O-MACS, a novel member of the medium-chain acyl-CoA synthetase family, specifically expressed in the olfactory epithelium in a zone-specific manner Yuichiro Oka, Ko Kobayakawa, Hirofumi Nishizumi, Kazunari Miyamichi, Satoshi Hirose, Akio Tsuboi and Hitoshi Sakano Department of Biophysics and Biochemistry, Graduate School of Science, The University of Tokyo, CREST Program of Japan Science and Technology Corporation, Japan In rodents, the olfactory epithelium (OE) can be divided into four topographically distinct zones, and each member of the odorant receptor (OR) gene family is expressed only in one particular zone. To study the functional significance of the zonal structure of the OE, we searched for genes expressed in a zone-specific manner by using the differential display method. Among the clones isolated from the rat OE, we characterized a novel olfactory protein termed O-MACS, a member of the medium-chain acyl-CoA synthetase family. The o-macs gene encodes a protein of 580 amino acids, sharing 56–63% identity with other MACS family proteins. RT-PCR analysis demonstrated that the o-macs gene is expressed only in the OE, unlike other MACS family genes. In situ hybridization revealed that the o-macs transcripts are present in the neuronal cell layer of olfactory sensory neu- rons (OSNs) as well as in the supporting and basal cell layers in the most dorso-medial area (zone 1) of the OE. Develop- mental analysis revealed that the o-macs gene is already expressed on embryonic day 11.5, before the onset of the OR gene expression, in a restricted area within the rat olfactory placode. Recombinant O-MACS protein tagged with c-Myc and His6 demonstrated an acyl-CoA synthetase activity for fatty acid activation, and protein localization to mitochon- dria like other MACS family proteins. The present study indicates that this novel protein may play important roles in processing odorants in a zone-specific manner, or the zonal patterning of the OE during development. Keywords: differential display method; olfactory epithelium; odorant receptor; odorant processing; medium-chain acyl- CoA synthetase. The olfactory system of mammals can recognize a variety of different odorants with G-protein-coupled odorant recep- tors (ORs) [1–3]. A multigene family encoding hundreds of related OR molecules was first identified in rat [4]. It has been reported that each olfactory sensory neuron (OSN) expresses only one OR gene in a mono-allelic manner [5–8]. In situ hybridization revealed that the olfactory epithelium (OE) can be divided into four topographically distinct zones, and that each OR gene is expressed in one particular zone [9,10]. Furthermore, OSNs expressing the same OR gene project their axons to a pair of glomeruli on the lateral and medial sides of the olfactory bulb (OB) [11–13]. Thus, odorant stimuli that activate a specific set of OSNs in the OE are converted to a topographic map of activated glomeruli on the OB [14]. Although the zone-to-zone correlation between the OE and the OB has been proposed, the mechanisms that control the zone-specific expression of and zonal projection for ORs are still largely unknown. In order to study the functional significance of the zonal structure, we have isolated genes that are expressed differentially between the most dorso-medial and the most ventro-lateral zones of the rat OE by using the differential display (DD) method. In the present study, we have analyzed a novel gene termed o-macs, a member of the medium-chain acyl-CoA synthetase (MACS) gene family. In situ hybridization revealed that o-macs is expressed specifically in the olfactory system, in the most dorso-medial area (zone 1) of the OE. It is known that acyl-CoA synthetase is involved in the initial step of fatty acid metabolism, i.e., the reaction of fatty acid with CoA to produce acyl-CoA on the outer membrane of mitochondria. Acyl-CoA