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Monooxygenase X, a Member of the Copper-dependent Monooxygenase Family Localized to the Endoplasmic Reticulum

2004; Elsevier BV; Volume: 279; Issue: 46 Linguagem: Inglês

10.1074/jbc.m407486200

ISSN

1083-351X

Autores

Xiaonan Xin, Richard E. Mains, Betty Eipper,

Tópico(s)

Metal-Catalyzed Oxygenation Mechanisms

Resumo

Based on sequence comparisons, MOX (monooxygenase X), is a member of the copper monooxygenase family that includes dopamine β-monooxygenase (DBM) and peptidylglycine α-hydroxylating monooxygenase (PHM). MOX has all of the residues expected to be critical for copper binding, and its cysteine residues can yield the intramolecular disulfide bond pattern observed in DBM. Although DBM and PHM function within the lumen of the secretory pathway, the published sequence for human MOX lacks a signal sequence, suggesting that it does not enter this compartment. We identified an upstream exon that encodes the signal sequence of human MOX. A retained intron yields minor amounts of transcript encoding MOX without a signal sequence. MOX transcripts are widely expressed, with the highest levels in the salivary gland and ovary and moderate levels in brain, pituitary, and heart. Despite the presence of a signal sequence, exogenous MOX is not secreted, and it localizes throughout the endoplasmic reticulum in both endocrine or nonendocrine cells. Neither appending green fluorescent protein to its C terminus nor deleting the hydrophobic domain near its C terminus facilitates secretion of MOX. MOX is N-glycosylated, is tightly membrane-associated, and forms oligomers that are not disulfide-linked. Based on its sequence and localization, MOX is predicted to hydroxylate a hydrophobic substrate in the endoplasmic reticulum. Based on sequence comparisons, MOX (monooxygenase X), is a member of the copper monooxygenase family that includes dopamine β-monooxygenase (DBM) and peptidylglycine α-hydroxylating monooxygenase (PHM). MOX has all of the residues expected to be critical for copper binding, and its cysteine residues can yield the intramolecular disulfide bond pattern observed in DBM. Although DBM and PHM function within the lumen of the secretory pathway, the published sequence for human MOX lacks a signal sequence, suggesting that it does not enter this compartment. We identified an upstream exon that encodes the signal sequence of human MOX. A retained intron yields minor amounts of transcript encoding MOX without a signal sequence. MOX transcripts are widely expressed, with the highest levels in the salivary gland and ovary and moderate levels in brain, pituitary, and heart. Despite the presence of a signal sequence, exogenous MOX is not secreted, and it localizes throughout the endoplasmic reticulum in both endocrine or nonendocrine cells. Neither appending green fluorescent protein to its C terminus nor deleting the hydrophobic domain near its C terminus facilitates secretion of MOX. MOX is N-glycosylated, is tightly membrane-associated, and forms oligomers that are not disulfide-linked. Based on its sequence and localization, MOX is predicted to hydroxylate a hydrophobic substrate in the endoplasmic reticulum. The copper/ascorbate-dependent monooxygenases constitute a small, but essential family of enzymes that use molecular oxygen and ascorbate to catalyze the hydroxylation of their substrates (EC 1.14.17.x) (1Prigge S.T. Mains R.E. Eipper B.A. Amzel L.M. Cell Mol. Life Sci. 2000; 57: 1236-1259Crossref PubMed Scopus (369) Google Scholar). The defining member of this family of enzymes was dopamine β-monooxygenase (DBM, 1The abbreviations used are: DBM, dopamine β-monooxygenase; PHM, peptidylglycine α-hydroxylating monooxygenase; MOX, monooxygenase