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Biochemical and Biophysical Research Communications 338 (2005) 1204–1215

BBRC

Nitric oxide synthase in the nervous system and ink gland of the cuttlefishSepia officinalis: Molecular cloning and expressionq

Vladimir Scheinker a,1, Gabriella Fiore b,1, Carlo Di Cristo c, Anna Di Cosmo c,Marco d�Ischia d, Grigori Enikolopov a, Anna Palumbo b,*

a Cold Spring Harbor Laboratory, 1 Bungtown Road, P.O. Box 100, Cold Spring Harbor, NY 11724, USAb Zoological Station ‘‘Anton Dohrn,’’ Villa Comunale, I-80121 Naples, Italy

c Department of Biological and Environmental Sciences, University of Sannio, Via Port�Arsa, 11, I-82100 Benevento, Italyd Department of Organic Chemistry and Biochemistry, University of Naples ‘‘Federico II,’’ Via Cinthia 4, I-80126 Naples, Italy

Received 11 October 2005Available online 21 October 2005

Abstract

Nitric oxide (NO) signaling is involved in numerous physiological processes in mollusks, e.g., learning and memory, feeding behavior,neural development, and defence response. We report the first molecular cloning of NOS mRNA from a cephalopod, the cuttlefish Sepia

officinalis (SoNOS). SoNOS was cloned using a strategy that involves hybridization of degenerate PCR primers to highly conserved NOSregions, combined with RACE procedure. Two splicing variants of SoNOS, differing by 18 nucleotides, were found in the nervous systemand the ink gland of Sepia. In situ hybridization shows that SoNOS is expressed in the immature and mature cells of the ink gland and inthe regions of the nervous system that are related to the ink defence system.� 2005 Elsevier Inc. All rights reserved.

Keywords: Sepia officinalis; Nitric oxide synthase; Splicing; Molecular cloning; Ink gland; Central nervous system

Nitric oxide (NO) acts as a crucial endogenous regulatorof blood flow, a neurotransmitter in the central and periph-eral nervous system, and a pathophysiological mediator ofinflammation and host defence in mammals. Its discoverytwo decades ago stimulated extensive research in nearlyevery field of biology and medicine aimed at understandingthe function of NO and the regulation of its production inthe organism. NO is generated from L-arginine and molec-ular oxygen by NO synthase (NOS) [1]. There are three dis-tinct NOS isoforms in mammals: neuronal NOS or NOS1(nNOS), inducible NOS or NOS2 (iNOS), and endothelial

0006-291X/$ - see front matter � 2005 Elsevier Inc. All rights reserved.

doi:10.1016/j.bbrc.2005.10.069

q Abbreviations: BH4, tetrahydrobiopterin; CaM, calmodulin; CNS,central nervous system; NOS, NO synthase; nNOS, neuronal NOS; iNOS,inducible NOS; eNOS, endothelial NOS; RACE, rapid amplification ofcDNA ends; RT, reverse transcriptase; SoNOS, Sepia officinalis NOS;TdT, terminal deoxynucleotidyl transferase.* Corresponding author. Fax: +39 81 7641355.E-mail address: palumbo@szn.it (A. Palumbo).

1 Both authors contributed equally to the manuscript.

NOS or NOS3 (eNOS). All NOS isoforms are homodimerswith subunits of 130–160 kDa. The size of NOS polypep-tides ranges from 130 to 160 kDa and all three isoformsrequire homodimerization for enzymatic activity. All threeisoforms have binding sites for NADPH, FMN, and FADin the carboxyterminal part (the reductase domain), andbinding sites for tetrahydrobiopterin (BH4) and heme inthe aminoterminal part (the oxygenase domain). Thereductase and oxygenase domains are linked by a calmod-ulin (CaM)-binding site. Occupation of this site promoteselectron transfer from the cofactors in the reductasedomain to the heme in the oxygenase domain, leading toproduction of NO. nNOS and eNOS isoforms are activatedin response to increased intracellular calcium concentra-tion, when calcium–CaM complex, in combination withBH4, binds to the enzyme and initiates NO production.iNOS activity, however, is independent of the temporalinflux of calcium and is mainly regulated at the transcrip-tional level.

V. Scheinker et al. / Biochemical and Biophysical Research Communications 338 (2005) 1204–1215 1205

Parallel to the studies of the mammalian enzymes, therehas been a steady interest in the functions of NO in inver-tebrates (for marine invertebrates see reference [2]), withstudies on porifera, cnidaria, platyhelmintes, arthropoda[3–13], mollusca [14–16], echinodermata, and chordata. Inthe cuttlefish Sepia officinalis NO signaling pathway hasbeen implicated in manipulative behavior [17], modulationof statocyst activity [18], regulation of blood flow and pres-sure [19], and ink defence response [20–23].

We have previously demonstrated that a calcium/CaM-dependent NOS activity is present in the central nervoussystem (CNS) [21] and in the ink gland [20] of Sepia. Theink gland produces a black melanin pigment which is usedby the animal for defence. Immunohistochemical studiesconfirmed the presence of NOS in CNS regions related toink defence as well as in the peripheral nervous system(e.g., in neural pathways controlling contraction of theink sac sphincters and wall muscles during ejection ofink) [21]. NOS immunopositivity was also detected in otherCNS regions, including those involved in feeding, highermotor control, learning, vision, and olfaction [24]. More-over, NO was found to regulate the metabolic activity ofthe ink gland, affecting the activity of tyrosinase, the extentof melanin production, and the secretion of ink constitu-ents from mature cells of the gland, mediated by acGMP-dependent signaling pathway [22,23].

