Báo cáo khoa học: Planarian peptidylglycine-hydroxylating monooxygenase, a neuropeptide processing enzyme, colocalizes with cytochrome b561 along the central nervous system pptx

Planarian peptidylglycine-hydroxylating monooxygenase, a neuropeptide processing enzyme, colocalizes with cytochrome b561 along the central nervous system Akikazu Asada1, Hidefumi Orii1, Kenji Watanabe1 and Motonari Tsubaki1,2,3 Department of Life Science, Graduate School of Life Science, University of Hyogo (formerly Himeji Institute of Technology), Hyogo, Japan CREST, Japan Science and Technology Agency (JST), Saitama, Japan Department of Molecular Science and Material Engineering, Graduate School of Science and Technology, Kobe University, Hyogo, Japan Keywords peptidylglycine a-hydroxylating monooxygenase; cytochrome b561; planarian; neuroendocrine vesicle; neuropeptide amidation Correspondence M Tsubaki, Department of Molecular Science and Material Engineering, Graduate School of Science and Technology, Kobe University, Rokkodai-cho, Nada-ku, Kobe, Hyogo 657-8501, Japan Fax: +81 78 803 6582 Tel: +81 78 803 6582 E-mail: mtsubaki@kobe-u.ac.jp Note The nucleotide sequence of planarian PHM in this article has been submitted to the DDBJ ⁄ EMBL ⁄ GenBank databases with accession number AB195502 (Received 27 September 2004, revised December 2004, accepted 14 December 2004) Planarians are one of the simplest animal groups with a central nervous system Their primitive central nervous system produces large quantities of a variety of neuropeptides, of which many are amidated at their C terminus In vertebrates, peptide amidation is catalyzed by two enzymes [peptidylglycine a-hydroxylating monooxygenase (PHM) and peptidyl-ahydroxylglycine a-amidating lyase] acting sequentially In mammals, both enzymatic activities are contained within a single protein that is encoded by a single gene By utilizing PCR with degenerate oligonucleotides derived from conserved regions of PHM, we succeeded in cloning a full-length cDNA encoding planarian PHM The deduced amino acid sequence showed full conservation of five His residues and one Met residue, which bind two Cu atoms that are essential for the activity of PHM Northern blot analysis confirmed the expression of a PHM mRNA of the expected size Distribution of the mRNA was analyzed by in situ hybridization, showing specific expression in neurons with two morphologically distinct structures, a pair of the ventral nerve cords and the brain The distribution of PHM was very similar to that of cytochrome b561 This indicates that the ascorbate-related electron transfer system operates in the planarian central nervous system to support the PHM activity and that it predates the emergence of Plathelminthes in the evolutionary history doi:10.1111/j.1742-4658.2004.04528.x Neuropeptides in the brain, in the nervous system, and in various endocrine cells are synthesized in the rough endoplasmic reticulum as large precursor proteins After transit to vesicles and during axonal transportation along axons, several processing enzymes residing in the vesicles process the peptides to convert them to mature forms C-terminal a-amidation of the peptides occurs in the late stage [1] and is probably a rate-limiting step in many instances [2] Over half of peptide hormones or neuropeptides are amidated in vertebrates; in insects, greater than 90% of such peptides show the presence of a C-terminal amide moiety [3] This C-terminal amide is very important in their functions, as its absence often disrupts the activity or receptor-binding properties of the peptide ligands [4] Indeed, most neurotransmitters thus far identified are amidated peptides in cnidarians Abbreviations AsA, ascorbic acid; CNS, central nervous system; DBH, dopamine b-hydroxylase; DBHL, dopamine b-hydroxylase-like protein; PAL, peptidyla-hydroxylglycine a-amidating lyase; PAM, peptidylglycine a-amidating monooxygenase; PHM, peptidylglycine a-hydroxylating monooxygenase; MDA, monodehydroascorbic acid; TBH, tyramine b-hydroxylase; VNC, ventral nerve cords 942 FEBS Journal 272 (2005) 942–955 ª 2005 FEBS A Asada et al (coral, jellyfish, sea anemones), which possess the most primitive form of nervous system, suggesting that the archetype nervous systems may have relied on amidated peptides [5] The C-terminal a-amidation of neuropeptides is catalyzed by two enzymes: peptidylglycine a-hydroxylating monooxygenase (PHM; EC 1.14.17.3) and peptidyl-a-hydroxyglycine a-amidating lyase (PAL; EC 4.3.2.5) consecutively [4,6,7] In vertebrates, PHM and PAL are expressed together within one polypeptide chain and denoted as peptidylglycine a-amidating monooxygenase (PAM) [8–10] In some animals, such as Drosophila [11] and Calliactis [12], PHM is encoded by a distinct gene In the mollusc, Lymnaea, a multifunctional PAM containing four different copies of PHM and a single PAL has been found [13] Endogenously, this zymogen was converted to yield a mixture of monofunctional isoenzymes However, in the nematoda, Caenorhabditis elegans, both types of enzyme are present, based on analyses carried out using the SMART web-based resource (http://smart.embl-heidelberg.de/) [14] Planarians – nonparasitic free-living flatworms of the phylum Plathelminthes, class Turbellaria – are phylogenetically very distant from vertebrates but have acquired a central nervous system (CNS), mesodermal tissues, and a bilateral structure during evolution [15] Planarians use neuropeptides [16,17] as major neurotransmitters Planarian neuropeptides (substance P-, FMRFamide-related peptides) are extensively a-amidated [16–19], as observed in other invertebrates Therefore, it is of interest to examine which form (PAM, PHM, or a different form) does exist in planarians to characterize their primitive CNS as the putative ancestor of the sophisticated CNS of higher animals The monooxygenase reaction catalyzed by PHM (or the PHM domain) requires copper, ascorbate, and molecular oxygen, as found for its close family member, dopamine b-hydroxylase (DBH) [2] Ascorbic acid (AsA), which is accumulated in a relatively concentrated manner inside the neuroendocrine secretory vesicles [20], is converted to monodehydroascorbic acid (MDA) radical after supplying the electron equivalent to the catalytic copper center of the enzyme As there is no transmembrane transport of AsA or MDA radical in the vesicles [21], it has been postulated that a common transmembrane electron carrier, cytochrome b561, conveys a reducing equivalent from the extravesicular AsA to the intravesicular MDA radical [21–27] Cytochrome b561, first observed in chromaffin vesicles of bovine adrenal medullae [28,29] and later found to be distributed in many neuroendocrine tissues [30–33], is structurally and functionally very different FEBS Journal 272 (2005) 942–955 ª 2005 FEBS Planarian PHM from other cytochromes [34] It spans the vesicle