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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

1999; Elsevier BV; Volume: 274; Issue: 15 Linguagem: Inglês

10.1074/jbc.274.15.10533

1083-351X

Laurence Fayadat, Patricia Niccoli‐Sire, Jeanne Lanet, Jean‐Louis Franc,

Porphyrin Metabolism and Disorders

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 NHS-SS-biotin was blocked by the addition of 50 mm NH4Cl in PBS-CM. The cells were then scraped in 600 μl of extraction buffer containing 50 mm Tris-HCl, pH 7.4, 0.15 m NaCl, 1% Triton X-100, 0.3% deoxycholic acid, and protease inhibitor mixture. The cells were then tumbled for 20 min at 4 °C with vortexing every 2 min and centrifuged for 3 min at 10,000 × g. The supernatant was incubated for 2 h with (mAb 47 + mAb 15)-protein A-Sepharose complexes. These mAbs had been complexed with protein A by incubation overnight at 4 °C. Immune complexes were retrieved by a brief centrifugation (10,000 × g, 10 s) and washed four times with 1 ml of hTPO extraction buffer. The immunoprecipitated hTPO was separated from the protein A-Sepharose pellet by heating 5 min at 100 °C after the addition of 10 μl of 10% SDS and 500 μl of hTPO extraction buffer. The suspension was diluted with 500 μl of hTPO extraction buffer and centrifuged for 3 min at 10,000 ×g. The supernatant was incubated 2 h with avidin-agarose at room temperature. Biotinylated surface hTPO and intracellular hTPO were separated by centrifugation (10 s, 10,000 × g). The beads were washed four times with hTPO extraction buffer and once with PBS, resuspended in Laemmli sample buffer (18Laemmli U.K. Nature. 1970; 227: 680-685Crossref PubMed Scopus (207227) Google Scholar), and then boiled for 5 min. The totality of supernatant corresponding to the cell surface fraction and only 110 of the supernatant corresponding to the intracellular fraction were prepared for SDS-PAGE analysis. The radioactivity was visualized and quantified by a phosphor imager (Fuji BAS 1000). CHO-hTPO cells were incubated in Ham's F-12 medium supplemented with 10 mm sodium butyrate, 1% FBS, and 10 μCi of [14C]aminolevulinic acid for 48 h. After 48 h, intracellular and cell surface radiolabeled hTPO molecules were separated by cell surface biotinylation, immunoprecipitated, and subjected to SDS-PAGE as described above. 14C-Heme-labeled hTPO was treated with acetone/HCl as described previously for other hemoproteins (19Kuras R. de Vitry C. Choquet Y. Girard-Bascou J. Culler D. Büschlen S. Merchant S. Wollman F.A. J. Biol. Chem. 1997; 272: 32427-32435Abstract Full Text Full Text PDF PubMed Scopus (83) Google Scholar) with small modifications. CHO-hTPO cells were labeled with [14C]aminolevulinic acid as described above. The14C-heme-labeled hTPO was immunoprecipitated and separated from protein A-Sepharose pellet by heating 5 min at 100 °C after the addition of 10 μl of 10% SDS. Then 40 μl of hTPO extraction buffer was added, and the suspension was centrifuged (3 min, 10,000 ×g). The supernatant was supplemented with 1 ml of acetone containing 0.2% HCl (12 n) and 20 μg of bovine serum albumin and incubated for 30 min at 4 °C. After centrifugation (10 min, 10,000 × g), the supernatant was discarded and the pellet was dried using a Speed Vac. This pellet was dissolved in 100 μl of Laemmli buffer, heated 5 min at 100 °C, and subjected to SDS-PAGE. Enzymatic activity was investigated as described by Neary et al. (20Neary J.T. Soodak M. Maloof F. Methods Enzymol. 1984; 107: 445-475Crossref PubMed Scopus (16) Google Scholar) with slight modifications. The medium was removed, and cells were washed twice with ice-cold PBS buffer. The incubation mixture contained bovine serum albumin (5 mg/ml in PBS) and Na125I (106cpm/ml). The reaction was initiated by the addition of H2O2 to obtain a final concentration of 0.5 mm. Cells were incubated for 20 min at room temperature. At the end of this incubation time, the medium was transferred to cold reaction tubes, and the cell surface was washed once with 0.5 ml of PBS. Then 1 ml of ice-cold 20% (w/v) trichloracetic acid supplemented with 