X; DBHR, dopamine β-hydroxylase-related; h, human; m, mouse; GFP, green fluorescent protein; CSFM, complete serum-free medium; DBHL, dopamine β hydroxylase-like; PHMcc, peptidylglycine α-hydroxylating monooxygenase, catalytic core; PAM, peptidylglycine α-amidating monooxygenase; EST, expressed sequence tag; ER, endoplasmic reticulum; TES, N-tris (hydroxymethyl)methyl-2-aminoethanesulfonic acid.1The abbreviations used are: DBM, dopamine β-monooxygenase; PHM, peptidylglycine α-hydroxylating monooxygenase; MOX, monooxygenase X; DBHR, dopamine β-hydroxylase-related; h, human; m, mouse; GFP, green fluorescent protein; CSFM, complete serum-free medium; DBHL, dopamine β hydroxylase-like; PHMcc, peptidylglycine α-hydroxylating monooxygenase, catalytic core; PAM, peptidylglycine α-amidating monooxygenase; EST, expressed sequence tag; ER, endoplasmic reticulum; TES, N-tris (hydroxymethyl)methyl-2-aminoethanesulfonic acid. also known as dopamine β-hydroxylase or DBH; EC 1.14.17.1) (2Ikeno T. Hashimoto S. Kuzuya H. Nagatsu T. Mol. Cell Biochem. 1977; 18: 117-123Crossref PubMed Scopus (7) Google Scholar, 3Stewart L.C. Klinman J.P. Annu. Rev. Biochem. 1988; 57: 551-592Crossref PubMed Google Scholar) (see Fig. 1). Hydroxylation of the β-carbon of dopamine consumes 1 mol of oxygen and 2 mol of ascorbate, yielding norepinephrine plus 2 mol of semidehydroascorbate. DBM was purified, sequenced, and studied in detail before it was cloned (4Dhariwal K.R. Black C.D.V. Levine M. J. Biol. Chem. 1991; 266: 12908-12914Abstract Full Text PDF PubMed Google Scholar, 5Kent U.M. Fleming P.J. J. Biol. Chem. 1987; 262: 8174-8178Abstract Full Text PDF PubMed Google Scholar). The second member of this family, peptidylglycine α-hydroxylating monooxygenase (PHM; EC 1.14.17.3), catalyzes the α-hydroxylation of the C-terminal Gly residue in many different secreted peptides (6Cuttitta F. Anat. Rec. 1993; 236: 87-93Crossref PubMed Scopus (51) Google Scholar, 7Eipper B.A. Mains R.E. Annu. Rev. Physiol. 1988; 50: 333-344Crossref PubMed Scopus (241) Google Scholar, 8Eipper B.A. Mains R.E. Glembotski C.C. Proc. Natl. Acad. Sci. U. S. A. 1983; 80: 5144-5148Crossref PubMed Scopus (328) Google Scholar). The 315-amino acid catalytic core of PHM, defined by truncation mutagenesis, is 28% identical to the corresponding region of DBM (9Southan C. Kruse L.I. FEBS Lett. 1989; 255: 116-120Crossref PubMed Scopus (68) Google Scholar). Structural studies on the catalytic core of PHM defined the active site and revealed key roles for six copper-binding ligands (10Eipper B.A. Park L.P. Dickerson I.M. Keutmann H.T. Thiele E.A. Rodriguez H. Schofield P.R. Mains R.E. Mol. Endocrinol. 1987; 1: 777-790Crossref PubMed Scopus (126) Google Scholar, 11Eipper B.A. Quon A.S.W. Mains R.E. Boswell J.S. Blackburn N.J. Biochemistry. 1995; 34: 2857-2865Crossref PubMed Scopus (102) Google Scholar, 12Prigge S.T. Kolhekar A.S. Eipper B.A. Mains R.E. Amzel L.M. Science. 1997; 278: 1300-1305Crossref PubMed Scopus (303) Google Scholar). Enzymes homologous to DBM and PHM are not found in yeast or bacteria.Although the reactions catalyzed by DBM and PHM use very different substrates and produce very different products, the chemistry involved is similar and both enzymes produce products stored in regulated secretory granules and used for intercellular communication. Both enzymes are essential for survival. Genetically engineered mice lacking DBM are not viable (13Thomas S.A. Matsumoto A.M. Palmiter R.D. Nature. 