Identification and characterization of a novel IκB-ɛ-like gene from Lamprey (Lampetra japonica) with a role in immune response

Fish & Shellfish Immunology 35 (2013) 1146e1154

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Identification and characterization of a novel IkB-3 -like gene from Lamprey (Lampetra japonica) with a role in immune response Peng Su a, b,1, Xin Liu a, b,1, Yinglun Han a, b, Zhen Zheng a, b, Ge Liu a, b, Jing Li a, b, Qingwei Li a, b, * a b

College of Life Science, Liaoning Normal University, Dalian 116029, China Institute of Marine Genomics & Proteomics, Liaoning Normal University, Dalian 116029, China

a r t i c l e i n f o

a b s t r a c t

Article history: Received 23 April 2013 Received in revised form 2 July 2013 Accepted 14 July 2013 Available online 2 August 2013

Nuclear factor of kappa B (NF-kB) is a stimuli-activated transcription factor, regulates the expression of a diverse array of genes. Inhibitor of kappa B-epsilon (IkB-3) is an inhibitor of NF-kB, which retains NF-kB in an inactive state in the cytoplasm. Lampreys (Lampetra japonica) belong to the lowest class of vertebrates with little information about its IkBs. We have identified a cDNA sequence IkB-3 -like in the lamprey and the deduced amino acid sequence of IkB-3-like. It contains a conserved DSGxxS motif and six consecutive ankyrin repeats, which are necessary for signal-induced degradation of the molecule. Phylogenetic analysis indicated it had high sequence homology with IkB-3s from other vertebrates. FACS analysis showed that IkB-3-like located in cytoplasm of leukocytes. The degradation of IkB-3-like could be observed in leukocytes of L. japonica stimulated with lipopolysaccharide. These results indicate that IkB-3 proteins are conserved across vertebrates and the NF-kB-like signaling pathway may exist in the oldest agnatha. Ó 2013 Published by Elsevier Ltd.

Keywords: IkB-3 NF-kB Lamprey Signaling pathway

1. Introduction Members of the nuclear factor of kappa B (NF-kB) family, NF-kB1 (p50 and its precursor p105), NF-kB2 (p52 and its precursor p100), RelA (p65), RelB and c-Rel are maintained as inactive homo- or heterodimers in the cytoplasm by inhibitors of kappa B (IkBs) [1,2]. The same as inhibitor of kappa B-alpha (IkB-a) and inhibitor of kappa B-beta (IkB-b), inhibitor of kappa B-epsilon (IkB-3) is a typical member of IkBs and plays important roles in nuclear translocation of NF-kB that regulates the expression of various immune factors [3,4]. In the N-terminal region of IkB-3, there is a classical motif, DSGxxS, which is highly similar to those in IkB-a and IkB-b. Moreover, IkB-3 contains six consecutive ankyrin repeats [5], which are also found in other classical members of the IkB family. The IkBs are characterized by the presence of multiple ankyrin repeats that mediate binding to NF-kB dimers and can interfere with nuclear

* Corresponding author. 1 Liu Shu Nan Jie, Liaoning Normal University, Dalian 116029, China. Tel.: þ86 0411 82156555; fax: þ86 0411 85827799. E-mail address: [email protected] (Q. Li). 1 These authors contributed equally to this work. 1050-4648/$ e see front matter Ó 2013 Published by Elsevier Ltd. http://dx.doi.org/10.1016/j.fsi.2013.07.026