10−4m KI was added to each tube. After 20 min at 4 °C, the suspension was centrifuged (2000 ×g, 6 min). The supernatant was discarded, and the acid-insoluble iodinated material obtained was washed three times with 2 ml of 10% trichloracetic acid. The radioactivity remaining in the pellet was counted. Total porphyrin concentration was determined fluorometrically as described by Sassa (21Sassa S. J. Exp. Med. 1976; 143: 305-315Crossref PubMed Scopus (216) Google Scholar). This method is based on the conversion of the heme moiety of the protein into its fluorescent porphyrin derivative by the removal of heme under acidic reducing conditions. Our previous data (15Fayadat L. Niccoli-Sire P. Lanet J. Franc J.L. Endocrinology. 1998; 139: 4277-4285Crossref PubMed Scopus (44) Google Scholar) revealed that in CHO cells, only 2% of hTPO molecules reach the cell surface, and the rest (98%) are degraded. To determine if the heme moiety had to be inserted into hTPO for its exit from the ER, we first used SA, a specific inhibitor of 5-aminolevulinic dehydratase, which catalyzes the formation of porpholibinogen from 5-aminolevulinate in the heme biosynthesis pathway (22Ponka P. Blood. 1997; 89: 1-25Crossref PubMed Google Scholar, 23Richardson D.R. Ponka P. Vyoral D. Blood. 1996; 87: 3477-3488Crossref PubMed Google Scholar). CHO-hTPO cells were incubated for 16 h, 3 days, or 6 days with or without 250 μm SA. CHO cells tolerated SA well at this concentration for up to 6 days with no change in cell morphology. In a first set of experiments, we determined the effects of SA on the total porphyrin concentration in the cells as described by Sassa (Ref. 21Sassa S. J. Exp. Med. 1976; 143: 305-315Crossref PubMed Scopus (216) Google Scholar; see "Experimental Procedures"). Compared with control, with SA the total porphyrin concentration decreased by 20, 50, and 50% for incubation times of 16 h, 3 days, and 6 days, respectively. Then we studied the effect of SA on hTPO enzymatic activity and delivery at the cell surface (Fig.1). After 16 h of incubation with SA the delivery of hTPO at the cell surface decreased by 37%, and the activity was decreased by 79%. After 3 and 6 days, the cell surface delivery decreased by 25 and 35%, and the activity decreased by 68 and 92%, respectively. Note that whatever the incubation time with SA, the decrease in delivery at the cell surface remains stable (about 30%) and is similar to or a little lower than the decrease in total porphyrin synthesis and that the decrease in hTPO activity is higher (68 and 92%) than the decrease in total porphyrin synthesis (20–50%). To confirm that SA impaired only heme biosynthesis without affecting intracellular transport of protein(s) not containing heme, we measured delivery at the cell surface of Na+-K+-ATPase. The trafficking of Na+-K+-ATPase was unaffected by SA (data not shown). To verify that the concentration of heme in CHO-hTPO cells is not a limiting factor (that could explain the low percentage of hTPO reaching the plasma membrane) we enhanced heme biosynthesis pathway by adding to the culture medium ALA, FeTf, or hemin for 48 h (Fig.2). For cell surface biotinylation assays, cells were incubated for 48 h with 35S-(Met + Cys) with or without those components. In cell surface activity experiments, a negative control was performed with methylmercaptoimidazole. Adding ALA or FeTf to the culture medium had no effect on activity and delivery at the cell surface, contrary to the association ALA plus FeTf and hemin, which increased delivery at the cell surface by 20% (Fig. 2 B) and the enzymatic activity by 20% for ALA plus FeTf and 120% for hemin (Fig. 2 A). Note that a 48-h treatment with ALA plus FeTf leads to a 10-fold higher concentration of total porphyrin in the cells than in control. The moderate increase (20%) in the delivery at the cell surface with ALA plus FeTf or with hemin shows that heme biosynthesis in CHO cells is only slightly limiting. Together with the SA data (Fig. 1), the first conclusion is that the insertion of heme is important for hTPO intracellular trafficking and delivery at