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Full length article
Characterization of a novel antimicrobial peptide with chiting-biding domain from Mytilus coruscus Q2
Chuan-li Qin a, 1, Wei Huang a, 1, Shi-quan Zhou b, Xin-chao Wang a, Hui-hui Liu a, Mei-hua Fan a, Ri-xin Wang a, Peng Gao a, Zhi Liao a, * a b
Key Laboratory for Marine Living Resources and Molecular Engineering, College of Marine Science, Zhejiang Ocean University, Zhoushan, 316022, PR China Laboratory of Immunogenomics, Zhoushan Hospital, Zhoushan, 316022, PR China
a r t i c l e i n f o
a b s t r a c t
Article history: Received 27 June 2014 Received in revised form 1 September 2014 Accepted 14 September 2014 Available online xxx
Using reverse phase high performance liquid chromatography (RP-HPLC), a novel antimicrobial peptide with 55 amino acid residues was isolated from the hemolymph of Mytilus coruscus. This new antimicrobial peptide displays predominant antimicrobial activity against fungi and Gram-positive bacteria. The molecular mass and the N-terminal sequence of this peptide were analyzed by Mass Spectrometry and Edman degradation, respectively. This antimicrobial peptide, with molecular mass of 6621.55 Da, is characterized by a chitin-biding domain and by 6 Cysteine residues engaged in three intra-molecular disulfide bridges. The full-length of cDNA sequence of this new peptide was obtained by rapid amplification of cDNA ends (RACE) and the encoded precursor was turn out to be a chitotriosidase-like protein. Therefore, we named the precursor with mytichitin-1 and the new antimicrobial peptide (designated as mytichitin-CB) is the carboxyl-terminal part of mytichitin-1. The mRNA transcripts of mytichitin-1 are mainly detected in gonad and the expression level of mytichitin-1 in gonad was up-regulated and reached the highest level at 12 h after bacterial challenge, which was 9-fold increase compared to that of the control group. These results indicated that mytichitin-1 was involved in the host immune response against bacterial infection and might contribute to the clearance of invading bacteria. © 2014 Published by Elsevier Ltd.
Keywords: Mytilus coruscus Antimicrobial peptide Chitin-biding domain Chitinase
1. Introduction Antimicrobial peptides (AMPs), which constitute a first line of host defense against pathogens, are widely expressed in organisms and have been linked to innate immunities in invertebrates. Marine molluscs are widespread, extremely diverse, and constantly under an enormous microbial challenge from the ocean environment. In order to defend themselves against such conditions, marine molluscs have developed very effective mechanisms that are part of their innate immunity [1]. AMPs are the major component of the innate immune system in molluscs [2e4] and antibacterial activity was first reported in mollusks in the ’80s [5]. Most of the AMP researches of marine mussels has focused on species Mytilus, including Mytilus edulis and Mytilus galloprovincialis, whereas the isolation and characterization of true AMPs from marine mussels date back to 1996 [6,7].
* Corresponding author. Tel./fax: þ86 580 2550753. E-mail addresses: [email protected], [email protected] (Z. Liao). 1 Equally contribution to this work.
Among the various natural AMPs, those containing pairs of cysteine residues forming intra-molecular disulfide bridges are particularly common [8e11]. In the last decades, AMPs have been isolated and characterized from marine mussels and classified into seven groups: defensins [6], mytilins [12], myticins [13,14], mytimycin [15], mytimacin [16], big defencin [16], and myticusin [17]. These AMPs have also commonly been identified and characterized in other marine bivalve mollusk. For example, defensins have been described in oysters Crassostrea virginica and Crassostrea gigas [18,19] and abalone Haliotis discus [4]; mytilins and myticins were also described in clams Ruditapes decussates [20]. These peptides belong to the cysteine-rich family of AMPs with strong activity against bacteria, fungus and virus [21e23], making them not only a promising template molecule for investigating the relationship between protein structure and function, but also a source of new leads for the treatment of bacterial disease [23]. The molecular diversity of AMPs increases the antimicrobial capabilities of mussels against a high diversity of pathogens. However, AMPs have been explored only in a few species. Therefore, there is still a great potential to unveil new molecules in this phylum. Mytilus coruscus is one of the most important economic
http://dx.doi.org/10.1016/j.fsi.2014.09.019 1050-4648/© 2014 Published by Elsevier Ltd.