2002; 374: 643-646Crossref Scopus (462) Google Scholar). Mice lacking PHM develop massive edema at about embryonic day 14, with no live progeny produced (14Czyzyk T.A. Morgan D.J. Peng B. Zhang J. Karantzas A. J. Neurosci. Res. 2003; 74: 446-455Crossref PubMed Scopus (13) Google Scholar). Drosophila lacking a functioning PHM gene generally die as late embryos (15Jiang N. Kolhekar A.S. Jacobs P.S. Mains R.E. Eipper B.A. Taghert P.H. Dev. Biol. 2000; 226: 118-136Crossref PubMed Scopus (79) Google Scholar). Both enzymes function in the lumen of the secretory pathway, and both require adequate supplies of substrate, reduced ascorbate, and copper. The vesicular monoamine transporters (VMAT1 and VMAT2) couple uphill transport of dopamine to efflux of protons, to deliver dopamine to the lumen of the secretory pathway (16Nirenberg M.J. Liu Y. Peter D. Edwards R.H. Pickel V.M. Proc. Natl. Acad. Sci. U. S. A. 1995; 92: 8773-8777Crossref PubMed Scopus (124) Google Scholar, 17Ugarte Y.V. Rau K.S. Riddle E.L. Hanson G.R. Fleckenstein A.E. Eur. J. Pharmacol. 2003; 472: 165-171Crossref PubMed Scopus (22) Google Scholar). The peptidylglycine substrates of PHM are produced from prepropeptides synthesized in the endoplasmic reticulum and subject to endo- and exoproteolytic processing as they progress through the secretory pathway (6Cuttitta F. Anat. Rec. 1993; 236: 87-93Crossref PubMed Scopus (51) Google Scholar, 18Oyarce A.M. Eipper B.A. J. Biol. Chem. 2000; 275: 3270-3278Abstract Full Text Full Text PDF PubMed Scopus (11) Google Scholar).A third family member, monooxygenase X (MOX), was identified in a search for genes whose expression was altered in senescent human fibroblasts (19Chambers K.J. Tonkin L.A. Chang E. Shelton D.N. Linskens M.H. Funk W.D. Gene (Amst.). 1998; 218: 111-120Crossref PubMed Scopus (16) Google Scholar). Although key active site residues are conserved, the published sequence for human MOX has no signal sequence, making it difficult to see how it could function in a manner similar to DBM or PHM. A screen for genes involved in neural crest development yielded chicken MOX (also known as dopamine β-hydroxylase-related, DBHR) (20Knecht A.K. Bronner-Fraser M. Dev. Biol. 2001; 234: 365-375Crossref PubMed Scopus (8) Google Scholar), which includes an N-terminal signal sequence. A potential homolog identified in the mouse genome (BAA95089), also includes an N-terminal signal sequence. No substrate has been identified for human or chicken MOX.Because both DBM and PHM produce essential signaling molecules, it was tempting to speculate that MOX plays a similarly important role in signaling. Although very little is known about the MOX protein, transcripts encoding hMOX are prevalent in brain, kidney, and lung, with levels increasing in some lines of senescent fibroblasts (19Chambers K.J. Tonkin L.A. Chang E. Shelton D.N. Linskens M.H. Funk W.D. Gene (Amst.). 1998; 218: 111-120Crossref PubMed Scopus (16) Google Scholar, 20Knecht A.K. Bronner-Fraser M. Dev. Biol. 2001; 234: 365-375Crossref PubMed Scopus (8) Google Scholar). In the developing chick embryo, MOX transcripts are expressed in newly differentiating neural crest cells, most migrating neural crest cells, and in non-neuronal tissues such as the myotome. The properties of the MOX protein have not been explored.We undertook these studies to determine whether a human MOX signal sequence could be identified. Monooxygenase family members present in Caenorhabditis elegans and Drosophila were identified. We next determined the sites of expression of MOX in adult mouse tissues and compared the pattern of MOX expression to that of DBM and PHM. Using several antisera to MOX, we characterized the expression of exogenous MOX in endocrine and non-endocrine cells. The unique features of MOX suggest that it functions in the endoplasmic reticulum.MATERIALS AND METHODSIsolation of RNA and Reverse Transcription-PCR—Tissues dissected from adult male and female C57/bl6 mice were extracted with TRIzol reagent (Invitrogen) for preparation of