localization signals (NLS) [6,7]. The C-terminal halves of p105 and p100 also have multiple ankyrin repeats that make them to serve an IkB-like function [8,9]. In the C-terminal region, IkB-a and IkB-b contain a PEST sequence, which is essential for them to be degraded in response to extracellular stimuli [10]. However, unlike the classical IkB-a and IkB-b, IkB-3 does not possess a C-terminal PEST sequence [5]. Although the IkBs are similar in structure, they differ from one another in preferential NF-kB binding and are subject to differentially transcriptional regulation by NF-kB family members. For example, IkB-a tends to bind RelA/p50 heterodimers, but IkB-3 prefers to bind RelA/RelA as well as c-Rel/RelA dimers [11]. Recent research suggests a non-redundant role for IkB-3 in the regulation of c-Rel-dependent B lymphocyte survival mechanisms [12]. Compared with IkB-a, IkB-3 is degraded with relatively slow kinetics, which may be due to the absence of a C-terminal PEST region [5]. IkB-3 plays an important role in the differential modulation of NF-kB-responsive genes during B cell differentiation [13]. The differential regulation of IkB-3 may also be effected through interactions with the protein phosphatase 6 (PP6) ternary complex and ankyrin repeat subunit (ARS)-A of PP6 [14,15]. Lampreys are surviving representative of the jawless vertebrates, which are considered to have appeared around 450e500

P. Su et al. / Fish & Shellfish Immunology 35 (2013) 1146e1154 Table 1 Primers. Name

Sequences

Cloning of the full-length cDNA 30 RACE of IkB-3 -like 50 -TCGGCCATCAGTTCCATGTGCGAAGC-30 50 RACE of IkB-3 -like 50 -AGACCCCCGACTCAATGTCCGTGCT-30 Cloning of IkB-3 -like fragment (upstream) 50 -CGCGGATCCATGAGCGTCTCTCGTGGAGATGCC-30 (downstream) 50 -CCCAAGCTTGACCCCCGACTCAATGTCCGTGCT-30

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million years ago [16]. Because of their unique position at the interface between the jawless and jawed vertebrates, they are the key species to study the evolution of immune system [17]. They both possess the innate and adaptive immune response system [18]. In earlier studies, immunologists had struggled to understand the molecular mechanism of the lamprey adaptive immune system, which lacks the hallmark components necessary for adaptive immunity in mammals [19]. Then immune molecules such as CD45, CD9, BCAP and CD98 were identified from lamprey, which make the question of the stage jawless vertebrates be concerned

Fig. 1. Nucleotide and deduced amino acid sequence of IkB-3-like cDNA from L. japonica. The start and stop codons are shown in box. Two Potential phosphorylation sites (Serine 26 and 30) are shown in bold type. The polyadenylation signal (AATAAA) is underlined.

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again in the evolution of their immune systems [20]. As research continues, scientists found that lamprey and hagfish use variable lymphocyte receptors (VLRs) comprised of leucine-rich-repeat (LRR) segments to recognize antigens as a substitute for immunoglobulin-based receptors used by vertebrates [21]. Three types of VLR genes, VLRA, VLRB and VLRC have been identified in lamprey [22e24]. VLRAþ and VLRBþ lymphocytes resemble mammalian T and B cells [25]. However, few studies about the issue of intracellular signal pathways of lamprey have been reported. In this study, we described the identification and cloning of an IkB-3 -like gene from Lampetra japonica (L. japonica). The connection between this protein and lymphocyte signal transduction was also investigated.

2.2. Separation of leukocytes, cell culture and lipopolysaccharide (LPS) stimulation L. japonica peripheral blood was collected from the caudal subcutaneous sinus. Leukocytes were enriched by the method of FicollePaque gradient centrifugation with the FicollePaque medium (the concentration is 1.092 g/ml). After centrifugation the leukocytes were collected and cultured in RPMI 1640 medium containing 10% fetal calf serum. The leukocytes were stimulated for 0, 2, 4, 6, 8, 24 h with 1 mg/ml of LPS (SigmaeAldrich, St. Louis, MO, USA) in complete RPMI medium. All cell cultures were incubated at 37  C in a 5% CO2 atmosphere. 2.3. Cloning of IkB-3 -like cDNA from L. japonica

2. Materials and methods 2.1. Animals Adult L. japonicas (length: 48e60 cm, weight: 112e274 g) were obtained from the Songhua River region of Heilongjiang province, China, in December and kept in freshwater at 4e16  C before stimulation.

