covalent attachment of heme. Two-day incubation of primocultures of thyroid cells with catalase led to a 30% decrease in TPO activity at the cell surface. In conclusion, we provide compelling evidence for an essential role of 1) heme incorporation in the intracellular trafficking of hTPO and of 2) H2O2 generated at the apical pole of thyroid cells in the autocatalytic covalent heme binding to the TPO molecule. Thyroperoxidase (TPO) is a glycosylated hemoprotein that plays a key role in thyroid hormone synthesis. We previously showed that in CHO cells expressing human TPO (hTPO) only 2% of synthesized hTPO reaches the cell surface. Herein, we investigated the role of heme moiety insertion in the exit of hTPO from the endoplasmic reticulum. Peroxidase activity at the cell surface and cell surface expression of hTPO were decreased by ∼30 and ∼80%, respectively, with succinyl acetone, an inhibitor of heme biosynthesis, and were increased by 20% with holotransferrin and aminolevulinic acid, precursors of heme biosynthesis. Results were similar with holotransferrin plus aminolevulinic acid or hemin, but hemin increased cell surface activity more efficiently (+120%) relative to the control. It had been suggested (DePillis, G., Ozaki, S., Kuo, J. M., Maltby, D. A., and Ortiz de Montellano, P. R. (1997)J. Biol. Chem. 272, 8857–8960) that covalent attachment of heme to mammalian peroxidases could be an H2O2-dependent autocatalytic processing. In our study, heme associated intracellularly with hTPO, and we hypothesized that there was insufficient exposure to H2O2 in Chinese hamster ovary cells before hTPO reached the cell surface. After a 10-min incubation, 10 μm H2O2 led to a 65% increase in cell surface activity. In contrast, in thyroid cells, H2O2 was synthesized at the apical cell surface and allowed covalent attachment of heme. Two-day incubation of primocultures of thyroid cells with catalase led to a 30% decrease in TPO activity at the cell surface. In conclusion, we provide compelling evidence for an essential role of 1) heme incorporation in the intracellular trafficking of hTPO and of 2) H2O2 generated at the apical pole of thyroid cells in the autocatalytic covalent heme binding to the TPO molecule. thyroperoxidase human TPO Chinese hamster ovary monoclonal antibody endoplasmic reticulum lactoperoxidase myeloperoxidase succinyl acetone aminolevulinic acid fetal bovine serum sulfosuccinimidyl-2- (biotinamide) ethyl-1,3′-dithiopropionate phosphate-buffered saline holotransferrin polyacrylamide gel electrophoresis Thyroperoxidase (TPO)1is a membrane-bound, glycosylated hemoprotein that plays a key role in the biosynthesis of thyroid hormones. It catalyzes the iodination of thyroglobulin and the coupling of some of the iodotyrosyl residues to produce thyroxine and 3,3′,5-triiodothyronine (1Nunez J. Pommier J. Vitam. Horm. 1982; 39: 175-229Crossref PubMed Scopus (108) Google Scholar, 2Rawitch A.B. Taurog A. Chernoff S.B. Dorris M.L. Arch. Biochem. Biophys. 1979; 194: 244-257Crossref PubMed Scopus (79) Google Scholar, 3Taurog A. Braverman L.E. Utiger R.D. The Thyroid. J. B. Lippincott Co., Philadelphia1991: 51-97Google Scholar). Kimura et al. (4Kimura S. Ikeda-Saito Proteins Struct. Funct. Genet. 1988; 3: 113-120Crossref PubMed Scopus (139) Google Scholar) proposed that His407 or His414 is the proximal heme binding site. On the other hand, His586may be a critical residue for the enzymatic activity of the protein by being the distal heme binding site (5Banga J.P. Mahadevan D. Barton G.J. Sutton B.J. Saldanha J.W. Odell E. McGregor A.M. FEBS Lett. 1990; 266: 133-141Crossref PubMed Scopus (37) Google Scholar). Recently, Taurog and Wall (6Taurog A. Wall M. Thyroid. 