RNA. cDNA was prepared from total RNA (1 μg) using Superscript II reverse transcriptase (Invitrogen). This cDNA (5% of the product) was amplified using primers specific for mMOX, mDBH, mPHM, and mDBHL. For mMOX: forward primer, 5′-TCACTGCACTCTGGAGTGCCT; reverse primer, 5′-GGTACCCACCCATCTCTCCA. For mDBM: forward primer, 5′-CTGAGGGCAATGAGGCCCTG; reverse primer, 5′-GCCATCCCTGGCGAGCACAG. For mPHM: forward primer, 5′-CGCCAGCCTTGCCCTTGCCC; reverse primer, 5′-TGAGTATGGACTCGGTAGGC. Because an mDBHL transcript was not detected after 38 cycles of amplification, we used a nested PCR with 30 cycles for first primer pair and 25 cycles for the second pair: first forward primer, 5′-CGGATCAGCACCTTCTGGATG; first reverse primer, 5′-CCAGCTCATTGGTCACATAG; nested forward primer, 5′-ACCAAAGATGAGTCGGGAGCA; nested reverse primer, 5′-TTGCAGAGCTTGCACACCC.To study the tissue-specific expression of human MOX mRNA and to verify the N-terminal splice variants, we designed three forward primers: 5′-ACCTATCCGCACCGGACCCT for N-terminal hMOX long form; 5′-GGACCTGATTCCCCAGTTGGA for N-terminal hMOX short form; 5′-GCACTGTGAGAGTGATCTGGG for the common region. A reverse primer (5′-GCCTCTCTGTATCACTGGCTC) in the common region was paired with the three forward primers to generate PCR products specific for long form (620 bp), the short form (600 bp), and both forms (250 bp). Human tissue total RNA was purchased from Clontech, and cDNA was prepared as described above. Amplification with β-actin-specific primers was used as an internal standard (21McPherson C.E. Eipper B.A. Mains R.E. Gene (Amst.). 2002; 284: 41-51Crossref PubMed Scopus (59) Google Scholar).Construction of Expression Vectors—To generate an expression vector encoding full-length mMOX, we used a pair of primers with appended restriction enzyme sites (underlined): the forward primer included a Pci1 site, 5′-TACATGTGCGGCTGGCCACTGCT; the reverse primer included a NotI site, 5′-AGCGGCCGCGTACCCACCCATCTCTCCA. Mouse brain first strand cDNA was used as the PCR template, and the product was subcloned into the PCR II vector (TA-cloning vector, Invitrogen) and digested with Pci1 and NotI (New England Biolabs) to release the 1.8-kb mMOX insert. The pEAK10 expression vector (Edge Biosystems, Gaithersburg, MD) was digested with NcoI and NotI to receive the mMOX insert. The insert was sequenced in its entirety by the Molecular Core at the University of Connecticut Health Center. To visualize mMOX in live cells, we fused GFP to the C terminus of mMOX (MOX-GFP). To evaluate the role of the C-terminal hydrophobic region (Leu597-Leu613), mMOX truncated at Arg596 was fused to GFP (MOX-Arg596-GFP) in the pEGFP-N2 vector (Clontech). Vectors encoding these MOX-GFP fusion proteins were prepared from the mMOX pEAK10 vector using a forward primer that contained the pEAK10 HindIII site just upstream of the translational start site, 5′-TCTCAAGCCTCAGACAGTGGTTCA. Both reverse primers included a KpnI site: for MOX-GFP, 5′-CGGGGTACCGCAAGCCCTGGCTGCTCAGGA; for MOX596s-GFP, 5′-CGGGGTACCACCGGAGGGAGAAAATGCCGTG. The PCR products were subcloned into the TA-cloning vector; inserts released with HindIII and KpnI were inserted into pEGFP-N2 vector digested with these same enzymes and sequenced.MOX Antibodies—Four MOX peptides were synthesized, and each was linked to keyhole limpet hemocyanin using glutaraldehyde: CT45 was raised to mMOX(243SSNFNDSVLDFGHE256); the mMOX(448KDRAVMTWGGLSTR461) and mMOX(346SLFHTIPPGMPEF358) peptides yielded no useable antibodies; CT164 was raised to mMOX(90YFTNADRELEKDAQQDY106). Production of antisera was carried out by Covance (Denver, PA). pEAK RAPID cells transiently transfected with vectors encoding GFP or mMOX were used to characterize the antisera. CT45 recognized native, but not SDS-denatured