Increasing Diversity of Human Thyroperoxidase Generated by Alternative Splicing
2003; Elsevier BV; Volume: 278; Issue: 6 Linguagem: Inglês
10.1074/jbc.m209513200
ISSN1083-351X
AutoresMireille Ferrand, Valérie Le Fourn, Jean‐Louis Franc,
Tópico(s)Antioxidant Activity and Oxidative Stress
ResumoThe human thyroperoxidase (hTPO) gene is composed of 17 exons. The longest complete cDNA sequence determined so far contains a full-length hTPO (TPO1) encoding a 933-amino acid polypeptide. Several mRNA species encoding for hTPO isoforms are present in normal thyroid tissues, including TPO2 with exon 10 deleted and TPOzanelli with exon 16 deleted. In the present study, we established the existence of two new single-spliced transcripts, TPO4 and TPO5, lacking exons 14 and 8, respectively. Upon transfecting the TPO4 cDNA into Chinese hamster ovary cells, it was observed that TPO4 is able to reach the cell surface, is enzymatically active, and is able to be recognized by a panel of 12 monoclonal antibodies directed against hTPO, whereas TPO5 does not fold correctly and is unable to reach the cell surface. In normal tissues, the expression of TPO4 mRNA was examined by performing quantitative reverse transcription PCR. This deleted TPO mRNA amounted to 32 ± 11% of the total TPO mRNAs. In the same tissues, the TPO2, TPOzanelli, and TPO5 amounted to 35 ± 12%, 36 ± 14%, and ∼10%, respectively. The sum of these four species (not including TPO1) was more than 100%, possibly due to the presence of multispliced mRNAs. This possibility was tested, and three new variants were identified: TPO2/3, lacking exons 10 and 16, TPO2/4, lacking exons 10 and 14, and an unexpected variant, TPO6, corresponding to the deletion of exons 10, 12, 13, 14, and 16. In conclusion, these results indicate the existence of five new transcripts. One of them, TPO4, codes for an enzymatically active protein, whereas TPO5 is unable to fold correctly. The functional significance of the other newly spliced mRNA variants still remains to be elucidated, but these results might help to explain the heterogeneity of the hTPO purified from the thyroid gland. The human thyroperoxidase (hTPO) gene is composed of 17 exons. The longest complete cDNA sequence determined so far contains a full-length hTPO (TPO1) encoding a 933-amino acid polypeptide. Several mRNA species encoding for hTPO isoforms are present in normal thyroid tissues, including TPO2 with exon 10 deleted and TPOzanelli with exon 16 deleted. In the present study, we established the existence of two new single-spliced transcripts, TPO4 and TPO5, lacking exons 14 and 8, respectively. Upon transfecting the TPO4 cDNA into Chinese hamster ovary cells, it was observed that TPO4 is able to reach the cell surface, is enzymatically active, and is able to be recognized by a panel of 12 monoclonal antibodies directed against hTPO, whereas TPO5 does not fold correctly and is unable to reach the cell surface. In normal tissues, the expression of TPO4 mRNA was examined by performing quantitative reverse transcription PCR. This deleted TPO mRNA amounted to 32 ± 11% of the total TPO mRNAs. In the same tissues, the TPO2, TPOzanelli, and TPO5 amounted to 35 ± 12%, 36 ± 14%, and ∼10%, respectively. The sum of these four species (not including TPO1) was more than 100%, possibly due to the presence of multispliced mRNAs. This possibility was tested, and three new variants were identified: TPO2/3, lacking exons 10 and 16, TPO2/4, lacking exons 10 and 14, and an unexpected variant, TPO6, corresponding to the deletion of exons 10, 12, 13, 14, and 16. In conclusion, these results indicate the existence of five new transcripts. One of them, TPO4, codes for an enzymatically