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An Inverse Correlation between Expression of a Preprocathepsin B-related Protein with Cysteine-rich Sequences and Steroid 11औ-Hydroxylase in Adrenocortical Cells

2003; Elsevier BV; Volume: 278; Issue: 19 Linguagem: Inglês

10.1074/jbc.m301477200

1083-351X

Kuniaki Mukai, Fumiko Mitani, Hideko Nagasawa, Reiko Suzuki, Tsuneharu Suzuki, Makoto Suematsu, Yuzuru Ishimura,

Steroid Chemistry and Biochemistry

A cDNA encoding a secretory protein hitherto unknown was cloned from mouse adrenocortical cells by subtractive hybridization between the cells without and with expressing steroid 11औ-hydroxylase (Cyp11b-1), a marker for the functional differentiation of cells in the zonae fasciculata reticularis (zFR). The deduced protein consisting of 466 amino acids contained a secretory signal, epidermal growth factor-like repeats, and a proteolytically inactive cathepsin B-related sequence. The amino acid sequence was 897 identical with that of human tubulointerstitial nephritis antigen-related protein. Among the mouse organs examined, adrenal glands prominently expressed its mRNA. The mRNA and its encoded protein were detected in the outer adrenocortical zones that do not express Cyp11b-1, i.e. the zona glomerulosa and the undifferentiated cell zone, while being undetectable in zFR that express Cyp11b-1. The new protein was designated as adrenocortical zonation factor 1 (AZ-1). Clonal lines with different levels of AZ-1 expression were established from Y-1 adrenocortical cells that originally express Cyp11b-1 but little AZ-1. Analyses of the clonal lines revealed that Cyp11b-1 is detected in the clonal lines maintaining little AZ-1 expression and becomes undetectable in those expressing AZ-1. On the other hand, irrespective of the AZ-1 expression, all clones expressed cholesterol side-chain cleavage enzyme, which occurs throughout the cortical zones. These results demonstrated that adrenocortical cells expressing AZ-1 do not express Cyp11b-1, whereas those with little AZ-1 express this zFR markerin vitro and in vivo, implying a putative role of AZ-1 in determining the zonal differentiation of adrenocortical cells. A cDNA encoding a secretory protein hitherto unknown was cloned from mouse adrenocortical cells by subtractive hybridization between the cells without and with expressing steroid 11औ-hydroxylase (Cyp11b-1), a marker for the functional differentiation of cells in the zonae fasciculata reticularis (zFR). The deduced protein consisting of 466 amino acids contained a secretory signal, epidermal growth factor-like repeats, and a proteolytically inactive cathepsin B-related sequence. The amino acid sequence was 897 identical with that of human tubulointerstitial nephritis antigen-related protein. Among the mouse organs examined, adrenal glands prominently expressed its mRNA. The mRNA and its encoded protein were detected in the outer adrenocortical zones that do not express Cyp11b-1, i.e. the zona glomerulosa and the undifferentiated cell zone, while being undetectable in zFR that express Cyp11b-1. The new protein was designated as adrenocortical zonation factor 1 (AZ-1). Clonal lines with different levels of AZ-1 expression were established from Y-1 adrenocortical cells that originally express Cyp11b-1 but little AZ-1. Analyses of the clonal lines revealed that Cyp11b-1 is detected in the clonal lines maintaining little AZ-1 expression and becomes undetectable in those expressing AZ-1. On the other hand, irrespective of the AZ-1 expression, all clones expressed cholesterol side-chain cleavage enzyme, which occurs throughout the cortical zones. These results demonstrated that