Given the important roles that NO signaling plays in thenervous system and the ink gland of the cuttlefish, wesought to determine the structure and the properties ofthe S. officinalis NOS (SoNOS). We here report the molec-ular cloning and properties of SoNOS, the first example ofa cloned cephalopod NOS. The pattern of SoNOS distribu-tion supports its role in the ink defence response of thecuttlefish.

Materials and methods

Animals. Specimens of S. officinalis were collected in the bay of Naplesand kept in a controlled environment for 2 days. Dissections were per-

Table 1Primers used for cloning

Primer Targeted amino acid sequence Sequence (5 0–30)

R1 EDIFG CCRAADATRTCYTCR2 HEDIFG CCRAADATRTCYTCRTGR3 FHEDIFG CCRAADATRTCYTCRTGR4 YHEDIFG CCRAADATRTCYTCRTGR5 30-UTR CAACTACGCTGATAGTGR6 SDTVEM AATCATCTCGACGGTGTR7 VLQDVYFGF AGCCAAAGTAGACATCTF1 — CCAGTGAGCAGAGTGA(F2 FRSFWQQ TTYMGNWSNTTYTGGCAF3 FRSFWQQ TTYMGNWSNTTYTGGCAF4 FRSFWQQ CNTTYMGNWSNTTYTGGF5 FRSFWQQ TTYMGNWSNTTYTGGCAF6 FRSFWQQ CNTTYMGNWSNTTYTGGF7 50-UTR GCGACGGTCAGCTGTCTF8 EMLAVPRR GAAATGTTGGCTGTACC

formed using cold anesthesia. The organs were collected and immediatelyfrozen in liquid nitrogen.

Isolation and sequencing of cDNA clones. Total RNA was isolated fromSepia optic lobes and ink glands using the guanidine isothiocyanate/acid–phenol method [25]. Reverse transcriptase (RT)-PCR strategy combinedwith rapid amplification of cDNA ends (RACE) procedure was used toclone the first fragment of SoNOS. To increase the specificity of the RTreaction, RNA was reverse transcribed by priming with the oligodeoxy-nucleotide primer R1 (Table 1) which targets a highly conserved stretch ofamino acids EDIFG in the NADPH-binding region of NOS. Onemicrogram of total RNA from the optic lobes was reverse transcribed withMuLV Reverse Transcriptase (Applied Biosystems) by priming with R1,in a 20 ll volume according to the manufacturer�s procedure. cDNA waspurified using PCR Purification Kit (Qiagen) and tailed with dATP usingterminal deoxynucleotidyl transferase (TdT) (Invitrogen), according to themanufacturer�s instructions. The first round of PCR amplification wasperformed with the oligo(dT)-containing primer F1 and a nested primerR2 that targeted the same conserved amino acid region HEDIFG; thisround did not produce any bands visible on agarose gels after electro-phoresis. An aliquot of the first PCR product was used as a template forthe second round of PCR amplification. A set of degenerate primers F2–F6 was designed based on another conserved sequence in the NADPH-binding region of NOS (FRSFWQQ). These primers were used togetherwith two degenerate reverse primers corresponding to the amino acidsequence FHEDIFG (R3) or YHEDIFG (R4), for the second round ofPCR. A 450 bp product was detected by electrophoresis in all reactionscontaining the primers F2–F6 and R3, but no products were found in thereactions with the F2–F6/R4 primers.

The PCR product generated with primers F6 and R3 was purifiedand cloned using TOPO TA Cloning Kit (Invitrogen) and 14 clones weresequenced, all giving similar results. Based on the sequence of the clonedfragment, new forward and reverse primers were designed and used withSMART RACE Amplification Kit (BD Biosciences) [26] to reach the 5 0-and 3 0-ends of the SoNOS mRNA. To confirm that the obtainedsequence corresponds to the full-length mRNA, we performed the 5 0-RACE procedure using SoNOS-specific reverse primer R5 for reversetranscription, tailed the cDNA with dATP and TdT, and used fourdifferent reverse primers positioned 100–400 bp upstream of the pre-sumptive 5 0-end for the following PCR. After cloning, 10 clones for eachproduct were analyzed, all of them having the same 5 0-ends. The full-length clone of SoNOS was obtained by RT-PCR using F7 and R5primers which targeted the 5 0- and 3 0-untranslated regions, respectively.Three overlapping clones of SoNOS encompassing the entire codingregion were obtained from the ink gland using RT-PCR with primersgenerated according to the sequence of SoNOS from the optic lobes. Allsequences were analyzed using programs within the Wisconsin GeneticsComputer Group (GCG) Sequence Analysis Software Package. Amino

3 0-nucleotide position in SoNOSb

37053702

RAA 3699RTA 3699ACAATTTAGAACGAGACAC 4061CAGA 3606TGAAGC 3446T)16 —RCA 3280ACA 3280CAA 3280GCA 3280CAG 3280GATCGAAACAG 40AAGAC 3318