is then transported into the matrix for b-oxidation of acyl-group. Among the MACS family proteins with the acyl-CoA synthetase activity, two murine proteins, MACS1 and SA, were detected in the liver and Correspondence to H. Sakano, Department of Biophysics & Biochemistry, Graduate School of Science, The University of Tokyo, 2-11-16 Yayoi, Bunkyo-ku, Tokyo, Japan. Fax: +81 3 5689 7240; Tel.: +81 3 5689 7239; E-mail: sakano@mail.ecc.u-tokyo.ac.jp Abbreviations: AceCS, acetyl-CoA synthetase; AST-IV, aryl sulfo- transferase IV; DIG, digoxigenin; DD, differential display; GST, glutathione S-transferase; IVD, isovaleryl-CoA dehydrogenase; LACS1, long-chain acyl-CoA synthetase 1; MACS, medium-chain acyl-CoA synthetase; OB, olfactory bulb; OE, olfactory epithelium; OMP, olfactory marker protein; OP, olfactory placode; OR, odorant receptor; OSN, olfactory sensory neuron; PST, phenol sulfotrans- ferase; RE, respiratory epithelium; VNE, vomeronasal epithelium; VLACS, very long chain acyl-CoA synthetase. Enzymes: Medium-chain acyl-CoA synthetase (EC 6.2.1.2). Notes: DNA databank of Japan (DDBJ) accession number for the O-MACS sequence is AB096688. (Received 24 December 2002, revised 10 March 2003, accepted 14 March 2003) Eur. J. Biochem. 270, 1995–2004 (2003) Ó FEBS 2003 doi:10.1046/j.1432-1033.2003.03571.x Table 1. Summary of differential display screening. The cDNA pools from zone 1 and zone 4 of the rat olfactory epithilium (OE) were compared with the differential display (DD) method. Among bands displayed, about 0.5% bands showed difference between the two zones. The DNA fragments were cloned and subjected to the sequencing analysis, indicating that they are classified into six categories (Intracellular enzymes, Secreted/membrane proteins etc.) based on their predicted structures and functions. The number of clones in each category is indicated in parentheses where cases are not named in table. The expression zone of each clone analyzed by in situ hybridization is indicated in ÔZoneÕ columns; ND, not detected in the OE; NP, not performed. The expression cell layers are indicated in ÔCell layerÕ columns as S, supporting cells; N, olfactory sensory neurons; B, basal cells; L, lamina propria. *, Patched expression. The expression in tissues other than the OE is also shown in ÔCell layerÕ column as BG, Bowman’s gland; LNG, lateral nasal grand; RE, respiratory epithelium. Clone preferentially amplified in zone 1 in DD Zone Cell layer Clone preferentially amplified in zone 4 in DD Zone Cell layer Intracellular enzymes Rat phenol sulfotransferase 1 S Rat 1-Cys-peroxiredoxin 2<3<4 S,N,B Rat paraoxonase 1 S Rat oxidative 17-b hydroxysteroid deydrogenase type 6 ND – Rat very long chain acyl-CoA synthetase 1>2>3 S,N,B Rat nucleoside diphosphate kinase ND – Rat glutathione transferase subunit 8 ND – Rat aryl sulfotransferase IV 2<3<4 S,N,B Rat RY2D1 odorant metabolizing protein ND BG Rat Isovaleryl-CoA dehydrogenase 2,3,4 S* Rat o-macs 1 S,N,B,L Rat glutathione S-transferase-Yb 2<3<4 S,N,B Mouse carbonyl reductase 2 like 1>2>3>4 S,N Rat glutathione S-transferase P ND – Mouse crystalline mu like ND – Rat cytochrome P-450 isozyme 5 ND – Mouse spermine synthase (Sms) like ND – Rat aldehyde dehydrogenase 1 ND – Rat epoxide hydrolase ND – Rat membrane lipid desaturase 1<2<3<4 S Secreted/membrane proteins Rat RY2G5 potential ligand binding protein ND BG Rat RYF3 (vomeromodulin) ND LNG Rat mucin-like protein 1,2,3 S Rat RYA3 potential ligand binding protein ND BG Rat cocaine, amphetamine-regulated transcript 1>2 N Rat RYD5 potential ligand binding protein ND BG Rat protocadherin-7c 1>2>3 N* Rat neuromedin U ND – Rat secretogranin III 1<2<3<4 N Rat Apolipoprotein E ND L Rat osteopontin ND Bone Rat Na + /K + -ATPase a subunit 1<2<3<4 N Mouse MOR10 like 4 N* Mouse Ym1 like 2,3,4 S* Signal transduction Rat guanine nucleotide releasing protein ND – Rat IGF binding protein 5 ND – Rat FGF receptor activating protein 1 ND – Rat PKC delta-binding protein ND – Rat cellular retinoyl-binding protein ND – Mouse rho (NET1A) like ND – Transcription factor (0) Mouse GATA-2 like ND – Mouse forkhead box C1 like ND – Mouse BSPRY like ND – 1996 Y. Oka et al. (Eur. J. Biochem. 