Molecular cloning and responsive expression of macrophage expressed gene from small abalone Haliotis diversicolor supertexta

Fish & Shellfish Immunology (2008) 24, 346e359

available at www.sciencedirect.com

journal homepage: www.elsevier.com/locate/fsi

Molecular cloning and responsive expression of macrophage expressed gene from small abalone Haliotis diversicolor supertexta Guo-Dong Wang a, Ke-Feng Zhang a, Zi-Ping Zhang b, Zhi-Hua Zou a, Xi-Wei Jia a, Shu-Hong Wang a, Peng Lin a, Yi-Lei Wang a,* a

The Key Laboratory of Science and Technology for Aquaculture and Food Safety, Fisheries College, Jimei University, Xiamen, Fujian 361021, China b Department of Chemistry & Biochemistry, Texas State University, San Marcos, TX 78666, USA Received 19 September 2007; revised 4 December 2007; accepted 13 December 2007 Available online 23 December 2007

KEYWORDS Abalone; Perforin-like protein; Macrophage expressed gene; Expression; Innate immunity

Abstract The complete cDNA sequence of macrophage expressed gene (saMpeg1), a perforin-like molecule, was isolated from small abalone (Haliotis diversicolor supertexta) by homology cloning and rapid amplification of cDNA ends (RACE). The full-length cDNA of saMpeg1 was 2781 bp, consisting of a 50 -terminal untranslated region (UTR) of 252 bp, a 30 -terminal UTR of 342 bp with a signal sequence TAA and a poly (A) tail, and an open reading frame of 2184 bp. The deduced protein (saMpeg1) was composed of 728 amino acids, and contains the cytolytic ‘‘helix-turn-helix’’ domain of perforin (residues 171e218), of which the alpha-helices are amphipathic as are those of perforin. A putative single transmembrane domain is located at residues 667e689, and a modified furin cleavage site (KRRRK; residues 689e693) immediately follows. The result of real time quantitative PCR showed that saMpeg1 was highly expressed at 8 h and 96 h post-injection of the Gram-negative bacterium Vibrio parahaemolyticus, but there was no change after TBT exposure. The structural similarity to mammalian perforin and the different gene expression level to bacterial infection and TBT exposure suggest that saMpeg1 may play a role in the immune response against microorganisms in small abalone. ª 2008 Elsevier Ltd. All rights reserved.

Introduction The marine aqueous milieu is, in contrast to the terrestrial environment, much richer in bacterial [1] and viral organisms * Corresponding author. Tel.: þ86 592 6182723; fax: þ86 592 6181420. E-mail address: [email protected] (Y.-L. Wang).

[2]. What makes marine invertebrate animals keep themselves free from microbial infection is a very important question for aquaculture. However, research on the immune response of gastropods (phylum Mollusca) is limited. Gastropods have both cellular and humoral defense systems, the cellular one being mediated by coelomocytes and the humoral component by agglutinins and opsonins that bind non-self material. At least two types of circulating molluskan

1050-4648/$ - see front matter ª 2008 Elsevier Ltd. All rights reserved. doi:10.1016/j.fsi.2007.12.008

Cloning and responsive expression of macrophage expressed gene

Materials and methods

immune cells can be distinguished [3]: amoeboid blood cells that are large and highly phagocytic and granular cells of various sizes [4]. Gastropod leukocytes accumulate around wounds resembling accumulations in injured vertebrate tissue [5]. Cytotoxic responses in vitro have also been reported in mollusks [6]. Lebel et al. [7] have established that two types of hemocytes are present in the blood of the abalone Haliotis tuberculata. At the molecular level, research on the immune genes of gastropods has been increasing rapidly in the recent years [8e12], but results are still lacking for small abalone, which is one of the most important culture species in China. Macrophage expressed gene (Mpeg) is a homolog of two mammalian proteins that share homology with mammalian perforin, a cytolytic and immune-regulatory protein of lymphocytes. One of the mammalian proteins, Mpeg1, is expressed in mature macrophages and prion-infected mouse brains [13], while the other, Epcs50, is expressed in ectoplacental cone cells of the invading placenta [14]. The Mpeg, which was identified in two other abalones (pink abalone, Haliotis corrugata, and red abalone, Haliotis rufescens) [15], and one sponge Suberites domuncula [16], is suggested to be involved in innate immunity. Small abalone Haliotis diversicolor supertexta is one of the most commercially important cultured abalone in southern China. Since late 2000, farmers have experienced mass mortality of abalone during grow-out stage, and settlement failure of larvae during nursery stage [17]. Wang et al. [18] reported some biochemical immune factors expressed in response to bacterial challenge in this species but more work needs be done to understand the small abalone immune system for controlling disease outbreaks. Tributyltin (TBT) is the most common organotin derivative used in antifoulant paints for water pipes and vessels. Despite the prohibition of its use, it is still used in many industrial processes. TBT is known to interfere with hormone metabolism [19e25], especially its androgenic effect leading to male imposex in gastropods [26e29]. Beyond the effects on hormone metabolism, TBT can perturb the immune system as demonstrated in several species [30e34]. In this article, we report the identification of a gene from small abalone whose sequence was similar to mammalian macrophage expressed protein, as well as the other abalone (H. corrugata and H. rufescens) protein. The similarity is especially high within the perforin domain. This gene is named as saMpeg1 according to the general terminology for macrophage expressed protein [15]. The differential expression of saMpeg1 response to bacterial infection and tributyltin (TBT) chloride exposure were also described.

Table 1

347

Animals Adult small abalones, H. diversicolor supertexta (body length 4.50  0.50 cm, weight 7.55  2.10 g), were collected from a local commercial farm (Futian, Dongshan, Fujian Province). Animals were maintained in polyethylene tanks, each containing 20 animals in 50 L aerated sandfiltered seawater at 23e25  C, and fed with kelp. The culture medium was renewed with fresh seawater everyday. Animals were left undisturbed for 2 weeks to acclimate to their environment before bacterial and TBT challenge.