the cell surface. We found that SA has no noticeable effect on hTPO stability because the half-life of hTPO remained unchanged with or without SA (data not shown). Concerning the importance of insertion of heme in other proteins for the exit from the ER, similar results have been obtained with MPO (13Arnljots K. Olsson I. J. Biol. Chem. 1987; 262: 10430-10433Abstract Full Text PDF PubMed Google Scholar). In addition, heme insertion into cytochromeb 558 plays an important role in its subunit assembly (24Yu L. Zhen L. Dinauer M.C. J. Biol. Chem. 1997; 272: 27288-27294Abstract Full Text Full Text PDF PubMed Scopus (129) Google Scholar). Hemin increased the quantity of hTPO at the cell surface level by 20% and increased the cell surface activity by 120%. These values suggest that some of the hTPO molecules at the cell surface are inactive because they lack heme. That hTPO binds hemin added in culture medium agrees with the conclusion of Fan et al. (25Fan J.L. Patibandla S. Kimura S. Rao T. Desai R.K. Seetharamaiah G.S. Kurosky A. Prabhakar B.S. J. Autoimmun. 1996; 9: 529-536Crossref PubMed Scopus (17) Google Scholar) and Guoet al. (26Guo J. McLachlan S. Hutchison S. Rapoport B. Endocrinology. 1998; 139: 999-1005Crossref PubMed Scopus (36) Google Scholar). It could be hypothesized that heme associates with hTPO in the ER and that part of this heme is dissociated from the enzyme after hTPO exits from the ER, especially at the cell surface. In mammalian peroxidases, the heme moiety is covalently linked to the protein backbone (11Fenna R. Zeng J. Arch. Biochem. Biophys. 1995; 316: 653-656Crossref PubMed Scopus (129) Google Scholar, 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). As mentioned above, TPO, LPO, MPO, and EPO belong to the mammalian peroxidase family. Spectral analysis (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. Moreover, Kooter et al. (27Kooter I.M. Pierik A.J. Merkx M. Averill B.A. Moguilevsky N. Bollen A. Wever R. J. Am. Chem. Soc. 1997; 119: 11542-11543Crossref Scopus (46) Google Scholar) also indicated that the prosthetic group of mammalian peroxidase was 1,5-dihydroxymethyl-modified heme b attached by covalent bonds to the protein. For type l and m (heme moiety of MPO) heme, the heme moiety is covalently linked to the protein through two ester bonds that link aspartate and glutamate residues (conserved in all mammalian peroxidases) of the protein with the heme hydroxymethyl groups. For MPO, there is an additional vinyl sulfonium link involving a methionine residue (not conserved in TPO and LPO) of the protein and the 2-vinyl moiety of the heme. Total dissociation of noncovalently bound heme from protein is classically obtained using acidic treatment. However, for some hemoproteins, SDS treatment leads to heme removal (19Kuras R. de Vitry C. Choquet Y. Girard-Bascou J. Culler D. Büschlen S. Merchant S. Wollman F.A. J. Biol. Chem. 1997; 272: 32427-32435Abstract Full Text Full Text PDF PubMed Scopus (83) Google Scholar). To see if heme was covalently linked to hTPO in CHO-hTPO cells and if acidic and SDS treatment provided the same results, we incubated CHO-hTPO cells in the presence of [14C]ALA for 48 h. After immunoprecipitation, the 14C-heme-labeled hTPO was treated by acetone/HCl or SDS/2-mercaptoethanol and then analyzed by SDS-PAGE. The result obtained is showed in Fig. 3. After quantification by phosphor imaging no difference was found between the two treatments. Therefore, we can assume that in CHO-hTPO cells heme can covalently link to hTPO and that after denaturation by SDS and reduction only covalently bound heme remains associated with this protein. Recently, DePillis 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 mechanism for autocatalytic heme modification and covalent binding in LPO and other mammalian peroxidases. In the presence of H2O2, heme could be autocatalytically modified and could subsequently be linked covalently to the protein. We hypothesize that in CHO cells there is an insufficient exposure to H2O2before hTPO reaches the cell surface. This would explain the dissociation of the heme