1 AAAACGTGAAAGGCGACGGTCAGCTGTCTGATCGAAACAGTGAAACGCGGCGAAGCATAG 60

61 AGATTCTGCAAGAACAGGATCACATTCAAGTCATTGTCAAAGGTGACGTGACAGTGCACA 120

121 GTGGTTCGTACGCCCCAAATGCAAATGACAAACTAGAGCGAGGTAAGGCAAACGGCGTGG 180

181 AGGGCGAGGGTTATGGTCAGACTGTCAATGTTAGCACGAAAGGCACACAGTCCGAAACGA 240

241 CTGCCGAAAAATCCGAAAAAGCACCAGACATCATAAACGGAACGAATAAAAGTCGGTCGC 300

301 GGAGCGGCAGCCGACACAGTTTGTTTAGCGAGAATGTGTGCCGCAGCTCCCCGGAAAGAA 360

361 GGGGCTCAAGTCCCGCCAAATATTATAAGCTGAAATCTTACCATAATGACAAGGTCACTA 420

421 CGGATATGCTTCATTTAAAAAATAGCGAGCCAGTTGCGTGTTCCCCAAACGGTTGCAAAG 480 1 M L H L K N S E P V A C S P N G C K 18

481 GATCTGTTATCTATTCAAGATTGAAAAAACCTAGGGCTGAATCACGCCCGAAAGAGGAGG 54019 G S V I Y S R L K K P R A E S R P K E E 38

541 TTTTAATTCAAGCTAAAGCATTTATGGACGAATATTACACGTCTATCAAAAGATTAAATA 60039 V L I Q A K A F M D E Y Y T S I K R L N 58

601 CCCCGTCATATCACAAACGTTGGCAAGAAGTGATGCAGTCAATCGAGAAAACTGGTTTGT 66059 T P S Y H K R W Q E V M Q S I E K T G L 78

661 ACGAATTGAATAAGAGCGAGCTTACTTTTGGTGCCAAAACAGCCTGGAGGAATGCCCCGC 72079 Y E L N K S E L T F G A K T A W R N A P 98

721 GGTGCATCGGAAGAATACAATGGTCAAAATTACAGGTGTTTGATGCCCGTCATATAAAAA 78099 R C I G R I Q W S K L Q V F D A R H I K 118

781 CGGCCCGAGGCATGTTTGAGGCAATTTGCAACCATATAAAATATGGGACAAACAAAGGAA 840119 T A R G M F E A I C N H I K Y G T N K G 138

841 ATATCAGGTCAGCAATTACCATTTTTCCTCAAAGAACTGACGGTAAACACGATTTCCGAA 900139 N I R S A I T I F P Q R T D G K H D F R 158

901 TATGGAACGCCCAAATTATCCGATATGCAGGCTACAAAAACCCTGACGGTAGCATCACTG 960159 I W N A Q I I R Y A G Y K N P D G S I T 178

961 GAGATCCCGTAAGCTTGGAATTCACAGAGATTTGCCAGAAACTTGGCTGGAAAGGGAAAG 1020179 G D P V S L E F T E I C Q K L G W K G K 198

1021 GTGGCATGTTTGATGTTCTCCCATTAGTGTTGCAAGCAGACGGCCAAGATCCGGAATGGT 1080199 G G M F D V L P L V L Q A D G Q D P E W 218

1081 TTGAAATACCGAAAGATTTAGTCATGGAAGTTCCCATCAAGCATCCCAATTATCCATGGT 1140219 F E I P K D L V M E V P I K H P N Y P W 238

1141 TTGAAGAGCTGGGATTAAAATGGTTCGCTTTTCCGGGAGTATCAAGTTTGCTGTTCGACT 1200239 F E E L G L K W F A F P G V S S L L F D 258

1201 GTGGTGGCCTTGAATTCACGGCCGCCCCTTTCAATGGCTGGTACATGGGCACTGAGATCG 1260259 C G G L E F T A A P F N G W Y M G T E I 278

1261 GTGCGCGAAATTTATGTGATGAATTCAGATACAACTTAGCCAAGACCTTTGGCAAACGCA 1320279 G A R N L C D E F R Y N L A K T F G K R 298

1321 TGGGGTTGGATACAAATCGTGATTCGTCTCTGTGGAAAGATCGTGTCGTCGTGGAAGTGA 1380299 M G L D T N R D S S L W K D R V V V E V 318

1381 ACGTTGCCATACTGTATAGCTTTCAGTCAATGCACGTCACGATATCCGATCACCATACAG 1440319 N V A I L Y S F Q S M H V T I S D H H T 338

1441 CGGCTGAATCCTTCATGAAACATTTTGAAAATGAACAGCGTGTTCGAGGTGGATGTCCGG 1500339 A A E S F M K H F E N E Q R V R G G C P 358

1501 CTGATTGGGTATGGATAGTGCCACCAATCAGCGGAAGTTTGACCCCCGTTTATCATCAGG 1560359 A D W V W I V P P I S G S L T P V Y H Q 378

1561 AGATGCTCAATTATTTCCTGAAACCTTCTTATGAGTACCAGGTAGAACCATGGAAGACAC 1620379 E M L N Y F L K P S Y E Y Q V E P W K T 398

1621 ACACCTGGAAGAAAGACAGAGAAAAATCGAAGCAGTTAGGAGACAGACCAAAAAGGAAAT 1680399 H T W K K D R E K S K Q L G D R P K R K 418

1681 TTGGTTTCCGAGATATTGCCAGGGCTGTGAAATTCTCGGCTAAACTGATGGGTAAGGCCC 1740419 F G F R D I A R A V K F S A K L M G K A 438

1741 TGGCGCGTCGTGTCAAGTGCACGATTCTGTATGGTACTGAGACTGGCAAATCCGAGAGAT 1800439 L A R R V K C T I L Y G T E T G K S E R 458

A

Fig. 1. Nucleotide and predicted amino acid sequence of SoNOS. (A) SoNOSa. The amino acid sequence is shown in the single-letter code. The initiationcodon is shown in bold. The asterisk denotes the termination of the coding region. (B) Region of difference between SoNOSa and SoNOSb. The six aminoacids of difference in SoNOSb are indicated in bold. The nucleotide sequences have been deposited in the GenBank under Accession Nos. AY582749 forSoNOSa and AY582750 for SoNOSb.