Please cite this article in press as: Qin C-l, et al., Characterization of a novel antimicrobial peptide with chiting-biding domain from Mytilus coruscus, Fish & Shellfish Immunology (2014), http://dx.doi.org/10.1016/j.fsi.2014.09.019
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shellfish and widely distributed in eastern coast in China. Several antimicrobial peptides have been characterized from M. coruscus in our previous work, including mytilins (FJ973154 ~ FJ973155 for mytilin-A and -B; GU324718~GU324723 for mytilin3~8), myticins (GU324724~GU324732 for myticin1~9), and a big antimicrobial peptide named myticusin-1 [17]. Here we present the isolation and biochemical characterization a novel antimicrobial peptide, mytichitin, from hemolymph of M. coruscus. 2. Material and methods 2.1. Animals and hemolymph collection Adult mussels (M. coruscus) were collected locally from Zhoushan (Zhejiang province, P. R. China) and were maintained in tanks with fresh sea water at 25 C. Hemolymph was prepared using the methods described by Mitta [12], the plasma was clarified by centrifugation (12,000 g, 20 min, 4 C) and then kept at 80 C until further analysis. 2.2. Antimicrobial assays Bacteria and fungus (about 1e2 * 108 cfu/mL) used in present study were obtained from China General Microbiological Culture Collection Center (CGMCC, Beijing, P. R. China). The bacterial strains were Escherichia coli (CGMCC1.1583), Vibrio Parahaemolyticus (CGMCC1.1616), Pseudomonas aeruginosa (CGMCC1.0102), Proteus vulgaris (CGMCC1.1527), Vibrio.harveyi (CGMCC1.1601), Bacillus subtilis (CGMCC1.1630), Staphylococcus aureus (CGMCC1.128), Sarcina luteus (CGMCC28001), and Bacillus megaterium (CGMCC1.1487). The fungus used here were Candida albicans (CGMCC2.2086) and Monilia albican (CMCC(F)98001). Antimicrobial activity was monitored by a liquid growth inhibition assay according to the method described previously [17]. During the various steps of peptide purification, antibacterial activity was monitored on the Gram-positive strain S. luteus and the Gramnegative strain E. coli. To determine the minimal inhibitory concentration (MIC), serial doubling dilutions were carried out following the protocol described by Mitta [12]. The MIC values are expressed an interval (aeb), where (a) represents the highest peptide concentration tested at which bacteria are still growing and (b) the lowest concentration that causes 100% growth inhibition. 2.3. Peptide purification All HPLC purification steps were carried out on a Waters Delta 600 HPLC system equipped with a Waters 2487 absorbance detector. Fractions were collected and assayed for antimicrobial activity. The plasma sample was directly subjected to a Sunfire™ prep C8 column (10 250 mm Waters) equilibrated with deionized water containing 0.1% trifluoroacetic acid. The sample was eluted with a gradient of 0e5% acetonitrile containing 0.1% trifluoroacetic acid over 5 min followed by 5e60% over 40 min and 60e95% over 5 min at a flow rate of 2.5 mL/min. The eluted fractions were collected manually, dried under vacuum, and reconstituted in MilliQ water (prepared by Millipore synergy system, France). The presence of antibacterial activities was detected by the liquid growth inhibition assays described above. Active fractions were applied to an analytical Vydac C18 RP HPLC column (218TP54, 4.6 250 mm) and eluted at the flow rate of 0.75 mL/min using 5e45% acetonitrile/water/0.1% trifluoroacetic acid as a linear gradient. To obtain the peptides with high purity, an additional step was performed on a SunFire™ C18 Column (100 Å, 3.5 mm,
3 150 mm, Waters) developed in the same diphasic gradients as above but at a flow rate of 0.25 mL/min. The eluted fractions with antimicrobial activities were vacuum-dried and used for determination of molecular mass and amino acid sequences. 2.4. Peptide sequencing and modeling The molecular mass of peptide was determined using Matrixassisted laser desorption/ionization-time of flight-mass spectrometry (Voyager-DE STR biospectometry workstation, Applied Biosystems). Purified peptide was submitted to reduction and alkylation, and Edman degradation was performed with a normal automatic cycle program using an Applied Biosystems Model 491 gas-phase sequencer. The 3-Dimensional structure of peptide was predicted from amino acid sequences by SWISS-MODEL platform on line (http:// swissmodel.expasy.org/interactive). 2.5. The full-length cDNA cloning The amino acid sequence detected from Edman sequencing was searched against the dbEST database of Mytilus using tblastn search tool (http://blast.ncbi.nlm.nih.gov/Blast.cgi). The matched EST sequence with high similarity was used as the template for designing specific primer of PCR amplification. Using Rapid Amplification cDNA Ends (RACE) method; the full length of cDNA was obtained by SUPERSWITCH™ RACE cDNA Synthesis Kit (Shanghai, China) according to the manufacturer's instructions. For 3’-RACE, a sense primer (5’-TAAAATGTGGCATGAATGG-3’) and a 3’Adaptor primer were used; Meanwhile, The antisense (5’GGGTCTTGTATGGAATCC-3’) corresponding to the 3’-end of amplified the cDNA fragment, together with 5’-Adaptor primer was used to obtain the cDNA 5’-end. The full-length cDNA was confirmed by specific primers (sense: 5’-CAGACCTACACTGTTCACGATGAT-3’, antisense: 5’- GTTCTCAATCTAATATTCCAAAGTCA-3’) designed in the 5’- and 3’-UTR, respectively, from sequences retrieved through RACE assays. The PCR was performed in 25 mL volume, including of 2.5 mL 10 SUPERSWITCH™ Hot Start Buffer, 0.1 mL of each primer (10 mМ), 2 mL dNTP Mix (2.5 mM), 0.5 mL of the cDNA template, and 0.5 