adrenocortical cells expressing AZ-1 do not express Cyp11b-1, whereas those with little AZ-1 express this zFR markerin vitro and in vivo, implying a putative role of AZ-1 in determining the zonal differentiation of adrenocortical cells. the zona glomerulosa adrenocortical zonation factor 1 FLAG-tagged AZ-1 cholesterol side-chain cleavage enzyme steroid 11औ-hydroxylase aldosterone synthase digoxigenin epidermal growth factor glyceraldehyde-3-phosphate dehydrogenase tubulointerstitial nephritis antigen TIN-ag-related protein steroidogenic factor 1 the zona fasciculata the zonae fasciculata-reticularis the undifferentiated cell zone Tris-buffered saline The adrenal cortex of mammals consists of three major zones that contain both functionally and morphologically distinct cells; they are the zona glomerulosa (zG),1the zona fasciculata (zF), and the zona reticularis (1Orth D.N. Kovacs W.J. DeBold C.R. Wilson J.D. Foster D.W. Williams Textbook of Endocrinology. W. B. Saunders Co., Philadelphia1992: 489-619Google Scholar). The cells in zG, the outermost zone of the cortex, secrete aldosterone, the strongest mineralocorticoid; and those in zF, the middle zone, produce glucocorticoids such as corticosterone in most rodents and cortisol in other mammals including human. Finally, the cells in the zona reticularis, the innermost portion of the cortex, also secrete glucocorticoids in many mammals including rats and mice and produce adrenal androgens in human and some other mammals. In addition to these three, a zone composed of 3–4 layers of small round cells has been recognized between zG and zF in various animals (2Cain A.J. Harrison R.G. J. Anat. 1950; 84: 196-226PubMed Google Scholar, 3Cater D.B. Lever J.D. J. Anat. 1954; 88: 437-454PubMed Google Scholar, 4Deane H.W. Greep R.O. Am. J. Anat. 1946; 79: 117-145Crossref PubMed Scopus (49) Google Scholar, 5Hall K. Korenchevsky V. 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The zone thus has been named the undifferentiated cell zone (zU) (12Mitani F. Mukai K. Miyamoto H. Suematsu M. Ishimura Y. Biochim. Biophys. Acta. 2003; 1619: 317-324Crossref PubMed Scopus (68) Google Scholar) after the functionally undifferentiated nature of its component cells (13Mitani F. Ogishima T. Miyamoto H. Ishimura Y. Endocr. Res. 1995; 21: 413-423Crossref PubMed Scopus (30) Google Scholar, 14Mitani F. Miyamoto H. Mukai K. Ishimura Y. Endocr. Res. 1996; 22: 421-431Crossref PubMed Scopus (33) Google Scholar, 15Mitani F. Mukai K. Ogawa T. Miyamoto H. Ishimura Y. Steroids. 1997; 62: 57-61Crossref PubMed Scopus (21) Google Scholar, 16Mitani F. Mukai K. Miyamoto H. Ishimura Y. Endocr. Res. 1998; 24: 983-986Crossref PubMed Scopus (9) Google Scholar, 17Mitani F. Mukai K. Miyamoto H. Suematsu M. Ishimura Y. Endocrinology. 1999; 140: 3342-3353Crossref PubMed Scopus (78) Google Scholar, 18Miyamoto H. Mitani F. Mukai K. Suematsu M. Ishimura Y. Endocr. Res. 2000; 26: 899-904Crossref PubMed Scopus (13) Google Scholar). Development and maintenance of the adrenocortical zones require many cellular processes including regulation of the steroidogenic gene expression and regulation of cell renewal and arrangements. Steroidogenic factor 1 (19Parker K.L. Schimmer B.P. Endocr. Rev. 1997; 18: 361-377Crossref PubMed Scopus (556) Google Scholar) (SF-1, also referred to as Ad4BP (20Morohashi K.I. Omura T. FASEB J. 1996; 10: 1569-1577Crossref PubMed Scopus (189) Google Scholar)) is a transcription factor essential for embryonic development of steroidogenic organs including adrenal cortex and gonads (21Luo X. Ikeda Y. Parker K.L. Cell. 1994; 77: 481-490Abstract Full Text PDF PubMed Scopus (1383) Google Scholar). SF-1 also plays a pivotal role in the earlier steps of the adrenocortical steroidogenesis over the entire adrenal cortex by controlling expression of cholesterol side-chain cleavage enzyme (Cyp11a) and steroid 21-hydroxylase (22Morohashi K. Iida H. Nomura M. Hatano O. Honda S. Tsukiyama T. Niwa O. Hara T. Takakusu A. Shibata Y. Omura T. Mol. Endocrinol. 