membrane [35] and has a favorable midpoint potential for interacting with AsA and the MDA radical [36–39] Very recently, we have succeeded in cloning the planarian cytochrome b561 cDNA [40] The cytochrome b561 gene product was specifically expressed in the planarian nervous system [40], suggesting that the AsA-related electron transfer system, which supports amidation of the neuropeptide, might predate or coincide with the emergence of the CNS in animals Thus, as the second objective of our present study, we examined the colocalization of PHM with cytochrome b561 in the planarian In the present study, we successfully accomplished the two objectives raised above, namely we obtained an entire planarian PHM cDNA and identified its specific expression in the planarian CNS as being colocalized with cytochrome b561 Results Cloning of planarian PHM By using degenerate oligonucleotide primers (PAMS4 ⁄ PAM-A3) (Fig 1) coding for the conserved region of PHMs of various animals, we cloned a 265 bp PCR product from a cDNA library of the head portion of the planarian The deduced amino acid sequence of the PCR product (67 amino acid residues, excluding the primer regions) was highly homologous (containing 10 fully conserved amino acid residues) to other PHMs and therefore consistent with a partial sequence of the planarian PHM cDNA To obtain a longer nucleotide sequence, we carried out nested PCR on the firststrand planarian head cDNA by using new specific primers (spPAM-S2 and spPAM-S3) and SK20 vector primer and a universal primer A PCR product ( 560 bp) was cloned and sequenced The clone was found to encode the 3¢ end of the PHM cDNA, including a stop codon (TAG), the 3¢ untranslated region (3¢-UTR), and the poly(A+) tail Stepwise dilution screening of the planarian head cDNA library by PCR using two specific primers (spPAM-S3 and spPAM-A3; PCR probe size, 144 bp) was then conducted One plaque was screened, in vivo excised, recloned, and sequenced completely This clone ( 1300 bp) was found to encode a major part of the planarian PHM gene, except for the NH2-terminal region Several cycles of PCR cloning, using specific primers (PPHMA4, PPHM-A3, and PPHM-A5) and vector primers, followed by sequencing, gradually narrowed the uncovered 5¢ end region Finally, the most 5¢ end PCR product was obtained, cloned, and sequenced to identify an 943 Planarian PHM A Asada et al Fig Schematic representation of the cDNA coding for planarian peptidylglycine a-hydroxylating monooxygenase (PHM) and positions of degenerate and specific primers used in the present study The broad white bar and the two flanking small white bars represent the coding region and the noncoding regions of the planarian PHM cDNA, respectively; whereas the two flanking broad lines outside indicate the k ZAP II vector The two gray boxes in the planarian PHM cDNA represent the conserved sequences [(P ⁄ V)FAFR(T ⁄ V)H(T ⁄ A)H for positions 231– 239 of human peptidylglycine a-amidating monooxygenase (PAM) and EMCN(F ⁄ L)Y(I ⁄ M ⁄ L) for positions 308–315 of human-PAM, respectively], for which two degenerate primers (PAM-S4 and PAM-A3) were designed The degenerate and specific primers used were indicated by horizontal arrows above the planarian PHM cDNA The PCR clones and partial cDNA clones obtained were indicated below the planarian PHM cDNA in the order of nucleotide sequence determination from top to bottom Arrows indicate the direction of nucleotide sequence determination PO8 indicates the M13 reverse primer in-frame stop codon (TAG) at the most 5¢ end and the putative initiation codon (ATG) followed by an open reading frame To obtain the entire planarian PHM cDNA, stepwise dilution screening of the planarian head cDNA library with PCR was again conducted by using two specific primers (PPHM-S4 and PPHM-A3) One plaque, containing the entire planarian PHM cDNA gene, was obtained, completely sequenced and analyzed The strategy used to obtain the planarian PHM cDNA sequence is summarized in Fig The whole planarian PHM cDNA (1384 bp) contained a single open reading frame (1149 bp), which was preceded by the 5¢-UTR of 87 bp and followed by the 3¢-UTR of 127 bp and the first 21 nucleotides of the poly(A+) tail (Fig 2) This indicated clearly that planarian PHM is coded in a gene separate from that of PAL Northern blot Northern blot analysis of total RNA from the asexual planarian was carried out The cDNA probe coding for a major region of planarian PHM hybridized with an mRNA giving a single band of  1.4 kb (Fig 3), being consistent with the expected size based on the cloned cDNA This result also confirmed that there is no PAM-like gene (i.e a fused gene with PHM and PAL) in the planarian that will generate an mRNA of  kb, as found for vertebrate PAMs Deduced amino acid sequence of planarian PHM The deduced amino acid sequence of planarian PHM predicted a protein with a length of 382 residues 944 (Fig 2) and a theoretical molecular weight of 43701.34 A hydrophobic segment (13 amino acid residues) at the N terminus satisfied the consensus rules for the signal sequences, with a cleavage site most likely to occur after amino acid 15, based on analyses conducted by using SignalP WWW and PSORT WWW servers An adjacent propeptide, present in vertebrate PAMs (including human, rat, bovine and Xenopus) was absent, as observed for molluscan PHM [13] The deduced planarian PHM amino acid sequence was compared with those of PHMs and PHM domains of PAMs from several representative species (including Schistosoma, Drosophila, Calliactis, C elegans, rat, bovine and human) by a multiple sequence alignment (Fig 4) Five histidine and one methionine residue in the central portion of PHM were considered to bind two Cu atoms (CuA and CuB, also termed CuH and CuM, respectively), which are essential for the catalytic reaction of PHM These six residues (indicated by #) were also conserved in planarian PHM (Fig 4) Furthermore, it is known that well-conserved Cys residues form putative disulfide bonds to fix the tertiary structure of the PHM domain Eight of the 10 cysteine residues were conserved in planarian-PHM, as found for other PHM sequences (except for C elegans-PAM) and are marked with + in Fig Additionally, corresponding amino acid residues to the residues shown to interact with bound peptide–substrate, such as Arg240, Asn316 and Tyr318 in rat PHM [7,41] were also conserved in planarian PHM (marked with ! in Fig 4) Percentage amino acid identities between these representative PHMs and PAMs were calculated for their PHM core regions and then tabulated (Table 1) The planarian PHM sequence showed around 40% amino FEBS Journal 272 (2005) 942–955 ª 2005 FEBS A Asada et al Planarian PHM Fig Nucleotide and deduced amino acid sequences of planarian peptidylglycine a-hydroxylating monooxygenase (PHM) The nucleotides are numbered from the 5¢ to the 3¢ end, and the amino acid