active protein, whereas TPO5 is unable to fold correctly. The functional significance of the other newly spliced mRNA variants still remains to be elucidated, but these results might help to explain the heterogeneity of the hTPO purified from the thyroid gland. thyroperoxidase human TPO gene-specific primer reverse transcription Chinese hamster ovary fetal bovine serum minimum Eagle's medium phosphate-buffered saline monoclonal antibody bovine serum albumin epidermal growth factor myeloperoxidase Thyroperoxidase (TPO)1 is the key enzyme in the process of thyroid hormone synthesis. The human TPO gene is about 150 kbp in size, is located on chromosome 2, locus 2p25, and consists of 17 exons and 16 introns (for a review, see Ref. 1McLachlan S.M. Rapoport B. Endocr. Rev. 1992; 13: 192-206PubMed Google Scholar). The complete sequence of the human TPO coding region is known (2Kimura S. Kotani T. McBride O.W. Umeki K. Hirai K. Nakayama T. Ohtaki S. Proc. Natl. Acad. Sci. U. S. A. 1987; 84: 5555-5559Crossref PubMed Scopus (257) Google Scholar, 3Libert F. Ruel J. Ludgate M. Swillens S. Alexander N. Vassart G. Dinsart C. EMBO J. 1987; 6: 4193-4196Crossref PubMed Scopus (149) Google Scholar, 4Magnusson R.P. Chazenbalk G.D. Gestautas J. Seto P. Filetti S. DeGoot L.J. Rapoport B. Mol. Endocrinol. 1987; 1: 856-861Crossref PubMed Scopus (111) Google Scholar). The full-length 3048-bp transcript (TPO1) codes for a protein consisting of 933 amino acids, which have a large extracellular domain, a transmembrane domain consisting of 60 residues, and a short intracytoplasmic tail consisting of 60 residues. Two other transcripts have been described, namely TPO2, in which exon 10 is spliced out, and TPOzanelli (TPO3), in which exon 16 is spliced out. TPO2 and TPO3 have been found to occur in normal thyroid tissues as well as in Graves' tissues (2Kimura S. Kotani T. McBride O.W. Umeki K. Hirai K. Nakayama T. Ohtaki S. Proc. Natl. Acad. Sci. U. S. A. 1987; 84: 5555-5559Crossref PubMed Scopus (257) Google Scholar, 5Elisei R. Vassart G. Ludgate M. J. Clin. Endocrinol. Metab. 1991; 72: 700-702Crossref PubMed Scopus (25) Google Scholar, 6Zanelli E. Henry M. Charvet B. Malthièry M. Biochem. Biophys. Res. Comm. 1990; 170: 735-741Crossref PubMed Scopus (32) Google Scholar). These two forms code for proteins consisting of 876 and 929 residues, respectively. TPO2 is rapidly degraded after its synthesis, does not reach the cell surface, and does not have any enzymatic activity (7Niccoli P. Fayadat L. Paneels V. Lanet J. Franc J.L. J. Biol. Chem. 1997; 272: 29487-29492Abstract Full Text Full Text PDF PubMed Scopus (31) Google Scholar), whereas TPO3 is able to reach the cell surface and shows enzymatic activity (8Niccoli-Sire P. Fayadat L. Siffroi-Fernandez S. Malthièry Y. Franc J.L. Biochemistry. 2001; 40: 2572-2579Crossref PubMed Scopus (12) Google Scholar). After being purified from the human thyroid gland, TPO is known to show up in SDS-PAGE under reducing conditions as a double band of 105 and 110 kDa. The relative intensity of these bands varies from one gland to another, and it has been established that TPO2 does not correspond to one of these bands (9Gardas A. Lewartowska B. Sutton B.J. Pasieka Z. McGregor A.M. Banga J.M. J. Clin. Endocrinol. Metab. 1997; 82: 3752-3757PubMed Google Scholar, 10Cetani F. Costagliola S. Tonacchera M. Panneels V. Vassart G. Ludgate M. Mol. Cell. Endocrinol. 1995; 115: 125-132Crossref PubMed Scopus (10) Google Scholar), certainly because it is too rapidly degraded after its synthesis (7Niccoli P. Fayadat L. Paneels V. Lanet J. Franc J.L. J. Biol. Chem. 1997; 272: 29487-29492Abstract Full Text Full Text PDF PubMed Scopus (31) Google Scholar). The difference in molecular weight between TPO1 and TPO3 (four amino acids) does not explain the existence of two bands, and glycosylation is not responsible for this heterogeneity either (11Giraud A. Franc J.L. Long Y. Ruf J. J. Endocrinol. 