MOX; no signal was detected on Western blots of transiently transfected cells, but MOX could be visualized in paraformaldehyde-fixed transfected cells or immunoprecipitated from cell extracts. CT164 visualized a band of the expected mass only in cells expressing mMOX; samples prepared by denaturation at 37 °C or 50 °C exhibited substantially less aggregation than samples prepared by denaturation at 100 °C.Transfection of Cells—AtT-20 corticotrope tumor cells were cultured in Dulbecco's modified Eagle's medium containing 10% fetal calf serum, 10% NuSerum (Collaborative Research, Waltham MA). pEAK RAPID cells were cultured in Dulbecco's modified Eagle's medium containing 10% fetal calf serum. For transfection, cells were grown to 80% confluence and rinsed with complete serum-free medium (CSFM) (22Steveson T.C. Zhao G.C. Keutmann H.T. Mains R.E. Eipper B.A. J. Biol. Chem. 2001; 276: 40326-40337Abstract Full Text Full Text PDF PubMed Scopus (21) Google Scholar, 23El Meskini R. Mains R.E. Eipper B.A. Endocrinology. 2000; 141: 3020-3034Crossref PubMed Scopus (26) Google Scholar). Expression vectors were incubated with LipofectAMINE 2000 (4 μg of DNA/10 μl of LipofectAMINE 2000; Invitrogen) for 20 min at room temperature, mixed with Opti-MEM (Invitrogen) and incubated with cells for 4–6 h. Two days after transfection, cells were harvested for analysis.Subcellular Fractionation—Transfected cells were harvested into ice-cold homogenization buffer (150 mm sucrose, 60 mm KCl, 2.5 mm MgCl2, 20 mm HEPES-KOH, pH 7.5) (0.5 ml/well of a 6-well plate) containing protease inhibitors and processed with a ball-bearing homogenizer (H&Y Enterprises, Redwood City, CA). Differential centrifugation yielded a series of pellets (P): P1, 4000 × gav for 5 min; P2, 14200 × gav for 15 min, a fraction enriched in secretory granules; P3, 355000 × gav for 15 min, a fraction enriched in lighter membrane compartments (22Steveson T.C. Zhao G.C. Keutmann H.T. Mains R.E. Eipper B.A. J. Biol. Chem. 2001; 276: 40326-40337Abstract Full Text Full Text PDF PubMed Scopus (21) Google Scholar, 23El Meskini R. Mains R.E. Eipper B.A. Endocrinology. 2000; 141: 3020-3034Crossref PubMed Scopus (26) Google Scholar); and the final supernatant (cytosol). Equal proportions of each fraction were subjected to Western blot analysis.Metabolic Labeling, Extraction, Immunoprecipitation, and Western Blot Analysis—Replica wells of pEAK RAPID cells were transiently transfected with mMOX.pEAK10. After 48 h, cells were rinsed with CSFM, incubated in medium lacking Met for 10 min, and then incubated in medium containing [35S]Met (0.7 mCi/ml) for 30 min (Pulse). After the pulse labeling, cells were rinsed with CSFM and either harvested immediately (Pulse) or further incubated in CSFM for the designed amount of time (Chase). Cells were extracted in 0.5 ml of TM buffer (20 mm sodium N-tris(hydroxymethy)methyl-2-aminoethanesulfonic acid, 10 mm mannitol, pH 7.4, containing protease inhibitors) (24Oyarce A.M. Eipper B.A. J. Cell Sci. 1995; 108: 287-297Crossref PubMed Google Scholar). Following centrifugation at 150,000 × g for 30 min, the supernatant (soluble fraction) was collected and the pellets (particulate fraction) were solubilized with 0.5 ml of TMT buffer (TM with 1% Triton X-100), followed by centrifugation at 150,000 × g for 30 min, and the supernatant was kept in fresh tubes. Spent chase medium was collected, and protease inhibitors were added. For immunoprecipitation, samples were incubated with MOX antibody CT45 or CT164 for 2 h on ice; antigen/antibody complexes were isolated using Protein A-Sepharose beads. Bound proteins were dissociated by boiling the resin in 1× Laemmli sample buffer and fractionated on 4–15% SDS gels (Bio-Rad); gels were soaked in