270) Ó FEBS 2003 kidney with different substrate specificities [15]. In contrast to these MACS family proteins, O-MACS is detected neither in the liver nor the kidney, but specifically in the OE in a zone-specific manner. Although O-MACS demonstra- ted MACS activity for the straight and saturated-fatty acids like other MACS proteins, its substrate preference was shown to be fatty acid lengths of C 6 –C 12 . Here we report the initial characterization of the novel protein, O-MACS, and discuss its possible roles in olfaction and in the development of the olfactory system. Experimental procedures cDNA cloning of o-macs Studies were performed in accordance with the guidelines for animal experiments at the University of Tokyo. Three- week-old-male Wistar rats were anesthetized with pento- barbital sodium (10 mg per animal) and decapitated. Olfactory epithelia were dissected and embedded. After sections (40-lm thick) were prepared, tissue pieces corresponding to zone 1 and zone 4 were excised with scalpels and collected in 1.5 mL tubes. Total RNA was extracted with RNeasy kit (Qiagen, Hilden, Germany). After DNase I digestion, cDNAs were synthesized with three sets of poly(dT) primers, gT 15 A, gT 15 CandgT 15 g, using SuperScript TM II reverse transcriptase (Invitrogen, Groningen, Netherlands). Fluorescent DD screening was performed as described [16]. The prospective cDNA fragments were cloned into a pGEM-T vector (Promega, Madison, WI, USA) and sequenced. The Full-length cDNA of rat o-macs was isolated with a direct cDNA selection method [17] from the full-length cDNA pools prepared with SMART TM PCR cDNA synthesis kit (Clontech, Palo Alto, CA, USA). The isolated cDNA was then cloned into a pGEM-T vector. RT-PCR analysis Total RNAs were extracted from tissues of 3-week-old Wistar rat by RNeasy kit (Qiagen) and subjected to cDNA synthesis. The PCR primers for o-macs, SA, KS, KS2 and MACS1 are as follows: o-macs,5¢-atgaaggttctcctccgctg-3¢ (forward), 5¢-gcctttcgggacaaggagcc-3¢ (reverse); SA 5¢-aggt gttttcagcgcctagc-3¢ (forward), 5¢-caccattactctgtctcctc-3¢ (re- verse); KS 5¢-ccttctggggcactgagatg-3¢ (forward), 5¢-agaac gcatgcagccgaggg-3¢ (reverse); KS2 5¢-tggtagctacctggga agcc-3¢ (forward), 5¢-gaagcaccagactcattctg-3¢ (reverse); MACS1 5¢-gagttggagctccaagctgg-3¢ (forward), 5¢-tgatccctgtt cgcatgcag-3¢ (reverse). In situ hybridization Olfactory epithelia were dissected from Wistar rats on embryonic (e) days 12, 14, 16, 18 and postnatal (P) day 21. Paraformaldehyde (PFA)-fixed sections (for P21 animals) or fresh frozen sections (for embryos) (10 lm) were hybridized with digoxigenin (DIG)-labeled antisense RNA probes for the o-macs, OMP, OCAM, NCAM,and NeuroD genes. cDNA fragments for the rat OMP, OCAM, NCAM,andNeuroD were prepared by RT-PCR using pairs of primers: OMP 5¢-gagtagagagcctgaagcag-3¢ Table 1. (Continued). Others Rat muscle myosin light chain 2 ND – Rat alpha-tropomyosin 2 ND – Mouse Mpv-17 like protein like 1 S,N,B,L Rat RNA binding protein (transformer-2-like) ND – Mouse CAAA01033971 region 1 S,N,B,L Rat interferon-inducible protein ND – Human reticulon3 (RTN3) like ND – Rat metallothionein-1, pseudogene a ND – Chick vitelline membrane outer layer protein like ND – Rat mitchondria ATP synthase beta subunit ND – EST (6) ND – Mouse HMG 17 like ND – Repeat (2) NP – Mouse heat shock protein 20-like protein like ND – Mouse interferon regulatory factor 6 like ND – Mouse palate unknown like ND RE Human T-plastin like ND – EST (8) ND – Repeat (6) NP – No homology (10) ND – Clone #B10g 3<4 S Others (21) ND – Ó FEBS 2003 Zone-specific expression of olfactory MACS protein (Eur. J. Biochem. 