An IkB-3 -like homolog in L. japonica was identified in leukocyte cDNA library of L. japonica we constructed before with the analysis by Basic Local Alignment Search Tool X (BLASTx) in the National Center for Biotechnology Information (NCBI). Total RNAs from leukocytes of L. japonica were extracted based on the Catrimox-14Ô RNA Isolation Kit (TaKaRa Biotechnology, Dalian, China) and then converted to cDNA by reverse transcriptase (Promega, Madison, WI,

Fig. 2. Alignment of the amino acid sequences of IkB-3 from various vertebrates. The conserved DSGxxS motif is indicated with black box. Six consecutive ankyrin repeats are marked with solid black arrows. The conserved sequences are highlighted in black.

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USA). Full-length cDNA was amplified using the 30 -RACE or 50 -RACE Core Set Kit (TaKaRa Biotechnology, Dalian, China) with the 30 -RACE primer and 50 -RACE primer listed in Table 1. The PCR products were cloned into pMD18-T Simple Vector using DNA Ligation kit (TaKaRa Biotechnology, Dalian, China) for subsequent DNA sequencing.

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centrifugation, washed, and resuspended in banding buffer (20 mM TriseHCl, pH 8.0, 50 mM NaCl, and 20 mM imidazole). The cells in banding buffer were lysed by sonicating for 30 min on ice and centrifuged again at 16,000  g for 10 min at 4  C. The soluble supernatant was collected and purified with Ni affinity chromatography (GE Healthcare, New York, NY, USA). The concentration of IkB-3-like recombinant protein determined by the Bradford method and the purity of the sample was analyzed by 12% SDS-PAGE.

2.4. Amino acid sequence analysis and phylogenetic analysis The amino acid sequence deduced from the full-length cDNA of IkB-3 -like of L. japonica was analyzed by online tool at http://www. expasy.org/tools/scanprosite. A total of 24 amino acid sequences alignments of IkB family including IkB-3-like were performed using ClustalX 1.81 with default settings. The result was converted into a mega format and imported into MEGA 3.1 to construct a phylogenetic tree with the NJ method and 1000 bootstrapped replicates.

2.6. Production of polyclonal antibodies Polyclonal antibodies against the truncated IkB-3-like recombinant protein were raised in male New Zealand white rabbits. Each animal was immunized for four consecutive injections with a gap of 2 weeks per immunization. For the first immunization, 400 mg of purified IkB-3-like recombinant protein in 500 ml PBS was incorporated and emulsified with an equal volume of Freund’s complete adjuvant (SigmaeAldrich St. Louis, MO, USA). For the subsequent three immunizations, 200 mg of the protein in 500 mg PBS was incorporated and emulsified with an equal volume of Freund’s incomplete adjuvant (SigmaeAldrich St. Louis, MO, USA). Peripheral blood was centrifuged at 7100  g for 5 min and the antiserum was collected. Then polyclonal antibodies (anti-IkB-3-like) were purified from antiserum by protein G affinity chromatography (GE Healthcare, New York, NY, USA). The concentration of purified

2.5. Expression and purification of IkB-3 -like recombinant protein A 435-bp fragment of the open reading frame (ORF) of IkB-3 -like cDNA (GenBank: KC335304 nucleotides 355e789) encoding 145 amino acids flanked by a HandIII and a BamHI restriction site, was amplified and subcloned into the pET-32a expression vector. The recombinant protein was expressed in Escherichia coli BL21 (DE3) by induction with 0.1 mM isopropyl-1-thio-b-D-galactopyranoside (IPTG) for 3.5 h. Subsequently, the cells were harvested by