1206 V. Scheinker et al. / Biochemical and Biophysical Research Communications 338 (2005) 1204–1215

1801 ACGCCCGGACATTGTGTGAAATATTCAGGCATGCTTTTGATGCAAAGGTCATGTGCATGG 1860459 Y A R T L C E I F R H A F D A K V M C M 478

1861 AGGACTATGATATCATTGACTTAGAACATGAGGCTCTTGTCCTGATGGTGACAAGCACAT 1920479 E D Y D I I D L E H E A L V L M V T S T 498

1921 TTGGAAACGGCGATCCACCAGAGAACGGATGGGCTTTCGCCGACGCTCTTTACGAGATGC 1980499 F G N G D P P E N G W A F A D A L Y E M 518

1981 GGCATCCGAACGGCGTGTCTGAACCTGAGATACAGAAAACGTCTTATATTCGAATGAGTG 2040519 R H P N G V S E P E I Q K T S Y I R M S 538

2041 TATCAAGTGAGACCGATATTACTATTAAGGAAGATATAAAAGAGGAACAAACATTACAGA 2100539 V S S E T D I T I K E D I K E E Q T L Q 558

2101 TGGAATCGGGACCCCTAGGAAATGTCAGGTACTCTGTTTTCGGGCTTGGGTCGCGTGCCT 2160559 M E S G P L G N V R Y S V F G L G S R A 578

2161 ATCCCAACTTTTGCGCCTTTGGTCATTTTCTCGACAAAGTGTGCAGCGAATTGGGAGCTG 2220579 Y P N F C A F G H F L D K V C S E L G A 598

2221 AAAGAATTTCACCAATGGTCGAAGGGGACGAACTATGTGCTCAAGAAGAATCATTCAAGG 2280599 E R I S P M V E G D E L C A Q E E S F K 618

2281 AATGGGCGAATGCCGTCTTCAAGGCGGCGTGTGATACATTCTGCTTGCAAGATGACGTCA 2340619 E W A N A V F K A A C D T F C L Q D D V 638

2341 ATATCCAAGAAGCAACAGGAGCCCTACACGGCAACGATCAGTCTTGGTCACTGGGAAAAT 2400639 N I Q E A T G A L H G N D Q S W S L G K 658

2401 TCCGATTAACACCGCAAGATGGCGTCAAAGAAATGGAGATTTCCACTGCTCTCTCTGGTT 2460659 F R L T P Q D G V K E M E I S T A L S G 678

2461 TGCACGGCAAGAATATAATGAACGCAACTGTCATAGAAAGACACAATCTCCAATCACCCA 2520679 L H G K N I M N A T V I E R H N L Q S P 698

2521 TATCGGAACGACAGACAATTCTTCTCAAGTTAGACACAAGGGGATCCTCAAGTTTTACTT 2580699 I S E R Q T I L L K L D T R G S S S F T 718

2581 ACGCTCCCGGAGACCACGTCGGAATTTATCCGGCCAACCGACCCGATCAAGTTGAAACTG 2640719 Y A P G D H V G I Y P A N R P D Q V E T 738

2641 TTTTATCACGGTTGCACAATGCGCCACCTGCCGACCAGGTTGTTAAGCTTGAAGTTCTTC 2700739 V L S R L H N A P P A D Q V V K L E V L 758

2701 AAGAACGATCACCACCACTTGGCCCTGCAAAGTCCTGGACTGGTTTTGAACGTTTCCCAA 2760759 Q E R S P P L G P A K S W T G F E R F P 778

2761 TTTGTACATTGCGAACGGCCTTCACCCGTTACCTGGACATTTCTATCACTCCAAGTAAGA 2820779 I C T L R T A F T R Y L D I S I T P S K 798

2821 ATCTGCTACAGTTATTCGCCGTCCTAGCCACGAGCGACTCGGACGGAGAACGACTGGACA 2880799 N L L Q L F A V L A T S D S D G E R L D 818

2881 CTCTAGCAAAGGACTCACAAGCTTATGAAAACTACAAACAGTACCATAGCCCGAATTTAG 2940819 T L A K D S Q A Y E N Y K Q Y H S P N L 838

2941 CAGAAATGTTGAAAGATTTCCCGTCTTTGAAGATCCCGCCAACGTTATTGCTGACACAAC 3000839 A E M L K D F P S L K I P P T L L L T Q 858

3001 TTCCGCTTTTGCAACAACGCTTCTATAGCGTCAGTTCTTCACCGAAATTCCACCCAGGAG 3060859 L P L L Q Q R F Y S V S S S P K F H P G 878

3061 AGGTTCACCTGACAATTGCCATTGCAAAATATATTAAACCAAATGGAGTCATTCATCACG 3120879 E V H L T I A I A K Y I K P N G V I H H 898

3121 GTATTTGTTCAACGTGGTTACAAACATGCCCTGTAGGTGAACAGGTGCCGTGTGTCATAC 3180899 G I C S T W L Q T C P V G E Q V P C V I 918