1998; 8: 185-191Crossref PubMed Scopus (17) Google Scholar) proposed His239 as the proximal binding site and His494 as the distal binding site. During the past 40 years, the nature of the heme prosthetic group of TPO has been debated. Hossaya and Morrison (7Hossaya T. Morrison M. J. Biol. Chem. 1967; 242: 2828-2836Google Scholar), Krinsky and Alexander (8Krinsky M.M. Alexander N.M. J. Biol. Chem. 1971; 246: 4755-4758Abstract Full Text PDF PubMed Google Scholar), and Ohtaki et al. (9Ohtaki S. Nakagawa H. Nakamura S. Nakamura M. Yamazaki I. J. Biol. Chem. 1985; 260: 441-448Abstract Full Text PDF PubMed Google Scholar) reported that the prosthetic group of TPO might be a ferriprotoporphyrin IX. Rawitch et al. (2Rawitch A.B. Taurog A. Chernoff S.B. Dorris M.L. Arch. Biochem. Biophys. 1979; 194: 244-257Crossref PubMed Scopus (79) Google Scholar) showed significant differences between the pyridine hemochromogene of TPO and horseradish peroxidase, suggesting that the heme in TPO is not ferriprotoporphyrin IX. TPO, lactoperoxidase (LPO), myeloperoxidase (MPO), saliva peroxidase, eosinoperoxidase, and intestinal peroxidase belong to the mammalian peroxidase family. These proteins share related protein primary structure (10Cals M.M. Mailliart P. Brignon G. Anglade P. Dumas B.R. Eur. J. Biochem. 1991; 198: 733-739Crossref PubMed Scopus (114) Google Scholar, 11Fenna R. Zeng J. Arch. Biochem. Biophys. 1995; 316: 653-656Crossref PubMed Scopus (129) Google Scholar) and are alike in spectral properties. Furthermore, their prosthetic heme moieties are not readily extracted by conventional approaches. Anderson et al. (12Andersson L.A. Bylkas S. Wilson A.E. J. Biol. Chem. 1996; 271: 3406-3412Abstract Full Text Full Text PDF PubMed Scopus (40) Google Scholar) considered that spectral similarities between LPO and other mammalian peroxidases indicated similar prosthetic heme moieties and a covalent attachment of the heme group to the protein. They suggested that the heme moiety of TPO is probably a type l heme, like LPO. This heme is covalently linked to the protein through ester bonds that link aspartate and glutamate residues (conserved in MPO, LPO, and TPO) and the hydroxymethyl group of heme. Heme is inserted into the protein certainly in the endoplasmic reticulum, as for MPO (13Arnljots K. Olsson I. J. Biol. Chem. 1987; 262: 10430-10433Abstract Full Text PDF PubMed Google Scholar). This insertion seems necessary for the proteolytic processing leading to the formation of an enzymatically active mature MPO and for the exit of MPO from the ER and its subsequent targeting to the lysosomes (14Nauseef W. McCormick S. Yi H. Blood. 1992; 80: 2622-2633Crossref PubMed Google Scholar). We had proposed a model for the folding, degradation, and intracellular trafficking of human TPO (hTPO) in CHO cells (15Fayadat L. Niccoli-Sire P. Lanet J. Franc J.L. Endocrinology. 1998; 139: 4277-4285Crossref PubMed Scopus (44) Google Scholar). We showed that after synthesis only 2% of the hTPO molecules exited from the ER and reached the cell surface. The remaining 98% are more or less rapidly degraded depending on their folding state. In the present work, we looked into the role of the heme moiety insertion in the exit of hTPO from the ER. We also investigated a possible H2O2-dependent autocatalytic modification of heme, responsible for its subsequent covalent attachment to TPO, as described by the group of Montellano (16DePillis G. Ozaki S. Kuo J.M. Maltby D.A. Ortiz de Montellano P.R. J. Biol. Chem. 1997; 272: 8857-8860Abstract Full Text Full Text PDF PubMed Scopus (91) Google Scholar) for LPO. Effectively in thyroid cells, H2O2 is synthesized at the apical cell surface level and then could be implicated in this covalent linking of the heme prosthetic group to TPO. Sigma supplied the following: succinyl acetone (SA), hemin, aminolevulinic acid (ALA), holotransferrin (FeTf), catalase, and BT2cAMP. Penicillin and streptomycin were from Life Technologies, Inc. Fetal bovine serum (FBS) and protease inhibitor mixture tablets (Complete) were from Boehringer Mannheim (Le Meylan, France). Na125I was obtained from Amersham Pharmacia Biotech (Les Ullis, France). Expre35S35S protein labeling mix (referred to as “35S-(Met + Cys)”) and14C-aminolevulinic acid were from NEN Life Science (Paris, France). Streptavidin-agarose and sulfosuccinimidyl-2-(biotinamide) ethyl-1,3′-dithiopropionate (NHS-SS-biotin) were from Pierce. Protein A-Sepharose 4B was from Zymed Laboratories Inc. (San Francisco, CA). Monoclonal antibodies (mAbs) raised against hTPO were given by J. Ruf. Stock solutions of hemin (2 mm) in phosphate-buffered saline (PBS) were prepared immediately before experiments and dissolved by the addition of 0.15m NaOH to obtain a pH range between 7.0 and 7.5. Succinyl acetone (25 mm in PBS), holotransferrin (1 mg/ml in PBS), and catalase (1% in PBS) solutions were freshly prepared for each experiment and filter-sterilized before the addition in culture medium. As described (15Fayadat L. Niccoli-Sire P. Lanet J. Franc J.L. Endocrinology. 1998; 139: 4277-4285Crossref PubMed Scopus (44) Google Scholar), full-length 3060-kilobase pair human TPO cDNA (kindly provided by B. Rapoport) was cloned into the eukaryotic transfer vector pcDNA3, and after stable transfection a CHO cell line (CHO-hTPO cells) expressing hTPO was obtained. The cells were cultured in Ham's F-12 medium supplemented with 10% FBS, penicillin (100 IU/ml), and streptomycin (100 μg/ml), and expression of hTPO was stimulated 48 h before experiments with sodium butyrate (5 mm). Primocultures of porcine thyroid cells were performed as described (17Chambard M. Verrier B. Gabrion J. Mauchamp J. J. Cell Biol. 1983; 96: 1172-1177Crossref PubMed Scopus (85) Google Scholar). Porcine thyroid cells were isolated by discontinuous trypsin-EGTA treatment and suspended in Dulbecco's modified Eagle's medium. The cells were seeded in 9.6-cm2 dishes and incubated at 36 °C in a water-saturated atmosphere of 5% CO2, 95% air. Culture media were changed every 2–3 days. After 3–5 days, a monolayer was obtained, and the cells were stimulated with 10−4m Bt2cAMP for 5 days. Confluent cells were radiolabeled for up to 6 days with 100 μCi/ml 35S-(Met + Cys) in cysteine- and methionine-free Dulbecco's modified Eagle's medium supplemented with 10% FBS, and 10 mm sodium butyrate. Cell monolayers were then washed twice with ice-cold PBS, pH 8.0, supplemented with 1 mm CaCl2 and 1 mm MgCl2 (PBS-CM) before the cross-linker (NHS-SS-biotin) was added at 0.5 mg/ml in PBS-CM for 20 min on ice. Cross-linker was removed, and the same operation was repeated once. Then the medium was removed, and the remaining reactive was by the addition of mm in The cells were then in of mm pH acid, and protease inhibitor The cells were then for 20 min at °C with every min and for min at The was incubated for h with + A-Sepharose These had been with protein by incubation at were by a 10 and washed with 1 of hTPO The hTPO was from the protein A-Sepharose by 5 min at 100 °C after the addition of 10 of 10% and of hTPO The was with of hTPO and for min at The was incubated h with at surface hTPO and intracellular hTPO were by The were washed with hTPO and with PBS, in PubMed Scopus Google Scholar), and then for 5 The of to the cell surface and only of the to the intracellular were prepared for The was and by a cells were incubated in Ham's F-12 medium supplemented with 10 mm sodium FBS, and 10 of acid for 48 After 48 intracellular and cell surface radiolabeled hTPO molecules were by cell surface and to