Bacterial infection and TBT exposure For each trial (bacterial infection, TBT exposure and their own controls), three replicates were prepared holding 20 abalones per tank. In the bacterial infection experiment, abalones were challenged by injecting either 50 mL of Vibrio parahaemolyticus (isolated from diseased abalone, [35]) in 0.9% NaCl (6.7  l07 cells/mL), or 50 mL of 0.9% NaCl (as control) into their pleopod muscle. After injection, the abalones were returned to their original tanks containing seawater at the same temperature. The hepatopancreas of control or challenged abalone was collected at 8, 24, 48, 96 h post-challenge. Samples were collected in a 1.5 mL centrifuge tube and frozen at 80  C until used. In the TBT exposure experiment, TBTCl (Sigma) was dissolved at a 2 g (Sn) L1 concentration in 100% ethanol as stock solution. The stock solution was then diluted in fresh seawater to a final concentration of 1.75 mg (Sn) L1 for exposure. In control, an equal volume of fresh seawater containing 0.1% of 100% ethanol was used instead of TBT solutions. Fresh seawater with specific TBT concentration was exchanged everyday. The hepatopancreas was taken from both challenged and control abalones at 2, 48, 96, 192 h post-challenge and frozen at 80  C until used.

RNA extraction and cDNA preparation Total RNA was extracted from hepatopancreas as described in the previous studies [36,37]. A further purification step was performed with an RNeasy kit (Qiagen, Germany) in order to digest and remove any contaminating genomic DNA for real time quantitative PCR. Single-strand cDNA was synthesized from 2e5 mg total RNA in a final volume of 20 mL containing 50 ng random primers, 50 mmol L1 TriseHCl, pH 8.3, 75 mmol L1 KCl, 3 mmol L1 MgCl2, 50 mmol L1 DTT, 0.75 U RNasin, 0.2 mmol L1 each of

Oligonucleotide primers used for real time quantitative PCR

Gene/accession no. saMpeg1/EF529460 28S rRNA/EF529461

Oligonucleotide Forward primer Reverse primer Forward primer Reverse primer

Sequence 0

Position 0

5 -GACCCTGACTTGGCGTAAGAAT-3 50 -CTCACCGCCCTGTGCATT-30 50 -CGGAGGAAAAGAAACTAACAAGGA-30 50 -TTCGGTGCTGGGCTCTTC-30

2148e2169 2191e2208 13e36 64e81

348

G.-D. Wang et al.

Figure 1 The deduced amino acid sequences of small abalone Mpeg1. The signal sequence (residues 1e19) has a single underline. Arrows show beta-sheet and cross bands show alpha-helices. The double underline (residues 150e350) shows the membrane attack complex/perforin-homology domain. Downward pointing arrows show the helix-turn-helix perforin-homology domain. The single transmembrane domain of 23 residues is boxed and is followed by the furin cleavage site KRRRK (dashed underline). Large dots indicate Cys residues of the Cys-rich domain. Two potential protein kinase A phosphorylation sites are S235 and S692 (^). Six potential Nlinked glycosylation sites are shown with asterisks. Two prokaryotic membrane lipoprotein lipid attachment sites are Y527 and A668 ( ). The two alpha-helices that are homologous to the perforin helix-turn-helix domain are labeled alpha-1 and alpha-2.

Cloning and responsive expression of macrophage expressed gene

Figure 1

dATP, dCTP, dGTP, dTTP, and 200 U MMLV reverse transcriptase (Promega). Reactions were incubated at 37  C for 1 h, terminated by heating at 95  C for 5 min, and subsequently stored at 20  C.

Sequencing the saMpeg1 cDNA The genes coding for macrophage expressed proteins from red abalone, H. rufescens (GenBank accession no. AY485640) and pink abalone, H. corrugata (GenBank accession no. AY485641) [15] have several highly similar regions in common. One pair of primers, forward primer (50 -ACATC GACAGTCGACATGTTG-30 ) and reverse primer (50 -TTTACACG TTCCGTCATCCA-30 ), was designed against nt 390e411 and nt 1516e1536 of red abalone Mpeg cDNA sequence, respectively; and used to amplify a Mpeg cDNA fragment of about 1075 bp from small abalone. The PCR programs were carried out at 94  C for 3 min, followed by 35 cycles of 94  C for 30 s, 60  C for 30 s, 72  C for 2 min and a final extension step at 72  C for 10 min. The PCR products were gel-purified and sequenced. Based on the known Mpeg cDNA of two abalones, Mpeg50 and Mpeg30 primers were designed for amplification of cDNA ends (RACE). The universal primer used for 50 -RACE and 30 -RACE was long primer (50 -CTAAT ACGACTCACTATAGGGCAAGCAGTGGTATCAACGCAGAGT-30 ). Both 50 -RACE and 30 -RACE were carried out using a Smart RACE cDNA Amplification Kit (Clontech, USA) according to the manufacturer’s instructions. The PCR programs were carried out at 94  C for 3 min, followed by 35 cycles of 94  C for 30 s, 63.2  C (50 -RACE)/68.12  C (30 -RACE) for 30 s, 72  C for 2 min and a final extension step at 72  C for 10 min. The PCR products were resolved by electrophoresis on 1% agarose gel. The fragments of interest were excised and then purified by a Gel Extraction Kit (Generay, China). The purified fragments were then cloned into pMD18-T vectors (Takara, Japan), propagated in Escherichia coli (JM109) competent cells. The plasmids isolated from positive clones were sequenced.