mL SUPERSWITCH™ Hot Start DNA polymerase for 41 cycles on a Thermal Cycler (Bio-Rad, USA). Each cycle consisted of 30 s at 94 C, 30 s at 60 C, and 120 s at 72 C, with a final extension of 5 min. The PCR products were subjected to 1.0% agarose gel electrophoresis, purified, cloned into pMD19-T vector (TaKaRa), and transformed into competent DH5a. The resulting cDNA sequences were analyzed using the software of DNAstar lasergen 7.0 after nucleic acid sequencing. Signal peptides were predicted using the SignalP 4.0 [24] online tool (http:// www.cbs.dtu.dk/services/SignalP/) and conserved domains were predicted using simple modular architecture research tool (SMART) software (http://smart.embl-heidelberg.de/). Following peptide signal removal, the theoretical MW and pI were determined on Expasy-ProtParam online tool (www.expasy.org/tools/protparam. html). Multiple sequence alignments were performed with DNAMAN (version 7.0.2.176). Phylogenetic analyses based on the amino acid sequences were conducted by MEGA 6.0 using Maximum Likelihood algorithm with 1000 bootstrap replicates. 2.6. Quantitative analysis of mytichitin-1 expression The tissue-specific expression and the temporal expression of transcripts in mussels challenged with S. luteus were determined by quantitative real-time PCR. Mussels of 5e6 cm shell length were collected from East China Sea. Total RNA was individually purified
Please cite this article in press as: Qin C-l, et al., Characterization of a novel antimicrobial peptide with chiting-biding domain from Mytilus coruscus, Fish & Shellfish Immunology (2014), http://dx.doi.org/10.1016/j.fsi.2014.09.019
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from previously homogenized hemocytes, digestive gland, mantle, posterior abductor muscle, gill, foot, and gonad. After reverse transcription, the expression of transcripts in tissues was measured by SYBR Green quantitative real-time PCR assay in Applied Biosystem 7500 Real-time PCR System. Two specific primers, mytichitin-RT-F (5’-CCTAAAATGTGGCATGAATGG-3’) and mytichitin-RT-R (5’-GCAAGCGTAGTCACAAAATCC-3’), were used for
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amplifying gene fragment of 158 bp, which was sequenced to verify the PCR specificity. Amplifications were carried out at a final volume of 20 mL, containing 1.0 mL cDNA sample, 10 mL SYBR Green Real-time PCR master mixtures (Takara), 0.4 mL ROX II, 1.0 mL of each primer and 7.6 mL H2O. The following thermal profile was used: an initial step of 5 min denaturation at 95 C, followed by 40 cycles at 95 C for 30 s, 50.3 C for 30 s, and 72 C for 40 s. Amplification
Fig. 1. Purification and identification of mytichitineCB. A, the plasma sample collected from M. coruscus was isolated by Sunfire™ prep C8 column and the target fraction (denoted by “*”) with antibacterial activity was collected for further purification. B, further purification of the target elution by analytical Vydac C18 column and the elusion with antibacterial activity (denoted by “*”) was collected for structural analysis. C, mass spectrometry analysis yielded a single mass of 6297.55 Da; N-terminal sequences (29-AA) determined by Edman degradation are indicated with character abbreviation.
Please cite this article in press as: Qin C-l, et al., Characterization of a novel antimicrobial peptide with chiting-biding domain from Mytilus coruscus, Fish & Shellfish Immunology (2014), http://dx.doi.org/10.1016/j.fsi.2014.09.019
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products were analyzed with a 65 C/95 C melting curve. The expression values were normalized using the b-actin of M. coruscus as housekeeping gene (McActin-F: 5’-ATGAAACCACCTACAACAGT3’; McActin-R: 5’- TAGACCCACCAATCCAGACG-3’). Results are given as the mean with standard deviation of three technical replicates. Comparisons between groups were made by one-way analysis of variance followed by a Duncan test for identification of the statistically distinct groups. 3. Results 3.1. Isolation and identification of new antimicrobial peptide from M. coruscus hemolymph Hemolymph was collected from M. coruscus. After cells and debris were removed by centrifugation, the supernatant was applied on a Sunfire™ prep C8 column and eluted with a gradient of 5e60 % acetonitrile, yielding the chromatogram shown in Fig. 1A. The eluted fractions were assayed for their antibacterial activity against two bacterial strains, S. luteus and E. coli. The fractions containing antibacterial activity (denoted by asterisk in Fig. 1A) were collected, vacuum dried, and subjected to the second C18 reverse-phase column for further purification (Fig. 1B). Finally, the antibacterial peptide (denoted by asterisk in Fig. 1B) was purified to homogeneity by reverse-phase HPLC. The molecular mass of this new antimicrobial peptide determined by MALDI-TOF was 6297.55 Da (Fig. 1C). The alkylated peptide was sequenced by Edman degradation, and a 29-residue Nterminal partial sequence was obtained (Fig. 1C). 