270) 1997 (forward), 5¢-ggttaaacaccacagaggcc-3¢ (reverse); OCAM 5¢-gagaagtggtgtcccctcaa-3¢ (forward), 5¢-cctccatcatcttgctt ggt-3¢ (reverse); NCAM 5¢-cttcctgtgtcaagtggcag-3¢ (for- ward), 5¢-gttggcagtggcattcacga-3¢ (reverse); and NeuroD 5¢-aagacgcatgaaggccaatg-3¢ (forward), 5¢-catgatgcgaatggct atcg-3¢ (reverse). Hybridization, washing, antibody reaction and color detection were performed as described previously [17], except that Proteinase K digestion was omitted for embryonic samples. The hybridized sections were photo- graphed with an Olympus AX70 microscope (Olympus Optical, Tokyo, Japan) after counterstaining with 0.01% methyl green. Recombinant protein expression, purification and MACS activity assay The coding sequences of the rat o-macs and murine MACS1 genes were prepared by RT-PCR using a pair of primers: EcoRI-rO-MACS-U (5¢-GAATTCatgaaggttctcctccactg-3¢) and rO-MACS-XhoI-D (5¢-CTCGAGtgcccgtccccactcctggt-3¢) for o-macs; EcoRI-mMACS1-U (5¢-GAATTCatgcagtggc tgaagagttt-3¢) and mMACS1-XbaI-D (5¢-TCTAGAtagct gaccaaactccttg-3¢)forMACS1, and cloned into the EcoRI/ XhoIorEcoRI/XbaI site of a pcDNA3.1/myc-His vector version A (Invitrogen), respectively. Plasmids were trans- fected into HEK293T or HeLa cells using the nonliposomal transfection reagent FUGENE6 (Roche Diagnostics, Mannheim, Germany) [18]. For purification, transfected HEK293T cells were cultured for 60 h. After washing with NaCl/P i , cells were lysed in lysis buffer [50 m M NaH 2 PO 4 (pH 8.0), 150 m M NaCl, 0.1% Triton X-100, 20 m M imidazole, 16 lgÆmL )1 benzamidine HCl, 10 lgÆmL )1 aprotinin, 10 lgÆmL )1 leupeptin, 10 lgÆmL )1 pepstatin A, 1 m M phenylmethanesulfonyl fluoride], and subjected to sonication. The His6-tagged proteins in cell lysate were bounded with Ni-NTA (Qiagen) and eluted with elution buffer (50 m M NaH 2 PO 4 , 150 m M NaCl,0.1%Tween20,and1 M imidazole). After eluent was dialyzed against buffer A(50 m M NaH 2 PO 4 , 150 m M NaCl), the protein solution was concentrated with Centricon Ò YM-50 (Millipore, Bedford, MA, USA). The protein content was determined with protein assay kit (Bio-Rad, Hercules, CA, USA) using bovine serum albumin (BSA) as a control. For Western blotting, 5 lg of purified proteins were subjected to SDS/PAGE (8%), and immunostained with monoclonal antibodies for c-Myc (1 : 1000; Sigma) and alkaline phosphatase-conjugated anti-mouse IgG (1 : 1000, Promega). Acyl-CoA synthetase activities were assayed with a spectrometric method as described [19] using the rO-MACS (1 lg) or mMACS1 (0.1 lg) protein in a 100-lL reaction containing 0.5 m M of substrate. Fig. 1. Comparison of MACS (medium-chain acyl-CoA synthetase) family proteins. (A) Pre- dicted amino acid sequences are compared for the rat (r), mouse (m) and human (h) O-MACS. Dots indicate identical amino acid residues with the rat O-MACS. Sequences for the AMP-binding motif are shaded. (B) Den- drogram of acyl-CoA synthetase family proteins. The tree was generated with a CLU- STAL X program using amino acid sequences of the mouse very long-chain acyl-CoA synthe- tase (mVLACS), human long-chain acyl-CoA synthetase 1 (hLACS1), mouse medium- chain acyl-CoA synthetase proteins (mSA, mMACS1, mKS, and mKS2), mouse acetyl- CoA synthetase 1 (mAceCS1), and O-MACS proteins from rat (r), mouse (m) and human (h). O-MACS belongs to the subtree of the MACS proteins (shaded). (C) Genomic organization of the mouse o-macs and other MACS family genes. They are linked in tan- dem to the 7F1 region of the mouse chromo- some 7. The o-macs gene is composed of 14 exons including 12 coding exons. (D) Tenta- tivelinkagemapofthehumanMACSgene family. The human o-macs gene is located on chromosome 12, whereas other MACS family genes are clustered on chromosome 16. Bars indicate exons. 1998 Y. Oka et al. (Eur. J. Biochem. 