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Molecular cloning and tissue distribution of ferritin inPacic white shrimp ( Litopenaeus vannamei )*

Shu-Ling Hsieh a, Yi-Chun Chiu b , Ching-Ming Kuo b ,*a Department of Nutrition and Health Science, Fooyin University, Kaohsiung, 831, Taiwan, ROC

b Marine Research Station, Institute of Cellular and Organismic Biology, Academia Sinica, Ilan 262, Taiwan, ROC

Received 14 July 2005; revised 28 October 2005; accepted 5 December 2005Available online 20 March 2006

Abstract

Ferritin, a cytosolic iron storage protein composed of 24 subunits of heavy chain and light chain, is an intracellular protein pri-marily involved in iron metabolism. It can sequester up to 4500 ferric ions in its inner core to protect cells against toxicity of iron.Ferritin is known to play important roles in detoxication and is also involved in immunity processes. In this study, a full-lengthferritin cDNAwas cloned from the haemocyte of the Pacic white shrimp, Litopenaeus vannamei : it comprises 1249 bp, including132 bp in the 5 0-untranslated region, 510 bp in the open reading frame which encodes 170 amino acid residues, and 607 bp in the 3 0-untranslated region. Alignments of the deduced amino acid sequence showed that the Pacic white shrimp ferritin shares 74%,69%, 62%, 67%, 50% and 48% identity with craysh, tick, brine shrimp, oyster, human and rat, respectively. The tissue-specicexpression pattern was examined by reverse transcription polymerase chain reaction and real-time quantitative PCR. The ferritinmRNA is expressed in various tissues of the shrimp in the order of haemocyte, midgut gland, brain ganglion, gill, hepatopancreas,abdominal ganglion, eyestalk, muscle, thoracic ganglion, and heart.

2005 Elsevier Ltd. All rights reserved.

Keywords: Ferritin; Cloning and expression; Shrimp; Detoxication; Immunity

Ferritins, members of ferritin-like diiron-carboxylate protein superfamily, are the primary iron storage proteins of most living organisms. In vertebrates, the ferritin protein contains 24 small subunits ( w 20 kDa) consisting of heavy(H) and light (L) chains [1]. The subunits display different immunological reactivities, spectroscopic characteristics,

surface charges and iron incorporation [2]. However, the increased level of ferritin is known as a nonspecic marker of inammatory processes and neoplasms, e.g., human breast cancer [3] and renal cell carcinoma [4].Ferritin subunits H and L, which differ in rates of iron uptake and mineralisation, have been characterised. Fe(II)

oxidation in the H subunits takes place initially at the ferroxidase center, which is a carboxylate-bridged diiron centerlocated within the subunit four-helix bundle. Literature also suggested that H-ferritin may suppress proliferation of

* The cDNA sequence for Pacic white shrimp ferritin has been deposited in DDBJ/EMBL/GenBank with accession no.: AY955373 .* Corresponding author. Marine Research Station, Institute of Cellular and Organismic Biology, Academia Sinica, 23-10, Dah-Uen Road,

Jiau-Shi, Ilan 262, Taiwan, ROC. Tel.: 886 3 9880544x14; fax: 886 3 9871035. E-mail address: [email protected] (C.-M. Kuo).

1050-4648/$ - see front matter 2005 Elsevier Ltd. All rights reserved.doi:10.1016/j.fsi.2005.12.003

www.elsevier.com/locate/fsiFish & Shellsh Immunology 21 (2006) 279 e 283
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T cells [5e 7], E-rosette formation [8], and colony formation by normal human macrophages [9]. In a complementaryrole, negatively charged residues on the inner surface of the L subunits protein shell promote ferrihydrite nucleation.L-ferritin subunits have a degenerated ferroxidase site and the gene duplication to encode L-ferritin subunits is foundonly in vertebrates. Although numerous studies on ferritin have been performed in vertebrates, mammals in particular,very little is known about the sequence and distribution of the transcripts in decapod crustaceans. In this study, a cDNAencoding ferritin from Pacic white shrimp (