99

IκB-α human IκB-α monkey

85

IκB-α cattle 100

98

IκB-α pig IκB-α mouse

100 99 100

IκB-α

IκB-α rat IκB-α chicken IκB-α salmon

100 98

34

IκB-α Atlanticcod IκB-ε human IκB-ε cattle

100

IκB-ε mouse 74

100

IκB-ε

IκB-ε rat IκB-ε zebrafish

97

100

IκB-ε salmon

100

IκB-β human

63

IκB-ε lamprey

IκB-β monkey

71

IκB-β cattle

100

IκB-β

IκB-β mouse 79

97

IκB-β rat IκB-β zebrafish IκB-ε oyster IκB-α oyster

oyster

Fig. 3. Phylogenetic relationship of IkBs. A neighbor-joining tree was constructed using the amino acid sequences of IkB-3, IkB-a and IkB-b. Numbers at the node indicate bootstrap confidence values derived from 1000 replications. The GenBank accession numbers are as follows: IkB-3: human (Homo sapiens), AAC51216.1; mouse (Mus musculus), NP_032716.2; rat (Rattus norvegicus), NP_954542.1; mole-rat (Heterocephalus glaber), EHB10886.1; cattle (Bos taurus), NP_001124218.1; salmon (Salmo salar), NP_001133852.1; zebrafish (Danio rerio), NP_001073558.1; lamprey (Lampetra japonica), KC335304; oyster (Crassostrea gigas), EKC37831.1. IkB-a: human (Homo sapiens), NP_065390.1; monkey (Macaca mulatta), NP_001244679.1; cattle (Bos taurus), NP_001039333.1; pig (Sus scrofa), NP_001005150.1; mouse (Mus musculus), NP_035037.2; rat (Rattus norvegicus), NP_001099190.2; chicken (Gallus gallus), NP_001001472.2; salmon (Salmo salar), ACI69528.1; cod (Gadus morhua), ADG85744.1; oyster (Crassostrea gigas), EKC30840.1. IkB-b: human (Homo sapiens), NP_002494.2; monkey (Macaca mulatta), NP_001245086.1; cattle (Bos taurus), NP_001069340.1; mouse (Mus musculus), NP_035038.2; rat (Rattus norvegicus), NP_110494.2; molerat (Heterocephalus glaber), EHA98161.1; zebrafish (Danio rerio), NP_001122267.1.

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anti-IkB-3-like was adjusted to 1 mg/ml and stored at 20  C in 50% glycerol. The antibody titer was determined by enzyme-linked immunosorbent assay (ELISA). The specificity of the antibodies was confirmed by western blot assay using the IkB-3-like recombinant protein and the lysates of leukocytes from L. japonica. 2.7. Western blotting Leukocytes were lysed in lysis buffer containing 1% 3[(3-cholamidopropyl) dimethylammonio]-1-propanesulphonate (CHAPS) (SigmaeAldrich St. Louis, MO, USA), protease inhibitor cocktail (Roche, Mannheim, Germany) and phenylmethanesulphonyl fluoride (PMSF) (SigmaeAldrich St. Louis, MO, USA). The lysates were centrifuged for 20 min at 13,000 g at 4  C and then 20 ml supernatant was added to 20 ml of 2 loading buffer and boiled at 100  C for 5 min. The proteins were analyzed by SDSPAGE under reduced conditions, transferred to a polyvinylidene difluoride (PVDF) membrane (Millipore Corporation, Billerica, MA, USA) and blocked with 5% fat-free milk in PBS plus 0.05% Tween (PBS-T). After being washed three times in PBS-T, the membrane was incubated with anti-IkB-3-like antibody (0.5 mg/ml) in PBS-T and 5% fat-free milk for 1 h at 37  C. After washing, the membrane was incubated with horseradish peroxidase (HRP)-conjugated goat anti-rabbit IgG in PBS-T and 5% fat-free milk for 30 min at 37  C. The signals were revealed using enhanced chemiluminescence (ECL) kit (Pierce, Rockford, IL, USA). Mouse anti-bActin (Zhongshanjinqiao, Beijing, China) was used to normalize the amount of protein per lane. For quantification, the optical density of the bands was corrected for those of the b-Actin and normalized as internal controls. 2.8. Fluorescence-activated cell sorting (FACS) analysis Leukocytes isolated from L. japonica blood were washed three times in banding buffer (PBS containing 10% FCS and 1% sodium azide), followed by fixing with 0.01% formaldehyde and permeabilizing with 0.1% NP-40 in order to ensure the antibodies getting into the cells. The leukocytes were incubated with the rabbit anti-IkB-3-like polyclonal antibodies (1 mg/ml) in 3% BSA/PBS for 1 h, and then washed another three times. The cells were stained with FITC-conjugated goat anti-rabbit IgG for the subsequent analysis in a FACS Aria flow cytometer (BD Biosciences, San