residues are numbered starting with the first ATG in the open reading frame The translation termination codon is indicated by an asterisk The sequence obtained by the first degenerate PCR is singly underlined, whereas the sequences corresponding to the two specific primers PPHM-S1 and PPHM-A1, used for production of the PCR probe for the Northern blot analysis, are underlined by broken lines 28S 18S cyt.b561 mRNA 1.4 kb acid identity with all the sequences listed in Table The closest similarity was found to Schistosomas PHM (46.0%) and the most distant one was C elegans PAM (33.6%) To further understand the evolutionary relationship, we conducted a detailed phylogenetic analysis of planarian PHM in comparison with other PHMs or PHM domains and DBHs [including tyramine b-hydroxylases (TBHs) and DBH-like proteins (DBHLs)] from various animals (28 sequences in total) The analysis showed that planarian PHM was very distant from DBH, TBH and DBHL (Fig 5) Planarian PHM was classified as a member of a subfamily consisting of PHMs from Drosophila, Heterodera, C elegans and Schistosoma and was distinct from PHM domains of vertebrate PAMs and cnidarian PHMs (Fig 5) Expression of PHM along the planarian CNS Fig Northern blot analysis of peptidylglycine a-hydroxylating monooxygenase (PHM) mRNA in the planarian A PCR probe produced by two specific primers – PPHM-S1 and PPHM-A1 – was used for Northern blot analysis Total RNA (10 lg) derived from the whole planarian body was probed with the planarian PHM cDNA FEBS Journal 272 (2005) 942–955 ª 2005 FEBS The planarian CNS is basically constituted by an anterior ‘brain’ and a pair of longitudinal ventral nerve cords (VNCs) that run through the length of the body The expression of the planarian PHM gene, as shown by in situ hybridization, was specific in these neurons (Fig 6A,B) The planarian cytochrome b561 gene was also expressed to a similar extent in the brain and in a pair of VNCs (Fig 6C,D), in accordance with our previous observation [40] The pattern of gene expression 945 Planarian PHM A Asada et al Fig Multiple alignment of peptidylglycine a-hydroxylating monooxygenase (PHM) or PHM-domain sequences of planarian, Schistosoma, Drosophila, Calliactis, Caenorhabditis elegans, rat, bovine and human origin Amino acid residues common to all sequences are denoted by asterisks below the sequences, whereas conservative residues are indicated by colons (:) or a full stop (.) The five His and one Met residue that form complexes with two Cu2+ atoms are indicated by # in the uppermost line, whereas eight conserved Cys residues that form four putative Cys-Cys disulfide bridges and three conservative residues that might interact with the bound peptide substrate are marked with + and !, respectively The sequences were obtained from S mansoni (Schistosoma-PHM; AY172995), Drosophila (Drosophila-PHM; AY069103), Calliactis (Calliactis-PHM; AF036337), C elegans (C.elePHM; AC025726 and C.ele-PAM; U80438), rat (Rat-PAM; X59687 and M82845), bovine (Bovine-PAM; M18683), and human (Human-PAM; S75038) origin PAM, peptidylglycine a-amidating monooxygenase 946 FEBS Journal 272 (2005) 942–955 ª 2005 FEBS A Asada et al Planarian PHM Table Percentage identity of amino acid residues between peptidylglycine a-hydroxylating monooxygenases (PHMs) and PHM domains of peptidylglycine a-amidating monooxygenase (PAMs) from various animal species (including Schistosoma-PHM, Drosophila-PHM, CalliactisPHM, C elegans-PHM, C elegans-PAM, rat-PAM, bovine-PAM, and human-PAM) were calculated by using the FASTA program of the DDBJ server (DNA Data Bank of Japan, Mishima, Japan) for the corresponding PHM core regions The PHM core regions were identical to those used in Fig PlanariaPHM Planaria-PHM Schistosoma-PHM Drosophila-PHM Calliactis-PHM C.ele-PAM C.ele-PHM Rat-PAM Bovine-PAM Human-PAM SchistosomaPHM DrosophilaPHM CalliactisPHM C.elePAM C.elePHM RatPAM BovinePAM HumanPAM 100 46.0 100 41.5 40.9 100 43.3 39.5 45.4 100 33.6 35.7 37.5 41.3 100 38.9 41.0 47.0 39.0 38.0 100 40.0 36.7 40.5 42.3 39.3 38.7 100 40.0 37.0 40.5 42.7 42.3 38.7 93.8 100 41.6 37.6 44.4 42.3 41.4 38.4 94.6 95.7 100 for these two proteins showed close similarities to those of neuron-specific proteins, including a synaptotagmin homologue, DjSYT [42], a homologue of PC2 (prohormone convertase 2) [15], a synapsin homologue, a carboxypeptidase E homologue [43] and a homologue of the nicotinic acetylcholine receptor a7-1 subunit [44] All of these proteins are related to neurotransmission, including neurotransmitters, receptors ⁄ channels and synaptic vesicles [44] Expression of PHM in the planarian brain The closer view of the head region by in situ hybridization using the planarian PHM antisense probe showed that numerous cells, which are considered to be neurons, were clustered around the inverted U-shaped axon bundles and that these cells were specifically stained (Fig 7A; red) No appreciable segmental structure was observed inside the inverted U-shaped structure, where the neuropil-containing axons and dendrites locate (Fig 7A) Higher magnification views showed that indeed only the neuronal cell bodies were stained with the PHM probe (Fig 7D; brown) Immunohistochemical staining of the head region, by using antiserum against planarian cytochrome b561, showed that both the inverted U-shaped axon bundles and the surrounding neurons were stained (Fig 7B,C; red) A higher magnification view (Fig 7E), and its merged image with that stained for nuclei (Fig 7F; blue), confirmed this observation Expression of PHM in the planarian eyes The planarian eye is composed of two types of cells, namely photoreceptor cells and pigmented cells [45] The pigment cells form an eye cup and the photorecepFEBS Journal 272 (2005) 942–955 ª 2005 FEBS tor cells project their microvilli into the inside of the cup, as indicated by immunohistochemical staining with antiplanarian arrestin (green) (Fig 7H,J) Planarian PHM was expressed only in the photoreceptor cells adjacent to the eye cup In particular, signals of the PHM were observable in the region surrounding the nuclei (brown in Fig 7G) The staining image with antiplanarian cytochrome b561 was, however, slightly different from that of planarian arrestin; only the outside of the eye cup was stained (Fig 7J), suggesting that the expression level of cytochrome b561 in the microvilli is not so significant Discussion This report describes the successful molecular cloning of planarian PHM cDNA In mammals, PHM and PHL are fused as a bifunctional protein – PAM – that is encoded by a single gene In some invertebrates, such as Drosophila [11] and Calliactis [12], PHM is encoded by a distinct gene In the mollusc, Lymnaea, a multifunctional PAM was found, which comprised four different copies of PHM and a single copy of PAL [13] Very recently it was reported that a human parasite S mansoni, which belongs to