1992; 132: 317-323Crossref PubMed Scopus (33) Google Scholar). The presence of other isoforms and/or the occurrence of endoproteolysis might explain the existence of these different species. The aim of the present study was to search for the presence of new TPO transcripts that might help to explain this heterogeneity. Reverse transcription was carried out from the total RNAs, and PCRs were performed with various pairs of primers. Two new single spliced species (TPO4 and TPO5) were identified. After quantification of the four spliced isoforms TPO2, TPO3, TPO4, and TPO5 in normal thyroid tissues, the possible existence of multispliced isoforms was hypothesized. The existence of two isoforms with double splicing and one isoform with five spliced exons was established. Frozen normal thyroid tissue was used in these experiments. Tissues were homogenized and prepared using thePromega kit (SV total RNA isolation system) according to the manufacturer's instructions, and the preparation was then treated with DNase. The RNA concentration was determined from the spectrophotometric absorption at 260 nm, and the RNAs were aliquoted and stored in water at −80 °C until further use. The absorption ratio (260/280) was between 1.7 and 2.0 with all the preparations. Depending on the experiments, reverse transcription was carried out using either 0.5 μg of oligo(dT)12–18, 1 μg of random hexamers, or 2 pmol of gene-specific primer (GSP). A 40-μl reverse transcription reaction mixture containing hexamers, GSP, or oligo(dT)12–18, 0.9 μg of RNA, 0.25 mm dNTP mix, 10 mmdithiothreitol, and 4 units of RNase recombinant inhibitor (Invitrogen) was incubated at 42 °C for 2 min when GSP and oligo(dT)12–18 were used, or at 25 °C for 10 min when random hexamers were used. Superscript II RNase H− reverse transcriptase (0.2 units) (Invitrogen) was then added, and the mixture was incubated at 42 °C for 50 min. The reaction was inactivated by heating the preparation at 70 °C for 15 min. The mixture then treated with 2 units of RNase H (Escherichia coli) at 37 °C for 20 min. Reaction mixtures (50 μl) consisted of 2 units of Fast Start Taq polymerase (Roche Molecular Biochemicals), Taq buffer, 0.3 μmoligonucleotide primers, 300 μm dNTP, 2 mmMgCl2, and 5 μl of GC-rich solution (Roche Molecular Biochemicals). A DNA sample was added to this mixture, and PCR was performed with the following profile: 5 min at 95 °C for an initial denaturation followed by 35 cycles of 30 s of denaturation at 94 °C, 30 s of annealing at temperatures depending on the primers, and a 45-s extension at 72 °C, ending with a 5-min final extension at 72 °C and a soak at 4 °C. The products obtained were electrophoresed on agarose gel in TAE (40 mm Tris-acetate, 1 mm EDTA, pH 8.3) buffer and stained with ethidium bromide. When necessary, the various PCR products were extracted from the gel using Qiaquick gel extraction kit (Qiagen) according to the manufacturer's instructions, and sequence analysis (Genomexpress, Grenoble France) or subcloning were then performed. Subcloning of the purified PCR products was performed using the TOPO TA cloning kit (Invitrogen). Size analysis of the fragment inserted and sequence analysis of this insert in the vector were performed with the various clones obtained. To study the relative levels of expression of the various hTPO mRNA variants, RT-PCR was performed as described in Ref. 12Spencer W.S. Christensen M.J. Biotechnique. 