anti-IκB-ε-like antibody control OD 450nm

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Fig. 5. Indirect ELISA analysis of IkB-3-like IgG with IkB-3-like recombinant protein. Serially diluted (1:10,000e1:320,000) polyclonal antibodies were tested against IkB3-like recombinant protein by ELISA. Pre-immune IgG was used as a negative control (n ¼ 3). Error bars indicate standard error of mean (s.e.m.).

Jose, CA, USA). Leukocytes without antibody treatment, leukocytes incubated with FITC-conjugated secondary antibodies, and leukocytes incubated with the polyclonal antibodies from pre-immune rabbit serum before FITC-conjugated secondary antibodies, were used as negative controls and assayed in parallel. The data were analyzed using the FlowJo software (Tree Star Inc., Canada). 2.9. Data analysis The results were analyzed with the SAS proprietary software release 8.02 and Student’s two-sample t test. 3. Result 3.1. Cloning, amino acid sequence analysis of L. japonica IkB-3 -like cDNA We identified an EST fragment whose nucleotide sequence shared high sequence homology with the homologs from other vertebrate species IkB-3 in a leukocyte cDNA library of L. japonica

Fig. 4. Expression and purification of L. japonica IkB-3-like recombinant protein. (A) Expression of the truncated form of IkB-3-like recombinant protein in E. coli BL21 (DE3), and it is indicated with black arrow. Lane 1, total protein of uninduced cells; lane 2, total protein of induced cells harboring pET-32a (control); lane 3, total protein of induced cells harboring pET-32a-IkB-3-like; lane 4, soluble fraction of the lysate of induced cells harboring pET-32a-IkB-3-like; lane 5, insoluble fraction of the lysate of induced cells harboring pET-32a-IkB3-like. (B) Purified IkB-3-like recombinant protein. Elution concentrations of recombinant protein are 100 mM (lane 1), 200 mM (lane 2), 300 mM (lane 3) and 500 mM (lane 4), respectively.

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3.2. Phylogenetic analysis of L. japonica IkB-3 -like To investigate the evolutionary relationship of L. japonica IkBwith its counterparts, we constructed a phylogenetic tree using neighbor-joining (NJ) method based on the amino acid sequences of IkB-3 and main IkB members, such as IkB-a and IkB-b (Fig. 3). The tree suggested that except Crassostrea gigas IkB-3 and IkB-a, the other members of the IkB family might be classified into three clusters (IkB-a, IkB-b and IkB-3). The L. japonica IkB-3-like was clustered in the branch of IkB-3 sequences. In addition, the tree showed that IkB-3 was more closely related to IkB-a than to IkB-b. The high bootstrap values supported the precision of topology. According to the analysis of NJ tree, the IkB-3-like identified in L. japonica should have a common ancestor with those of higher vertebrates.