as described hTPO was with as described previously for other R. de J. D. S. S. J. Biol. Chem. 1997; 272: Full Text Full Text PDF PubMed Scopus Google Scholar) with cells were with acid as described hTPO was and from protein A-Sepharose by 5 min at 100 °C after the addition of 10 of 10% Then 40 of hTPO was and the was The was supplemented with 1 of acetone and 20 of bovine serum and incubated for min at After the was and the was a This was dissolved in 100 of 5 min at 100 and to activity was investigated as described by et al. M. PubMed Scopus Google Scholar) with The medium was removed, and cells were washed twice with ice-cold The incubation mixture bovine serum (5 mg/ml in PBS) and Na125I The was by the addition of H2O2 to obtain a of 0.5 were incubated for 20 min at the of this incubation the medium was to and the cell surface was washed with 0.5 of Then 1 of ice-cold 20% acid supplemented with 10−4m was added to each After 20 min at the was 6 The was and the obtained was washed with of 10% The remaining in the was was as described by S. J. PubMed Scopus Google Scholar). This is on the of the heme moiety of the protein into its by the of heme (15Fayadat L. Niccoli-Sire P. Lanet J. Franc J.L. Endocrinology. 1998; 139: 4277-4285Crossref PubMed Scopus (44) Google Scholar) that in CHO cells, only 2% of hTPO molecules the cell and the are the heme moiety had to be inserted into hTPO for its exit from the we a inhibitor of catalyzes the formation of from in the heme biosynthesis P. Blood. 1997; PubMed Google Scholar, P. D. Blood. 1996; PubMed Google Scholar). cells were incubated for days, or 6 days with or μm CHO cells at this for up to 6 days with in cell In a of we the of on the in the cells as described by S. J. PubMed Scopus Google with with the decreased by and for incubation of days, and 6 days, Then we the of on hTPO enzymatic activity and at the cell surface After h of incubation with the of hTPO at the cell surface decreased by and the activity was decreased by After and 6 days, the cell surface decreased by and and the activity decreased by and that the incubation with the decrease in at the cell surface stable and is similar to or a the decrease in synthesis and that the decrease in hTPO activity is and the decrease in synthesis that only heme biosynthesis intracellular of not heme, we at the cell surface of The trafficking of was by not that the of heme in cells is not a could the of hTPO the we heme biosynthesis by to the culture medium or hemin for 48 h cell surface cells were incubated for 48 h with 35S-(Met + Cys) with or In cell surface activity a was performed with or to the culture medium had on activity and at the cell to the plus and hemin, increased at the cell surface by 20% and the enzymatic activity by 20% for plus and for hemin that a treatment with plus to a of in the cells in control. The increase in the at the cell surface with plus or with hemin that heme biosynthesis in CHO cells is only with the the is that the insertion of heme is for hTPO intracellular trafficking and at the cell surface. We that has on hTPO the of hTPO with or not the of insertion of heme in other proteins for the exit from the similar been obtained with MPO (13Arnljots K. Olsson I. J. Biol. Chem. 1987; 262: 10430-10433Abstract Full Text PDF PubMed Google Scholar). In heme insertion into plays an role in its L. L. J. Biol. Chem. 1997; 272: Full Text Full Text PDF PubMed Scopus (129) Google Scholar). increased the of hTPO at the cell surface level by 20% and increased the cell surface activity by These that some of the hTPO molecules at the cell surface are heme. hTPO hemin added in culture medium with the of et al. J.L. S. Kimura S. T. A. J. 1996; PubMed Scopus (17) Google Scholar) and al. J. S. S. B. Endocrinology. 