3.2. Cloning of cDNA encoding the new antimicrobial peptide When we used the 29-residue N-terminal sequence of the new antibacterial peptide to search against Mytilus EST database, one matched EST sequence (gbjES407161.1j) with 73% identity was obtained. Based on the partial N-terminal amino acids sequence and matched EST sequence, we designed and synthesized specific primer to amplify the cDNA of the new antibacterial peptide from M. coruscus and the complete cDNA was determined using RACE technique. Five distinct variants with high sequence identity (>95%) were obtained (showed in Supplementary Fig. 1), in which, the represent cDNA sequence (gij568218204) is shown in Fig. 2. The complete 1588-bp cDNA obtained revealed an open reading frame of 1341 bp. This open reading frame encodes a protein composed of 446 amino acids with a putative N-terminal 24-residues signal peptide (Fig. 2), and this sequence turns out to be a chitotriosidase-like protein after sequence analysis. A comprehensive search of none-redundant protein sequences data revealed significant homology of this sequence (>35%) with chitotriosidase/chitinases from other species. Furthermore, domain analysis showed that the obtained sequence has one GH18_chitolectin_chitotriosidase domain (cd02872) and one Chitin-binding domain (smart00494). Therefore, we propose the name of mytichitin-1 for this new sequence. The mature peptide of mytichitin-1 consists of a 422-amino acid residue with a calculated molecular mass of 48,005.79 Da and an estimated pI of 9.37. The 29-residues fragment (Thr392-Arg420) of mytichitin-1 perfectly matches the amino acid sequences determined by Edman degradation. Moreover, the 55-residues (containing 6 cysteines) of the carboxyl terminus of mytichitin-1 shows a theoretical molecular mass of 6303.38 Da, which is identical to the experimental molecular mass (6297.55 Da, measured by MALDI-TOF) of the antimicrobial peptide purified in this article, considering the fact that forming of 3 disulfides will lose 6 Da. These results indicate that the isolated antibacterial peptide in present study corresponds to the carboxyl terminus of mytichitin-1.
Fig. 2. cDNA and deduced amino acid sequence of mytichitin-1 (A) and sequence alignment (B) of mytichitineCB with chitinebiding domain (smart00494). The predicted signal peptide is shown in bold. The asterisk (*) indicates the stop codon. A polyadenylation signal (AATAAA, denoted by bold Italic) emerged in the 3’-UTR at position 15 upstream of the poly-(A)-tail. The motif eDXXDXDXE- is boxed. The sequence of mytichitin-CB is underlined. The numbers at right panel indicate the serial number of amino acids and nucleotides.
Additionally, the 55-residues of the carboxyl terminus of mytichitin-1 contains a chitin-biding domain (Fig. 2B), therefore, we named the antimicrobial peptide presented in this article as mytichitin-CB. The tertiary structure modeling by SWISS-MODEL was successful for mytichitin-CB and model with the highest score was shown in Fig. 3A. The predicted tertiary structure of mytichitin-CB is characterized by several bsheets, including Asn25-Ala31, Arg34-Cys40, Val45-Asn47, and Tyr51-Asp53 as determined by Swiss-PdbViewer (v4.0.1). 3.3. Activity spectrum of mytichitin-CB The activity spectrum of mytichitin-CB was investigated against a variety of bacterial strains (Table 1). In liquid growth inhibition assays, the purified mytichitin-CB had marked activity against the Gram-positive strains including B. subtilis, S. aureus, S. luteus; and B. megaterium (MIC5 mM). Additionally, mytichitin-CB had weaker effects on the Gram-negative strains including E. coli, V. Parahaemolyticus, P. aeruginosa, and V. harveyi (MIC > 50 mM). 3.4. Multiple sequences alignment and phylogenetic analysis Blast analysis revealed that mytichitin-1 shared a high homology with chitotriosidase/chitinase from other species. The deduced
Please cite this article in press as: Qin C-l, et al., Characterization of a novel antimicrobial peptide with chiting-biding domain from Mytilus coruscus, Fish & Shellfish Immunology (2014), http://dx.doi.org/10.1016/j.fsi.2014.09.019
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curves for both two genes, indicated that the amplification were specific (Data not shown). Real-time qPCR demonstrated that mytichitin-1 mRNA amount was measured mainly in the gonad (in both male and female, Fig. 6A, C), and to a lesser extent, in the digestive gland. Temporal change of mytichitin-1 mRNA in gonad after microbial challenge was shown in Fig. 6B. Compared to the control group injected with only PBS solution, the expression level of mytichitin-1 mRNA in the challenge group increased and reached the highest level at 12 h (about 9-fold) post-injection after S. luteus challenge (P < 0.01). 4. Discussion Fig. 3. Comparison of the structures of mytichin-CB (A) and tachycitin (B) and 3D structure-based sequence alignment of mytichitin-CB and tachycitin with regard to the region corresponding to Cys-40eAsp53 of tachycitin (C). The putative key residues of tachycitin for chitin-biding are denoted by “*”.
amino acid sequence of mytichitin-1 was aligned with other reported chitotriosidase/chitinase and several conserved features were found in mytichitin-1 (Fig. 4) including two conserved domains, chitin catalytic domain and chitin-biding domain. The motif eDGLDL(P/I/F)DL(W)- regarding chitin degradation were also conserved in mytichitin-1 [25]. In addition, a short link region with abundant Ser/Thr between chitin catalytic domain and chitinbiding domain was observed in most sequences. The sequence similarity and the common structure features suggested that mytichitin-1 was a new counterpart of chitotriosidase/chitinase. The phylogenetic tree of invertebrate and vertebrate chitotriosidase/chitinase (Fig. 5) included six major clades with high bootstrap value: acidic mammalian chitinase from mammal, acidic mammalian chitinase from bird, chitinase1/2 from fish, chitotriosidase from mammal, chitotriosidase from mollusk, and chitinase from arthropod. Despite low bootstrap values, mytichitin-1 was firstly clustered with chitotriosidase-like protein from the bivalve C. gigas, and then grouped with chitotriosidase from mollusk and amphioxus.