349

(continued).

described in our previous study [37]. In general, primers for saMpeg1 and 28S rRNA were designed using primer3 at http://frodo.wi.mit.edu/cgi-bin/primer3/primer3_www.cgi (Table 1) and tested to ensure amplification of single discrete bands with no primer-dimers. An aliquot of 2 mg of RNA pre-treated with DNAse I was used as a template for total cDNA synthesis in 25 mL reactions with random hexamers using the Superscript First-Strand Synthesis System for RTPCR (Invitrogen). For real time PCR, an amount of cDNA corresponding to 25 ng of input RNA was used in each reaction. Reactions were performed with the SYBR Green PCR Master Mix (Applied Biosystems), and analyzed in the ABI 7500 real time system. The cycling conditions for both saMpeg1 and 28S RNA were as follows: 15 min at 95  C, followed by 40 cycles (15 s at 95  C, 60 s at 60  C). Melting curves were also plotted (60e90  C) in order to make sure that a single PCR product was amplified for each pair of primers. A standard curve was constructed for each experiment using a 10fold dilution of the cloned saMpeg1 and 28S amplifications. The comparative threshold cycle (CT) method (user bulletin #2, the ABI PrismR 7700 Sequence Detector (PE Applied Biosystems)) was used to calculate the relative concentrations. This method involves obtaining CT values for saMpeg1 normalizing to the housekeeping gene, 28S rRNA (GenBank accession no. EF529461); and comparing the relative expression level. Experiments were performed routinely with three challenged abalones and three control abalones with values presented as 2DDCT for the expression levels of saMpeg1 normalized with 28S rRNA (DCT Z CT of saMpeg1 minus CT of 28S rRNA, DDCT Z CT of challenged sample minus DCT of the control sample). Data were expressed as mean and standard error of the mean (SEM) unless otherwise stated. Three separate individuals at each time were tested, each assayed in triplicate. Statistical analysis of the normalized CT values was performed with Student’s t-test using SPSS. Differences were considered significant at p < 0.05 (two-tailed test).

Analysis of sequences Real-time quantitative PCR The analysis of saMpeg1 expression after challenge was performed by real time quantitative PCR using SYBR Green I as

The signal sequence was identified using the program SignalP (http://www.cbs.dtu.dk/services/SignalP). The PredictProtein server (http://cubic.bioc.columbia.edu/

Mus-Mpeg1

1 ----------------------------MNSFMALVLIWMIIACA

17

Homo-Mpeg1

1 ----------------------------MNNFRATILFWAAAAWA

17

Mus-Pfp1

1 ----------------------------MNSFIVTVLIWTTVAYA

17

Mus-Epcs50

1 ----------------------------MNSFIVTVLIWTTVAYA

17

Suberites-Mpeg

1 MVCLTRQIGSLTDTHPTNKLVMVGGGGGAGGLLSLVSGALSGGGG

45

Haliotis-abMpeg1

1 ----------------------------MLGFVLVVSIVASVSGG

17

Haliotis-saMpeg1

1 ----------------------------MLSFVLVVSGVASVLGG

17

cons

1

.

45

Mus-Mpeg1

18 E--ADKPLGETGTTGFQIC-KNALKLPVLEVLPGGGWDNLRNVDM

59

Homo-Mpeg1

18 K--SGKPSGEMDEVGVQKC-KNALKLPVLEVLPGGGWDNLRNVDM

59

Mus-Pfp1

18 E--EEKALKEIHEYGFQKC-QNALNLPVLEVLPGGGWDNLRNMDM

59

Mus-Epcs50

18 E--EEKALKEIHEYGFQKC-QNALNLPVLEVLPGGGWDNLRNMDM

59

.:

:

Suberites-Mpeg

46 NIRAGAPRNMYPRGDPRNCLSGNPKLNILQVVPGIGWDNLRNSET

90

Haliotis-abMpeg1

18 ELLDSVKKPEFPKGDVRACYGDNKKLERFEVLPGQGWDNLRNVDA

62

Haliotis-saMpeg1

18 EVLHSGNKSEFPIGDVRACYRDNKKLDRFEVLPGQGWDNLRNVDA

62

cons

46 :

90

Mus-Mpeg1

60 GRVMDLTYTNCKTTEDGQYIIPDEVYTIPQKESNLEMNSEVLESW

104

Homo-Mpeg1

60 GRVMELTYSNCRTTEDGQYIIPDEIFTIPQKQSNLEMNSEILESW

104

Mus-Pfp1

60 GQVMDLTYNNCKTTEDGQYIIPDDVFTIPQKESNLEMNSEILDSW

104

Mus-Epcs50

60 GQVMDLTYNNCKTTEDGQYIIPDDVFTIPQKESNLEMNSEILDSW

104

. : *

.

:*

::*:** ******* :

Suberites-Mpeg

91 GILTSFSYSQCKVTYDRRYLIPDETFAIPIKTSTIDYQAELFDHW

135

Haliotis-abMpeg1

63 GLVVVYNYSRCRTTEDGRYLIPDTVNTIPLKASKLNVYAELISHW

107

Haliotis-saMpeg1

63 GLVVVYNYSRCRTTEDGRYLIPDTVNTIPLKTSKLNVYAELISHW

107

cons

91 * :

:*::. *

135

Mus-Mpeg1

105 MNYQSTTSLSINTELAL--F-SRVNGKFSTEFQRMKTLQVKDQAV

146

Homo-Mpeg1

105 ANYQSSTSYSINTELSL--F-SKVNGKFSTEFQRMKTLQVKDQAI

146

Mus-Pfp1

105 ENYRSTTSFSINLNLDY--R-PRVNGKFSSEFQRIKTTQVRDQAV

146

Mus-Epcs50

105 ENYRSTTSFSINLNLDY--R-PRVNGKFSSEFQRIKTTQVRDQAV

146

Suberites-Mpeg

136 DAYKSVTSRSINAGF--DFF-GKIGG-------------------

158

Haliotis-abMpeg1

108 SSYTSTTAHGVNVDAGLKYDSVQVSGKFSSGYESVKSKQIGDKSY

152

Haliotis-saMpeg1

108 SNYTSTTAHGVNVDAGLHFDSVKVSGKFSSGYEDVKSKQIGDRSY

152

cons

136

180

.*..*:.* * :*:***

* * *: .:*

::.*

:** * *.::