Enhance and Amplify (PerkinElmer Life Sciences/Amersham Biosciences), dried, and exposed to film. For co-immunoprecipitation experiments, pEAK RAPID cells transiently expressing mMOX were extracted as above and MOX was isolated using CT164. Immunoprecipitates were fractionated by SDS-PAGE and visualized using antisera to MOX (CT164, 1:1000), calnexin (BD Biosciences, 1:1000), or calreticulin (BD Biosciences, 1:2500).To assess glycosylation, immunoprecipitates of metabolically labeled soluble and particulate MOX were heated at 100 °C for 5 min in 0.5% SDS, 1% β-mercaptoethanol, and then diluted 2-fold with 50 mm sodium citrate buffer (pH 5.5). After treatment with endoglycosidase H (500 units/μl, New England Biolabs) at 37 °C for 1 h, samples were analyzed using 4–15% SDS-PAGE and fluorography. pEAK RAPID cells transiently transfected with the mMOX.pEAK10 vector for 48 h were pretreated with 2 μg/ml of tunicamycin for 3.5 h. Cells were then incubated in medium containing [35S]Met and processed as described above.Membrane Fractionation and Sodium Carbonate Extraction—Isolation of intracellular membranes and extraction with sodium carbonate was described (25Zhai L. Chaturvedi D. Cumberledge S. J. Biol. Chem. 2004; 279: 33220-33227Abstract Full Text Full Text PDF PubMed Scopus (199) Google Scholar). pEAK RAPID cells were transfected with pEAK10-MOX, pCIS-PAM-3, or pCIS-PAM-1 expression vectors for 48 h and harvested into TM buffer. After a 5-min centrifugation at 5,000 × g to remove debris, the supernatants were centrifuged at 355,000 × g for 30 min to separate the soluble fractions and pellets. Pellets were resuspended in 100 mm sodium carbonate, pH 11.0, for 30 min on ice, followed by centrifugation at 355,000 × g for 1 h to generate sodium carbonate insoluble fraction, which was then sonicated into TMT buffer, and a sodium carbonate soluble fraction. Equal amounts of each fraction were subjected to Western blot analysis with CT164 for MOX, GFP polyclonal antibody (AbCam, 1:2500) for MOX-GFP and MOX-Arg596-GFP, and PAM polyclonal antibody 1761 (1:1000) for PAM-3 and PAM-1.Immunofluorescence—AtT-20 cells transiently transfected with mMOX.PEAK10 or mMOX.GFP vectors were fixed with 4% formalin 48 h after transfection. Following permeabilization with 1% Triton X-100 in phosphate-buffered saline containing 2 mg/ml bovine serum albumin and blocking with 2 mg/ml bovine serum albumin in phosphate-buffered saline, MOX-GFP was visualized directly and MOX was visualized using affinity-purified or crude CT164 (1:1000) and Cy3-anti-rabbit-IgG (Jackson ImmunoResearch Laboratory Inc., 1:1000). At the same time, various marker proteins were visualized: BiP (Affinity BioReagents, rabbit polyclonal, 1:100); ACTH (NOVACASTRA, monoclonal antibody, 1:500); and syntaxin 6 (BD Transduction Laboratories, monoclonal antibody, 1:300). Images were obtained under oil using a 60× lens on a Nikon Eclipse TE300 microscope with a Hamamatsu Orca ER Digital charge-coupled device camera operated in Open Lab (Improvision, Lexington, MA); z-stacks were subjected to deconvolution using Volocity software (Improvision).Sucrose Gradient Fractionation—Continuous sucrose gradients were prepared using solutions of 5 and 20% sucrose in TMT buffer; a cushion of 50% sucrose in the same buffer was placed below the 1878 μl of gradient. Samples of metabolically labeled MOX (250 μl of a 30-min Pulse sample) and culture medium from a stably transfected DBM cell line were placed above the gradient. Before application to the gradients, samples were either treated with dithiothreitol (1 mm for DBM; 10 mm for MOX; 30 min at 37 °C) or not. Gradients were centrifuged for 5 h at 214,000 × g in a Ti-55 swinging bucket rotor (22Steveson T.C. Zhao G.C. Keutmann