3.5. Quantitative analysis of mytichitin-1 expression In order to establish the expression patterns of the mytichitin-1 transcripts in tissues, real-time qPCR was employed to quantify the expression of hemocyte, digestive gland, mantle, posterior adductor muscle, gill, foot, and gonad. The amplification specificity for mytichitin-1 and b-actin was determined by analyzing the dissociation curves. Only one peak presented in the dissociation
Table 1 Activity spectrum of mytichitin-CB against bacteria and fungus. Organism Bacteria:gramenegative Escherichia coli Vibrio Parahaemolyticus Pseudomonas aeruginosa Vibrio harveyi Bacteria:gramepositive Bacillus subtilis Staphylococcus aureus Sarcina luteus Bacillus megaterium Fungus Candida albicans Monilia albican
Mytichitin-CB (mmol/L) >100.0 50.0e100.0 50.0e100.0 >100.0 1.56e3.13 3.13e6.25 1.56e3.13 3.13e6.25 6.25e12.5 12.5e25.0
In present study, a novel antimicrobial peptide named mytichitin-CB was purified from M. coruscus plasma. Mytichitin-CB contains 55 residues including 6 cysteines in its sequence, which was further verified by its cDNA analysis. The result of Edman degradation with reduced and alkylated sample, together with the comparison of theoretical and experimental molecular mass, indicates the forming of three disulfides in mytichitin-CB. In present work, mytichitin-CB was active in vitro against Gram-positive bacteria but showed no or limited activities against Gramnegative bacteria, which is similar to the action of other AMPs from mussel, such as mytilins (mytilin A and G1) [12], myticins (myticin A and B) [13], and MGDs [6]. In addition, some defensins from insect [26] and Oyster C. gigas [19] were also found to be specific to Gram-positive bacteria. Unfortunately, the mechanism of selective inhibition against Gram-positive bacteria is unclear. Some researchers suggested that this selective function may results from different composition in cell wall of Gram-positive (lipoteichoic acid) and Gram-negative bacteria (Lipopolysaccharide) [27], the antimicrobial peptide charge [28], and the ratio of Arg/Lys of AMPs [29]. Although mytichitin-CB shows a strong activity against Grampositive bacteria and fungi, there is no sequence homology between mytichitin-CB and those characterized antimicrobial peptides to date. A chitin-biding domain was detected in mytichitin-CB. The antimicrobial activity is initially identified for chitin-binding proteins extracted from plants [30,31], which commonly comprise single or multiple copies of the chitin-binding domain. Some chitin-biding-domain-containing antimicrobial peptides were also found in arthropod. For example, Penaeidins, a family of antimicrobial peptides characterized in the shrimp Penaeus vanname [32], and Tachycitin, a 73-residue polypeptide having antimicrobial activity presented in the hemocyte of horseshoe crab Tachypleus tridentatus [33,34]. For invertebrates, the chitin-binding domain was assumed to comprise about 65 residues involving a high percentage of cysteine and aromatic residues in a similar manner to the plant chitin-binding domain [35]. We compared modeling structure of mytichitin-CD with the structure of tachycitin (1DQC), and the structural similarity between segment Cys40eAsp53 of mytichitin-CB and segment Cys40eAsp54 (proposed as an essential chitin-biding region by Suetake et al. [34]) of tachycitin, both comprising the antiparallel bεta-sheet and a hairpin loop, was examined. As shown in Fig. 3, the residues of Asn47 and Phe51 of mytichitin-CB are located at perfectly corresponding positions to the residues of Asn47 and Val52 (essential chitin-biding residues [34]) of tachycitin. Therefore, one could assume that the region of Cys40eAsp53 from mytichitin-CB serves as an essential chitin-binding site. It is suggested that the chitinbinding ability of Penaeidin and Tachycitin could not only be essential for their antifungal activity but could also be involved in chitin assembly or wound healing [36]. However, there is no significant sequence homology between mytichitin-CB and those chitin-biding-domain-containing antimicrobial peptides from
Please cite this article in press as: Qin C-l, et al., Characterization of a novel antimicrobial peptide with chiting-biding domain from Mytilus coruscus, Fish & Shellfish Immunology (2014), http://dx.doi.org/10.1016/j.fsi.2014.09.019
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Fig. 4. Multiple sequence alignment of mytichitin-1 (gij568218204) with chitotriosidase/chitinase from other species. Conserved residues are shaded in dark gray. Amino acids of the catalytic sites are shown with asterisk (*). The link region and the chitin-biding domain are shown with dash line and solid line, respectively. Gaps are indicated by dot to improve the alignment. The GenBank accession numbers, protein names, and the species are shown in left panel.