270) Ó FEBS 2003 Immunocytochemistry Transfected HEK293T or HeLa cells were cultured over- night on cover slides and stained with 500 n M of Mito- TrackerGreen TM (Molecular Probes) at 37 °C for 1 h. Cells were fixed with 4% paraformaldehyde in NaCl/P i for 10 min at room temperature. After washing three times with NaCl/P i , cells were permeabilized with 0.2% Triton X-100 for 5 min, and blocked in NaCl/P i containing 1% BSA for Fig. 2. In situ hybridization of the olfactory and vomeronasal epithelia. Coronal sections of the rat OE (A) and VNE (B) were hybridized with DIG-labeled antisense probes of OMP, OCAM and o-macs.The o-macs gene is specifically expressed in the zone 1 (OCAM negative) of theOE.Thesenseprobeofo-macs was used as a negative control. Enlarged regions are boxed in the low-magnification figures. The o-macs transcripts are detected in all cell layers; supporting cell layer (s), OSN layer (n), basal layer (b), and lamina propria (lp). The o-macs mRNA was not detected in the apical (a) or basal (b) layer of the VNE. Scale bars indicate 1 mm and 100 lm for the low magnification figures of the OE and VNE, respectively. Bars in the high magnification figures indicate 10 lm. Fig. 3. OE-specific expression of the o-macs gene. (A) RT-PCR ana- lysis of the transcripts from the o-macs and other MACS family genes. Total RNA was isolated from the rat OE, brain, thymus, lung, heart, liver, kidney, testis and spleen. The o-macs gene is expressed specifically in the OE, while other MACS gene transcripts are found in the liver and kidney. The OE-specific expression of o-macs was also confirmed with Southern hybridization shown below the RT-PCR profile. (B) In situ hybridization of the rat OE. Coronal sections of the OE were hybridized with DIG-labeled antisense probes of o-macs, OMP, MACS1 and SA.TheMACS1 and SA transcripts are found in the respiratory epithelium (dotted lines). Scale bar indicates 100 lm. Ó FEBS 2003 Zone-specific expression of olfactory MACS protein (Eur. J. Biochem. 270) 1999 15 min. Monoclonal antibodies for c-Myc (1 : 1000; Sigma) were used for immunostaining. The immunoreactivity was detected with Rhodamine-conjugated anti-mouse IgG (1 : 1000; Chemicon Int, Temecula, CA, USA), and ana- lyzed with a confocal microscope (Fluoview FV500, Olym- pus). Other procedures are as described [18]. Results Cloning of cDNA encoding the OE-specific medium-chain acyl-CoA synthetase, O-MACS To isolate genes expressed in a zone-specific manner in the rat OE, RNA transcripts were compared between the most dorso-medial and ventro-lateral areas, zones 1 and 4, respectively. Two pieces of OE tissues, one from zone 1 and the other from zone 4, were isolated for the total RNA preparation. With three different sets of oligo(dT) anchor primers, cDNA pools were synthesized and subjected to the DD of PCR products. Using 500 arbitrary primer sets, we examined about 50 000 bands, 266 of which amplified differently between the two zones, 1 and 4. Cloning and sequencing of the differently amplified bands revealed 112 independent genes: 37 genes for zone 1 and 75 genes for zone 4. In situ hybridization of the rat OE with DIG-labeled RNA probes demonstrated the region-specific signals for 20 genes. In Table 1, these genes are classified into six different categories based on their predicted structures and functions. Among prospective clones, a novel gene, termed o-macs, specifically expressed in zone 1 was chosen for further analysis. To characterize the rat o-macs gene, we isolated the full- length cDNA using a direct cDNA selection method. The isolated clone of 2084 base pairs contained one open reading frame encoding a protein of 580 amino acid residues (Fig. 1A). A BLASTN search identified the mouse and human o-macs orthologues. Identities of the deduced amino acid sequences are 96% between the rat and mouse and 85% between the rat and human. We also found