H.T. Mains R.E. Eipper B.A. J. Biol. Chem. 2001; 276: 40326-40337Abstract Full Text Full Text PDF PubMed Scopus (21) Google Scholar). For calibration, a sample containing cytochrome c, ovalbumin, bovine serum albumin, catalase, and apoferritin (200 μg each) was analyzed simultaneously. After centrifugation, samples were collected starting from the top of the gradient; 1× Laemmli sample buffer was used to recover protein that reached the bottom of the gradient. MOX was immunoprecipitated from each fraction using CT45 and fractionated on a 10% SDS gel. For DBM, equal aliquots of each fraction were fractionated on a 10% SDS gel and detected with a DBM antibody (1:1000, Ab3125).RESULTSDistinguishing Features among Copper Monooxygenase Family Members—Although their catalytic cores are highly homologous, the topologies predicted for MOX, DBM, and PAM are distinctly different (Fig. 1). Although mouse MOX is predicted to have a cleaved signal sequence, human MOX lacks a signal sequence (19Chambers K.J. Tonkin L.A. Chang E. Shelton D.N. Linskens M.H. Funk W.D. Gene (Amst.). 1998; 218: 111-120Crossref PubMed Scopus (16) Google Scholar). Both mouse and human MOX have a stretch of 17 hydrophobic residues near their C termini. DBM has a signal anchor, and its disulfide-linked dimers form tetramers (26Robertson J.G. Adams G.W. Medzihradszky K.F. Burlingame A.L. Villafranca J.J. Biochemistry. 1994; 33: 11563-11575Crossref PubMed Scopus (36) Google Scholar). PAM is a Type 1 membrane protein with an N-terminal signal sequence and a transmembrane domain near its C terminus (27Husten E.J. Eipper B.A. Arch. Biochem. Biophys. 1994; 312: 487-492Crossref PubMed Scopus (29) Google Scholar). Similar disulfide bonding patterns have been identified in DBM and PAM, and these Cys residues are conserved in MOX (Fig. 1).Both the human genome and the Drosophila genome contain three copper monooxygenase family members (Table I). Consistent with their size and predicted topologies, the catalytic core of MOX is more similar to DBM than to PHM. The mouse genome contains a fourth copper monooxygenase family member, DBHL, which is slightly more similar to MOX than to DBM. Although the intron/exon boundaries of mDBHL are similar to those of hMOX and hDBM, a gene resembling mDBHL could not be identified in the human genome. The functions of two of the Drosophila copper monooxygenases have been studied: the catalytic core of dPHM is 39% identical to that of hPHM, and the catalytic core of Drosophila tyramine β-hydroxylase is 50% identical to that of hDBM (Table I). The third family member in Drosophila is almost equally identical to hMOX and hDBM and is designated dMOX. The C. elegans genome encodes only two family members. C. elegans PHM is 38% identical to hPHM. The protein encoded by C. elegans cosmid H13N06 (19021–24000 bp) is 48% identical to hDBM, identifying it as part of the catecholamine biosynthetic pathway. In C. elegans, as in Drosophila, the major function of this enzyme is hydroxylating tyramine to produce octopamine (28Monastirioti M. Linn Jr., C.E. White K. J. Neurosci. 1996; 16: 3900-3911Crossref PubMed Google Scholar).Table IThe copper monooxygenase family (catalytic core)% IdentityhMOXhDBMhPHM%MOX human1003925Mouse814226Drosophila353424DBM human3910028Mouse378027TBH Drosophila345025C. elegans354828PHM human2528100Mouse252895Drosophila252639C. elegans202338DBHL mouse413723 Open table in a new tab Knowledge of the crystal structure of PHMcc and biochemical modification of DBM provide another means of analyzing MOX (Table II). The six ligands that interact with the two essential copper atoms in PHM are completely conserved among all family members. The active site residues that interact with the peptidylglycine substrate have been identified (1Prigge S.T. Mains R.E. Eipper B.A. Amzel L.M. Cell Mol. Life Sci. 2000; 57: 1236-1259Crossref PubMed Scopus (369) Google Scholar, 12Prigge S.T. Kolhekar A.S. Eipper B.A. Mains R.E. Amzel L.M. Science. 