Litopenaeus vannamei) was cloned, and the expression of ferritin mRNA

in different tissues was further examined by RT-PCR and real-time quantitative PCR.Pacic white shrimp ( L. vannamei ), 19.69 2.5 g in body weight, and 14.7 2.2 cm in body length, were col-

lected from an aquacultural farm in northern Taiwan. They were maintained at 34 ppt of salinity and temperaturesof 27e 28 C, and fed with a high-protein commercial feed (Hsin-Da Feed Co., Pingtung, Taiwan) under an ambientphotoperiod regime. Total RNA was isolated from frozen tissue by RNAzol isolation system (Biotecx Laboratories,USA) following the manufacturers instructions. First strand cDNA synthesis by RT-PCR was accomplished by usingSuper-Script II RNase H reverse transcriptase (Promega Corporation, Madison, WI, USA) to transcribe poly(A) RNA

G G G G G A G T G C T C C G G G T C A C C A G T G T G T G G A C G A G T A C C T T C A A G C G A A C C T C T G G A A A T 60

C C T C T C C T T T G G A C C T T T T C C A T T A A C T T C A T T C A C C T C A C G T C A T C T T C A C G A A T C C A G 120

A A G A C G A T C A A G A T G G C C A G C C A A G T C C G C C A G A A C T A C C A T G A G G A C T G T G A A G C C T C C 180

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A T C G T T C T C C A G C A G A T T G C A G C T C C T T C C A T G C A A G A A T G G G G C A C T G G T C T G G A A G C C 420

96 I V L Q Q I A A P S M Q E W G T G L E A

C T T C A G G C T G C T C T T G A C C T G G A G A A G C A G G T C A A T C A G T C T C T C C T T G A A C T T C A T G G C 480

16 L Q A A L D L E K Q V N Q S L L E L H G

A C T G C A A G T G G C A A C A A C G A T C C T C A T C T C A C C A A G C T T C T T G A A G A T G A G T A T C T G G A G 540

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C C A G C T G G T C T T G G A G A G T A C T T G T T T G A T A A G G A A C T C C A C T A A A T C C A C A G A A A T A T T 660

70 P A G L G E Y L F D K E L H *

G A A A A T C A A G C C C T A T T C T T A T G T A A A T T T A G G G A A T G T G T T A T C A G T A A C A A A A A G A A A 720

A A A G T C A A C C G T A C A T A A C C A C T G C T T T T T T A C A C C T G A A G C A T C A C A A A G G T T A C A T T G 780

T C T T T T G G A T A G T A T A G G A C T T T G C A C A C C A A T C A G T A A C A T C A G T T G T A T G T A C A A A T G 840

G T G C T T T A T T A G T A T A A G A C A C T T C C A T C C A A A T T C T G T A A T T T T G G T T G A A C T T C A G T A 900

A A T G G A T T A A T A G T C T G G T A A A C A A C C T A A A G C A G T T A G A A T G G G A A T T G A G A T T T G A T A 960

G T T A A T T T G A T T T G T G G T A C A C C A C T C A A C A T G T T G A T T A T T G T A A T T C T A T A G A T C G G A 1020

A T T A G T G C A A C A G T G T T A G A T C C T T T G A C A A C A A A T A T A A G T A T G T T A C T A T T C T T A T C C 1080

A G G G T G A T T T A T T G G T T G T A A A T C T T G C A T T T A A T C T G C T T G T T T A G T A A C A A C A T T G C A 1140

T C T G G T G T T G G C T T C T G G A A A C T G G A T G T T T C A T C A C T C C A T T T T A T C A T T A C A T T A A A G 1200

G G T C C C C A G C T A A A A A A A A A A A A A A A A A A A A A A A A A A A A A A A A A A A A A A 1249

Fig. 1. The nucleotide and deduced amino acid sequences of ferritin cDNA from Pacic white shrimp ( Litopenaeus vannamei , GenBank accessionno.: AY955373 ). The amino acid sequence is shown below the nucleotide sequence in single letter code. It contains 170 deduced amino acids. Thetranslation start and termination codons are marked as M and an asterisk (*), respectively. The polyadenylation signal is underlined.