Figure 2 Comparison of the saMpeg1 aa sequence with Mpeg homologs and mouse Epcs50. Species and gene names are abbreviated at the left and represent Mus musculus macrophage expressed gene 1, GenBank accession no. BC129844; Homo sapiens macrophage expressed gene 1, GenBank accession no. BC112230; M. musculus pore forming protein-like, GenBank accession no. NM_019540; M. musculus Epcs50, GenBank accession no. AF250839; Suberites domuncula macrophage expressed protein, GenBank accession no. AJ890501; Haliotis corrugata macrophage expressed protein, GenBank accession no. AY485640; Haliotis diversicolor supertexta macrophage expressed protein, GenBank accession no. EF529460. Sequence alignment was performed using the T-Coffee at http://www.ch.embnet.org/index.html. Consistency scores are shown by color as: BAD AVG GOOD.

Mus-Mpeg1

147 TTRVQVRNRIYTVKTTPTSELSLGFTKALMDICDQLEKNQTKMAT

191

Homo-Mpeg1

147 TTRVQVRNLVYTVKINPTLELSSGFRKELLDISDRLENNQTRMAT

191

Mus-Pfp1

147 TTRAQVRNLVYTVKANPNAELNLGFKKELMEICDRLEKNQTKMAT

191

Mus-Epcs50

147 TTRAQVRNLVYTVKANPNAELNLGFKKELMEICDRLEKNQTKMAT

191

Suberites-Mpeg

159 ------------------------------EILSLLKSNDTESAD

173

Haliotis-abMpeg1

153 TTRVQLRYVRYSAKLQPDASLHPTFKTRLLSIAGSLQLNKTDQAR

197

Haliotis-saMpeg1

153 TTRVQLRYVRYTAKLQPDAALHPTFKSRLLSIAGSLQLNKTDQAR

197

cons

181

225

Mus-Mpeg1

192 YLAELLILNYGTHVITSVDAGAALVQEDHVRSSFLLDNQNSQNTV

236

Homo-Mpeg1

192 YLAELLVLNYGTHVTTSVDAGAALIQEDHLRASFLQDSQSSRSAV

236

.* . *: *.*

*

Mus-Pfp1

192 YLAELLILNYGTHIITSVDAGATLVQEDHVKSSYLKNNKGNRAAV

236

Mus-Epcs50

192 YLAELLILNYGTHIITSVDAGATLVQEDHVKSSYLKNNKGNRAAV

236

Suberites-Mpeg

174 YAAQILIRDYGTHCITSIDAGAVLIKEDNLKSTIMSNYKGRADSL

218

Haliotis-abMpeg1

198 YDSELLVRDFGTHVVTSVDAGAALVQEDQVSSEFVNSRKFTKNQI

242

Haliotis-saMpeg1

198 YDSELLVRDFGTHVITDVDAGAALVQEDQVSAEFVNSRKFSKSQI

242

cons

226 * :::*: ::***

:

270

Mus-Mpeg1

237 TASAGIAFLNIVNFKVETDYISQTS--LTKDYLSNRTNSRVQSFG

279

Homo-Mpeg1

237 TASAGLAFQNTVNFKFEENYTSQNV--LTKSYLSNRTNSRVQSIG

279

Mus-Pfp1

237 AASAGFTFAKVVNFKIETGFEYQNN--LATGYLQNRTGSRVQSIG

279

Mus-Epcs50

237 AASAGFTFAKVVNFKIETGFEYQNN--LATGYLQNRTGSRVQSIG

279

Suberites-Mpeg

219 STAAGVEFYDMLKLRASAGFSSYSGDSDLKAYRQNRTSSRLYTYG

263

Haliotis-abMpeg1

243 TAGASASLFGIFSIDVSYHSSTSNE--VKKAYEQSRSSSQIDTLG

285

Haliotis-saMpeg1

243 TAGASASLFGIFSIDVSYHSSTSNE--VQTAYEQHRSSSQIDTLG

285

cons

271 ::.*.

. * . *:.*:: : *

315

Mus-Mpeg1

280 GVPFYP-GITLETWQKGITNHLVAIDRAGLPLHFFIKPDKL-PGL

322

Homo-Mpeg1

280 GVPFYP-GITLQAWQQGITNHLVAIDRSGLPLHFFINPNML-PDL

322

:

..:

*.:****.*::**:: :

.

.

: . :

Mus-Pfp1

280 GIPFYP-GMTIETWQKGIINHLVAVDRAGLPLHFFIKPEKL-PAF

322

Mus-Epcs50

280 GIPFYP-GMTIETWQKGIINHLVAVDRAGLPLHFFIKPEKL-PAF

322

Suberites-Mpeg

264 GPPYKL-GMNLSRWENDLMNNLVATDRSGKPSHSLSTTQSLKPEV

307

Haliotis-abMpeg1

286 GPMFKASNFTANDWTNEVDHELVAVDRSGDPLFFLINSASL-PEL

329

Haliotis-saMpeg1

286 GPMFKASNFTANDWTSEVDHELVAVDRSGDPLLFLINSVSL-PEL

329

cons

316 *

360

:

.:. . * . : :.*** **:* *

Figure 2

predictprotein) was used to determine secondary structures and post-translational modification. The TMHMM program (http://cbs.dtu.dk/services/TMHMM/) was used to locate transmembrane segments. Pepwheel diagram (http://kael. net/helical_old.htm) was used to show the amphipathic

: ..