3181 GAGCGGCACCTAATTTCCACATGCCTGAAGACGGTACACGGCCAATCATAATGGTTGGAC 3240919 R A A P N F H M P E D G T R P I I M V G 938

3241 CGGGAAGTGGTATTGCACCATTCCGGAGTTTTTGGCAACAAAGAAAGATTGACAAAGAAA 3300939 P G S G I A P F R S F W Q Q R K I D K E 958

3301 TGTTGGCTGTACCAAGACATGGAGAAAAGAAAGGCTGGGGCAGCATGACAATTTATTTTG 3360959 M L A V P R H G E K K G W G S M T I Y F 978

3361 GATGCCGCGATCGGAACATTGACAACATCTACGAAAATGAACTCAAACAGTATCAAGAAG 3420979 G C R D R N I D N I Y E N E L K Q Y Q E 998

3421 AAGACGTGCTTCAAGATGTCTACTTTGGCTTCTCTCGAGAACCTGGCAAGAAAAAGACCT 3480999 E D V L Q D V Y F G F S R E P G K K K T 1018

Fig. 1. (continued)

V. Scheinker et al. / Biochemical and Biophysical Research Communications 338 (2005) 1204–1215 1207

3541 GTGGACACTTCTATGTCTGCGGTGATGTGCAAATGGCATCCGACGTGTCTGACACCGTCG 36001039 G G H F Y V C G D V Q M A S D V S D T V 1058

3601 AGATGATTCTTAAAGAAGATGCTCCGATGTCATCGGAGGAGGCCAAAAACTATGTGCTCA 36601059 E M I L K E D A P M S S E E A K N Y V L 1078

3661 AATTACGGGATGCGAACCGTTTCCATGAAGATATCTTTGGAGTTACTGTGAAAAGATCAA 37201079 K L R D A N R F H E D I F G V T V K R S 1098

3721 CGCCCAGCTCCGTGGATACATCTGACAAAGTTCATCTGCGATCAAAGGGGCGATTCTGGA 37801099 T P S S V D T S D K V H L R S K G R F W 1118

3781 AAGAAAATAAAATTCTGGAATGCGACGAGGAATTGCTAAAGAAATAATGACGTCACCGAA 38401119 K E N K I L E C D E E L L K K * 1133

3841 AACAATATGGCTGGTCGGTCGACTGAGTTTTCAGCATTTATTCACTAACGATTCAACCAA 3900

3901 ATGACGACGATAATGAAGGAAATTAAAAAGCAAAAGAAAACGACAACTTTAAAGCTGGGC 3960

3961 TTGTTTTTCATTGTATAATTTCTCTATTTCCCCCCAAAACGTTATAATATTATACTTTAT 4020

4021 ATTTCATTTTTATTTTCATTATGGTGTCTCGTTCTAAATTGTCACTATCAGCGTAGTTGG 4080

4081 GGAAAATGGCTACCTTTAATCAAATGAAATAACAATAACCCATTCCTAAATTCATTCATA 4140

4141 ACATTTATGTTTTAAAAAAATGTATTATTTTTGTTTCGTTTGTAGTTAGACTTTATTCTT 4200

4201 CCTGTAGACCAGGAATGACATCGGACAAAAATAGATTTATTTTCATAGCTTGACAGAAAA 4260

4261 AAAAAAAAAAAA 4272

SoNOSa

3301 TGTTGGCTGTACCAAGACATGGAGAAAAGAAAGGCTGGGGCAGCATGACAATTTATTTTG 3360959 M L A V P R H G E K K G W G S M T I Y F 978

SoNOSb

3301 TGTTGGCTGTACCAAGACGTAAGTTCCATGATCCTTATGGAGAAAAGAAAGGCTGGGGCA 3360959 M L A V P R R K F H D P Y G E K K G W G 978

B

3481 ACGTACAGCACCTGCTGAAGAAGAACTCCAAATCCGTGTGCGACGCCATTGTCCGAGAAG 35401019 Y V Q H L L K K N S K S V C D A I V R E 1038

Fig. 1. (continued)

1208 V. Scheinker et al. / Biochemical and Biophysical Research Communications 338 (2005) 1204–1215

acid sequences were analyzed using ClustalW (multiple sequence align-ments) and BioEdit [27].

Expression studies. The full-length cDNAs coding for SoNOSa andSoNOSb carrying HA (hemmagglutinin) epitope on the C-end were clonedinto pcDNA3 expression vector (Invitrogen). Enzymatic activity ofSoNOS after transfection into 293 cells was determined as describedpreviously [28]. Formation of stable NOS dimers was analyzed by low-temperature SDS–PAGE as previously described [29]. The Western blotswere analyzed using anti-HA antibodies (Sigma).