3-like

1.6

Relative IκB-ε-like/β-Actin

constructed before. A 3643 bp full-length cDNA of IkB-3 -like was successfully isolated by subsequent 30 -and 50 -RACE using primers designed from the EST fragment. The cDNA has a 1272 bp ORF, which encodes a polypeptide with 424 amino acid residues, a 354bp 50 -untranslated region (UTR) and a 2014-bp 30 -UTR with polyadenylation signal (AATAAA) (Fig. 1). The L. japonica IkB-3 -like cDNA sequence was submitted to GenBank database with the accession number of KC335304. The deduced amino acid sequence of IkB-3 -like cDNA from L. japonica had a high sequence identity with the sequences of IkB-3 from other vertebrates. The alignment reveals a strongly conserved DSGxxS motif, which plays a significant role in inhibition of NF-kB, at the N-terminus of the protein. The L. japonica IkB-3-like contains six consecutive ankyrin repeats that are the main feature of IkB family. Like the classical IkB-3, the PEST region is also absent in the C-terminal region of L. japonica IkB-3-like (Fig. 2).

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1.4 1.2 1 0.8 0.6 0.4 0.2 0

Time (h)

0

2

4

6

8

24

IκB-ε-like β-Actin Fig. 7. Degradation of IkB-3-like (1). Immunoblots of total cell lysates from L. japonica leukocytes stimulated with LPS (1 mg/ml, 0e24 h) were performed with Abs directed against L. japonica IkB-3-like. Total IkB-3-like quantification was determined by measuring the band density and then normalizing against internal controls. Data represent mean percentages  SE, Error bars indicated (s.e.m), n ¼ 3.

3.3. Expression and purification of L. japonica IkB-3 -like SDS-PAGE analysis revealed that most of the target proteins existed in the soluble fraction of the E. coli BL21 (DE3) cell lysate, indicating that it was mainly expressed as soluble protein (Fig. 4A). The expressed L. japonica IkB-3-like was about 36 kDa, and this was in accordance with the molecular mass predicted from the cDNA sequence. After affinity chromatography, a relatively pure protein was finally obtained. The target proteins were eluted with different

Fig. 6. Protein expression analysis of L. japonica IkB-3-like. (A) Western blot analysis of the specificity of rabbit anti-IkB-3-like polyclonal antibodies. Line 1, IkB-3-like recombinant protein; line 2, leukocytes from L. japonica. (B) Flow cytometric analysis for surface-expressed and intracellular IkB-3-like. Leukocytes were treated first with anti-IkB-3-like Ab and then with FITC-labeled goat anti-rabbit IgG Ab (red). Leukocytes were treated first with isotype-matched IgG Ab and then with FITC-labeled goat anti-rabbit IgG Ab (blue). Leukocytes incubated without antibodies were used as a control for the first Ab (black). Intracellular IkB-3-likes were detected in permeabilized cells. (For interpretation of the references to color in this figure legend, the reader is referred to the web version of this article.)

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concentrations of imidazole (e.g. 100 mM, 200 mM, 300 mM and 500 mM), and the best concentration was 200 mM (Fig. 4B).

higher than 1: 160,000 (Fig. 5) using pre-immunized rabbit IgG as negative control.

3.4. Titer and specificity analysis of polyclonal antibodies

3.5. Expression of L. japonica IkB-3 -like in leukocytes

The polyclonal antiserum was prepared successfully through the procedure described in the Materials and methods. The polyclonal antibodies against recombinant L. japonica IkB-3-like protein were purified from rabbit antiserum by protein G. The titers of the obtained antibodies were analyzed by ELISA. The result of ELISA showed the titer of rabbit anti-IkB-3-like polyclonal antibodies was

The specificity of the antibody and expression of L. japonica IkBin leukocytes were determined by western blot analysis. The antibody could detect both the truncated L. japonica IkB-3-like protein and the native L. japonica IkB-3-like protein in leukocytes (Fig. 6A). In order to study the cellular localization of IkB-3-like, the normal leukocytes and permeabilized leukocytes were separately