1994; 8: 643-653PubMed Google Scholar). Based on these features, SF-1 is unlikely to be a key regulator for the zonal differentiation of the steroidogenesis. Regarding factors regulating expression of the steroidogenic genes for the last steps of the syntheses, we previously suggested that AP-1 transcription factors were necessary for the spatially restricted expression of Cyp11b-1 in zFR (23Mukai K. Mitani F. Shimada H. Ishimura Y. Mol. Cell. Biol. 1995; 15: 6003-6012Crossref PubMed Scopus (32) Google Scholar, 24Mukai K. Mitani F. Agake R. Ishimura Y. Eur. J. Biochem. 1998; 256: 190-200Crossref PubMed Scopus (32) Google Scholar). Other regulatory factors playing a crucial role in the zone-specific steroidogenesis of zFR have been unknown. Furthermore, no regulatory factor for the functional differentiation of the rest of the cortex, zG and zU, has been identified so far. Therefore, molecular mechanisms underlying the zonal differentiation of the adrenocortical steroidogenesis remain to be solved. The goal of this study was to explore unidentified factors that control the functional differentiation of adrenocortical cells. To this end, we used the mouse adrenocortical cell lines that we established recently (25Mukai K. Nagasawa H. Agake-Suzuki R. Mitani F. Totani K. Yanai N. Obinata M. Suematsu M. Ishimura Y. Eur. J. Biochem. 2002; 269: 69-81Crossref PubMed Scopus (15) Google Scholar). They are derived from the adrenal glands of transgenic mice (26Obinata M. Genes Cells. 1997; 2: 235-244Crossref PubMed Scopus (62) Google Scholar,27Obinata M. Biochem. Biophys. Res. Commun. 2001; 286: 667-672Crossref PubMed Scopus (33) Google Scholar) carrying a temperature-sensitive large T-antigen gene of simian virus 40, being at different degrees of differentiation from one another. In the present study, a subtractive cDNA cloning employing the adrenocortical cell lines, named AcA101 and AcA201, as well as a conventional cell line Y-1, was carried out. Among the cell lines, AcA101 is the most undifferentiated one which expresses neither Cyp11b-1 nor Cyp11b-2, whereas Y-1 is the most differentiated one that expresses Cyp11b-1 with a responsiveness to ACTH stimuli (28Schimmer B.P. Methods Enzymol. 1979; 58: 570-574Crossref PubMed Scopus (89) Google Scholar). A cDNA cloned from AcA101 cells by a subtractive hybridization encodes a protein termed adrenocortical zonation factor-1 (AZ-1), the subject of this paper. AZ-1 is a unique secretory protein with a preprocathepsin B-related structure carrying epidermal growth hormone (EGF) motifs. This study demonstrates that expression of AZ-1 in adrenocortical cells is inversely correlated with expression of Cyp11b-1 in vitro and in vivo. Mouse adrenocortical AcA101 and AcA201 cells were cultured at 33 °C, a permissive temperature for the mutant SV40 T-antigen protein, under the conditions described previously (25Mukai K. Nagasawa H. Agake-Suzuki R. Mitani F. Totani K. Yanai N. Obinata M. Suematsu M. Ishimura Y. Eur. J. Biochem. 2002; 269: 69-81Crossref PubMed Scopus (15) Google Scholar). Mouse adrenocortical Y-1 cells (28Schimmer B.P. Methods Enzymol. 