* * .

(continued).

character of helices. Protein multiple-alignments were performed with T-Coffee (http://www.ch.embnet.org/ index.html). Maximal parsimony dendrograms based on T-Coffee alignments were drawn using njplot (http://pbil. univ-lyonl.fr/software/njplot.html; [38]).

352

G.-D. Wang et al.

Mus-Mpeg1

323 PG-PLVKKLSKTVETAVRHYYTFNTHPGCTNVDSPNFNFQANMD-

365

Homo-Mpeg1

323 PG-PLVKKVSKTVETAVKRYYTFNTYPGCTDLNSPNFNFQANTD-

365

Mus-Pfp1

323 PR-HLVEQLAKTVETAAESYYNFNTYPGCTNINSPNFDFQANTD-

365

Mus-Epcs50

323 PR-HLVEQLAKTVETAAESYYNFNTYPGCTNINSPNFDFQANTD-

365

Suberites-Mpeg

308 TTSQEVFLLRRLVKSAVSQYYHYNTHTGCKNPKAPNLDHQTNNGA

352

Haliotis-abMpeg1

330 PN-SVLYQLQNLVEETILHYYEFNTYRGCTELDSPNFSPAANLD-

372

Haliotis-saMpeg1

330 PN-SVLYQLQNLVQETILHYYEFNTYRGCTELDSPNFSPAANLD-

372

cons

361 .

405

Mus-Mpeg1

366 DDSCDAKVTNFTFGGVYQECTELSGD---VLCQNLEQKNLLTGDF

407

Homo-Mpeg1

366 DGSCEGKMTNFSFGGVYQECTQLSGNRDVLLCQKLEQKNPLTGDF

410

:

: . *: :

** :**: **.: .:**:.

:* .

Mus-Pfp1

366 DGSCDGKLVNAPFGGVYQLCRQLSGHDYDDMCLDFHQKNPLTGDF

410

Mus-Epcs50

366 DGSCDGKLVNAPFGGVYQLCRQLSGHDYDDMCLDFHQKNPLTGDF

410

Suberites-Mpeg

353 PGVCKEPSANYTFGGVFQSCRS----NGNDICGKLLQKNPLTGGY

393

Haliotis-abMpeg1

373 DGTCKSPYTNLTFGGVYQTCSMSSGSNNGDLCSGLDQVNPKTGGH

417

Haliotis-saMpeg1

373 DGTCKSPYTNLTFGGVFQTCSMSSGSNNGDLCSGLDQVDPKTGGH

417

cons

406

**..

450

Mus-Mpeg1

408 SCPPGYTPVHLLSQTHEEGYSRLEC--KKKCTLKIFCKTVCED--

448

Homo-Mpeg1

411 SCPSGYSPVHLLSQIHEEGYNHLEC--HRKCTLLVFCKTVCED--

451

. *.

.* .****:* *

:*

: * :

Mus-Pfp1

411 SCPPDYTPVHLLSQTHEKGYTGMEC--RDKCILKIFCRTSCKD--

451

Mus-Epcs50

411 SCPPDYTPVHLLSQTHEKGYTGMEC--RDKCILKIFCRTSCKD--

451

Suberites-Mpeg

394 SCPKNFKALLLQLGTERSHKMRRVCVWKRKCTFFVFN---CHDVD

435

Haliotis-abMpeg1

418 TCPDGYESVELHTGRLSDSKSVHSC--H-SCWLFFKC---CHD--

454

Haliotis-saMpeg1

418 TCPDGYEPVELHTGRLSNSKSVHSC--H-SCWLFFKC---CHD--

454

cons

451 :** .: .: *

495

Mus-Mpeg1

449 --VFRV--AKAEFRAYWCVAAGQVPDNSGLLFGGVFTDKTINPMT

489

Homo-Mpeg1

452 --VFQV--AKAEFRAFWCVASSQVPENSGLLFGGLFSSKSINPMT

492

.

*

: .* : .

*.*

Mus-Pfp1

452 --EFRV--AKAEVSSYWCAASSQVPENSGILFGGVFTDKSINPMT

492

Mus-Epcs50

452 --EFRV--AKAEVSSYWCAASSQVPENSGILFGGVFTDKSINPMT

492

Suberites-Mpeg

436 DCTFVPSVEIASYQTYWCAPNKKNPPKFGYMFGGIYSNDIQNPIT

480

Haliotis-abMpeg1

455 --NYYH--SEATYIMHWCAATGPVSQDSGYLFGGLYTSQLNNPLT

495

Haliotis-saMpeg1

455 --NYYH--SEATYIMHWCAATGSVSQESGYLFGGLYTSQLNNPLT

495

cons

496

540

:

*

Figure 2

.**..

(continued).

. . * :***:::..

**:*

Cloning and responsive expression of macrophage expressed gene

353

Mus-Mpeg1

490 NAQSCPAGYIPLNLFESLKVCVSLDYELGFKFSVPFGGFFSCIMG

534

Homo-Mpeg1

493 NAQSCPAGYFPLRLFENLKVCVSQDYELGSRFAVPFGGFFSCTVG

537

Mus-Pfp1

493 NEPSCPTSYFPLKLFENVKVCVSLDHELGPKYSVPFGGFFSCTKG

537

Mus-Epcs50

493 NEPSCPTSYFPLKLFENVKVCVSLDHELGPKYSVPFGGFFSCTKG

537

Suberites-Mpeg

481 RSCSCPTHFLPLRMGERATVCVSEDYELGHQFSLPFGGFFSCVSG

525

Haliotis-abMpeg1

496 QGKTCPVNFYTRTLGKDLHICISDDYELGMKYSMPFGGFISCTTG

540

Haliotis-saMpeg1

496 QRKDCPVNFYTRTLGKDLHICISDDYELGMKYSMPFGGFISCTSG

540

cons

541 .