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with oligo-d(T) 18 primer: 5 0-GCATGCGCGCGGCCGCGGAGGTTTTTTTTTTTTTT-3 0. Reaction conditions rec-ommended by the manufacturer were followed. Full-length ferritin cDNA of L. vannamei was obtained by the pro-cedures of reverse transcription polymerase chain reaction (RT-PCR) and rapid amplication of cDNA (RACE).The L. vannamei ferritin degenerated PCR forward primer was 5 0-ATCCAGAAGACGATCAAG-3 0 (Ferritin-F),and the reverse primer was 5 0-GTTCTCCAGCAGATTGCAG-3 0 (Ferritin-R). The PCR reaction conditions includedan initial step at 94 C for 3 min, followed by 30 cycles at 94 C for 1 min, 50 C for 1 min, and 72 C for 2 min. Anal extension step was performed at 72 C for 10 min.

For 50-RACE, 5 mg of total RNA was reverse-transcribed with the rst nested F5 01R primer (5 0-GGAACTCTATGCGAGCTACG-3 0), which is specic to the L. vannamei ferritin gene. The 5 0RACE was performed using a SmartRace cDNA amplication kit (Clontech, USA). The primer sets consisted of F5 01R, 0.01 mg PT1 (50-GTTGCCGACGACGAGCCTACTTTTTTTT-3 0), and 0.1 mg PT2 (50-GTTGCCGACGACGAGCCTAC-3 0) for the rst-run PCR,and F5 02R (50-TACCATGAGGACTGTGAAGC-3 0) and PT2 for the second-run PCR. The PCR conditions describedwere also followed for RT-PCR, except that the annealing temperature was elevated to 55 C. The procedure for 5 0-RACE was also applied for 3 0-RACE. Total RNAwas reverse-transcribed after annealing with the PT1 anchor primer.Two successive PCRs were performed with the primer set F3 01F (50-CAGCAGATTGCAGCTCCTTC-3 0) and 0.1 mgof PT1 primer and serially diluted anchored PT2 primer for the rst run and F3 02F(5 0-ACCAAGCTAAAGCGTGCT-3 0)and PT2 for the second run. Amplied fragments were cloned and sequenced as described above. The PCRproducts were examined for specic amplication on 1% agarose gel electrophoresis in 0.5 TBE. Ligations,transformations, and plasmids were prepared following the method described by Hsieh et al. [10]. Amino acidsequences translated from the cDNA sequence were compared with sequences of the public database, NCBI-BLAST(http://www.ncbi.nlm.nih.gov/blast ). The BLAST sequence analysis program ( http:/www.ncbi.nlm.nih.gov/BLAST/ )was used for initial sequence comparisons. Multiple alignment of ferritin from the Pacic white shrimp ( L. vannamei ,GenBank accession no.: AY955373 ), craysh ( Pacifastacus leniusculus , X90566 ), oyster (Crassostrea gigas ,

Pacific white shrimp -MASQ V RQNYHEDCEA S IN K QINMELYASYVYLSMAYYF E RDDVALPGF A 49Oyster M AE SQ CRQNYHQES EAGIN R QINMELYA CYT YQSMAYYFDRDDVALPGFS 50Tick M A AT QP RQNYH VK CEA R INKQINMELYASYVY T SMAYYFDRDDVALPGF H 50Brine shrimp M AL S RC RQNF HEES EAAINKQINMELYASY A YL A MFT YFDRDDVA S PGF A 50Crayfish -MASQIR HNYHEDCE P -INKQIN L EF YASYVY M SMGHYFDRDD IS LPG A S 49Rat -M T SQIRQNY STEV EAA V NRLV NLH LR ASY T YLS LCFF FDRDDVAL E G VG 49Human -M S SQIRQNY ST D V EAA V NSLV NLY LQASY T YLS LGF YFDRDDVAL E G V S 49

Pacific white shrimp KFFK DSSDEEREHA QIF MKYQNKRGGRI VLQQIA A PS MQ EWGTGLE AL QA 99Oyster KFFK N SSDEEREHAEKLMKYQNKRGGRVVLQDI K KP DR DEWGTGLDAMQ V 100Tick KFFK K SS E EEREHAEKLMKYQN M RGGRVVL RP I QKP A QDEWG A GLDAMQA 100Brine shrimp KFF E E A S K EEREHAEKL I KYL NKRGGRV IYHP I EKP MKQ EWGSC LE AMED 100Crayfish KFFK DSSDEEREH GQKLMKYQNKRG A RI VLQ A IA A PS LQ EWG NLH DAL QA 99Rat HFF R ELAE EK RE GAER LL KLQNE RGGR ALF QD VQ KPSQDEWG KT LE AMEA 99Human HFF R ELAE EK RE GYER LL K M QNQRGGR ALF QDI K KP AE DEWGKTP DAMK A 99