In situ hybridization. CNS and ink glands were fixed overnight at roomtemperature by immersion into 4% paraformaldehyde in PBS. Tissueswere dehydrated in ethanol, cleared in xylene, and embedded in paraffin(Paraplast). In situ hybridization experiments were conducted on 7 lmsections with digoxigenin-11-UTP-labeled riboprobes (Roche Diagnostics,Basel, Switzerland), according to the manufacturer�s instructions and aspreviously described [30]. The antisense SoNOS digoxigenin riboprobewas prepared from the cDNA fragment encoded by the last 150 aminoacids and the 3 0-UTR of SoNOS. Eighty nanograms of probe per slide at60 �C was used. Control experiments were run in parallel using the cor-responding sense probe.

Results

Cloning of SoNOS cDNA

Several regions of the NOS coding sequences are highlyconserved across species and homology cloning has beensuccessfully used to isolate NOS genes from diverse ani-

mal genomes. We attempted to clone the Sepia NOSsequelog from Sepia optic lobes using oligo(dT) or ran-dom hexamers as primers for cDNA synthesis, with sub-sequent PCR with degenerate primers targeted against theconserved regions of NOSs (heme, CaM, FMN, andFAD-binding regions). However, these attempts werenot successful, reflecting a high degree of divergence ofthe Sepia NOS gene (see below) from its sequelogs inother species. We then increased the specificity of theRT reaction by using a degenerate primer R1 directedat a highly conserved amino acid sequence EDIFG inthe NADPH-binding domain of NOS (Table 1). ThecDNA product was tailed with oligo(dA) using TdT.The first PCR amplification was performed with oli-go(dT)-containing primer F1 and a nested degenerateprimer that targeted the same conserved amino acidregion HEDIFG (R2). The PCR product was furtheramplified in a second round of PCR using a set of degen-erate forward primers targeted to a highly conservedFRSFWQQ sequence in the NADPH-ribose-bindingdomain of NOS and two degenerate reverse primers tar-geted either to the YHEDIFG sequence (conserved inthe neuronal, endothelial, and inducible forms of mam-malian NOS) or FHEDIFG (present in the NOS geneof another mollusk, Lymnaea stagnalis [31]. This round

V. Scheinker et al. / Biochemical and Biophysical Research Communications 338 (2005) 1204–1215 1209

of PCR produced a fragment of about 450 bp with adeduced amino acid sequence bearing homology to NOSgenes from other species. Cloning of the PCR productrevealed two sequences (designated a and b) which dif-fered by a stretch of 18 nucleotides. We next used thecloned fragment to generate full-length clones of theSoNOS mRNA using the SMART RACE technique. 5 0-RACE procedure with four different primers located100–400 nt downstream of the 5 0-end of cloned cDNArevealed identical 5 0-ends, strongly suggesting that wehave cloned the true 5 0-end of the SoNOS mRNA.

There are several ATG codons at positions 426, 564, and633 in-frame with a long open reading frame (Fig. 1A).None of them corresponds precisely to the Kozak consen-sus sequence for translation initiation codons; however, thefirst ATG codon (position 426) is, apparently, the true ini-

Fig. 2. Amino acid alignment of SoNOSa and SoNOSb with other known NBioEdit. The introduced gaps are shown as hyphens. Identical and similar aminletters on a gray background, respectively. Sequences shown are from m(D. melanogaster, DroNOS), chordates (C. intestinalis, CioNOS), mammals (Ho

sites for heme, CaM, FMN, FAD pyrophosphate (FAD-PPi), FAD isoallo(NADPH-Ade), and the C-terminal conserved sequence necessary for NADPH

tiating codon because it would enable the translation of aCys-(X)4-Cys motif (positions 12–17) which is importantfor NOS dimer stability and binding of zinc [32,33], andis highly phylogenetically conserved. The full-lengthSoNOS mRNA is 4272 nt (SoNOSa) or 4290 nt (SoNOSb),with 425 nt and 445 nt of 5 0- and 3 0-untranslated regions,respectively (Fig. 1). It encodes proteins of 1133 (SoNOSa)and 1139 (SoNOSb) amino acids with a deduced molecularmass of approximately 129 kDa. The lack of other differ-ences in the analyzed full-length clones of SoNOS suggeststhat the two types of mRNAs represent splice variants ofthe SoNOS transcript rather than the products of two sep-arate genes.

Multiple alignment of the predicted SoNOS amino acidsequences with other known NOS sequences showed goodhomology throughout the sequence with clearly identifiable

OS sequences. SoNOSa and SoNOSb were aligned with other NOS usingo acid residues are shown by white letters on a black background and blackolluscs (L. stagnalis, LymNOS, and A. californica, AplyNOS), insectsmo sapiens, HuNOS1, HuNOS2, and HuNOS3). Putative cofactor-bindingxazine (FAD-Iso), NADPH ribose (NADPH-ribose), NADPH adeninebinding are overlined.

Fig. 2. (continued)

1210 V. Scheinker et al. / Biochemical and Biophysical Research Communications 338 (2005) 1204–1215

Fig. 4. RT-PCR SoNOS from the optic lobes and ink gland. Total RNAfrom the optic lobes and the ink glands was reverse transcribed with theoligo(dT) primer. (1) cDNA from the optic lobes was directly used forPCR with primers F8–R7. (2) cDNA from the ink glands was used first asa template for PCR with primers F8–R6, and 1 ll of the product of thisreaction was used for the nested PCR with primers F8–R7. Positions ofthe molecular size standards are indicated.