the same phylum (Plathelminthes) as planarian, but a different class (Trematoda), possessed a monofunctional PHM lacking a PAL domain [46] In the nematoda C elegans, both types of enzyme precursors, a monofunctional PHM and a bifunctional PAM, are present, based on analysis conducted using the SMART web-based resource (http://smart.embl-heidelberg.de/) (Figs and 5) In our present study, the cDNA probe coding for the major region of planarian PHM hybridized with an mRNA, giving a single band of  1.4 kb (Fig 2), being consistent with the expected size based on the 947 Planarian PHM Fig Phylogenetic relationships of peptidylglycine a-hydroxylating monooxygenases (PHMs) or PHM domains from various animals Phylogenetic relationships of PHMs [or PHM domains of peptidylglycine a-amidating monooxygenase (PAM)], dopamine b-hydroxylases (DBHs), tyramine b-hydroxylases (TBHs) and dopamine b-hydroxylase-like proteins (DBHLs) were determined as described in the text Amino acid sequences for Bovine-PAM (M18683), Bovine-DBH (J05160), Equus-PAM (D29625), Equus-DBH (AB029430), Human-PAM (S75038), Human-DBHL (BC018756), Human-DBH (X13256), Mouse-PAM (U79523), Mouse-DBH (S50200), Mouse-DBHL (AB065134), Mouse-MonX (monooxygenase X) (AK081586), Rat-PAM (M82845 and X59687), Rat-DBH (L12407), Ciona-PAM (AK113539), Xenopus-PAM (X62771), C.elePHM (AC025726), C.ele-PAM (U80438), C.elegans-TBH (Z99942), Chicken-DBHL (AF327450), planarian-PHM (this study), DrosophilaPHM (AY069103), Drosphila-TBH (AE003442), Schistosoma-PHM (AY172995), Lymnae-PHM4, Lymnae-PHM3, Lymnae-PHM2, Lymnae-PHM1 (AF109920), Calliactis-PHM (AF036337), Aplysia-PAM (AF140271) and Heterodera-PHM (AY242521) were obtained from the DNA Data Bank of Japan (Mishima, Japan) The phylogenetic tree was created from the distance matrix by using the neighborjoining method cloned cDNA Furthermore, we were unable to find any PCR clones longer than that shown in Fig In addition, there were four in-frame stop codons downstream of the terminator codon in the cloned PHM 948 A Asada et al cDNA (Fig 2) These results suggest that there is no PAM-like gene (i.e a fused gene containing both PHM and PAL) in the planarians, which would generate a mRNA of  kb, as found for higher vertebrate PAMs Therefore, PHM and PAL are coded for by different genes in the planarians and only later in the evolution these genes appear to have fused, forming a single gene coding for the bifunctional PAM enzymes that we know from vertebrates ˚ High-resolution (2.1 A) X-ray structures of rat PHM domain have been reported previously [41,47] The structure shows two copper-binding domains connected by a three-residue linker Domain binds one copper (CuH also termed as CuA) with three histidine Nd ligands (His107, His108 and His172), while domain binds the other copper (CuM, also termed as CuB) with two histidine Ne ligands (His242 and His244) and a methionine sulfur (Met314) [47] The two coppers ˚ are 11 A apart and face the interdomain space in such a way that the cleft between them is fully accessible to solvent and peptide substrates [47] As the peptide substrate, a-N-acetyl-diiodo-Tyr-Gly, binds in a pocket close to CuM, this leads to the proposal that dioxygen binds to the CuM site, as previously suggested (on the basis of spectroscopic and kinetic data) for the related enzyme, DBH [48,49] Therefore, it was proposed that the bound peptide substrate mediates the electron transfer from CuA to CuB, where the activation of molecular oxygen takes place On the basis of FT-IR spectroscopy of carbon monoxide-bound PHM and EXAFS data, however, a superoxide-channeling mechanism was proposed [50–52] This proposal is based on the finding that both copper sites are reactive towards CO and, by inference, O2 [50] The substrate-free enzyme in the reduced state binds a single CO, assignable to CuM–CO, with a frequency of 2092 cm)1 Binding of the peptidylglycine substrate caused the appearance of a second CO frequency at 2062 cm)1, assignable to CuH–CO As the binding of CO to the CuH center suggests the potential binding of O2 to the CuH center, it was suggested that dioxygen is first reduced to superoxide at the CuH center and that the subsequent electron transfer is mediated by a superoxide molecule, which channels from CuH to CuM [50] Tyr79 of rat PHM was considered to have an assisting role in the channeling of superoxide, formed at CuH, towards CuM The corresponding Tyr33 in planarian PHM probably has a similar role, if the latter scenario is indeed valid It was suggested that the evolution of the vertebrate CNS might have begun with free-living flatworms – planarians that evolved before the divergence of metazoans into invertebrate and chordate branches [53,54] FEBS Journal 272 (2005) 942–955 ª 2005 FEBS A Asada et al Planarian PHM Fig Whole mount in situ hybridization with a planarian peptidylglycine a-hydroxylating monooxygenase (PHM) antisense RNA probe (A, B) and a cytochrome b561 antisense RNA probe (C, D) (A, C) Dorsal view (B, D) Ventral view Anterior is left and posterior is right Positive signals for the PHM were detected in the brain (br), eyes (e), and ventral nerve cords (vnc) The distribution was very similar to that of the planarian cytochrome b561 (C, D) Planarians are among the simplest animals to develop a body plan of bilateral symmetry and axes of growth with gradients of genetic expression, enabling cephalization, dorsal and ventral surfaces, medial and lateral regions, and an aggregate of neural cells in the head that form a bilobed brain The brain, a neural structure located in the head, differs from a ganglion by the following characteristics, [53,54] namely (a) a brain serves the entire body, not just restricted segments, (b) it has functionally specialized parts, (c) it is bilobar with commissures, (d) neurons form the surface with axons in the central core, (e) interneurons are more numerous than primary motor or primary sensory neurons, and (f) multisynaptic rather than monosynaptic circuits predominate The planarian brain fulfills all the criteria listed above Neurons of the planarian brain are known to more closely resemble those of vertebrates than those of advanced invertebrates, exhibiting typical vertebrate features of multipolar shape, extensive dendritic branching, the presence of dendritic spines for synaptic contact, a single axon per neuron, expression of certain vertebrate-like neural proteins, and relatively slow, spontaneously generated electrical activity [54] Thus, the planarian CNS appears to offer insight into the origin of the vertebrate CNS by being the simplest and most remote animal to exhibit these vertebrate features [54] Agata and coworkers described in detail the structure of the CNS of the freshwater planarian Dugesia japonica by using several molecular markers, including prohormone convertase PC2 [15] and synaptotagmin [42] By using both in