1999; 27: 1044-1051Crossref PubMed Scopus (56) Google Scholar with some modifications. PCR reactions were performed as described above except that the volume of the mixture was 100 μl, and 9-μl aliquots were taken from the reaction after each consecutive cycle and loaded onto 2% agarose gel. After staining the products with ethidium bromide, bands were detected, and their intensity was quantified using an Image Station 440 (Kodak). To correct the difference in nucleotide length, the density of the smaller size band was multiplied by a factor corresponding to this difference. To determine whether the detection of a variant was really dependent on its initial proportion within the cDNA population, we performed PCR amplifications with various quantities of the three cloned variants, pcDNA3-TPO2, pcDNA3-TPO3, or pcDNA3-TPO4 in relation to that of pcDNA3-TPO1 (3:1, 1:1, and 1:3, respectively). In all the cases that were tested, the ratio between the isoforms after their amplification corresponded to their initial proportions. Full-length 3060-kb TPO1 cDNA kindly provided by B. Rapoport was cloned into the HindIII andXbaI sites of the eukaryotic transfer vector pcDNA3 (Invitrogen). The internal deletion of the cDNA corresponding to exon 14 or exon 8 was performed using a single PCR procedure (13Makarova O. Kamberov E. Margolis B. Biotechnique. 2000; 29: 970-972Crossref PubMed Scopus (196) Google Scholar, 14Wang J. Wilkinson M.F. Biotechnique. 2000; 29: 976-978Crossref PubMed Scopus (38) Google Scholar). The primer pair used was P-TPO4F or P-TPO5F (sense) and P-TPO4R or P-TPO5R (antisense) (see TableI). The PCR mixture contained 160 ng of each primer, 50 ng of the pcDNA-TPO1, 200 μm dNTPs, 10% (v/v) dimethyl sulfoxide, 2.5 units of PfuTurbo DNA polymerase (Stratagene), and the corresponding buffer in a total volume of 50 μl. The reaction was performed under the following conditions: denaturation at 95 °C for 30 s followed by 17 cycles of denaturation at 95 °C for 30 s, annealing at 55 °C for 1 min, and an extension at 68 °C for 17 min. PCR products were incubated with 10 units of DpnI for 2 h, and 5 μl of this solution was then transformed into 50 μl of MAX efficiency DH5α (Invitrogen). Parts of this transformant were spread onto LB agar plates. Correct pcDNA3-TPO4 and -TPO5 clones were evidenced by sequencing. pcDNA3-TPO4 and -TPO5 pure plasmid DNA preparations were obtained with the Wizard Midipreps kit (Promega, Madison, WI).Table IPrimer sequences for RT-PCRPrimerExonPositionSequencePE2F253–735′-CCTTCTTCCCCTTCATCTCGA-3′PE9Fa91392–14135′-GCAGTACGTGGGTCCCTATGAA-3′PE9Fb91382–14015′-AGGCCTTCCAGCAGTACGTG-3′PE9R91416–13975′-GGGATGTAATCCCTCAGGGTG-3′PE11F111833–18625′-AGCCATCGCCAGCAGGAGCGTGGCCGACAA-3′PE11R111794–17735′-GCAGAACTCCCTCCACTCATTG-3′PE12F122111–21405′-TGCCCATGGATGCCTTCCAAGTCGGCAAAT-3′PE15F152577–26065′-CGGAGGCTTCGCAGGTCTCACCTCGACGGT-3′PE15R152573–26035′-TCGAGGTGAGACCTGCGAAGCCTCCGATCA-3′PE17Ra172881–28515′-TTTGCCTGTGTTTGGAAAAGAGTCGTACGG-3′PE17Rb172793–27735′-TCTCGGCAGCCTGTGAGTATC-3′P-TPO4-F13-155′-GATGGGATTTCCAGCCTCCCCTCTGCAAAGACTCCGGGAGGCTCCCTCGG-3′P-TPO4-R15-135′-CCGAGGGAGCCTCCCGGAGTCTTTGCAGAGGGGAGGCTGGAAATCCCATC-3′P-TPO5-F7-95′-AACCCATGTTTTCCCATACAAATCATCACCCTGAGGGATTAC-3′P-TPO5-R9-75′-GTAATCCCTCAGGGTGATGATTTGTATGGGAAAACATGGGTT-3′ Open table in a new tab CHO cells (ECACC no. 85050302) were kept in Ham's F-12 medium supplemented with 10% FBS, penicillin (100 IU/ml), and streptomycin (0.1 mg/ml). Cells were transfected using LipofectAMINE (Invitrogen) with pcDNA3-TPO4 or pcDNA3-TPO5. Cells were incubated in a saturated atmosphere (5% CO2/95% air) at 37 °C. Stable transfectants were selected in the presence of geneticin (400 μg/ml) and subcloned using limiting dilutions. Positive TPO4 and TPO5 expressing cell lines were identified by performing Western blotting or immunoprecipitation after [35S](Met + Cys) labeling (EXPRE35S35S protein labeling mix, PerkinElmer Life Sciences). A significant level of TPO1, TPO4, and TPO5 expression was obtained by growing TPO1-CHO, TPO4-CHO, and TPO5-CHO cell lines as described previously (15Fayadat L. Niccoli-Sire P. Lanet J. Franc J.L. Endocrinology. 