other species (data not shown), indicating mytichitin-CB may represent a new class of antimicrobial peptide family with chitinbiding domain. Using RACE technique, the complete cDNA (named mytichitin1) of mytichitin-CB was cloned and a 446-residues precursor including a 24-residues putative signal peptide was detected. The sequence of mytichitin-CB is perfectly matched the carboxyl terminus of mytichitin-1, therefore, we believed that mytichitin-CB is released from the carboxyl-terminal part of mytichitin via protein cleavage. Many antimicrobial peptides are derived from larger precursors, and processing and generation of antibacterial peptides have been reported from several species. For example, in invertebrate, buforin I from the stomach gland cells of the Asian toad Bufo bufo is generated by a pepsin-mediated processing of the cytoplasmic histone H2A [37]. astacidin 1 is released from the carboxylterminal part of crayfish hemocyanin by a cysteine-like proteinase
[38]. In mice, the precursor a-defensin is cleaved by metalloproteinase, a matrilysin to produce a -defensin [39], and human defensin-5 is also processed by paneth cell trypsin [40]. The organization and processing of peptides from one large precursor molecule is an efficient way to synthesize different effector molecules and amplify the antibacterial response. Mytichitin-1 shows a high (about 35e40%) sequence similarity with chitotriosidase/chitinase from other species (Fig. 4). Both chitotriosidase and chitinase is member of the glycosyl_hydrolase_18 superfamily capable of hydrolyzing chitin [41]. In addition, chitotriosidase/chitinase is also regarded as an immune molecule capable of killing chitin-bearing pathogens [42]. It has been demonstrated that recombinant mammalian chitinase is a fungistatic factor capable of inhibiting hyphal growth of chitincontaining fungi, suggesting a physiological role in defense against chitin-bearing pathogens [43,44]. Moreover, previous
Please cite this article in press as: Qin C-l, et al., Characterization of a novel antimicrobial peptide with chiting-biding domain from Mytilus coruscus, Fish & Shellfish Immunology (2014), http://dx.doi.org/10.1016/j.fsi.2014.09.019
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Fig. 5. Phylogenetic tree constructed by maximum likelihood method based on the amino acid sequences of chitotriosidase/chitinase from animals. This tree was generated by MEGA 6.0 using Maximum Likelihood algorithm with 1000 bootstrap replicates. Mytichin-1 from M. couscus is denoted by “*”. Numbers at each branch node represent the percentage values given by bootstrap analysis.
studies have shown that the C-terminal chitin-biding domain of human ChT permitted the enzyme to bind to chitin [45,46], and play a key role in the fungistatic activity of human chitinase [47]. However, little information is available to date regarding the antimicrobial activity of separated chitin-biding domain from chitinase. Phylogenetic analysis showed that mytichtin-1 and reported chitotriosidase/chitinase from other species seem to form different phylogenetic groups. This result, together with the fact that the Cterminal of mytichitin-1 can be released as an antimicrobial peptide, led us to assume that mytichitin-1 may represent a special chitotriosidase-like protein with a different position in evolution from known chitotriosidase/chitinase. This assumption awaits, however, confirmation from the identification of more mytichitinlike proteins. Unlike most of the reported bivalve mollusc AMPs, the expression of mytichitin-1 transcript was predominantly expressed in gonad instead of hemocytes. Considering the fact that mytichitinCB was purified from hemolymph in this study, it is hard to avoid speculating that mytichitin-1 may synthesized in gonad and mytichitin-CB was released from the C-terminal of precursor to hemolymph via proteolytic cleavage after invading of pathogens.
However, the processing of mytichitin-CB from mytichitin is unknown. Several antimicrobial peptides have been characterized as sex-specific, such as male-specific Andropin and female-specific ceratotoxin from reproductive apparatus of Drosophila melanogaster [48,49]; and male-specific Scygonadin from reproductive tract of Scylla serrata [50]. Additionally, both Andropin and ceratotoxin is unresponsive to bacterial injections but induced by mating. Unlike these reported sex-specific antimicrobial peptides, mytichitin-CB was expressed in both female and male gonad and can be induced by bacterial challenge. In the present study, the expression level of mytichitin-1 mRNA was obviously up-regulated post bacterial challenge, and the peak expression level was detected at 12 h with more than 9-fold increase compared with that of control group. These results indicated that mytichitin-CB was a reproductive-related antimicrobial peptide and served as one of the acute phase proteins involved in the elimination of invasive pathogens. However, much is still left to be explored, including the mechanism and processing of mytichitin-CB from mytichitin, and the roles of mytichitin in Mytilus mating. In conclusion, mytichitin-CB is a new type of cysteine-rich peptides with no homologous sequences available in AMP
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Fig. 6. Quantitative analysis of mytichitin-1 expression. A, Tissue-specific expression of mytichitin-1. Vertical bars represent the mean ± SD of three technical replicates. The Y axis of each graph is scaled based on the highest level of expression. GIL: gills, GON: gonad, FOO: foot, MAN: mantle, ADD: abductor muscle, DIG: digestive gland, HAE: Hemocytes. B, Temporal expression of mytichitin-1 in gonad after challenge. Vertical bars represent the mean ± SD (n ¼ 3). C, The expression of mytichitin-1 in male and female mussel gonad.