situ hybridization and immunohistochemical techniques, they found that the CNS is composed of two morphologically distinct structures, two longitudinal VNC and an anterior brain The brain forms independent, inverted U-shaped lobes located dorsally to the VNC The brain contains FEBS Journal 272 (2005) 942–955 ª 2005 FEBS nine branched structures (first-ninth) on each side of the body, and nine branches project away from each lobe towards the periphery of the head In spite of this unique structure of the CNS in the planarian in comparison with those in vertebrates, the basic architecture of the planarian CNS has undergone functional regionalization, based on the comparative genomic analysis of planarian expressed sequence tags (ESTs) [44] and DNA microarray analysis [43] Furthermore, most of these nervous system-related genes were found to be shared among human, fruit fly and nematode These include genes for neurotransmission, such as the production of neurotransmitters, receptors ⁄ channels for the neurotransmitters, and synaptic vesicles In higher animals, such as human, neuropeptides are synthesized as preprohormones at the rough endoplasmic reticulum During translocation across the rough endoplasmic reticulum membranes, the prepart is removed and the prohormone region is sorted into the Golgi apparatus and subsequently into neurosecretory dense-core vesicles [6] In the Golgi apparatus and neurosecretory vesicles, the prohormones are processed to yield the bioactive neuropeptides The following processing steps are involved, namely (a) prohormone convertase, which cleaves at the C-terminal sites of certain dibasic and monobasic residues and liberates immature propeptides, (b) carboxypeptidase specific for basic residues, which removes the C-terminal basic amino acid residues, and (c) PHM and PAL, two consecutive enzymatic reactions where the C-terminal Gly residue is converted into an amide group [6] In planarians, a homolog of PC2 (prohormone convertase 2) [15] and a carboxypeptidase E homolog [43] were identified and both genes were expressed in the planarian CNS [15,43], although a homolog of PAL has not been identified These observations, together with our findings on PHM (present study) and cytochrome b561 949 Planarian PHM [40], indicate that the amidated neuropeptide-producing neurons had already acquired the neuroendocrine vesicles equivalent to those seen in the present-day neurons of higher animals by the time of the emergence of the Plathelminthes in the evolutionary history The eye of the planarian is one of the most ancestral and primitive types of visual systems in animals and is 950 A Asada et al of particular interest in light of the proposal that all eyes originate from a common evolutionary precursor [55] The planarian eyes consist of two cell types, namely pigment cells and photoreceptors [45] The pigment cells form a cup-shaped structure [56], while the photoreceptors are located outside the pigment eye cup and have two types of processes One type of process enters the eye cup and forms rhabdomeres, an assem- FEBS Journal 272 (2005) 942–955 ª 2005 FEBS A Asada et al bly of microvilli which may be associated with photopigments The other processes are optic nerve fibers projecting onto the brain [15] As both PHM and a homologue of PC2 (prohormone convertase), together with cytochrome b561, are expressed in visual neurons of the planarian, amidated peptides must have important mediators during the light perception in the planarian It should be noted that cytochrome b561 was present mainly in the region between the nucleus and the rhabdomeres of the photoreceptor cells, but little in the rhabdomeres The region where cytochrome b561 was present is termed ‘stalk’ and contains smooth endoplasmic reticulum, Golgi apparatus, and smooth surfaced and coated vesicles [57] Electron microscopic studies have also shown that, in the stalk region, efferent nerve fibers form synapses with dendritic spines protruding from stalks of the photoreceptor cells The structure appeared similar to that in the neuropil of the brain [57] In the stalk region of the photoreceptor cells, neuropeptides may be synthesized and work to modulate the transmission of light stimulus To clarify the role of the amidated neuropeptides in the planarian eyes and visual neurons, more detailed morphological and physiological studies, including identification of the neuropeptides, will be required Experimental procedures Organisms All planarians in this study were derived from a single worm of Dugesia japonica collected in the Irima River in Gifu, Japan and maintained as described (clonal strain: GI) [58] Cloning and sequencing of planarian PHM cDNA A cDNA library of the planarian made with k ZAP II vector (Stratagene, La Jolla, CA, USA) [15] was used as a template for PCR The degenerate primers used for PCR were Planarian PHM designed based on the conserved amino acid sequences in the PHM domain of human PAM, bovine PAM, rat PAM, Xenopus PAM, and C elegans PAM The primers were PAM-S4 (5¢-TTYGCITWYMGIGTICAYRCNCA-3¢) [coding for the conserved amino acid sequence of (P ⁄ V)FAFR(T ⁄ V)H(T ⁄ A)H; positions 231–238 of human PAM underlined in Fig 5] and PAM-A3 (5¢-RTACA TIADRTAIARRTTRCACATYTC-3¢) [coding for the conserved amino acid sequence of EMCN(F ⁄ L)Y(I ⁄ M ⁄ L); positions 308–315 of human PAM underlined in Fig 5] The reaction mixture consisted of lL each of 10· Taq buffer, 2.5 mm dNTPs, 10 lm forward primer, 10 lm reverse primer, and planarian head cDNA library mixture, lL of autoclaved H2O, and 0.05 lL of AmpliTaq DNA polymerase (Applied Biosystems) in a final volume of 10 lL Thermal cycling was performed with 40 cycles of the following step program: 94 °C for min, 43.5 °C for (primer annealing), and 72 °C for (primer extension) Final primer extension was made at 72 °C for The PCR products were separated in a 6.0% polyacrylamide gel The band with expected size (265 bp) was isolated and used as a template for re-amplification with the same set of primers (PAM-S4 ⁄ PAM-A3) The PCR product was cloned into pT7Blue T vector (Novagen) by using TA cloning and sequenced with a DNA sequencer (Model DSQ-1000 L; Shimadzu Corp., Kyoto, Japan) Based on the nucleotide sequence obtained, two specific forward primers – spPAMS2 (5¢-AAACTGCAGAGAAATTGGTAAGAAATCTCC-3¢) and spPAM-S3 (5¢-TCAGGATCCAGATTCTTTAGC TGCCAGATG-3¢) –were synthesized The former primer had a PstI site as an adaptor The 3¢ end of the cDNA was amplified by the 3¢-RACE method [59] using these primers and cDNA from the planarian head, cloned and sequenced This led to a clarification of the 3¢ half of the planarian PHM cDNA sequence including the terminal poly(A) sequence Based on the nucleotide sequence obtained, one specific reverse primer, spPAM-A3 (5¢-ATGAAGCTTAT CATTTCTTTGAACGCTTCG-3¢), was synthesized By using by using spPAM-S3 and spPAM-A3, the planarian