1998; 139: 4277-4285Crossref PubMed Scopus (44) Google Scholar). Cells were incubated in cysteine- and methionine-free MEM supplemented with 10% FBS, 10 mmsodium butyrate, and 100 μCi/ml [35S](Met + Cys). The incubation was carried out for 5, 16, or 48 h. In the pulse-chase experiments, cells were incubated for 1 h in Cys- and Met-free MEM supplemented with 10% dialyzed FBS and 10 mm sodium butyrate. Cells were then pulsed for 30 min in the presence of 100 μCi/ml [35S](Met + Cys). After the pulse, the labeling medium was removed, and the cell surface was washed three times with PBS and then replaced by Ham's F-12 medium supplemented with 10% FBS, 5 mm Met, and 5 mm Cys. Chases were performed for 30 min and 1, 3, 5, 16, and 24 h. After being metabolically labeled, cells were washed twice with PBS, harvested on ice by scraping them into 1 ml of PBS, and centrifuged at 200 ×g for 7 min. Cell pellets were resuspended in 600 μl of TPO extraction buffer (50 mm Tris-HCl, pH 7.4, 150 mm NaCl, 1% Triton X-100, 0.3% sodium deoxycholate, and protease inhibitors (CompleteTM, Roche Molecular Biochemicals)), vortexed every 2 min for 20 min, and centrifuged at 10,000 × g for 5 min. Radiolabeled supernatants were incubated for 2 h at room temperature with a pair of mAbs recognizing either a sequential region (mAb 47) or a conformational epitope (mAb 15) of the TPO molecule (16Ruf J. Toubert M.E. Czarnocka B. Durand-Gorde J.M. Ferrand M. Carayon P. Endocrinology. 1989; 125: 1211-1218Crossref PubMed Scopus (155) Google Scholar). These mAbs were previously complexed with protein A-Sepharose 4B (Zymed Laboratories Inc.) by incubating them overnight at 4 °C. Immune complexes were then retrieved by performing a brief centrifugation (10,000 × g, 10 s) and washed 4 times with 1 ml of TPO extraction buffer and once with 1 ml of PBS. Immunoprecipitated TPO was recovered from mAb-protein A-Sepharose 4B complexes by boiling the complexes for 5 min in 80 μl of electrophoresis buffer (62 mm Tris-HCl, pH 6.8, 2% SDS, 5% 2-mercaptoethanol, and 5% glycerol), and it was then analyzed by SDS-PAGE (7.5%). Protein-associated radioactivity was detected and quantified using a phosphorimaging device (Fudjix BAS 1000). TPO was also immunoprecipitated with a panel of mAbs directed against various antigenic domains of the TPO1 molecule (16Ruf J. Toubert M.E. Czarnocka B. Durand-Gorde J.M. Ferrand M. Carayon P. Endocrinology. 1989; 125: 1211-1218Crossref PubMed Scopus (155) Google Scholar). In this experiment, [35S](Met + Cys)-radiolabeled CHO-TPO cell lysates were immunoprecipitated for 4 h at 25 °C with 50 μg of each of the TPO-mAbs previously complexed with protein A-Sepharose 4B. TPO1-, TPO4-, and TPO5-CHO confluent monolayers were metabolically labeled for 18 h with 100 μCi/ml [35S](Met + Cys) in the presence of 10 mm sodium butyrate, and cell surfaces were biotinylated as described previously (15Fayadat L. Niccoli-Sire P. Lanet J. Franc J.L. Endocrinology. 