database. Mytichitin-CB has a chitin-biding domain and is produced from the C-terminal of mytichitin, a chitotriosidase-like protein of M. coruscus. The antimicrobial activities, and the rather specific and time-dependent expression in gonad, argue in favor of the participation of mytichitin-CB in immune defense. A more detailed study, including recombinant expression mytichitin-1 and mytichitin-CB, the subcellular location, the processing of mytichitin-CB from mytichitin-1, and the antibacterial mechanism need be performed to gain insight into the role of mytichitin in mussel immunity. These data are of great importance to understand how the mussel immune system responds to pathogens in the environment. Acknowledgments We are grateful to Dr. Hu Wei-jun (Hunan Normal University, Changsha, China) for mass spectrometry analysis and protein Nterminal sequencing. We thank Dr. Song xue-mei (Zhejiang academy of agricultural sciences, Hangzhou, China) for advices during cDNA cloning. Appendix A. Supplementary data Supplementary data related to this article can be found at http:// dx.doi.org/10.1016/j.fsi.2014.09.019. References [1] Tincu JA, Taylor SW. Antimicrobial peptides from Marine invertebrates. Antimicrob Agents Chemother 2004;48:3645e54. [2] Mercado L, Schmitt P, Marshall S, Arenas G. Gill tissues of the mussel Mytilus edulis chilensis: a new source for antimicrobial peptides. Electron J Biotechnol 2005;8:284e90. n F, C [3] Arenas G, Guzma ardenas C, Mercado L, Marshall SH. A novel antifungal peptide designed from the primary structure of a natural antimicrobial peptide purified from Argopecten purpuratus. Peptides 2009;30:1405e11. [4] De Zoysa M, Whang I, Youngdeuk L, Sukkyoung L, Lee JS, Lee J. Defensin from disk abalone Haliotis discus discus: molecular cloning, sequence characterization and immune response against bacterial infection. Fish Shellfish Immunol 2010;28:261e6. [5] Kubota Y, Watanabe Y, Otsuka H, Tamiya T, Tsuchiya T, Matsumoto JJ. Purification and characterization of an antibacterial factor from snail mucus. Comp Biochem Physiology C 1985;82:345e8. [6] Hubert F, Noel T, Roch P. A member of the arthropod defensin family from edible Mediterranean mussels (Mytilus galloprovincialis). Eur J Biochem 1996;240:302e6.
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Full length article
Characterization of a novel antimicrobial peptide with chiting-biding domain from Mytilus coruscus Q2
Chuan-li Qin a, 1, Wei Huang a, 1, Shi-quan Zhou b, Xin-chao Wang a, Hui-hui Liu a, Mei-hua Fan a, Ri-xin Wang a, Peng Gao a, Zhi Liao a, * a b
Key Laboratory for Marine Living Resources and Molecular Engineering, College of Marine Science, Zhejiang Ocean University, Zhoushan, 316022, PR China Laboratory of Immunogenomics, Zhoushan Hospital, Zhoushan, 316022, PR China
a r t i c l e i n f o
a b s t r a c t
Article history: Received 27 June 2014 Received in revised form 1 September 2014 Accepted 14 September 2014 Available online xxx
Using reverse phase high performance liquid chromatography (RP-HPLC), a novel antimicrobial peptide with 55 amino acid residues was isolated from the hemolymph of Mytilus coruscus. This new antimicrobial peptide displays predominant antimicrobial activity against fungi and Gram-positive bacteria. The molecular mass and the N-terminal sequence of this peptide were analyzed by Mass Spectrometry and Edman degradation, respectively. This antimicrobial peptide, with molecular mass of 6621.55 Da, is characterized by a chitin-biding domain and by 6 Cysteine residues engaged in three intra-molecular disulfide bridges. The full-length of cDNA sequence of this new peptide was obtained by rapid amplification of cDNA ends (RACE) and the encoded precursor was turn out to be a chitotriosidase-like protein. Therefore, we named the precursor with mytichitin-1 and the new antimicrobial peptide (designated as mytichitin-CB) is the carboxyl-terminal part of mytichitin-1. The mRNA transcripts of mytichitin-1 are mainly detected in gonad and the expression level of mytichitin-1 in gonad was up-regulated and reached the highest level at 12 h after bacterial challenge, which was 9-fold increase compared to that of the control group. These results indicated that mytichitin-1 was involved in the host immune response against bacterial infection and might contribute to the clearance of invading bacteria. © 2014 Published by Elsevier Ltd.
Keywords: Mytilus coruscus Antimicrobial peptide Chitin-biding domain Chitinase
1. Introduction Antimicrobial peptides (AMPs), which constitute a first line of host defense against pathogens, are widely expressed in organisms and have been linked to innate immunities in invertebrates. Marine molluscs are widespread, extremely diverse, and constantly under an enormous microbial challenge from the ocean environment. In order to defend themselves against such conditions, marine molluscs have developed very effective mechanisms that are part of their innate immunity [1]. AMPs are the major component of the innate immune system in molluscs [2e4] and antibacterial activity was first reported in mollusks in the ’80s [5]. Most of the AMP researches of marine mussels has focused on species Mytilus, including Mytilus edulis and Mytilus galloprovincialis, whereas the isolation and characterization of true AMPs from marine mussels date back to 1996 [6,7].