1979; 58: 570-574Crossref PubMed Scopus (89) Google Scholar) were cultured at 37 °C with Ham's F-10 medium supplemented with 107 heat-inactivated fetal bovine serum (Hyclone, Logan, UT), 107 heat-inactivated horse serum (Invitrogen), 200 units/ml penicillin, and 200 ॖg/ml streptomycin. The cells were incubated under a humidified atmosphere containing 57 CO2. Total RNA was extracted from AcA101, AcA201, and Y-1 cells with a modified single-step isolation method employing Trizol reagent (Invitrogen). Poly(A)+ RNA was prepared with an oligo(dT)-cellulose column (Amersham Biosciences) and treated with RNase-free DNase (Promega, Madison, WI). Poly(A)+ RNA (2 ॖg) from AcA101 cells was converted into cDNA with a NotI-oligo(dT) primer. A cDNA library of AcA101 cells was prepared using the λZipLox system (Invitrogen) according to the instructions from the manufacturer. Subtractive probes were prepared with the chemical cross-linking subtraction method using an RNA subtraction kit (Amersham Biosciences). Single-stranded cDNA (0.4 ॖg) of AcA101 cells was subtracted with poly(A)+ RNA (10 ॖg) of AcA201 or Y-1 cells by hybridization and chemical cross-linking reaction. To reduce signals from SV40 T-antigen mRNA, RNA encoding T-antigen was synthesized in vitro by using cloned T-antigen genes (25Mukai K. Nagasawa H. Agake-Suzuki R. Mitani F. Totani K. Yanai N. Obinata M. Suematsu M. Ishimura Y. Eur. J. Biochem. 2002; 269: 69-81Crossref PubMed Scopus (15) Google Scholar) and was added to the hybridization reaction. The resulting subtracted cDNA was labeled with [α-32P]dCTP (3000 Ci/mmol,Amersham Biosciences) using Sequenase version 2.0 (Amersham Biosciences). The AcA101 cDNA library (2 × 105plaque-forming units) was screened with the subtractive probes under stringent conditions. Positive plaques were isolated through a second screening process using digoxigenin-labeled cDNA (Roche Diagnostics) probes (not subtracted) from AcA101, AcA201, and Y-1 cells. Plasmids carrying a cDNA insert were recovered from λ phage clones utilizing the cre-lox systems. DNA sequencing was performed by the dideoxy termination method using Thermo SequenaseTM (Amersham Biosciences). Total RNA was prepared from adrenal glands, whole brains, hearts, kidneys, livers, skeletal muscles, spleens, and testes of C57BL/6 mice using Trizol reagent as described above. The RNA preparations (10 ॖg) were subjected to Northern blot analysis as described previously (24Mukai K. Mitani F. Agake R. Ishimura Y. Eur. J. Biochem. 1998; 256: 190-200Crossref PubMed Scopus (32) Google Scholar). Before transfer to positively charged nylon membranes (Roche Diagnostics), ribosomal RNAs were visualized by ethidium bromide to verify that the amounts of RNA loaded were comparable with each other (<157) and that degradation of the RNA preparations was undetectable under our experimental conditions. A cDNA fragment (position 1490–1810 of Fig.1A) was labeled with [α-32P]dCTP (3000 Ci/mmol, Amersham Biosciences) and High Prime (Roche Diagnostics). Hybridization signals were detected with a Kodak BioMax film with an intensifying screen. Genomic DNA was prepared from the liver of CL57BL/6 mice and was digested with EcoRI,BamHI, and HindIII. The digests (10 ॖg) were subjected to electrophoresis through 0.87 agarose and blotted on a positive charged nylon filter (Roche Diagnostics). A cDNA fragment of AZ-1 (position 1490–1810) was isolated and labeled with DIG-dUTP (Roche Diagnostics). Hybridization, stringent washing, and detection with color development were carried out according to the manufacturer's instructions (Roche Diagnostics) and as described previously (29Mukai K. Imai M. Shimada H. Okada Y. Ogishima T. Ishimura Y. Biochem. Biophys. Res. Commun. 