cDNA library was screened by stepwise dilution, as previously described [60] A single phage plaque containing Fig Expression of the peptidylglycine a-hydroxylating monooxygenase (PHM) gene in the planarian brain and eyes A horizontal section of the planarian head was subjected to in situ hybridization (brown) with the planarian PHM antisense RNA probe (A) and to immunocytochemical staining (red) with antibody against planarian cytochrome b561 (B) (C) The image for nuclei staining (blue) with Hoechst 33342 was superimposed on (B) (A), (B), and (C) are the same frame (D), (E), and (F) are a high magnification of (A), (B), and (C), respectively, and are the same frame White and yellow arrowheads point the same cell in (D), (E), and (F), respectively The expression signal for the PHM gene was observable in the cell body of neurons in the peripheral region of the brain Cytochrome b561 was observable in neuropil (np), composed of axons and dendrites of the neurons, as well as in their cell bodies (G) In situ hybridization of a horizontal section of eye with antisense PHM probe (brown) (H) Immunohistochemical staining with anti-arrestin (green) was superimposed on nuclei-staining (blue) with Hoechst 33342 The frame was the same as that in (G) The PHM gene was expressed in photoreceptor cells (I) Normarski image of a horizontal section of the eye was superimposed on the nuclei-staining (blue) with Hoechst 33342 (J) Double stained image with anti-arrestin immunoglobulin (green) and with anti-cytochrome b561 immunoglobulin (red) The frame is the same as that in (I) Note that cytochrome b561 present in the ‘stalk’ region adjacent to the opening of the pigment eye cup showed a marked contrast to the even distribution of arrestin in photoreceptor cells Scale bars,  0.1 mm FEBS Journal 272 (2005) 942–955 ª 2005 FEBS 951 Planarian PHM the PHM gene was isolated and subjected to subcloning into the plasmid vector by in vivo excision Unfortunately, the insert lacked the 5¢ end region of the PHM gene The 5¢ end of the PHM gene was amplified by the 5¢-RACE method [59] using two new specific reverse primers (PPHM-A3, 5¢-CAATAAAGCTTCCTCAGCTTCAAC AGA-3¢; and PPHM-A4, 5¢-ATTAAGCTTCTTTGGT GGGTTTCACTC-3¢), and the cDNA library as a template A nested PCR product obtained using the vector primer M13 reverse primer (PO8; Toyobo) and PPHM-A4, followed by SK20 (Stratagene) and PPHM-A3 primers, was cloned and sequenced As the PCR product also lacked the 5¢ end of the gene, a nested PCR [first with M13 reverse primer (PO8) and PPHM-A3, and second with SK20 and a new specific reverse primer, PPHM-A5 (5¢-CGAAGCTT TCCAGCAAAAAACGA-3¢)], was performed as described above The longest PCR fragment showed an in-frame stop codon at the most 5¢ end followed by a putative initiation codon Based on the sequence, a forward specific primer, PPHM-S4 (5¢-GGCACGAGGATAGAATTTGAGCA-3¢), which corresponded to the boundary region between the vector and the cDNA insert, was synthesized Stepwise dilution screening of the planarian cDNA library was then performed by using specific primers PPHM-S4 and PPHM-A3, as described above Finally, a single plaque containing a full-length PHM cDNA was obtained and completely sequenced on both strands Deduced amino acid sequence analysis The deduced amino acid sequence of planarian PHM was compared with those of PHM and PHM domains of PAMs from several representative species by multiple sequence alignment using the program clustal w 1.83 of the DDBJ server (DNA Data Bank of Japan, Mishima, Japan; http:// www.ddbj.nig.ac.jp/Welcome-e.html) The percentage amino acid identity between the PHM core regions of these sequences was calculated using the fasta program (DNA Data Bank of Japan) and tabulated The signal sequence and its cleavage site of planarian PHM were analyzed for the NH2-terminal sequence (40 amino acid residues) by using the SignalP WWW server (Center for Biological Sequence Analysis, BioCentrum-DTU, Technical University of Denmark, Denmark; http://www.cbs.dtu.dk/services/ SignalP/) [61] and for the whole sequence by using the PSORT WWW server (Human Genome Center, The Institute of Medical Science, The University of Tokyo; http:// psort.ims.u-tokyo.ac.jp/) Phylogenetic analysis The deduced amino acid sequences of PHMs (or PHM domains of PAMs) and DBHs (including TBHs and DBHLs) from various animals were multiply aligned by using the program clustal w 1.83, and the phylogenetic 952 A Asada et al distance between the protein sequences was calculated by using the Kimura protein distance method The calculations were made only to the core regions of PHM and DBH (e.g regions corresponding from Val88 to Thr345 of rat PAM and from Arg223 to Leu498 of bovine DBH) A phylogenetic tree was created from the distance matrix with the program treeview ppc (v 1.6.6) using either the neighbor-joining method or the alignment guide tree method Northern blotting The DNA probe was synthesized by PCR using two specific primers: PPHM-S1 (5¢-TGTAGATCTATTTCTGTTGAA GCTGAG-3¢) and PPHM-A1 (3¢-CAACTTCTGCTTAA AAAGATTTCGAATAG-5¢) (the corresponding locations are indicated in Fig 2, as underlining with broken lines) with planarian head cDNA library mixture as a template These two primers had a BglII and a HindIII restriction site, respectively The PCR product (1136 bp) was purified by electrophoresis on a 1% agarose gel Total RNA was prepared, from asexual planarians, by using the CsCl cushion method The RNA was glyoxylated, separated in a 1% agarose gel and transferred to a membrane (Hybond-N; Amersham Bioscience) The membrane was baked at 80 °C for h, prehybridized for h under hybridization conditions in the absence of probe and hybridized with 32P-labeled DNA at 65 °C for 18 h in 6· NaCl ⁄ Cit, 5· Denhardt’s solution, 100 lgỈmL)1 salmon testis DNA, 0.5% (v ⁄ v) SDS The probe was labeled by using a BcaBest random DNA labeling kit (Takara Shuzo Co., Kyoto, Japan) The membrane was washed twice for h in 2· NaCl ⁄ Cit, 0.1% (v ⁄ v) SDS, at 65 °C, and for h in 0.1· NaCl ⁄ Cit, 0.1% SDS at 65 °C, and analyzed by using a BAS 2000 image analyzer (Fuji Photo Film Co., Tokyo, Japan) Whole-mount in situ hybridization Planarians of 5–7 mm length were starved for 7–10 days before use Whole-mount in situ hybridization was performed, as described previously [62], by using a digoxigenin-labeled RNA probe derived from the planarian PHM cDNA clone or a digoxigenin-labeled RNA probe derived from the planarian cytochrome b561 cDNA [40] Immunohistochemistry Paraffin-embedded sections were prepared by the standard procedure, hybridized with the digoxigenin-labeled RNA probe and reacted with anti-digoxigenin immunoglobulin conjugated with alkaline phosphatase (Roche), as described previously [63] They were subjected to immunohistochemical staining, as described previously [64] Antiserum against planarian cytochrome b561 was prepared as