1998; 139: PubMed Scopus Google Scholar). It could be hypothesized that heme with hTPO in the ER and that of this heme is from the after hTPO from the at the cell surface. In mammalian the heme moiety is covalently linked to the protein R. Zeng J. Arch. Biochem. Biophys. 1995; 316: 653-656Crossref PubMed Scopus (129) Google Scholar, L.A. Bylkas S. Wilson A.E. J. Biol. Chem. 1996; 271: 3406-3412Abstract Full Text Full Text PDF PubMed Scopus (40) Google Scholar). As TPO, LPO, MPO, and belong to the mammalian peroxidase family. (12Andersson L.A. Bylkas S. Wilson A.E. J. Biol. Chem. 1996; 271: 3406-3412Abstract Full Text Full Text PDF PubMed Scopus (40) Google Scholar) indicated that the heme of TPO could be a type l heme like LPO. et al. M. A. R. J. Chem. 1997; Scopus Google Scholar) also indicated that the prosthetic group of mammalian peroxidase was heme by covalent bonds to the protein. type l and moiety of heme, the heme moiety is covalently linked to the protein through ester bonds that link aspartate and glutamate residues (conserved in mammalian of the protein with the heme hydroxymethyl MPO, there is an link a residue in TPO and of the protein and the moiety of the heme. of heme from protein is obtained for some treatment to heme R. de J. D. S. S. J. Biol. Chem. 1997; 272: Full Text Full Text PDF PubMed Scopus Google Scholar). heme was covalently linked to hTPO in cells and and treatment provided the same we incubated cells in the of for 48 After the hTPO was by or and then by The obtained is showed in After by was between the we that in cells heme covalently link to hTPO and that after by and only covalently heme associated with this protein. Recently, et al. (16DePillis G. Ozaki S. Kuo J.M. Maltby D.A. Ortiz de Montellano P.R. J. Biol. Chem. 1997; 272: 8857-8860Abstract Full Text Full Text PDF PubMed Scopus (91) Google Scholar) proposed a for autocatalytic heme modification and covalent binding in LPO and other mammalian In the of heme could be modified and could be linked covalently to the protein. We that in CHO cells there is an insufficient exposure to hTPO reaches the cell surface. This the of the heme from the protein after its exit from the ER. In cells, H2O2 in the intracellular is by peroxidase, catalase, or B. in and Scholar, K. J. Biol. Chem. 1998; Full Text Full Text PDF PubMed Scopus Google Scholar). the that the heme moiety of TPO could be covalently linked to the protein through an autocatalytic as for LPO (16DePillis G. Ozaki S. Kuo J.M. Maltby D.A. Ortiz de Montellano P.R. J. Biol. Chem. 1997; 272: 8857-8860Abstract Full Text Full Text PDF PubMed Scopus (91) Google Scholar), we incubated cells with or 10 μm H2O2 for 10 min or 20 μm hemin for 10 min or 48 h before hTPO activity 10-min exposure to 10 μm H2O2 led to a 65% increase in cell surface activity. This increase is similar to that obtained with a 10-min treatment with hemin, that hTPO activity in with the of covalent heme of the exposure to H2O2 this only on the activity of the hTPO molecules at the cell surface. an increase in covalent heme binding and then in activity could be obtained by the incubation with we incubated cells with or 10 μm H2O2 for activity and of hTPO at the cell surface were H2O2 activity increased at the cell surface decreased by 30% protein synthesis was by exposure to H2O2 not increase the enzymatic activity. This be to a of H2O2 on the cells an of a of H2O2 in the culture medium. as in and the hTPO at the cell surface 1) a linked heme that with H2O2 and then increase 2) heme the with or a covalently linked heme 6 covalent linking intracellularly and cell surface hTPO has a covalently linked heme, we investigated the incorporation of radiolabeled of heme into hTPO in the intracellular or at the cell surface by a cell surface After of the protein with a of (15Fayadat L. Niccoli-Sire P. Lanet J. Franc J.L. Endocrinology. 