1991; 180: 1187-1193Crossref PubMed Scopus (28) Google Scholar, 30Mukai K. Imai M. Shimada H. Ishimura Y. J. Biol. Chem. 1993; 268: 9130-9137Abstract Full Text PDF PubMed Google Scholar). To construct a plasmid for expression of recombinant proteins, oligonucleotide primers for PCR were prepared. A forward primer including the putative translational initiation codon was designed to contain an artificial BstXI site at position 48 of the sequence shown in Fig. 1A: 5′-CGCCAGTGTGCTGGAGGCACCATGTGGGGATGT-3′. A reverse PCR primer corresponding to the C terminus of the protein was designed to contain the sequence encoding FLAG epitope followed by an XbaI site: 5′-GCTCTAGACTACTTGTCATCGTCGTCCTTGTAGTCGTGGTGCCCCATGTCCTCC-3′. Another reverse PCR primer 5′-GCTCTAGATCAGTGGTGCCCCATGTCCTCC-3′ lacking the FLAG tag sequence was also designed. cDNA fragments (either the presence or absence of the FLAG sequence) that were amplified by PCR were inserted into pRc/CMV (Invitrogen). To avoid incorrect nucleotides incorporated in PCR, the DraIII fragment (position 69–1280 of Fig. 1A) carrying the PCR-amplified region was replaced by the authentic fragment prepared from the original cDNA. The resulting pR/C11.13FD1 and pR/C11.13D1 plasmids were used for templates in in vitrotranscription-translation reaction of T7 TnT-coupled Reticulocyte Lysate System (Promega) in the presence or absence of [35S]methionine (1000 Ci/mmol, Amersham Biosciences). Reaction mixtures were subjected to SDS-PAGE. Detection was carried out by autoradiography or immunoblotting using anti-FLAG M2 monoclonal antibody (Sigma) as described below. Peptides P1 (NH2-Cys-Ser-Gln-Gly-Arg-Pro-Glu-Gln-Tyr-Arg-Arg-His-Gly-Thr-COOH) and P2 (NH2-Cys-Gly-Arg-Val-Gly-Met-Glu-Asp-Met-Gly-His-His-COOH), corresponding to amino acid residues 386–398 and 456–466, respectively, were synthesized and each conjugated at the additional Cys residue to keyhole limpet hemocyanin (Pierce) through a thioether bond using maleimide-activated keyhole limpet hemocyanin (Pierce) as described in the instruction manual. The peptide-keyhole limpet hemocyanin conjugates were mixed (1:1 (w/w)) and emulsified with Freund's complete adjuvant and injected into IsaBrown hens 6 times intradermally at 2-week intervals. Blood was collected on the 35th day or thereafter. Antibodies were purified from antisera (10 ml) with a peptide-conjugated agarose gel column. The agarose gel was prepared by coupling a 1:1 (w/w) mixture of the two peptides using a SulfoLink coupling gel (Pierce) according to the instructions. Based on titration by an immunoblot analysis using a recombinant AZ-1 protein, the recovery of the purified antibody after the chromatography was ∼707. The EcoRI-BamHI fragment (position 1490–1810 of Fig. 1A) of the isolated cDNA clone was subcloned into pZL1 (Invitrogen). Mouse cDNAs encoding Cyp11b-1 (position 761–950) or Cyp11b-2 (761–953) (31Domalik L.J. Chaplin D.D. Kirkman M.S. Wu R.C. Liu W.W. Howard T.A. Seldin M.F. Parker K.L. Mol. Endocrinol. 1991; 5: 1853-1861Crossref PubMed Scopus (148) Google Scholar) were obtained by PCR with primer pairs as described previously (25Mukai K. Nagasawa H. Agake-Suzuki R. Mitani F. Totani K. Yanai N. Obinata M. Suematsu M. Ishimura Y. Eur. J. Biochem. 2002; 269: 69-81Crossref PubMed Scopus (15) Google Scholar) from the total RNA of Y-1 cells after reverse transcription, and they were cloned into pGEM4-Z (Promega). DIG-labeled antisense and sense RNA probes were synthesized with T7 and SP6 RNA polymerases, respectively, using DIG RNA labeling kit (Roche Diagnostics). Mouse adrenals were excised after transcardial perfusion with phosphate-buffered saline(−) containing 47 paraformaldehyde and were further fixed with the same fixative overnight at 4 °C. The adrenals were embedded with paraffin, and 4-ॖm sections were prepared using 3-aminopropyltriethoxysilane-coated glass slides. After deparaffinization with standard methods, in situhybridization was carried out as described previously (17Mitani F. Mukai K. Miyamoto H. Suematsu M. Ishimura Y. Endocrinology. 