previously FEBS Journal 272 (2005) 942–955 ª 2005 FEBS A Asada et al described [40] Antiserum against planarian arrestin [15], specific for the photoreceptor cells, was also used as the primary antibody Goat anti-mouse IgG(H+L) IgG conjugated with Alexa488 (Molecular Probes), and goat antirabbit IgG(H+L) IgG conjugated with Cy3 (Jackson Immuno Research Laboratories), were used as secondary antibodies The specimens were counter-stained for nuclei with Hoechst 33342 and observed under a BX60 microscope (Olympus) After fluorescent immunohistochemistry, the signals of in situ hybridization were detected by using Nitro Blue tetrazolium (Sigma) and 5-bromo-4-chloro-3indolyl phosphate (Sigma) [62] Acknowledgements This work was supported by Grant-in-Aids for Scientific Research on Priority Areas (Comprehensive Promotion of Study of Brain; 12050236 to M.T.) and (C) (12680659 and 14580676 to M.T.) from the Japanese Ministry of Education, Science, Sports and Culture References Bradbury AF & Smyth DG (1991) Peptide amidation Trends Biochem Sci 16, 112–115 Eipper BA, Milgram SL, Husten EJ, Yun H & Mains RE (1993) Peptidylglycine a-amidating monooxgenase: a multifunctional protein with catalytic, processing, and routing domains Protein Sci 2, 489–497 Jiang N, Kolhekar AS, Jacobs PS, Mains RE, Eipper BA & Taghert PH (2000) PHM is required for normal developmental transitions and for biosynthesis of secretory peptides in Drosophila Dev Biol 226, 118–136 Kulathila R, Merkler KA & Merkler DJ (1999) Enzymatic formation of C-terminal amides Nat Prod Report 16, 145–154 Grimmelikhuijzen CJP, Leviev I & Carstensen K (1996) Peptides in the nervous systems of cnidarians: structure, function, and biosynthesis Int Rev Cytol 167, 37–89 Eipper BA, Stoffers DA & Mains RE (1992) The biosynthesis of neuropeptides: peptide alpha-amidation Annu Rev Neurosci 15, 57–85 Prigge ST, Mains RE, Eipper BA & Amzel LM (2000) New insights into copper monooxygenases and peptide amidation: structure, mechanism and function Cell Mol Life Sci 57, 1236–1259 Ohsuye K, Kitano K, Wada Y, Fuchimura K, Tanaka S, Mizuno K & Matsuo H (1988) Cloning of cDNA encoding a new peptide C-terminal a-amidating enzyme having a putative membrane-spanning domain from Xenopus laevis skin Biochem Biophys Res Commun 150, 1275–1281 FEBS Journal 272 (2005) 942–955 ª 2005 FEBS Planarian PHM Stoffers DA, Barthe-Rosa C & Eipper BA (1989) Alternative mRNA splicing generates multiple forms of peptidyl-glycine a-amidating monooxygenase in rat atrium Proc Natl Acad Sci USA 86, 735–739 10 Fan X, Spijker S, Akalal D-BG & Nagle GT (2000) Neuropeptide amidation: cloning of a bifunctional alpha-amidating enzyme from Aplysia Mol Brain Res 82, 25–34 11 Kolhekar AS, Roberts MS, Jiang N, Johnson RC, Mains RE, Eipper BA & Tagher PH (1997) Neuropeptide amidation in Drosophila: separate genes encode the two enzymes catalyzing amidation J Neurosci 47, 1363– 1376 12 Hauser F, Williamson M & Grimmelikhuijzen CJP (1997) Molecular cloning of a peptidylglycine a-hydroxylating monooxygenase from sea anemones Biochem Biophys Res Commun 241, 509–512 13 Spijker S, Smit AB, Eipper BA, Malik A, Mains RE & Geraerts WPM (1999) A molluscan peptide a-amidating enzyme precursor that generates five distinct enzymes FASEB J 13, 735–748 14 Schultz J, Milpetz F, Bork P & Ponting CP (1998) SMART, a simple modular architecture research tool: identification of signalling domains Proc Natl Acad Sci USA 95, 5857–5864 15 Agata K, Soejima Y, Kato K, Kobayashi C, Umesono Y & Watanabe K (1998) Structure of the planarian central nervous system (CNS) revealed by neuronal cell markers Zool Sci 15, 433–440 16 Johnston RN, Shaw C, Brennan GP, Maule AG & Halton DW (1995) Localisation, quantitation, and characterisation of neuropeptide F- and FMRFamideimmunoreactive peptides in turbellarians and a monogenean: a comparative study J Comp Neurol 357, 76–84 17 Shaw C, Maule AG & Halton DW (1996) Platyhelminth FMRFamide-related peptides Int J Parasitol 26, 335– 345 18 Reuter M, Gustafsson MK, Sheiman IM, Terenina N, Halton DW, Maule AG & Shaw C (1995) The nervous system of Tricladida II Neuroanatomy of Dugesia tigrina (Paludicola, Dugesiidae): an immunocytochemical study Invert Neurosci 1, 133–143 19 Curry WJ, Shaw C, Johnston CF, Thim L & Buchanan KD (1992) Neuropeptide F: primary structure from the tubellarian, Artioposthia triangulata Comp Biochem Physiol C 101, 269–274 20 Stewart LC & Klinman JP (1988) Dopamine betahydroxylase of adrenal chromaffin granules: structure and function Annu Rev Biochem 57, 551–592 21 Beers MF, Johnson RG & Scarpa A (1986) Evidence for an ascorbate shuttle for the transfer of reducing equivalents across chromaffin granule membranes J Biol Chem 261, 2529–2535 953 Planarian PHM 22 Njus D, Knoth J, Cook C & Kelley PM (1983) Electron transfer across the chromaffin granule membrane J Biol Chem 258, 27–30 23 Russell JT, Levine M & Njus D (1985) Electron transfer across posterior pituitary neurosecretory vesicle membranes J Biol Chem 260, 226–231 24 Levine M, Morita K & Pollard H (1985) Enhancement of norepinephrine biosynthesis by ascorbic acid in cultured bovine chromaffin cells J Biol Chem 260, 12942–12947 25 Wakefield LM, Cass AEG & Radda GK (1986) Electron transfer across the chromaffin granule membrane Use of EPR to demonstrate reduction of intravesicular ascorbate radical by the extravesicular mitochondrial NADH:ascorbate radical oxidoreductase J Biol Chem 261, 9746–9752 26 Tsubaki M, Nakayama M, Okuyama E, Ichikawa Y & Hori H (1997) Existence of two heme B centers in cytochrome b561 from bovine adrenal chromaffin vesicles, as revealed by a new purification procedure and EPR spectroscopy J Biol Chem 272, 23206–23210 27 Kobayashi K, Tsubaki M & Tagawa S (1998) Distinct roles of two heme centers for transmembrane electron transfer in cytochrome b561 from bovine adrenal chromaffin vesicles, as revealed by pulse radiolysis J Biol Chem 273, 16038–16042 28 Spiro MJ & Ball EG (1961) Studies on the respiratory enzymes of the adrenal gland I The medulla J Biol Chem 236, 225–230 29 Ichikawa Y & Yamano T (1965) Cytochrome 559 in the microsome of the adrenal medulla Biochem Biophys Res Commun 20, 26–30 30 Duong LT, Fleming PJ & Russell JT (1984) An identical cytochrome b561 is present in bovine adrenal chromaffin vesicles and posterior pituitary neurosecretory vesicles J Biol Chem 259, 4885–4889 31 Pruss RM & Shepard EA (1987) Cytochrome b561 can be detected in many neuroendocrine tissues using a specific monoclonal antibody Neuroscience 22, 149–157 32 Weiler R, Cidon S, Gershon MD, Tamir H, HogueAngeletti R & Winkler H (1989) Adrenal chromaffin granules and secretory granules from thyroid parafollicular cells have several common antigens FEBS Lett 257, 457–459 33 Fried G (1978) Cytochrome b-561 in sympathetic nerve terminal vesicles from rat vas deferens Biochim Biophys Acta 507, 175–177 34 