from the protein after its exit from the ER. In most cells, H2O2 availability in the intracellular compartments is controlled by cytosolic selenium-containing GSH peroxidase, peroxisomal catalase, or PRX enzymes (28Halliwell B. Gutteridge J.M.C. Free Radicals in Biology and Medicine. Clarendon Press, Oxford1991: 86-187Google Scholar, 29Kang S.W. Chae H.Z. Seo M.S. Kim K. Baines I.C. Rhee S.G. J. Biol. Chem. 1998; 273: 6297-6302Abstract Full Text Full Text PDF PubMed Scopus (618) Google Scholar). To test the possibility that the heme moiety of TPO could be covalently linked to the protein through an autocatalytic process 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 CHO-hTPO cells with or without 10 μm H2O2 for 10 min or 20 μm hemin for 10 min or 48 h before assaying hTPO activity (Fig. 4). A 10-min exposure to 10 μm H2O2 led to a 65% increase in cell surface activity. This great increase is similar to that obtained with a 10-min treatment with hemin, indicating that hTPO activity increases in parallel with the extent of covalent heme binding. Because of the short exposure time to H2O2 (10 min), this compound can act only on the activity of the hTPO molecules at the cell surface. To check if an increase in covalent heme binding and then in activity could be obtained by extending the incubation time with H2O2, we incubated CHO-hTPO cells with or without 10 μm H2O2 for 24 h. Enzymatic activity and delivery of hTPO at the cell surface were measured (Fig. 5). With H2O2 activity increased slightly (10%) (Fig.5 A), delivery at the cell surface decreased by 30% (Fig.5 B) because protein synthesis was inhibited by H2O2. Hence, long exposure to H2O2 does not notably increase the enzymatic activity. This may be due to a toxic effect of H2O2 on the cells and/or an impossibility of maintaining a constant concentration of H2O2 in the culture medium. However, as shown in Figs. 4 and 5, the hTPO at the cell surface may have 1) a noncovalently linked heme that can react with exogenous H2O2 and then increase activity, 2) no heme (thus explaining the results with hemin), or 3) a covalently linked heme (Fig. 6 A). To determine if covalent linking occurs intracellularly and if cell surface hTPO has a covalently linked heme, we investigated the incorporation of [14C]ALA (a radiolabeled precursor of heme biosynthesis) into hTPO in the intracellular compartments or at the cell surface by using a cell surface biotinylation technique. After immunoprecipitation of the protein with a couple of mAbs (15Fayadat L. Niccoli-Sire P. Lanet J. Franc J.L. Endocrinology. 1998; 139: 4277-4285Crossref PubMed Scopus (44) Google Scholar), analysis was done by SDS-PAGE after denaturation of the protein with SDS and reduction with 2-mercaptoethanol (Fig. 7). Depending on the experiments, 50–70% of heme was recovered covalently linked to hTPO in the intracellular compartment, and 30–50% was recovered at the cell surface level. It was not possible to determine the relative quantity of the hTPO bearing or not bearing heme and of the hTPO with a heme covalently linked.Figure 5Effect of a 24-h incubation with H2O2 on the activity and delivery of hTPO at the cell surface. A, CHO-hTPO cells were cultured with or without H2O2 (10 μm) for 24 h before hTPO activity was assayed. B, CHO-hTPO cells were incubated as in A, but the cell culture medium was supplemented with 35S-(Met + Cys). After cell surface biotinylation and hTPO immunoprecipitation, the percentage of hTPO at the cell surface level was determined. Specific activity of hTPO at the cell surface after incubation for 24 h with or without H2O2 was determined. Statistically significant differences versus control were as follows:p < 0.01 (★★).View Large Image Figure ViewerDownload (PPT)Figure 6Model for the attachment of heme to hTPO in CHO-hTPO cells (A) and in thyroid cells (B).View Large Image Figure ViewerDownload (PPT)Figure 7Incorporation of [14C]ALA into hTPO present at the intracellular level or at the cell surface.Cells were incubated for 48 h with [14C]ALA. After cell surface biotinylation and immunoprecipitation,14C-hTPO