1999; 140: 3342-3353Crossref PubMed Scopus (78) Google Scholar). Concentrations of the antisense and sense probes (400 ng/ml) in the hybridization solutions were adjusted based on analysis by the agarose gel electrophoresis of the synthesized RNA. Paraffin sections of paraformaldehyde-fixed mouse adrenal glands were treated with affinity-purified chicken anti-AZ-1 antibody (10 ॖg/ml) or control chicken IgY (10 ॖg/ml, Sigma) overnight at 4 °C. The concentrations of the antibodies were adjusted after protein determination with Coomassie Brilliant Blue G-250. The secondary antibody used was rabbit anti-chicken IgY antibody conjugated with horseradish peroxidase (1:300; Promega). The peroxidase activity was visualized with 3,3′-diaminobenzidine tetrahydrochloride and hydrogen peroxide as described previously (32Ogishima T. Suzuki H. Hata J. Mitani F. Ishimura Y. Endocrinology. 1992; 130: 2971-2977Crossref PubMed Scopus (160) Google Scholar). pR/C11.13FD1, which was the same DNA construct as used for in vitro synthesis of FLAG-tagged AZ-1 (AZ-1F), or a control vector pRc/CMV was linearized by digestion withBglII. Y-1 cells (3 × 106) were transfected with the linearized DNA (10 ॖg) by calcium phosphate precipitation method using the Profection kit (Promega). The cells were selected for resistance to antibiotics G418 (400 ॖg/ml), and colonies were isolated and expanded for characterization as described below. A mock transfectant (clone 9) and an AZ-1F vector-transfectant (clone 14; see Fig.7), which showed the highest level of AZ-1F mRNA, were used for detection of the AZ-1 polypeptide. Cell extracts from them (3.6-cm2 well) were prepared with an SDS sample buffer (120 ॖl) consisting of 62 mm Tris-HCl, pH 6.8, 27 (w/v) SDS, 27 (v/v) 2-mercaptoethanol, and 0.017 bromphenol blue. The extracts were incubated at 100 °C for 5 min before electrophoresis. For immunoprecipitation, purified chicken anti-AZ-1 antibody (150 ॖl) was bound to anti-chicken IgY-agarose (150 ॖl of 507 suspension;Promega) by incubation for 1 h at 4 °C with gentle shaking. The beads were washed with 1 ml of Tris-buffered saline (TBS) 5 times. The supernatants of the used culture media (1 ml) were prepared by centrifugation to remove cells and cell debris and incubated with 30 ॖl of the gel suspension for 18 h at 4 °C with gentle shaking. The agarose gel beads were washed with TBS containing 0.17 Tween 20 (TBST) 4 times followed by a wash with TBS. They were resuspended in an SDS sample buffer and treated at 100 °C for 5 min. The immunoprecipitates and the cell extracts were subjected to 107 PAGE in the presence of SDS, and polypeptides were electrophoretically blotted onto Immobilon-P membranes (Millipore, Bedford, MA) according to standard procedures. The membranes were treated with anti-FLAG monoclonal M2 antibody (0.88 ॖg/ml) overnight at 4 °C. They were washed with TBST and then incubated with a secondary antibody solution of rabbit anti-mouse IgG conjugated with horseradish peroxidase (1:25,000 dilution with TBST) for 3 h at room temperature. When the affinity-purified chicken anti-AZ-1 antibody (1:200 dilution) was used for immunoblotting, membranes were treated with rabbit anti-chicken IgY conjugated with horseradish peroxidase (1:1000 dilution). Bound secondary antibodies were detected by enhanced chemiluminescence (Amersham Biosciences). An AZ-1F-vector transfectant (clone 14) was cultured in the presence of 10 ॖg/ml tunicamycin (Sigma) or vehicle (0.27 (v/v) methanol in the culture medium) for 18 h, and cell extracts were prepared as described above. For treatment with peptide N-glycosidase F, cell extracts of the transfectant containing 27 SDS were diluted by 10-fold with 10 mm Tris-HCl, pH 7.5, and incubated with 50 units/ml of N-glycosidase F (Roche Diagnostics) at 37 °C for 18 h. After the treatment, the SDS sample buffer was added to the reaction mixtures. These samples were analyzed by immunoblotting as described above. RT-PCR analysis was performed by the methods as described in the previous paper (25Mukai K. Nagasawa H. Agake-Suzuki R. Mitani F. Totani K. Yanai N. Obinata M. Suematsu M. Ishimura Y. Eur. J. Biochem. 2002; 269: 69-81Crossref PubMed Scopus (15) Google Scholar) using primer pairs as follows: (i) AZ-1F: 64f, 5′-ACCATGTGGGGATGTTGGCTGG-3′ (position 64–85, see Fig.1A) and FL1485r, 5′-GTCATCGTCGTCCTTGTAGTCG-3′ that corresponds to the FLAG peptide encoding sequence); (ii) AZ-1: 420f, 5′-GGACAACTGCAATCGATGCACC-3′ (420–441); 1003r, 5′-GGCTGTGCATCATACAACGAGG-3′ (1003–982); this primer pair was used for detection of mRNA of both endogenous Az-1 gene and AZ-1F; (iii) Cyp11b-1 (25Mukai K. Nagasawa H. Agake-Suzuki R. Mitani F. Totani K. Yanai N. Obinata M. Suematsu M. Ishimura Y. Eur. J. Biochem. 2002; 269: 69-81Crossref PubMed Scopus (15) Google Scholar); (iv) Cyp11a (25Mukai K. Nagasawa H. Agake-Suzuki R. Mitani F. Totani K. Yanai N. Obinata M. Suematsu M. Ishimura Y. Eur. J. Biochem. 2002; 269: 69-81Crossref PubMed Scopus (15) Google Scholar); and (v) glyceraldehyde-3-phosphate dehydrogenase (GAPDH) (25Mukai K. Nagasawa H. Agake-Suzuki R. Mitani F. Totani K. Yanai N. Obinata M. Suematsu M. Ishimura Y. Eur. J. Biochem. 2002; 269: 69-81Crossref PubMed Scopus (15) Google Scholar). Amplification conditions for PCR were 45 s at 94 °C, 45 s at 56 °C, and 2 min at 72 °C for appropriate cycle numbers followed by 7 min at 72 °C. Cycle numbers were 20 for AZ-1 (420f and 1003r), Cyp11a, and GAPDH and 25 for AZ-1F (64f and FL1485r), and Cyp11b-1. Experiments for comparison of relative amounts of mRNAs among the transfectants were performed within the exponential phase of the amplification reactions to obtain the linear response concerning the initial mRNA amounts. PCR products were analyzed by agarose gel electrophoresis followed by visualization with ethidium bromide. Intensities of the visualized products were determined by densitometric analysis and were normalized with that of GAPDH cDNA. For the subtractive cDNA cloning to identify genes whose transcripts are expressed in higher levels in undifferentiated adrenocortical cells than in differentiated cells, we prepared two probes as described under “Experimental Procedures”; one was obtained by a subtraction between AcA101 and AcA201 and the other was between AcA101 and Y-1. A cDNA library of AcA101 cells was prepared to represent the whole population of mRNA molecules. It was screened by hybridization of the two subtracted probes onto plaque lifts. We isolated cDNA clones whose hybridization signal was detected with both subtractive probes. Among the cDNA clones isolated, one was composed of 1926 nucleotides except for a poly(A) tail as shown in Fig.1A. The cDNA attracted our attention because the amino acid sequence of its encoded protein had a unique structure as described below. The protein was termed as AZ-1 since its expression level was inversely related to the degree of the functional differentiation of adrenocortical cells as described below. The cDNA contained an open reading frame encoding a polypeptide chain consisting of 466 amino acids, which was from a translational initiation codon at position 67 to a