1998; 139: 4277-4285Crossref PubMed Scopus (44) Google Scholar). Cells were washed twice with PBS supplemented with 1 mm CaCl2 and 1 mm MgCl2 and exposed to a 0.5 mg/ml EZ-link sulfo NHS-SS-Biotin (Pierce) for 20 min at 4 °C. The cross-linker was removed, and the procedure was repeated once. The biotin reagent was quenched by incubating the preparation with 50 mmNH4Cl in PBS for 10 min at 4 °C. Cells were washed with PBS and harvested. To recover the immunoprecipitated antigens, the complexes were supplemented with 10 μl of 10% SDS, boiled for 5 min, diluted with 600 μl of TPO-extraction buffer, and centrifuged (10,000 × g, 3 min). Supernatant containing the total TPO was incubated for 2 h with avidin-agarose (Pierce). Biotinylated surface TPO and intracellular TPO were separated by centrifugation (10,000 × g, 10 s). The beads were washed four times with TPO-extraction buffer and once with PBS, resuspended in electrophoresis buffer, and boiled for 5 min. The supernatants were analyzed by SDS-PAGE (7.5%). Microsomal fraction pellets, prepared as described previously (7Niccoli P. Fayadat L. Paneels V. Lanet J. Franc J.L. J. Biol. Chem. 1997; 272: 29487-29492Abstract Full Text Full Text PDF PubMed Scopus (31) Google Scholar), were solubilized by resuspending them in 15 mm Tris-HCl, pH 7.5, 150 mm NaCl, 1% Triton X-100, and 0.1 mm potassium iodide. Microsomes prepared from CHO cells transfected with pcDNA3 were used as a negative control. Microsomal fractions were centrifuged (10,000 × g, 2 min), and the supernatant was used for the enzymatic assay. Extracts containing approximately the same amount of protein were added to 1 ml of 40 mm guaiacol (Fluka Chimie, St. Quentin-Fallavier, France) and 67 mm sodium phosphate buffer, pH 7.5. The reaction was performed at room temperature and initiated by adding H2O2 to obtain a final concentration of 0.25 mm. Guaiacol oxidation was measured by absorbance at 470 nm and monitored spectrophotometrically every 15 s for 3 min. Cell surface enzymatic activity was assayed as in Ref. 17Fayadat L. Niccoli-Sire P. Lanet J. Franc J.L. J. Biol. Chem. 1999; 274: 10533-10538Abstract Full Text Full Text PDF PubMed Scopus (79) Google Scholar, with slight modifications. TPO1- and TPO4-CHO cells were incubated in Ham's F12 medium supplemented with 10 mm sodium butyrate and 20 μm hemin for 48 h. CHO cells transfected with pcDNA3 alone or TPO1-CHO cells were used as a negative or positive control, respectively. The medium was removed, and the cells were washed twice with PBS before being incubated with BSA (5 mg/ml in PBS) and Na125I (106 cpm/ml), with or without 2 mm2-mercapto-1-methylimidazole, as the control medium. The reaction was initiated by adding H2O2 to obtain a final concentration of 0.5 mm, and cells were incubated for 20 min at room temperature. The medium was then transferred to cold reaction tubes; the cell surface was washed out with 0.5 ml PBS, and the wash was added to the medium. Each tube was filled with 1 ml of ice-cold 20% (w/v) trichloroacetic acid supplemented with 10−4m KI, and incubated for 20 min at 4 °C before being 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% (w/v) trichloroacetic acid. The radioactivity remaining in the pellet was counted. A strategy was developed to search for new isoforms of hTPO. It is known that the various transcripts resulting from alternative splicing can have different lengths of the poly(A) tail (18Zhao J. Hyman L. Moore C. Microbiol. Mol. Biol. Rev. 1999; 63: 405-445Crossref PubMed Google Scholar), and, consequently, some isoforms cannot be detected by using oligo(dT)12–18during the RT experiments. In the present study, after extracting the total RNA from normal thyroid tissues, three different kind of primers were thus used in the first strand cDNA synthesis reaction depending on the experiment, namely random hexamers, GSPs, and oligo(dT)12–18. Random hexamers lead to the production of short cDNA fragments and can therefore be used to avoid secondary structure problems or when