1998; 139: 4277-4285Crossref PubMed Scopus (44) Google Scholar), was by after of the protein with and with on the of heme was covalently linked to hTPO in the intracellular and was at the cell surface It was not possible to the relative of the hTPO or not heme and of the hTPO with a heme covalently of a incubation with H2O2 on the activity and of hTPO at the cell surface. cells were cultured with or H2O2 for h before hTPO activity was cells were incubated as in but the cell culture medium was supplemented with 35S-(Met + After cell surface and hTPO the of hTPO at the cell surface level was activity of hTPO at the cell surface after incubation for h with or H2O2 was significant differences were as for the attachment of heme to hTPO in cells and in thyroid cells of into hTPO present at the intracellular level or at the cell were incubated for 48 h with After cell surface and in the intracellular or at the cell surface was by that covalent attachment of heme to hTPO we investigated the incorporation of into the hTPO present at the cell surface with or treatment with H2O2 for 10 min on the we between and more associated with hTPO the cells were for 10 min with 10 μm H2O2 before cell surface Furthermore, this also that the increase in cell activity at the cell surface level in the of H2O2 from a on the hTPO and for from a of an linked to this H2O2 leading to covalent heme binding is probably a of transfer in to CHO cells, thyroid cells an of H2O2 that is in the iodination at the apical cell surface and in thyroid hormone synthesis. This H2O2 is generated by an in the apical R. D. A.M. J. Pommier J. A. Eur. J. Biochem. 1996; PubMed Scopus Google Scholar). H2O2 synthesized at the apical cell surface of thyroid cells might to covalent attachment of heme we prepared primocultures of porcine thyroid cells and the of a 10-min incubation with 10 μm H2O2 and a treatment with 20 μm hemin or In culture thyroid cells monolayers with an apical pole the culture medium (17Chambard M. Verrier B. Gabrion J. Mauchamp J. J. Cell Biol. 1983; 96: 1172-1177Crossref PubMed Scopus (85) Google Scholar). increased hTPO activity by 30% is less the with CHO cells, heme is covalently linked to hTPO at the cell surface of the of H2O2 by thyroid This was by the of of H2O2 added for 10 min in the culture medium the increase with hemin that some hTPO at the cell surface not heme. some hTPO the cell surface heme. The increase with hemin also be to by the type of culture in H2O2 synthesized at the apical cell surface level is to be into the culture covalent heme binding in a less in thyroid These were by cells with catalase for 48 h the addition of after led to a 30% decrease in hTPO activity at the surface of thyroid We the catalase in the culture medium is to the H2O2 is then to the cell surface. addition not a of hTPO activity 1) some heme be linked covalently to hTPO intracellularly and 2) of the of hTPO at the cell surface and covalently heme with hTPO molecules synthesized before catalase This that heme insertion into hTPO is for the of protein at the cell surface and that the heme moiety is covalently to hTPO through the H2O2-dependent autocatalytic as for LPO (16DePillis G. Ozaki S. Kuo J.M. Maltby D.A. Ortiz de Montellano P.R. J. Biol. Chem. 1997; 272: 8857-8860Abstract Full Text Full Text PDF PubMed Scopus (91) Google Scholar). we a role for H2O2 generated at the apical of thyroid cells its role in and thyroid hormone apical H2O2 to TPO into an active through its in autocatalytic covalent attachment of heme to TPO. We B. for full-length hTPO, J. for hTPO for porcine thyroid cells, M. for and A. for the