that the rat O-MACS shares significant sequence identity (56–63%) with the murine MACS family proteins, e.g. SA, KS, KS2 and MACS1. Identity to the murine acetyl-CoA synthetase (AceCS) family was much less (20–30%). These observa- tions indicate that the O-MACS protein is a novel member of the acyl-CoA synthetase family, belonging to the subtree of the MACS family proteins (Fig. 1B). Fig. 4. The o-macs expression during develop- ment. Olfactory placode (OP) or OE sections of rat embryos were hybridized with the DIG-labeled antisense probes for the o-macs, NeuroD and NCAM genes. Boxes in the low- magnification figures indicate the enlarged areas in the high-magnification figures. The o-macs expression is restricted to the dorso- medial part of the OP and OE, whereas the NeuroD and NCAM transcripts are found in the entire OP and OE areas. The o-macs transcripts are found in all cell layers of the OP andOEincontrasttotheNCAM transcripts. Scale bars in the low and high magnification figures indicate 500 lmand50lm, respect- ively. D, dorsal; A, anterior; P, posterior; M, medial. 2000 Y. Oka et al. (Eur. J. Biochem. 270) Ó FEBS 2003 Using the mouse genome database, we then analyzed the genomic organization of the murine o-macs gene. The mouse o-macs is located in the 7F1 region of chromosome 7 and is composed of 14 exons including the 5¢-and3¢-non coding regions. To our surprise, o-macs is linked closely to other MACS family genes for SA, KS, KS2 and MACS1 (Fig. 1C). Transcriptional orientations of these genes are the same, and exon/intron organizations are conserved among them, suggesting that these genes were generated by repeated gene duplication during evolution from a common ancestral gene. It is interesting that the human o-macs gene is located on chromosome 12, whereas other members of the MACS gene family appear to be clustered on chromosome 16 (Fig. 1D), although the human genome database (NCBI) is still incomplete. The OE-specific expression of the o-macs gene We examined the zone-specificity of o-macs expression by in situ hybridization, using the olfactory-specific cell adhe- sion molecule (OCAM) as the zone 1-negative marker [20]. The gene probe for olfactory marker protein (OMP) was used to detect OSNs [21]. The o-macs transcripts were found only in the OCAM-negative region, i.e. zone 1 of the rat OE (Fig. 2A). The o-macs mRNA was not detected in the vomeronasal epithelium (Fig. 2B). The OE is composed of three layers of different cell types: the supporting cells, OSNs and basal cells (reviewed in [22]). Unlike other zone-specific olfactory genes, e.g. OR genes, o-macs is expressed in all three cell layers of the OE (Fig. 2A), although the level of expression in the OSN layer is somewhat lower than those in other cell-layers. The o-macs transcripts were also detected in the cells within the lamina propria in a graded manner from zone 1 to zone 2 (Fig. 2A). We then examined the expression of o-macs and other MACS family genes in various tissues. RT-PCR analysis revealed that o-macs is expressed specifically in the rat OE (Fig. 3A). In contrast, other MACS family genes, SA, MACS1, KS and KS2, are expressed mainly in the liver and kidney (Fig. 3A). Lower expression was detected in the OE for the SA and MACS1 genes, probably due to contamin- ation of the OE preparation with the respiratory epithelium (RE). In situ hybridization analysis confirmed that RNA transcripts for the SA and MACS1 genes are present only in the RE area of the sections, but not in the OMP positive OE (Fig. 3B). In contrast, the o-macs transcripts are not found in the OMP negative RE. Fig. 5. Isolation of recombinant O-MACS and MACS1 proteins. (A) Plasmid constructs for O-MACS and MACS1 proteins tagged with c-Myc and His6. Each cDNA of the rat o-macs or the murine MACS1 was cloned into pcDNA3.1/myc-His vector and was expressed under the control of CMV