3-like

Fig. 8. Degradation of IkB-3-like (2). Flow cytometric analysis for L. japonica leukocytes stimulated with LPS (1 mg/ml, 0e24 h). Leukocytes incubated without antibodies were used as a control for the first Ab (black line). Leukocytes were incubated with FITC-labeled goat anti-rabbit IgG Ab (A), isotype-matched IgG Ab (B), anti-IkB-3-like Ab (CeG) as the first Ab (gray filled histogram). In panel A, FITC-labeled goat anti-rabbit IgG Ab was used as both first Ab and second Ab. Data represent mean percentages  SE, Error bars indicated (s.e.m), n ¼ 3.

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incubated with anti-IkB-3-like antibodies and analyzed by FACS. The IkB-3-like was not detected on the surface of leukocytes (Fig. 6B). 3.6. L. japonica IkB-3 -like degraded in response to LPS To assess the expression of IkB-3-like in leukocyte activation, the time course of LPS-induced protein degradation was studied. Western blot analysis showed that the L. japonica IkB-3-like protein was degraded at, about 2 h following LPS stimulation. With continuous LPS stimulation, the level of L. japonica IkB-3-like protein expression was down-regulated within 8 h and was slightly up-regulated until 24 h (Fig. 7). The similar results were got by FACS analysis (Fig. 8). 4. Discussion Lampreys are the key species to study the evolutionary origin of adaptive immunity because of the unique position they occupy in chordate phylogeny. Here we isolated and characterized a fulllength IkB-3 -like cDNA from L. japonica for the first time. The deduced amino acid sequence showed a typical IkB family structure including a DSGxxS motif and six consecutive ankyrin repeats. The NF-kB-inhibitory protein IkB-a is phosphorylated by the IKK complex, which is composed of IKKa, IKKb and NEMO, on two serines of the DSGxxS motif [26]. Phosphorylated IkB-a undergoes ubiquitination and is degraded by the proteasome system, which thereby frees NF-kB to translocate to the nucleus and activate the transcription of genes [27]. The ankyrin repeats are involved in specific binding of the IkB proteins to NF-kB dimers [28], and mediate the nuclear localization of IkB proteins [29]. The PEST is an amino acid composition motif riched in proline (P), glutamic acid (E), serine (S), and threonine (T) residues; it has been considered to be related to protein degradation [30]. Phosphorylation of C-terminal PEST domain in the IkB-a affects intrinsic protein stability [31]. However, L. japonica IkB-3-like does not contain a C-terminal PEST sequence, which is consistent with mammalian IkB-3. The similar structure between L. japonica IkB-3-like and mammalian IkB-3 implied that they might have similar functions. Phylogenetic analysis suggested that the identified L. japonica IkB-3-like rescued from L. japonica was a member of the IkB-3 family and the ancestor of vertebrate IkB-3 was present in primitive metazoans prior to the formation of protostomia. In addition, the phylogenetic tree showed that IkB-3 was more closely related to IkB-a than to IkB-b. This observation indicated that IkB-3 and IkB-a might share a common ancestor. Furthermore, it is likely that IkB-3 and IkB-a use a similar mechanism for transit through the nuclear pore complex, as nuclear import of IkB-3 is not disrupted by the dominant-negative RanQ69L-GTP protein, which is similar to that of IkB-a [32]. The IkB-3-like protein identified from L. japonica appeared to be the most primitive form of IkB-3-like protein, and yet its structure seemed to have the capacity to fulfill the same functions of IkB-3 proteins in higher vertebrates. To further analyze IkB-3-like’s potential involvement in the L. japonica immunity, we obtained high potency anti-IkB-3-like polyclonal antibodies. The analysis of IkB-3-like protein localization showed that IkB-3-like was expressed intracellularly in L. japonica leukocytes. It further determined that the identified IkB-3-like protein was a member of the IkB-3 family. NF-kB is a stimuli-activated transcription factor that regulates a diverse array of genes involved in innate and adaptive immunity and inflammatory responses [33]. IkBs retain NF-kB dimers in the cytoplasm of resting cells and form IkB family, such as IkB-a, IkB-b, and IkB-3, which are classical IkBs. Lipopolysaccharide (LPS) induces the degradation of IkBs and the nuclear translocation of NF-kB