in the intracellular compartments or at the cell surface was analyzed by SDS-PAGE.View Large Image Figure ViewerDownload (PPT) To confirm that covalent attachment of heme to hTPO required H2O2, we investigated the incorporation of [14C]ALA into the hTPO present at the cell surface with or without treatment with H2O2 for 10 min (Fig.8). Depending on the experiment, we detected between 2- and 5-fold more [14C]ALA associated with hTPO when the cells were treated for 10 min with 10 μm H2O2 before cell surface biotinylation. Furthermore, this result also confirms that the increase in cell activity observed at the cell surface level in the presence of H2O2 (Fig. 4) results from a direct effect on the hTPO and not, for example, from a removal of an inhibitory ligand linked to this enzyme. Intracellular H2O2 leading to covalent heme binding is probably a by-product of electron transfer reactions. However, in contrast to CHO cells, thyroid cells have an additional physiological production of H2O2 that is involved in the iodination reactions at the apical cell surface and in thyroid hormone synthesis. This H2O2 is generated by an NADPH oxidase system in the apical membrane (30Gorin Y. Ohayon R. Carvalho D.P. Dème D. Leseney A.M. Kaniewski J. Pommier J. Virion A. Dupuy C. Eur. J. Biochem. 1996; 240: 807-814Crossref PubMed Scopus (28) Google Scholar). Thus, H2O2 synthesized at the apical cell surface of thyroid cells might contribute to covalent attachment of heme (Fig.6 B). To verify this, we prepared primocultures of porcine thyroid cells and examined the effects of a 10-min incubation with 10 μm H2O2 and a 48-h treatment with 20 μm hemin or 0.01% catalase. In these culture conditions, thyroid cells form monolayers with an apical pole oriented toward the culture medium (17Chambard M. Verrier B. Gabrion J. Mauchamp J. J. Cell Biol. 1983; 96: 1172-1177Crossref PubMed Scopus (85) Google Scholar). Hemin increased hTPO activity by 30% (Fig. 9), which is less than the percentage with CHO cells, because most heme is covalently linked to hTPO at the cell surface because of the production of H2O2 by thyroid cells. This was confirmed by the absence of effect of H2O2 added for 10 min in the culture medium (Fig. 9). However, the increase with hemin shows that some hTPO at the cell surface does not bear heme. Consequently, some hTPO may reach the cell surface without heme. The increase with hemin may also be attributed to by the type of culture used, primoculture in monolayer. H2O2 synthesized at the apical cell surface level is likely to be diluted into the culture medium, which would allow covalent heme binding in a less efficient way than in thyroid follicles. These results were confirmed by incubating cells with catalase for 48 h (with the addition of enzyme after 24 h), which led to a 30% decrease in hTPO activity at the surface of thyroid cells. We can assume the catalase in the culture medium is unable to pass the plasma membrane. Its H2O2 degradation action is then restricted to the cell surface. Catalase addition does not cause a greater inhibition of hTPO activity 1) because some heme can be linked covalently to hTPO intracellularly and 2) because of the half-life of hTPO at the cell surface level, and covalently bound heme remained with hTPO molecules synthesized before catalase addition. This study shows that heme insertion into hTPO is important for the delivery of protein at the cell surface and that the heme moiety is covalently bound to hTPO through the H2O2-dependent autocatalytic process 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). Also, we found a new role for H2O2 generated at the apical membrane of thyroid cells besides its classical role in TPO-catalyzed iodotyrosines and thyroid hormone formation; apical H2O2 helps to stabilize TPO into an active form through its contribution in autocatalytic covalent attachment of heme to TPO. We thank B. Rapoport for providing full-length hTPO, J. Ruf for providing hTPO mAb, O. Chabaud for providing porcine thyroid cells, M. Ferrand for helpful discussion, and A. Giraud for reading the manuscript.