Pacific white shrimp ALDLEK QVNQSLL E LHGT AS GNNDPHL TKL LEDE YLEEQV DSIKKIGD M I 149Oyster AL QLEKTVNQSLLDLHK V ADSHK D AQM CDFLE THY LEEQV NA IK E I S DHI 150Tick AL E LEKTVNQSLLDLHKLA TDH ND AQ LCDFLE S EYL A EQVK A IK ELS DYV 150Brine shrimp AL SM EKDVNE SLL K LHK V AS T RE DPHL TKY LEDEFL DEQVE SI YKI AH H V 150Crayfish ALDLE NE VNQSLLDL DAT AS KI NDPHL TNM LE GEFLEEQV E SI E KIG NL I 149Rat AL A LEK NL NQ A LLDLH A LGSAR T DPHLCDFLE SH FL DKE VKL IKK M G N HL 149Human A MA LEK KL NQ A LLDLH A LGSAR T DPHLCDFLE TH FL DEE VKL IKK M GDHL 149

Pacific white shrimp T K LKR A GP ----------AGLGEYLFDKEL H--- 170Oyster T QLKRVG----------- S GLGEY EY DRR LDS-- 171Tick TNLKRVG----------- P GLGEY M FDKE TLS D- 172Brine shrimp T R LR RVG----------- DGLG V YI FDK DLKN-- 171Crayfish T R LKR A GT ---------- S GLGE F LFDKEL KQRF LPSLTSHPN 182Rat TNL R RV A GP QPAQTGVAQA S LGEYLF ERLT L K HD 183Human TNL HRL GGP E --------AGLGEYLF ERLT L K HD 175

Fig. 2. Comparison of the amino acid sequences of ferritin. The different amino acid residues among the species are presented with a black back-ground. Deduced amino acid sequences were obtained from Pacic white shrimp ( Litopenaeus vannamei , GenBank accession no.: AY955373 ),craysh ( Pacifastacus leniusculus , X90566), Oyster (Crassostrea gigas , AY321299 ), tick ( Dermacentor andersoni , AY456681 ), brine shrimp( Artemia franciscana , AY062897 ), rat ( Rattus norvegicus , BC061525), and human ( Homo sapiens , BC062708).

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AY321299 ), tick ( Dermacentor andersoni , AY456681 ), brine shrimp ( Artemia franciscana , AY062897 ), rat ( Rattusnorvegicus , BC061525 ), and human ( Homo sapiens , BC062708 ) was performed with CLUSTAL W, a Windowsinterface for the CLUSTAL W multiple sequence alignment program [11].

Tissue specicity of ferritin gene expression in L. vannamei was determined by RT-PCR and real-time quantitativeRT-PCR. Total RNA from various tissues of Pacic white shrimp ( L. vannamei ) including eyestalk, brain ganglion,thoracic ganglion, abdominal ganglion, gill, midgut gland, haemocyte, muscle, heart and hepatopancreas wasextracted as described previously. First strand cDNA synthesis from 5 mg of each total RNA was performed usingan MMLV Reverse Transcriptase First Strand cDNA Synthesis kit (Life Sciences, USA). For RT-PCR, two oligonu-cleotide primers, FRT1: 5 0-GGGGGAGTGCTCCGGGTCAC-3 0and FRT2: 5 0-GTTCTCCAGCAGATTGCAG-3 0,were designed based on the nucleotide sequence corresponding to the nucleotide positions 1 e 20 and 364e 382 of L. vannamei ferritin cDNA, respectively. A set of b-actin primers, b-actin-F: 5 0-TAGGTGGTCTCGTGGATGCC30and b -actin-R: 5 0-GAGACCTTCAACACCCCCGC-3 0, served as a control for amount and quality of each cDNA.PCR reactions were conducted for 25 cycles with denaturation at 94 C for 1 min, annealing at 55 C for 30 s,and extension at 72 C for 30 s. In addition, level of ferritin mRNA also determined following the real-time quanti-tative RT-PCR analysis. Following ABI manufacturers protocols (Perkin e Elmer, Applied Biosystems, USA), theprimers for real-time quantitative RT-PCR analysis are FQPCRF: 5 0-GCATCGTTCTCCAGCAGATTG-3 0, and theFQPCRR: 5 0-AATGGGGCACTGGTCTGGA-3 0. The ABI Prism 7000 Sequence Detector System measured uores-cent emissions, which increase in direct proportion to the increase of amplied product during the PCR amplication.Fluorescence data were acquired during annealing or extension of reactions containing SYBR Green I (Roche).Relative quantities of mRNA were calculated with a known quantity of PCR fragments of ferritin and b -actin usingthe comparative threshold cycle number of each sample tted to a three-point standard curve.