promoter. E, EcoRI; Xh, XhoI; Xb, XbaI. (B) SDS/PAGE profiles of the purified rat O-MACS and murine MACS1 proteins. Eluents from Ni 2+ -column were subjected to 8% SDS/ PAGE and stained with Coomassie Brilliant Blue or with anti-c-Myc Igs. Dots indicate the recombinant proteins. Molecular size markers are indicated in kDa on the left. (C) MACS activities for fatty acids. Substrate specificities were examined for the purified rO-MACS and mMACS1. Enzymatic activities were determined with the spectro- metric method. C 2 , acetate; C 4 ,butanoate;C 6 ,hexanoate;C 8 , octanoate; C 10 ,decanoate,C 12 , dodecanoate, C 16 , palmitate, BA, benzoate. The data are means ± SD of triplicate assays. Ó FEBS 2003 Zone-specific expression of olfactory MACS protein (Eur. J. Biochem. 270) 2001 Expression of o-macs in the olfactory placode during development To study the o-macs expression during development, we performed in situ hybridization analysis of rat embryonic sections. The o-macs transcripts were first detected in the olfactory placode on embryonic day 11.5 (e11.5) speci- fically in the dorso-medial area of the OE (Fig. 4). The region-specific expression of o-macs was similarly found at e12, e14, e16, and e18. It should be noted that the o-macs transcripts were detected in all cell layers regardless of their positions in apical-basal axis (Fig. 4, high magnifi- cations). In contrast, OR gene transcripts started to appear at e14 only in the neuronal layer of OSNs (data not shown). Transcripts for the neuronal cell adhesion molecule (NCAM) and NeuroD, a transcription factor with a basic helix-loop-helix motif, were detected after e14 and e11.5, respectively, without the zonal specificity (Fig. 4). The present study demonstrates that zonal specification of the OE is already established as early as e11.5, prior to the expression of OR genes and before the invagination of the olfactory pit. O-MACS protein catalyzes the acyl-CoA synthesis of C 6)12 fatty acids We purified the recombinant O-MACS protein to exam- ine whether it has an acyl-CoA synthetase activity like other MACS family proteins. The recombinant plasmid carrying either the rat o-macs or the murine MACS1 cDNA was transfected into HEK293T cells. The recom- binant proteins contained the c-Myc and His6 tags (Fig. 5A). Each tagged, O-MACS or MACS1, protein was purified with Ni 2+ -column (Fig. 5B) and used to determine the chain-length preference of fatty acids. As showninFig.5C,therecombinantO-MACSmediated the CoA addition to the C 6)12 fatty acids, while the recombinant MACS1 demonstrated the preference for the C 6 and C 8 chain-lengths (Fig. 5C, ref [15]). Furthermore, MACS1 catalyzed the benzoic acid-CoA synthesis, whereas the O-MACS protein did not (Fig. 5C, ref [15]). It should be noted that copurified proteins did not contain the c-Myc tag (Fig. 5B) and showed no MACS activity (data not shown). O-MACS is localized to mitochondria We then studied the subcellular localization of the recom- binant O-MACS using MACS1 as a control. It has been reported that the MACS1 protein is localized to mitochon- dria in the mouse kidney [15]. The plasmid carrying either the rat o-macs or the murine MACS1 gene was transfected into HeLa cells, which were then stained with the mito- chondrial marker (MitoTrackerGreen TM ) and anti-(c-Myc) Igs. Recombinant proteins were tagged with both c-Myc and His6 (Fig. 5A). As red immuno-staining signals (for the recombinant proteins) were completely overlapped with green fluorescence (for mitochondria), it was concluded that the recombinant O-MACS, like MACS1, is localized to mitochondria in HeLa cells (Fig. 6). The same results were obtained also with HEK293T cells (data not shown). Discussion In order to study the functional significance of the zonal structure of the OE, we have searched for genes expressed in a zone-specific manner with the DD method. Among 