V. Scheinker et al. / Biochemical and Biophysical Research Communications 338 (2005) 1204–1215 1211

putative recognition sites for the co-factors and co-sub-strates heme, CaM, FMN, FAD pyrophosphate, FAD iso-alloxazine, NADPH ribose, and NADPH adenine (Fig. 2).However, the N-end of the SoNOS was relatively shortcompared with other known NOS sequences, with theexception of the NOS gene of another mollusk, L. stagnalis[31]. Of the three mammalian NOS proteins, the neuronalisoform is more closely related to SoNOS, with identityof 52%.

Functional expression of SoNOS

When expressed in cultured mammalian cells, SoNOSacDNA produced a polypeptide with the expected electro-phoretic mobility at 130 kDa. The expressed SoNOSa didnot demonstrate enzymatic activity as measured by thearginine-citrulline conversion assay (data not shown). Toexamine whether this may be related to the assembly of cat-alytically active homodimers, we analyzed expressedSoNOS under conditions which preserve the dimeric struc-ture of NOS [29]. Low temperature PAGE of the expressedSepia protein, analyzed alongside the Xenopus NOS as areference, showed that SoNOS migrates through the gelas a monomer at 130 kDa whereas Xenopus NOS(160 kDa for a monomer) migrates for the most part asan oligomer with a molecular weight higher than160 kDa (Fig. 3), thus suggesting that after expression inmammalian cells SoNOS is present in an inactive mono-meric state.

Tissue-specific expression of SoNOS

RT-PCR of RNA from optic lobes showed that bothforms SoNOSa and SoNOSb are present (Fig. 4, lane 1).The same reaction performed on the RNA from ink glandsrevealed poorly distinguishable bands reflecting the lowabundance of NOS mRNA in this organ (data not shown).However, the nested PCR performed with cDNA from theink glands showed a prevalence of SoNOSb in this organ(Fig. 4, lane 2).

Fig. 3. Low-temperature PAGE of SoNOSa and Xenopus NOS. HEK293cells were transfected with SoNOSa subcloned into pcDNA3 vector andXenopus NOS cloned into pCS2 vector. Both constructs contain HA-epitope at the C-end. Cell extracts were analyzed by low-temperatureSDS–PAGE analysis. Lane 1, SoNOSa; lane 2, Xenopus NOS. NOS andDiNOS refer to nNOS monomer and dimer with apparent molecularmasses of approx. 160 and 320 kDa, respectively. In Xenopus extracts thebands below the monomer reflect the proteolysis products.

NOS localization

Localization of NOS in Sepia CNS and ink gland wasdetermined by in situ hybridization with SoNOS antisenseprobe which encodes the last 150 amino acids and the 3 0-UTR of SoNOS. To reveal the hybridization patterns,the slides were processed for anti-digoxigenin immunohis-tochemistry. Adjacent sections were hybridized with anti-sense and sense (taken as a control) probes.

In the CNS, the analysis was restricted to those regionsthat are related to the ink defence system, e.g., the pallio-visceral lobe. The distribution of mRNA encoding theSoNOS in the CNS (Fig. 5) shows that SoNOS mRNA isexpressed in the small inner neurons of both the lateroven-tral palliovisceral and central palliovisceral lobes of Sepia(Figs. 5A and B). Their size ranged from 10 to 15 lm.Hybridization with the SoNOS sense probe did not pro-duce specific signals (Fig. 5C). These experiments wererepeated with at least three batches of tissue and threetimes with essentially the same results.

In the ink gland, SoNOS was mainly distributed alongthe trilayer of the outer glandular epithelium (Figs. 6Aand B). SoNOS-expressing cells were located in the inner-glandular epithelium (Fig. 6C). Hybridization with theSoNOS sense probe again did not produce a signal(Fig. 6D). These experiments were repeated with at least

Fig. 5. In situ hybridization of SoNOS RNA probe in the pallioviscerallobe of S. officinalis. Lobe sections were exposed to digoxigenin-containingprobes as indicated and then subjected to enzyme-linked digoxigeninimmunohistochemistry. (A) The SoNOS antisense probe labels thecytoplasm of small interneurons lining the neuropil of lateroventralpalliovisceral lobe. (B) Central palliovisceral lobe neurons showingcytoplasmic distribution of SoNOS mRNA. (C) Section exposed to theSoNOS sense probe showing no signal. Scale bars: 20 lm in (A–C).

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four batches of tissue and four times with essentially thesame results.

Discussion

During the past decade the number of cloned NOS genesfrom invertebrates has steadily increased. This includesNOSgenes from the cnidaria Discosoma striata (Accession No.:AY036119), the arthropoda Drosophila melanogaster [6],Rhodnius prolixus [34], Manduca sexta [35], Bombyx mori

[36], the tropical land crabGecarcinus lateralis [37], the mos-quito Anopheles stephensi [38,39], the sea urchin Arbacia

punctulata [40], and the chordate Ciona intestinalis (http://genome.jgi-psf.org). In mollusks, NOS gene was clonedfrom Helix pomatia [41], Aplysia californica (AccessionNo.: AAK83069), and the pond snail L. stagnalis where, inaddition to the full-length NOS mRNA, two pseudo-NOStranscripts have been identified [31,42,43].