*

585

Mus-Mpeg1

535 NPLVNSDTAK---DV--RAPSL-----------KKCPGGFSQHLA

563

Homo-Mpeg1

538 NPLVDPAISR---DL--GALSL-----------KKCPGGFSQHPA

566

**. : .

: :

:*:* *:*** ::::*****:**

Mus-Pfp1

538 NPLYGSFTSG---DL--EKSFL-----------PKCPGGFSQQLA

566

Mus-Epcs50

538 NPLYGSFTSG---DL--EKSFL-----------PKCPGGFSQQLA

566

Suberites-Mpeg

526 NVLAGNGSSE--------------FLNNPKDWPMRCPGGFTQHLA

556

Haliotis-abMpeg1

541 NPLAMNPKPKSKGDMNSALPSLHSFFQGSKTWPKHCPKGYSQHLA

585

Haliotis-saMpeg1

541 NPLAMSPKPE--GNKNSALPSLHSFFQSSKSWPKHCPKGYSQHLA

583

cons

586 * *

:** *::*: *

630

Mus-Mpeg1

564 VISDGCQVSYCVKAGIFTGGSLLPVRLPPYTKPPLMSQVATNTVI

608

Homo-Mpeg1

567 LISDGCQVSYCVKSGLFTGGSLPPARLPPFTRPPLMSQAATNTVI

611

.

Mus-Pfp1

567 AIIDGCQVSYCVKAGVFTRASLAPARLPPYTQLP-MSQLSTDSVE

610

Mus-Epcs50

567 AIIDGCQVSYCVKAGVFTRASLAPARLPPYTQLP-MSQLSTDSVE

610

Suberites-Mpeg

557 LTEKGCRVNFCVKAGSLLRASDLELVLPPFDPKPTLRKNSTSDLF

601

Haliotis-abMpeg1

586 YVDQGCEINYCLLAGSLSEVGLPKIRRPPFQTAPLLLPSTENHVV

630

Haliotis-saMpeg1

584 YVDQGCEINYCLKAGSLSEVGLPKIRRPPFQSAPLLLPSTENQVV

628

cons

631

675

Mus-Mpeg1

609 VTNSETARSWIKDPQTNQWKLGEPLELRRAMTVIHGDSNGMSGGE

653

Homo-Mpeg1

612 VTNSENARSWIKDSQTHQWRLGEPIELRRAMNVIHGDGGGLSGGA

656

Mus-Pfp1

611 MT-GESPISWVKDPYTLQWNLEE-----------SSKSGRLSGGS

643

Mus-Epcs50

611 MT-GESPISWVKDPYTLQWNLEE-----------SSKSGRLSGGS

643

Suberites-Mpeg

602 S--KPAVAP---------------------------SGSLPIKGV

617

Haliotis-abMpeg1

631 F--DPVTLTWRKNQEAMQF-----ISARVGEATPSSTGSGMSAGA

668

Haliotis-saMpeg1

629 F--DPLTLTWRKNQEAMQF-----INAQGGEARSSFAGSALFAGA

666

cons

676

720

.**.:.:*: :* :

.

.

Figure 2

**:

* :

: . :

..

(continued).

*

354

G.-D. Wang et al. Mus-Mpeg1

654 AAGITLGVTIALGIVITLAIYGTRKYKKKEYQEIEEQESLVGSLA

698

Homo-Mpeg1

657 AAGVTVGVTTILAVVITLAIYGTRKFKKKAYQAIEERQSLVPGTA

701

Mus-Pfp1

644 TAAITIVVILALGVLTAMAIYGNRRFKKNKPNGLPRQQALL-PFP

687

Mus-Epcs50

644 TAAITIVVILALGVLTAMAIYGNRSLRRINPMDFQDNRHCF-LFQ

687

Suberites-Mpeg

618 PPPLD-----NNRVIMYYPVLSSNTGQRD----------------

641

Haliotis-abMpeg1

669 AAGIAVVATLGCVVISTVIVVLIKRRRKS----------------

697

Haliotis-saMpeg1

667 AAGIAVVATFGCVVISTLIIVLIKRRRKS----------------

695

cons

721 .. :

765

Mus-Mpeg1

699 TDATV---------------------LNGEEDPSPA---------

::

:

.

::

713

Homo-Mpeg1

702 ATGDT---------------------TYQEQGQSPA---------

716

Mus-Pfp1

688 RVRVF---------------------PDGVQDPNPA---------

702

Mus-Epcs50

688 ELESFLMECRIQIQLNSSLKKQFLSSPDTGSSSSFSPFSSTSAFL

732

Suberites-Mpeg

642 ---------------NGVGR---ITEPFFCHSWDWSVDLSVAHQA

668

Haliotis-abMpeg1

698 ---------------SAGYRHLAIDDPLLSPQSNYGATGSDTVNV

727

Haliotis-saMpeg1

696 ---------------LATYRKLAIDDPLLSPQSNYGSTGSDAVSV

725

cons

766

810

Mus-Mpeg1

714 --------------------

. .