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complexes, such as RelA and p50 subunits [34,35]. Furthermore, in the previous studies, it was found that stimuli resulted in a rapid degradation of IkB-a, followed by a recovery period lasting up to a few hours [36]. In comparison, the signal-induced IkB-b and IkB-3 degradation and resynthesis occur at much slower kinetics [37]. In this report, we found that L. japonica IkB-3-like protein was degraded in cell lysates and was resynthesized after 24 h following LPS stimulation. The phenomenon, which is similar to that of mammals, implied that the IkB-3-like protein participated in the immune response of the lamprey and a NF-kB-like signaling pathway might exist in the oldest agnatha. As described above, stimulation leads to a loss of IkBs from cells and triggers the nuclear translocation of NF-kB in mammals. Transactivation by NF-kB, in turn, activates a variety of genes including IkB-a, IkB-b, and IkB-3, which can then restore the unstimulated inhibited state [38,39]. Unfortunately, we have not yet found the homolog of NF-kB in L. japonica. Thus further research is needed to shed more light in this field and increase our understanding of the mechanism by which L. japonica IkB-3-like protein fulfills its function as an immune molecule in L. japonica. Acknowledgments This work was supported by grants from Chinese National Natural Science Foundation Grants 31071991, 31170353 and 31271323. Liaoning Province Education Department of Higher Education projects L2011188. The authors thank Dr Alan K Chang, School of Life Science and Biotechnology, Dalian University of Technology, Dalian, China for helpful discussion and his contribution in the revision of the manuscript. Abbreviations

L. japonica Lampetra japonica NF-kB nuclear factor of kappa B IkBs inhibitors of kappa B IkB-a inhibitor of kappa B-alpha IkB-b inhibitor of kappa B-beta IkB-3 inhibitor of kappa B-epsilon NLS role of nuclear localization signals VLRs variable lymphocyte receptors LRR leucine-rich-repeat IPTG isopropyl-1-thio-b-D-galactopyranoside ELISA enzyme-linked immunosorbent assay CHAPS 3-[(3-cholamidopropyl) dimethylammonio]-1propanesulphonate LPS lipopolysaccharide References [1] Miyamoto S, Verma IM. Rel/NF-kappa B/I kappa B story. Adv Cancer Res 1995;66:255e92. [2] Ghosh S, May MJ, Kopp EB. NF-kappa B and Rel proteins: evolutionarily conserved mediators of immune responses. Annu Rev Immunol 1998;16: 225e60. [3] Bonizzi G, Karin M. The two NF-kappaB activation pathways and their role in innate and adaptive immunity. Trends Immunol 2004;25:280e8. [4] Hayden MS, Ghosh S. Signaling to NF-kappaB. Genes Dev 2004;18:2195e224. [5] Whiteside ST, Epinat JC, Rice NR, Israel A. I kappa B epsilon, a novel member of the I kappa B family, controls RelA and cRel NF-kappa B activity. EMBO J 1997;16:1413e26. [6] Huxford T, Huang DB, Malek S, Ghosh G. The crystal structure of the IkappaBalpha/NF-kappaB complex reveals mechanisms of NF-kappaB inactivation. Cell 1998;95:759e70. [7] Vallabhapurapu S, Karin M. Regulation and function of NF-kappaB transcription factors in the immune system. Annu Rev Immunol 2009;27:693e733. [8] Dobrzanski P, Ryseck RP, Bravo R. Specific inhibition of RelB/p52 transcriptional activity by the C-terminal domain of p100. Oncogene 1995;10:1003e7.

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