All invertebrate NOS proteins carry putative recogni-tion sites for the cofactors heme, BH4, CaM, FMN,FAD, and NADPH, with the exception of H. pomatia

NOS [41]. The latter protein does not contain consensussequences for the typical NOS cofactors and resembles anNOS from plant Arabidopsis thaliana, suggesting the exis-tence of a novel family of NO-producing enzymes [44].

Our study describes the first NOS sequence from a ceph-alopod,S. officinalis. The higher similarity to nNOS suggeststhat we have cloned the Sepia ortholog of the mammaliannNOS gene. Phylogenetic analysis of NOS sequences innon-mammalian species confirms the conserved nature ofNOS and particularly of the various cofactor-bindingdomains. SoNOS has a shorter N-terminal region thanmammalian nNOS, a feature sharedwith anothermolluskanenzyme, Lymnaea NOS [31] (Fig. 2). However, unlike Lym-

naea NOS, SoNOSs carry an N-terminal C-(X)4-C motifcommon to most of the known NOSs [32]. A characteristicfeature of the Lymnaea NOS gene is a region located adja-cent to the NADPH-ribose-binding site coding region andcomposed of 12 tandemly organized 21 bp long repeatsand thus formally defined as a minisatellite [31]. SoNOSlacks this minisatellite, which is replaced by an 18 nucleotideinsert.

We have found that in Sepia there are two alternativelyspliced SoNOS RNA isoforms. These SoNOSa andSoNOSb RNAs differ by 18 nucleotides, and as a resultby 6 amino acids; since this is the only difference betweenthe RNAs, they, most probably, correspond to the prod-ucts of alternative splicing. Although alternative splicingis a common mechanism in mammalian NOS [45,46], ithas been so far rarely reported for invertebrate nNOS, anoticeable exception being Drosophila NOS [7,47]. SoNOSisoforms are expressed to different extents in the ink glandand the CNS. Semi-quantitative RT-PCR of the SoNOSmRNA showed expression of both forms of SoNOSa andSoNOSb in the optic lobes and preferential expression ofSoNOSb in the ink glands, suggesting specific biologicalfunction for these isoforms. The structural and functional

Fig. 6. In situ hybridization of SoNOS RNA probe in the ink gland of S. officinalis. Sections were exposed to digoxigenin-containing probes and thensubjected to enzyme-linked digoxigenin immunohistochemistry. (A) Positive SoNOS cells were observed in the trilayer glandular epithelium (arrows). (B)SoNOS-expressing cells in the trilayer lining the lumen (arrows). (C) Positive SoNOS cells in the inner glandular layer. The inset shows a highermagnification of cells indicated by the arrow. (D) Section exposed to the SoNOS sense probe showing no signal. Scale bars: 20 lm in (A,B), 35 lm in (C,D).

V. Scheinker et al. / Biochemical and Biophysical Research Communications 338 (2005) 1204–1215 1213

differences between the brain and ink gland forms, as wellas between SoNOS and other NOS enzymes, should be asubject of further investigation.

Our in situ hydridization experiments confirmed previ-ous immunohistochemical data indicating that in the SepiaCNS NOS is expressed in the regions related to the inkdefence system [21]. However, in the ink gland, in contrastto the distribution of NOS immunoreactivity [20], RNAhybridization shows a broader distribution, marking boththe immature and mature parts of the ink gland. This dif-ference may reflect a differential expression of SoNOSRNA and protein in the ink gland or reflect the fluores-cence-quenching properties of melanin granules in maturecells obscuring the presence of NOS in the mature cellsof the gland.

Although SoNOS encodes a protein with all of the cru-cial catalytic and cofactor-binding sites present, we couldnot detect enzymatic activity in the arginine-citrulline assayafter transfection into cultured mammalian cells. Such lackof catalytic activity is not unprecedented for heterologouslyexpressed recombinant NOS proteins and may be relatedto the observed inability of SoNOS to dimerize under our

experimental conditions (Fig. 3). Dimerization is criticalfor the NOS enzymatic activity [33] and it is possible thatwhen the protein is expressed in a heterologous systemsome specific cofactors which are necessary for enzymaticactivity are missing; this interesting aspect of SoNOSdeserves further investigation.

Differences in the structure of SoNOS and other NOSproteins may affect the enzymatic activity of SoNOS as wellas the products of its catalysis. For instance, SoNOS iso-forms may be more predisposed than mammalian NOSsto generate reactive oxygen species along with NO. Nota-bly, we have previously demonstrated that nervous tissueof Sepia exhibits detectable basal protein nitration [48]and it is tempting to link protein nitration to a partiallyuncoupled NO synthesis by SoNOS.

Acknowledgments

We thank Rita Marino and Rita Graziano of the Service‘‘Technology for the Study of Gene Expression’’ for in situhybridization, the Service ‘‘Marine Resourches for Re-search’’ for assistance with living organisms. V.S. and

1214 V. Scheinker et al. / Biochemical and Biophysical Research Communications 338 (2005) 1204–1215

G.E. were supported by the National Institutes of Healthand the Seraph Foundation.

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