1997; 278: 1300-1305Crossref PubMed Scopus (303) Google Scholar). In addition, molecular modeling has predicted residues in DBM that may interact with its substrate, dopamine (1Prigge S.T. Mains R.E. Eipper B.A. Amzel L.M. Cell Mol. Life Sci. 2000; 57: 1236-1259Crossref PubMed Scopus (369) Google Scholar). When the homologous residues in MOX are identified, it is striking that all are hydrophobic. For example, Arg240, which binds the peptide carboxylate in PHMcc, is Gln395 in hDBM and Leu319 in hMOX. Similarly, Asn316 in PAM is another Leu in MOX. If the active site of MOX resembles that of PHM, its substrate is unlikely to be charged or highly polar.Table IICopper and substrate binding residues Rat PHM: CuA, CuB, and substrate interacting residues.View Large Image Figure ViewerDownload (PPT) Open table in a new tab Expression of MOX Is Widespread—We next set out to determine where MOX was expressed and to compare its expression pattern to those of PAM and DBM (Fig. 2). Total RNA prepared from the indicated adult mouse tissues was reverse-transcribed, and the cDNA used as a PCR template (Fig. 2). Genespecific primers were designed for PAM (420 bp), DBM (520 bp), MOX (420 bp), and DBHL (702 bp). MOX transcripts were readily identified in a wide variety of adult tissues. Levels were highest in the salivary gland and ovary, with lower levels in olfactory bulb, cerebellum/brain stem, parietal cortex, pituitary, atrium, ventricle, adrenal, thymus, testis, and kidney. Still lower levels of MOX transcript were detected in lung, lymph nodes, spleen, and liver (Fig. 2). Expression of DBM is much more restricted, with significant signal detected only in adrenal and detectable amounts in brainstem. As expected from similar studies in the rat, PAM transcripts are widely expressed, with highest levels in pituitary, atrium, ventricle, lung, and adrenal (Fig. 2). Expression of DBHL is very low and highly restricted. Signal was detected only in the thymus and the testis, and only after a nested PCR; the smaller PCR product detected in the testis could be nonspecific or a splice variant.Fig. 2MOX transcripts are expressed in a wide variety of adult tissues. cDNA prepared from the adult mouse tissues indicated was reverse-transcribed and used for PCR. Primer sets specific for mouse MOX, DBM, PAM, and DBHL were used to compare patterns of expression. PCR amplification: PAM, 25 cycles; DBM, 30 cycles; MOX, 35 cycles; DBHL with nested PCR, 30 cycles followed by 25 cycles. Actin levels in all samples were comparable (data not shown).View Large Image Figure ViewerDownload (PPT)hMOX Can Include an N-terminal Signal Sequence—Following the identification of human MOX (hMOX; GenBank™ accession number AY007239), homologous genes were identified in mouse (mMOX; GenBank™ accession number: BAA95089), and chicken (GenBank™ accession number AF327450) (20Knecht A.K. Bronner-Fraser M. Dev. Biol. 2001; 234: 365-375Crossref PubMed Scopus (8) Google Scholar). Based on SMART and Signal P analyses, both mouse MOX (mMOX) and Gallus MOX include N-terminal signal sequences (smart.embl-heidelberg.de; www.cbs.dtu.dk/services/SignalP-2.0); cleavage of the mMOX signal sequence is predicted to occur after Gly19 (Fig. 3A, open arrow) In addition to lacking a signal sequence, the published sequence for hMOX (19Chambers K.J. Tonkin L.A. Chang E. Shelton D.N. Linskens M.H. Funk W.D. Gene (Amst.). 1998; 218: 111-120Crossref PubMed Scopus (16) Google Scholar) appeared to lack another 67 a

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