termination codon at position 1465. Several nucleotides preceding the methionine codon were consistent with the consensus sequence for translational initiation (33Kozak M. Nucleic Acids Res. 1987; 15: 8125-8148Crossref PubMed Scopus (4168) Google Scholar). A typical polyadenylation signal was found at position 1907. The N-terminal 17-amino acid sequence was predictable as a signal peptide for the secretory pathway (34von Heijne G. J. Mol. Biol. 1984; 173: 243-251Crossref PubMed Scopus (462) Google Scholar). No hydrophobic region large enough to span a membrane was recognized. Possible N-glycosylation sites were present at Asn-77 and Asn-160. These features in the predicted amino acid sequence suggest that AZ-1 is a secretory protein. When searched in the GenBankTM/EMBL/DDBJ data base, the predicted amino acid sequence of the open reading frame had an identity of 897 with human tubulointerstitial nephritis antigen-related protein (TIN-ag-RP) (35Wex T. Lipyansky A. Bromme N.C. Wex H. Guan X.Q. Bromme D. Biochemistry. 2001; 40: 1350-1357Crossref PubMed Scopus (27) Google Scholar) (Fig. 1B), indicating that the identified cDNA encodes a mouse orthologue of the human protein. As Wexet al. (35Wex T. Lipyansky A. Bromme N.C. Wex H. Guan X.Q. Bromme D. Biochemistry. 2001; 40: 1350-1357Crossref PubMed Scopus (27) Google Scholar) discussed the features of amino acid sequence of TIN-ag-RP, the N-terminal one-third of AZ-1 polypeptide was rich in Cys residues and contained EGF-like repeats. The C-terminal two-thirds of AZ-1 polypeptide contained procathepsin B-related sequence with a Ser residue at position 228, which replaced a conserved Cys residue in the active site of cysteine proteinases (Fig. 1A), suggesting that AZ-1 does not have a proteinase activity. These structural features were also found in tubulointerstitial nephritis antigen (TIN-ag) (36Nelson T.R. Charonis A.S. McIvor R.S. Butkowski R.J. J. Biol. Chem. 1995; 270: 16265-16270Abstract Full Text Full Text PDF PubMed Scopus (32) Google Scholar, 37Kanwar Y.S. Kumar A. Yang Q. Tian Y. Wada J. Kashihara N. Wallner E.I. Proc. Natl. Acad. Sci. U. S. A. 1999; 96: 11323-11328Crossref PubMed Scopus (33) Google Scholar, 38Ikeda M. Takemura T. Hino S. Yoshioka K. Biochem. Biophys. Res. Commun. 2000; 268: 225-230Crossref PubMed Scopus (26) Google Scholar) in mammals and also in non-mammalian homologues including Caenorhabditis elegans F26E4.3 protein (39The Caenorhabditis elegans Sequencing and Consortium Science. 1998; 282: 2012-2018Crossref PubMed Scopus (3610) Google Scholar). To verify the size of the polypeptide encoded by the isolated cDNA, we carried out in vitro synthesis of AZ-1 using its cDNA as a template for a coupled transcription and translation reaction in the presence of [35S]Met (Fig.2A, left). As seen, the reaction with the cDNA insert (lane 2) gave a major product with an electrophoretic mobility of 46 kDa, which matched with the predicted molecular mass of the encoded protein. We also constructed a DNA encoding the AZ-1 polypeptide tagged with a FLAG peptide at the C terminus. The resulting FLAG-tagged polypeptide, AZ-1F, showed a band with a mobility of 47 kDa (lane 3). The increase of 1 kDa in molecular mass was consistent with an addition of the FLAG peptide of 8 amino acid residues to the native polypeptide. Additional bands with higher mobilities might be polypeptides where internal Met residues were used for translational initiation. The reaction with the control vector gave no signal under the experimental conditions (lane 1). Transcription-translation reaction products in the absence of radioactive amino acids were examined by immunoblotting employing an anti-