E B T A G M H Mu He Ht N

-Actin

Ferritin

A

0

10

20

30

40

50

60

70

80

90

E B T A G M H Mu He Ht

Tissue

B

A r b

i t r a r y u n

i t ( )

Fig. 3. Tissue specicity of ferritin gene expression in Pacic white shrimp ( Litopenaeus vannamei ). (A) Analysis by RT-PCR, E: eyestalk; B:brain ganglion; T: thoracic ganglion; A: abdominal ganglion; G: gill; M: midgut gland; H: haemocyte; Mu: muscle; He: heart; Ht: hepatopancreas;and N: negative control. Specic-PCR was performed using pacic white shrimp ferritin specic primer (Ferritin-RT1 and Ferritin-RT2) withcDNA of several tissues synthesised by RT-PCR. b-Actin gene was used as the internal control. (B) Ferritin mRNA levels were determinedby real-time quantitative PCR, measured values represent an arbitrary unit [Ferritin PCR value (b -actin quantitative PCR value) 3 100].Values are the mean SEM of six animals ( n 6).

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The cDNA sequence of L. vannamei ferritin was cloned by RACE e PCR using two overlapping 5 0 and 3 0 fragments of cDNA, and a preliminary full-length cDNA sequence of 1249 bp was obtained, including 132 bp in the 5 0-untranslatedregion, 510 bp in the open reading frame which encodes 170 amino acid residues, and 607 bp in the 3 0-untranslated region(Fig. 1, GenBank accession no.: AY955373 ). The calculated molecular mass of the mature protein (170 amino acids) is19.4 kDa with an estimated p I of 4.94. The L. vannamei ferritin amino acid sequence shows relatively high identities andsimilarities to deduced ferritin amino acid sequences of other animals: 74% and 86% to craysh, 69% and 79% to tick,62% and 78% to brine shrimp, 67% and 78% to oyster, 50% and 70% to human and 48% and 68% to rat, respectively(Fig. 2). The phylogenetic tree reveals that the L. vannamei ferritin is paraphyletic to the eukaryotic ferritin.

Tissue expression of ferritin mRNA was examined by RT-PCR and real-time quantitative RT-PCR ( Fig. 3). In a RT-PCR study, a 382-bp fragment was amplied with the ferritin specic primers (FRT1 and FRT2) that were derived fromthe cDNAof all tissuesexamined.The ferritin washighly expressed in haemocytes, but weakly in thoracicganglion andheart tissue of L. vannamei (Fig. 3A). The results derived from real-time quantitative RT-PCR analysis ( Fig. 3B) on L. vannamei ferritin were consistent with those of the RT-PCR analysis. Literatures indicated that ferritin is distributedin various tissues of a variety of species. In vertebrates, ferritin is mainly located in cytoplasm and blood, but in mostinsects, ferritin is present within the vacuolar system and lumen of the endoplasmic reticulum, or secreted in the hae-molymph [12]. In the present study, however, mRNA of ferritin was present in all the tissues of L. vannamei examined,including eyestalk,brain ganglion, thoracicganglion, abdominal ganglion, gill, midgut gland, haemocyte,muscle, heartand hepatopancreas. Among these organs, haemocyte is the major expression site as much as 2.39-fold higher thanhepatopancreas. Since there are differences in the function and expression between ferritin subunits [13,14] , furtherstudies are needed to clarify the tissue-specic expression and the function of ferritin subunits in L. vannamei .

Acknowledgements

We would like to thank Prof. Chu-Fang Lo, National University of Taiwan, Taiwan for valuable informationof shrimp EST.

References

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