713

Homo-Mpeg1

717 --------------------

716

Mus-Pfp1

703 --------------------

702

Mus-Epcs50

733 RAKAEVATERRFFITLWLCL

752

Suberites-Mpeg

669 IDK----------FAFA--K

676

Haliotis-abMpeg1

728 HVE-----------------

730

Haliotis-saMpeg1

726 NVE-----------------

728

cons

811

830

Figure 2

Results The saMpeg1 sequence The saMpeg1 open reading frame is 2184 bp in length. The 50 untranslated region locates 252 bp upstream of the putative start codon (ATG) (Fig. 1). The 30 untranslated region of 342 nucleotides ends at a poly (A) tail. A possible consensus signal sequence for polyadenylation, TAA, is located 311 bp upstream of the poly (A) tail (Fig. 1). Bioinformatic analysis employing BLAST searches of public domain DNA sequence databases showed that the nucleotide sequence of the saMpeg1 displayed a high degree of identity with H. corrugata and H. rufescens. Eighty eight percent of 2189 bp and 87% of 2216 bp were identical

(continued).

to Mpeg1 mRNA from H. corrugata and H. rufescens, respectively. saMpeg1 cDNA encodes a 728 amino acid protein with a putative signal sequence of 17 residues. The calculated molecular mass of the mature saMpeg1 of 711 residues is 79.8 kDa and the predicted pI is 6.69. saMpeg1 shares high similarity with abMpeg1 of H. corrugata (GenBank accession no. AAR82935) and H. rufescens (GenBank accession no. AAR82936) (both 89% identity and 94% similarity). Moreover, saMpeg1 also shares significant sequence similarity with similar proteins identified in both vertebrates and invertebrates (Fig. 2). For example, saMpeg1 shares 40% identity and 59% similarity with Mpeg1 from Homo sapiens (GenBank accession no. BC112230). The predicted amino acid sequences of Mpeg from a sponge S. domuncula (GenBank accession no. AF250839) share 34% identity and 51% similarity

Cloning and responsive expression of macrophage expressed gene

355

Figure 5 The change of saMpeg1 after TBT exposure. Data are presented as means  SEM of three separate individuals, each assayed in triplicate. There is no significant difference between treatment and control (p > 0.05).

Figure 3 Dendrogram of MPEG and Epcs50 from different organisms based on amino acid sequence comparisons. For legend see Fig. 2.

with asMpeg1. Comparison of various similar proteins using T-Coffee generates trees of phylogenetic relationship of the topology (Fig. 3). The phylogenetic tree reveals a grouping of metazoan perforin-related proteins into three branches, one comprising the abalone proteins, a second one containing the sponge protein, and finally the mammalian sequences in the third branch. BLAST searches of GenBank were used to find homologous protein domains. The saMpeg1 sequence possesses a ‘‘membrane attack complex/perforin-homology domain’’ (residues 150e350, shown in Fig. 1) containing the cytolytic helixturn-helix functional domain of perforin (residues 171e218,

shown in Fig. 2) [15,39], a cysteine-rich region containing 16 Cys residues (residues 357e594, shown in Fig. 1), a putative single transmembrane domain (TMS; residues 667e689, shown in Fig. 1), and a modified furin cleavage site (KRRRK; residues 689e693, shown in Fig. 1). In saMpeg1 sequences, there are six potential N-linked glycosylation sites, five upstream of the Cys-rich region, two potential sites for cAMP-dependent protein kinase phosphorylation (Fig. 1, positions S235 and S692) and two prokaryotic membrane lipoprotein lipid attachment sites (Fig. 1, positions Y527 and A668). Predictions of secondary structure show short beta-strands, alpha-helices, short loop regions, and longer unstructured regions (Fig. 1). The three longest helices (alpha 1e3) are predicted to be within the helix-turn-helix cytolytic perforin domain (Fig. 1).

Expression of saMpeg1 in response to V. parahaemolyticus injection and TBT exposure In order to measure the gene expression of saMpeg1 after bacterial challenge or TBT treatment, relative mRNA levels were measured using quantitative real time PCR. A significantly higher level of saMpeg1 expression was observed at 8 h and 96 h post-bacterial injection compared to the controls (Fig. 4) (p < 0.05, t-test). There was no significant difference between TBT treatment and controls (Fig. 5) (p > 0.05, t-test).

Discussion

Figure 4 The change of saMpeg1 after Vibrio parahaemolyticus challenge. Data are presented as means  SEM of three separate individuals, each assayed in triplicate. Asterisks show that there is significant difference between treatment and control (p < 0.05).

Mpegs are known in humans [40], mouse and mollusks [41], as well as in the sponge system [16,42]. The amino acid of saMpeg1 displayed high sequence similarity (89%) with the abMpeg1 from pink abalone and red abalone. Between saMpeg1 and mouse Mpeg1, there is 38% similarity. saMpeg1 and mouse Epcs50 are 37% similar. Mah et al. [15] suggest abalone abMpeg1, mouse Mpeg1 and mouse Epcs50 are a new gene family, Mpeg1 family. Because of high sequence similarity between saMpeg1 and the gene family, saMpeg1 belongs to the Mpeg1 family [15]. The members of the new family have putative signal sequences at the Ntermini. Similar to mouse Epcs50 and abMpeg1, saMpeg1