the mRNA has a short poly(A) tail. In the latter case, GSP can also be used. In the first set of experiments, which was designed to detect any alternative splicing between exons 9 and 17, reverse transcriptions were performed using random hexamers, and several different pairs of primers were then used in the PCR experiments. Two PCR products were obtained with one of these pairs located in exons 12 and 15, a band with an apparent size of 499 bp corresponding to the predicted full-sized mRNA of 493 bp and a smaller 375-bp band (Fig. 1). Sequence analysis of the latter band showed that this was a 362-bp species from which exon 14 had been specifically deleted. This new variant of hTPO was named TPO4 (GenBankTM accession number AY136822). Exon 14 codes for an extracellular part of the protein near its transmembrane domain. This part of the protein corresponds exactly to its EGF-like domain (3Libert F. Ruel J. Ludgate M. Swillens S. Alexander N. Vassart G. Dinsart C. EMBO J. 1987; 6: 4193-4196Crossref PubMed Scopus (149) Google Scholar). Juxtaposing exons 13 and 15 did not induce any changes in the reading frame and the corresponding full-length cDNA codes for a protein consisting of 889 amino acids. To detect any alternative splicing occurring between exons 2 and 9, reverse transcription was performed using a GSP located in exon 9 (PE9R), and a PCR experiment was then performed using a pair of primers located in exons 2 and 9 (PE2F and PE9R). Two bands were obtained, one with an apparent size of 1330 bp corresponding to the predicted full-size cDNA of 1311 bp, and a smaller one with an apparent size of 785 bp (Fig. 2). This band was purified and sequenced and found to correspond to a 793-bp species from which exon 8 had been deleted. This variant was named TPO5 (GenBankTM accession number AF533528). Exon 8 codes for an extracellular part of the protein located in its myeloperoxidase-like domain. Juxtaposing exons 7 and 9 did not lead to any changes in the open reading frame, and the corresponding full-length protein codes for a protein of 760 amino acids. Two hypothetically crucial residues are spliced out in TPO5, namely Arg-396, which may participate in the catalytic mechanism underlying the formation of compound I, and Glu-399, which may covalently bind to the heme prosthetic group through ester linkage (19Taurog A. Biochimie (Paris). 1999; 81: 557-562Crossref PubMed Scopus (70) Google Scholar). Two potential N-glycosylation sites (Asn-307 and Asn-342) are also spliced out. Contrary to what occurs in TPO5, the deleted exon in TPO4 mRNA codes for a whole domain (the EGF-like domain) that is not included in the main catalytic part of the molecule. It therefore seemed possible that this isoform might be active. We therefore examined the properties of the proteins corresponding to these two transcripts, focusing in particular on TPO4.Figure 2Identification of exon 8 deletion.Reverse-transcription was performed using the gene-specific primer PE9R, and TPO cDNAs were amplified by performing PCR as described under "Experimental Procedures" using PE2F and PE9R with an annealing temperature of 55 °C. PCR products were analyzed on a 1% agarose gel. Lane 1, DNA size marker; lanes 2 and3, RT-PCR products obtained using RNA from two different normal thyroid tissues.View Large Image Figure ViewerDownload Hi-res image Download (PPT) pcDNA3-TPO4 and pcDNA3-TPO5 were constructed from pcDNA3-TPO1 using a one stage PCR protocol compatible with the deletion of exon 14 or exon 8. CHO cells were transfected with pcDNA3-TPO4 or pcDNA3-TPO5, and several clones expressing significant levels of TPO4 or TPO5 were then isolated. After a metabolic labeling step using [35S](Met + Cys), immunoprecipitation was performed using the pair mAb15 + mAb47, and TPO4 and TPO5 showed up as bands with the predicted molecular weight on the SDS-PAGE analysis (Fig. 3,A and B). To determine whether TPO4 has a modified three-dimensional structure in comparison with TPO1, we used a panel of 12 mAbs directed against hTPO. All of these mAbs except one, mAb47, were directed against conformational epitopes. TPO1- and TPO4-CHO cells were labeled for 16 h with [35S](Met + Cys), and, after the extraction step, immunoprecipitations were performed with each of the 12 mAbs (Fig. 4, A andB). TPO1 as well as TPO4 immunoreactivity was observed with all the mAbs. However, mAbs 1, 24, and 59 showed a slight decrease in immunoreactivity with TPO4 as compared with TPO1 (Fig. 4 C). This seems to indicate that most of the TPO4 fold correctly in comparison with TPO1 and that none of the mAbs used were directed against the EGF-like domain. To determine whether the small differences observed affect the global half-life of the TPO4 synthesized in CHO cells, we performed a pulse-chase experiment. Cells were pulsed for 30 min with [35S](Met + Cys) and then chased for various times. Immunoprecipitation of TPO was performed, and samples were analyzed by SDS-PAGE. Quantification of these bands (Fig. 5) showed that TPO4 has a shorter half-life than TPO1, i.e. 5 versus 7.5 h. All of these events may affect the intracellular trafficking of TPO4 and hence its level of expression at the cell surface. To check whether TPO4 can reach the cell surface of the CHO cells, the cell surface expression of the two isoforms was determined after labeling CHO cells with [35S](Met + Cys) for 48 h and performing cell surface biotinylation (Fig. 6 A). Quantification of the bands obtained showed that 25% of the TPO1 and only 12% of the TPO4 were present at the cell surface (Fig. 6 B).Figure 5Rate of degradation of TPO1 and TPO4.Cells were pulsed for 30 min in the presence of 100μCi/ml of [35S](Met + Cys) in a Cys- and Met-free MEM supplemented with 10% FBS. After the pulse step, the medium was removed and replaced by Ham's F-12 medium supplemented with 5 mm Cys and Met. At the times indicated, after the extraction step, TPO from radiolabeled cell lysate was immunoprecipitated using the pair mAb 15 + mAb 47. Immunoprecipitated TPO1 (A) and TPO4 (B) were analyzed by SDS-PAGE. Bands corresponding to TPO1 (○) and TPO4 (●) were quantified by phosphorimaging. This figure gives the results of an experiment that is representative of four identical experiments performed.View Large Image Figure ViewerDownload Hi-res image Download (PPT)Figure 6Cell surface expression of TPO1 and TPO4. TPO1- and TPO4-CHO cells were metabolically labeled for 16 h with [35S](Met + Cys), and cell surface biotinylation was then carried out as described under "Experimental Procedures." The cells were lysed, and TPO was immunoprecipitated with the couple mAb 15 + mAb 47 prior to reprecipitating the TPO present at the cell surface by adding avidin-agarose. The tagged fraction and only one-tenth of the supernatant corresponding to the intracellular fraction were analyzed by SDS-PAGE (7.5%). A, supernatants corresponding to the intracellular fractions are shown inlane 1 (TPO1) and lane 3 (TPO4). Supernatants corresponding to cell surface fractions are shown in lane 2(TPO1) and lane 4 (TPO4). The bands were detected and quantified by phosphorimaging. B, the percentages of intracellular TPO (gray) and TPO expressed at the cell surface (black) were calculated. This figure gives the mean value of three different experiments.View Large Image Figure ViewerDownload Hi-res image
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