Báo cáo khoa học: O-MACS, a novel member of the medium-chain acyl-CoA synthetase family, specifically expressed in the olfactory epithelium in a zone-specific manner doc

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

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 situhybridization 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 situhybridization 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)

+ /K

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 saturademonstra-ted-fatty acids like other MACS proteins, its substrate preference was shown to be fatty acid lengths of C6–C12 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 ofo-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, gT15A, gT15C and gT15g, using SuperScriptTM 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 SMARTTM 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); MACS15¢-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 NeuroDgenes cDNA fragments for the rat OMP, OCAM, NCAM, and NeuroD were prepared by RT-PCR using pairs of primers: OMP 5¢-gagtagagagcctgaagcag-3¢

(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¢) for MACS1, and cloned into the EcoRI/

XhoI or EcoRI/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/Pi, cells were lysed in lysis buffer [50 mM NaH2PO4 (pH 8.0), 150 mM NaCl, 0.1% Triton X-100, 20 mMimidazole, 16 lgÆmL)1benzamidine HCl, 10 lgÆmL)1 aprotinin, 10 lgÆmL)1 leupeptin,

10 lgÆmL)1 pepstatin A, 1 mM 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 mMNaH2PO4, 150 mM NaCl, 0.1% Tw een 20, and 1Mimidazole) After eluent was dialyzed against buffer A (50 mMNaH2PO4, 150 mMNaCl), the protein solution was concentrated with Centricon 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 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-tive linkage map of the human MACS gene family The human o-macs gene is located on chromosome 12, whereas other MACS family genes are clustered on chromosome 16 Bars indicate exons.

Transfected HEK293T or HeLa cells were cultured

over-night on cover slides and stained with 500 nM of

Mito-TrackerGreenTM(Molecular Probes) at 37C for 1 h Cells were fixed with 4% paraformaldehyde in NaCl/Pi for

10 min at room temperature After washing three times with NaCl/Pi, cells were permeabilized with 0.2% Triton X-100 for 5 min, and blocked in NaCl/P 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

the OE The sense probe of o-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 lowmagnification 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 The MACS1 and SA transcripts are found in the respiratory epithelium (dotted lines) Scale bar indicates 100 lm.

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 and OE in contrast to the NCAM transcripts Scale bars in the lowand high magnification figures indicate 500 lm and 50 lm, respect-ively D, dorsal; A, anterior; P, posterior;

M, medial.

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¢- and 3¢-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 theo-macs gene

We examined the zone-specificity of o-macs expression by

in situhybridization, 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-macstranscripts 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 preparcontamin-ation 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 Ni2+-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.

Expression ofo-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 C6)12fatty 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 Ni2+-column (Fig 5B) and used to

determine the chain-length preference of fatty acids As

show n in Fig 5C, the recombinant O-MACS mediated

the CoA addition to the C6)12 fatty acids, while the

recombinant MACS1 demonstrated the preference for the

C6and C8chain-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 (MitoTrackerGreenTM) 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 MitoTrackerGreenTMfor 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 ·.

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 Since the o-macs expression

is restricted to zone 1 of the OE, knockout studies will be

helpful in examining whether O-MACS plays a role in

odorant processing in zone 1 or is involved in determining

the zonal structure during development

Acknowledgements

This work was supported by grants from Japan Science and

Technology (JST) Corporation, Ministry of Education, Culture and

Science, Mitsubishi Foundation, and Japan Foundation for Applied

Enzymology A T is supported by the Precursory Research for

Embryonic Science and Technology (PRESTO) program of JST We

thank Hitomi Sakano for critical reading of this manuscript.

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