356 has putative single TMS close to the C-termini. If saMpeg1 is a type of membrane protein, the prediction showed that the 666 residues upstream of the 23 residue TMS would be on the external side of a membrane compartment, while the 39 residues downstream of the TMS would make up the C-terminus on the luminal side of the membrane. Similar to pink and red abMpeg1 proteins, the saMpeg1 protein has a furin cleavage site immediately following this single TMS, whereas mouse Epcs50 does not have a consensus furin site. Cleavage at the furin site could increase the movement of saMpeg1 within the plane of the membrane. saMpeg1, abMpeg1, mouse Mpeg1, human Mpeg1 and mouse Epcs50 proteins share homologous domain with mouse perforin. The helix-turn-helix motif is what makes perforin a lytic protein of cytotoxic T lymphocytes and natural killer cells [39]. These amphipathic alpha-helices allow perforin to enter the lipid bilayer where it self-associates to form a pore which kills the target cell [13]. The region of greatest similarity between these four proteins and mouse perforin corresponds to the helix-turn-helix domain of perforin (Fig. 2, saMpeg1 residues 171e219). Helical wheel diagrams of alpha-helices 1e3 of saMpeg1 show that these three helices are amphipathic (Fig. 6). This structural similarity to perforin is further evidence that Mpeg1 proteins could be involved in cell killing and immune regulation [39]. Until recently, perforin was thought to be present only in cytotoxic T lymphocytes and natural killer cells and to be involved solely in killing virus-infected or transformed cells [39]. However, it is now known that perforin is also found free in the cytoplasm of human fetal and adult astrocytes in culture and in astrocytomas [43]. Perforin has also been detected in areas of inflammation in white and gray matter in brains of multiple sclerosis and Alzheimer’s disease victims [43]. Mutations that delete the normal function of perforin result in familiar hemophagocytic lymphohistiocytosis (FHL), a lethal inherited disorder of humans [39]. Mouse Mpeg1 is highly expressed in mature macrophages [13]. It is also upregulated in microglia in the brains of mice infected with experimental prion disease. Upregulation correlates with spongiform degeneration of the brain and it has been suggested that Mpeg1 could be directly involved in neuronal cell death [44]. Mouse Mpeg1 has been found in a collection of 15,000 full-length cDNA sequences [45]. Lastly, Mpeg1 protein has been found to be associated with isolated phagosomes from a mouse macrophage cell line [46]. The upregulation of saMpeg1 after bacterial challenge suggests that it plays a role in the process of eliminating and killing bacteria. The upregulation of the macrophage expressed gene is also found in the pink abalone, red abalone [15] and sponge [16]. However, the temporal expression pattern of saMpeg1 was different. In pink abalone and red abalone, a high level mRNA is expressed at 24 h post-bacterial challenge [15] and in sponge, a strongly increased expression was observed at day 1 after challenge, which rose further during the following 2 days [16]. However, there is no upregulation at 24 h and 48 h postchallenge in small abalone but it should be noted that there is a different challenge mode between small abalone (injection) and pink abalone, red abalone and sponge (exposed). As an effector molecule, the Mpeg expression is controlled by its regulators. Wiens et al. [16] suggested that

G.-D. Wang et al.

Figure 6 Alpha-helical wheel diagrams show that alphahelices (alpha-1, alpha-2, and alpha-3) and their nearby residues (Fig. 1) are amphipathic. Hydrophobic residues are shaded and numbers indicate the amino acid position.

Cloning and responsive expression of macrophage expressed gene the MyD88-dependent signaling pathway is the regulator of Mpeg. We also found that lipopolysaccharide (LPS)interacting protein, a molecule of the MyD88-dependent signaling pathway (unpublished data), was involved in the abalone response to bacterial challenge. We suggest that the activation of the signaling pathway may be dependent on the quantity of bacteria. The injection of bacteria activated the signaling pathway and resulted in the increase of saMpeg1 at 8 h. The increase of saMpeg1 may have killed most of the bacteria leading to downregulation of the signaling pathway and saMpeg1 decreased to the normal level. However, some bacteria may have survived in the abalone tissues. When saMpeg1 decreased to the normal level, the bacteria may have reproduced and reached such a high quantity that they activated the signaling pathway again resulting in the increased expression of saMpeg1 again at 96 h. Moreover, different invading microorganisms induce different toxicity [47,48] which may also work as the stimuli to upregulate the expression of saMpeg1. In mouse and abalone, this ancient protein family may function in cell killing and the regulation of the inflammatory response. The recombinant Mpeg from sponge displays a toxic effect on bacteria in vitro, supporting the notion that this protein plays an important role in killing bacteria. Although it is clear that the immune system is a target for TBT toxicity [34,49], our TBT exposure shows that there is no significant impact on the expression of saMpeg1. Most of the available information concerning the immunotoxicity of TBT to mollusks, mainly inhibition of phagocytosis, has been obtained from Mytilus edulis [50], Tapes philippinarum [51] and Mya arenaria [52]. In these reports, TBT decreases the number of hemocytes. Few reports pay attention to the relation between TBT and the genes related to immunity. Our group is in the process of identifying other different expression genes of small abalone after TBT exposure or bacterial challenge. We find that superoxide dismutase gene (Sod ) is downregulated after TBT exposure and there is no significant change after bacterial challenge (unpublished data). The SODs are a group of metalloenzymes that catalyse the conversion of reactive superoxide anions (O 2 ) to yield hydrogen peroxide (H2O2), and are considered to play a pivotal antioxidant role [53]. TBT is known as an inhibitor of oxidative phosphorylation, affecting cell metabolism by stimulating the production of adenosine triphosphate and by inhibiting its transformation into adenosine diphosphate [54]. As a result, malformations of the mitochondrial membranes are observed. Moreover, several studies examining the composition of hemocyte populations of gastropods suggested that hemocytes are composed of a mixture of different subpopulations of cells [55]. For example, only the small round hemocytes (RH) of Planorbarius corneus are able to lyse a human cell line in a natural killer (NK) cytotoxicity test indicating an early sharing of functional characteristics between RH and NK-related cells [56]. Considering Mpeg1 is expressed in mature macrophages [13] and perforin is mainly expressed in natural killer (NK) cells [39] suggest that TBT has an impact on antioxidant enzymes and their genes, even the survival of some subpopulations of hemocytes, but has little effect on perforin-like protein genes, like the macrophage expressed gene. In summary, we have cloned Mpeg1 from the small abalone. The sequence is similar to mammalian Mpeg1.

357

The expression of saMpeg1 was upregulated following bacterial infection but was not changed after TBT exposure which suggests that the saMpeg1 gene may play a role in the immune response.

Acknowledgments This work was supported by 863 High Technology Project (No. 2002AA629220) from the Chinese Ministry of Science and Technology, Xiamen, Fujian (502Z20055024) and Foundation of Key Laboratory of Science and Technology for Aquaculture and Food Safety, Fujian Province University (2007J201). We thank Mr. Scot Libants (Department of Fisheries & Wildlife, Michigan State University, USA) for critical reading of the manuscript.

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