Okuyama E, Yamamoto R, Ichikawa Y & Tsubaki M (1998) Structural basis for the electron transfer across the chromaffin vesicle membranes catalyzed by cytochrome b561: analyses of cDNA nucleotide sequences and visible absorption spectra Biochim Biophys Acta 1383, 269–278 35 Duong LT & Fleming PJ (1984) The asymmetric orientation of cytochrome b561 in bovine chromaffin granule membranes Arch Biochem Biophys 228, 332–341 954 A Asada et al 36 Flatmark T & Terland O (1971) Cytochrome b561 of the bovine adrenal chromaffin granules A high potential b-type cytochrome Biochim Biophys Acta 253, 487–491 37 Takeuchi F, Kobayashi K, Tagawa S & Tsubaki M (2001) Ascorbate inhibits the carbethoxylation of two histidyl and one tyrosyl residues indispensable for the transmembrane electron transfer reaction of cytochrome b561 Biochemistry 40, 4067–4076 38 Takigami T, Takeuchi F, Nakagawa M, Hase T & Tsubaki M (2003) Stopped-flow analyses on the reaction of ascorbate with cytochrome b561 purified from bovine chromaffin vesicle membranes Biochemistry 42, 8110– 8118 39 Takeuchi F, Hori H, Obayashi E, Shiro Y & Tsubaki M (2004) Properties of two distinct heme centers of cytochrome b561 from bovine chromaffin vesicles studied by EPR, resonance Raman, and ascorbate reduction assay J Biochem 135, 53–64 40 Asada A, Kusakawa T, Orii H, Agata K, Watanabe K & Tsubaki M (2002) Planarian cytochrome b561: conservation of a six-transmembrane structure and localization along the central and peripheral nervous system J Biochem 131, 175–182 41 Prigge ST, Kolhekar AS, Eipper BA, Mains RE & Amzel LM (1999) Substrate-mediated electron transfer in peptidylglycine a-hydroxylating monooxygenase Nat Struct Biol 6, 976–983 42 Tazaki A, Gaudieri S, Ikeo K, Gojobori T, Watanabe K & Agata K (1999) Neural network in planarian revealed by an antibody against planarian synaptotagmin homologue Biochem Biophys Res Commun 260, 426–432 ´ 43 Nakazawa M, Cebria F, Mineta K, Ikeo K, Agata K & Gojobori T (2003) Search for the evolutionary origin of a brain: planarian brain characterized by microarray Mol Biol Evol 20, 784–791 ´ 44 Mineta K, Nakazawa M, Cebria F, Ikeo K, Agata K & Gojobori T (2003) Origin and evolutionary process of the CNS elucidated by comparative genomics analysis of planarian ESTs Proc Natl Acad Sci USA 100, 7666– 7671 45 Sakai F, Agata K, Orii H & Watanabe K (2000) Organization and regeneration ability of spontaneous supernumerary eyes in planarians - eye regeneration field and pathway selection by optic nerves Zool Sci 17, 375–381 46 Mair GR, Niciu MJ, Stewart MT, Brennan G, Omar H, Halton DW, Mains RE, Eipper BA, Maule AG & Day TA (2004) A functionally atypical amidating enzyme from the human parasite Schistosoma mansoni FASEB J 18, 114–121 47 Prigge ST, Kolhekar AS, Eipper BA, Mains RE & Amzel LM (1997) Amidation of bioactive peptides: the structure of peptidylglycine a-hydroxylating monooxygenase Science 278, 1300–1305 FEBS Journal 272 (2005) 942–955 ª 2005 FEBS A Asada et al 48 Reedy BJ & Blackburn NJ (1994) Preparation and chracterization of half-apo dopamine-b-hydroxylase by selective removal of CuA Identification of a sulfur ligand at the dioxygen binding site by EXAFS and FTIR spectroscopy J Am Chem Soc 116, 1924–1931 49 Blackburn NJ, Pettingill TM, Seagraves KS & Shigeta RT (1990) Characterization of a carbon monoxide complex of reduced dopamine b-hydroxylase J Biol Chem 265, 15383–15386 50 Jaron S & Blackburn NJ (1999) Does superoxide channel between the copper centers in peptidylglycine monooxygenase? A new mechanism based on carbon monoxide reactivity Biochemistry 38, 15086–15096 51 Boswell JS, Reedy BJ, Kulathila R, Merkler D & Blackburn NJ (1996) Structural investigation on the coordination environment of the active-site copper centers of recombinant bifunctional peptidylglycine a-amidating enzyme Biochemistry 35, 12241–12250 52 Eipper BA, Quon ASW, Mains RE, Boswell JS & Blackburn NJ (1995) The catalytic core of peptidylglycine a-hydroxylating monooxygenase: investigation by site-directed mutagenesis, Cu X-ray absorption spectroscopy, and electron paramagnetic resonance Biochemistry 34, 2857–2865 53 Sarnat HB & Netsky MG (1985) The brain of the planarian as the ancestor of the human brain Can J Neurol Sci 12, 296–302 54 Sarnat HB & Netsky MG (2002) When does a ganglion become a brain? Evolutionary origin of the central nervous system Semin Pediatr Neurol 9, 240–253 55 Mannini L, Rossi L, Deri P, Gremigni V, Salvetti A, ´ Salo E & Batistoni R (2004) Djeyes absent (Djeya) controls prototypic planarian eye regeneration by cooperating with the transcription factor Djsix-1 Dev Biol 269, 346–359 FEBS Journal 272 (2005) 942–955 ª 2005 FEBS Planarian PHM 56 Asano Y, Nakamura S, Ishida S, Azuma K & Shinozawa T (1998) Rhodopsin-like proteins in planarian eye and auricle: detection and functional analysis J Exp Biol 201, 1263–1271 57 Carpenter KS, Morita M & Best JB (1974) Ultrastructure of the photoreceptor of the planarian Dugesia dorotocephala Cell Tissue Res 148, 143–158 58 Orii H, Agata K & Watanabe K (1993) POU-domain genes in planarian Dugesia japonica: the structure and expression Biochem Biophys Res Commun 192, 1395– 1402 59 Frohman MA (1990) RACE: rapid amplification of cDNA ends In PCR Protocols – a Guide to Methods and Applications (Innis MA, Gelfand DH, Sninsky JJ & White TJ, eds), pp 28–38 Academic Press, San Diego, Tokyo 60 Watanabe K, Sakai F & Orii H (1997) Stepwise dilution screening of a cDNA library by polymerase chain reaction Anal Biochem 252, 213–214 61 Nielsen H, Engelbrecht J, Brunak S & von Heijne G (1997) Identification of prokaryotic and eukaryotic signal peptides and prediction of their cleavage sites Protein Eng 10, 1–6 62 Tazaki A, Kato K, Orii H, Agata K & Watanabe K (2002) The body margin of the planarian Dugesia japonica: characterization by the expression of an intermediate filament gene Dev Genes Evol 212, 365–373 63 Kobayashi C, Kobayashi S, Orii H, Watanabe K & Agata K (1998) Identification of two distinct muscles in the planarian Dugesia japonica by their expression of myosin heavy chain genes Zool Sci 15, 861–869 64 Orii H, Ito H & Watanabe K (2002) Anatomy of the planarian Dugesia japonica I The muscular system revealed by antisera against myosin heavy chains Zool Sci 19, 1123–1131 955 ... specific forward primers – spPAMS2 (5¢-AAACTGCAGAGAAATTGGTAAGAAATCTCC-3¢) and spPAM-S3 (5¢-TCAGGATCCAGATTCTTTAGC TGCCAGATG-3¢) –were synthesized The former primer had a PstI site as an adaptor The 3¢... Cloning and sequencing of planarian PHM cDNA A cDNA library of the planarian made with k ZAP II vector (Stratagene, La Jolla, CA, USA) [15] was used as a template for PCR The degenerate primers... from that of PAL Northern blot Northern blot analysis of total RNA from the asexual planarian was carried out The cDNA probe coding for a major region of planarian PHM hybridized with an mRNA giving