112 prospective clones, 20 genes were shown to be expressed in a zone-specific or graded manner in the OE by in situ hybridization analysis (Table 1). Although many candidate clones were difficult to characterize further due to the limited levels of expression, we were able to identify some zone-specific genes coding for the intracellular enzymes, e.g. phenol sulfotransferase (PST), glutathione S-transferase (GST), aryl sulfotransferase IV (AST-IV), paraoxonase 1 (aryl-esterase), and 1-Cys peroxiredoxin (acidic calcium- independent phospholipase A2) (Table 1). It has been reported that PST may be involved in the olfactory perireceptor processes such as odorant clearance or xeno- biotic detoxification [23]. Other enzymes expressed in a zone-specific or graded manner may also be involved in detoxification or processing of odorants. Zonal distribution of such enzymes may reflect the zone-specific expression of OR molecules whose ligands need to be processed or detoxified in the OE. This idea may be applied to the following carboxylic acid-metabolizing enzymes with dif- ferent substrate specificities: the very long chain acyl-CoA synthetase (VLACS) expressed in a graded manner in zones 1 > 2 > 3; isovaleryl-CoA dehydrogenase (IVD) Fig. 6. Subcellular localization of the recom- binant O-MACS protein. Cells were stained with Rhodamine (red) for the c-Myc tag and with MitoTrackerGreen TM for mitochondria, and analyzed by a confocal microscope. Red fluorescent signals for the recombinant O-MACS protein were overlapped with the green fluorescent signals for mitochondria. The murine MACS1 tagged with c-Myc was also analyzed as a control. Bright views show the positions of cells. Magnification, 400 ·. 2002 Y. Oka et al. (Eur. J. Biochem. 270) Ó FEBS 2003 expressed in three zones 2, 3 and 4; and O-MACS, a novel protein characterized in this study that is specifically expressed in zone 1 (Table 1). Like other MACS proteins, O-MACS has the MACS activity for fatty acid activation, and is localized to mitochondria. Although O-MACS shares many biochemical characteristics with other MACS family proteins, only O- MACS is olfactory-specific and expressed in the OE in a zone-specific manner. This may suggest a unique function for O-MACS in the olfactory system. Electrophysiology and imaging studies demonstrated that fatty acids are received as odorants in the OE, and activate a particular set of glomeruli in the dorsal region of the OB [24–29]. It is possible that fatty acids are received by OSNs in zone 1 and metabolized by O- MACS which catalyzes the first step of the b-oxidation in mitochondria. It is also possible that O-MACS is involved in the clearance of excess fatty acids taken into the supporting cells and cells in the lamina propria. Some biotransformation enzymes for xenobiotics are known to be expressed in these types of cells [23,30–33]. Another possibility for the function of O-MACS is that it may play a role in generating the zonal segregation of the OE during development. Unlike other zone-specific genes, e.g. OR genes, o-macs is expressed not only in the neuronal cell layer of OSNs but also in the supporting and basal cell layers. Furthermore, the o-macs transcripts are detected early in development at e11.5, before the onset of the OR gene expression, in the dorso-medial part of the olfactory placode of the embryonic frontonasal region. Such an expression is maintained throughout the development of the OE, even after birth. Fatty acylation, e.g. for sonic hedgehog and NCAM, is known to regulate the protein function in neuronal development [34,35]. It is possible that O-MACS is involved in a similar protein modification by supplying specific acyl-CoA to the acyl-transferase. Specific acylation of protein factors may be important in generating the zonal structure of the OE. 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