* Corresponding author. Tel./fax: þ86 580 2550753. E-mail addresses: [email protected], [email protected] (Z. Liao). 1 Equally contribution to this work.
Among the various natural AMPs, those containing pairs of cysteine residues forming intra-molecular disulfide bridges are particularly common [8e11]. In the last decades, AMPs have been isolated and characterized from marine mussels and classified into seven groups: defensins [6], mytilins [12], myticins [13,14], mytimycin [15], mytimacin [16], big defencin [16], and myticusin [17]. These AMPs have also commonly been identified and characterized in other marine bivalve mollusk. For example, defensins have been described in oysters Crassostrea virginica and Crassostrea gigas [18,19] and abalone Haliotis discus [4]; mytilins and myticins were also described in clams Ruditapes decussates [20]. These peptides belong to the cysteine-rich family of AMPs with strong activity against bacteria, fungus and virus [21e23], making them not only a promising template molecule for investigating the relationship between protein structure and function, but also a source of new leads for the treatment of bacterial disease [23]. The molecular diversity of AMPs increases the antimicrobial capabilities of mussels against a high diversity of pathogens. However, AMPs have been explored only in a few species. Therefore, there is still a great potential to unveil new molecules in this phylum. Mytilus coruscus is one of the most important economic
http://dx.doi.org/10.1016/j.fsi.2014.09.019 1050-4648/© 2014 Published by Elsevier Ltd.
Please cite this article in press as: Qin C-l, et al., Characterization of a novel antimicrobial peptide with chiting-biding domain from Mytilus coruscus, Fish & Shellfish Immunology (2014), http://dx.doi.org/10.1016/j.fsi.2014.09.019
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shellfish and widely distributed in eastern coast in China. Several antimicrobial peptides have been characterized from M. coruscus in our previous work, including mytilins (FJ973154 ~ FJ973155 for mytilin-A and -B; GU324718~GU324723 for mytilin3~8), myticins (GU324724~GU324732 for myticin1~9), and a big antimicrobial peptide named myticusin-1 [17]. Here we present the isolation and biochemical characterization a novel antimicrobial peptide, mytichitin, from hemolymph of M. coruscus. 2. Material and methods 2.1. Animals and hemolymph collection Adult mussels (M. coruscus) were collected locally from Zhoushan (Zhejiang province, P. R. China) and were maintained in tanks with fresh sea water at 25 C. Hemolymph was prepared using the methods described by Mitta [12], the plasma was clarified by centrifugation (12,000 g, 20 min, 4 C) and then kept at 80 C until further analysis. 2.2. Antimicrobial assays Bacteria and fungus (about 1e2 * 108 cfu/mL) used in present study were obtained from China General Microbiological Culture Collection Center (CGMCC, Beijing, P. R. China). The bacterial strains were Escherichia coli (CGMCC1.1583), Vibrio Parahaemolyticus (CGMCC1.1616), Pseudomonas aeruginosa (CGMCC1.0102), Proteus vulgaris (CGMCC1.1527), Vibrio.harveyi (CGMCC1.1601), Bacillus subtilis (CGMCC1.1630), Staphylococcus aureus (CGMCC1.128), Sarcina luteus (CGMCC28001), and Bacillus megaterium (CGMCC1.1487). The fungus used here were Candida albicans (CGMCC2.2086) and Monilia albican (CMCC(F)98001). Antimicrobial activity was monitored by a liquid growth inhibition assay according to the method described previously [17]. During the various steps of peptide purification, antibacterial activity was monitored on the Gram-positive strain S. luteus and the Gramnegative strain E. coli. To determine the minimal inhibitory concentration (MIC), serial doubling dilutions were carried out following the protocol described by Mitta [12]. The MIC values are expressed an interval (aeb), where (a) represents the highest peptide concentration tested at which bacteria are still growing and (b) the lowest concentration that causes 100% growth inhibition. 2.3. Peptide purification All HPLC purification steps were carried out on a Waters Delta 600 HPLC system equipped with a Waters 2487 absorbance detector. Fractions were collected and assayed for antimicrobial activity. The plasma sample was directly subjected to a Sunfire™ prep C8 column (10 250 mm Waters) equilibrated with deionized water containing 0.1% trifluoroacetic acid. The sample was eluted with a gradient of 0e5% acetonitrile containing 0.1% trifluoroacetic acid over 5 min followed by 5e60% over 40 min and 60e95% over 5 min at a flow rate of 2.5 mL/min. The eluted fractions were collected manually, dried under vacuum, and reconstituted in MilliQ water (prepared by Millipore synergy system, France). The presence of antibacterial activities was detected by the liquid growth inhibition assays described above. Active fractions were applied to an analytical Vydac C18 RP HPLC column (218TP54, 4.6 250 mm) and eluted at the flow rate of 0.75 mL/min using 5e45% acetonitrile/water/0.1% trifluoroacetic acid as a linear gradient. To obtain the peptides with high purity, an additional step was performed on a SunFire™ C18 Column (100 Å, 3.5 mm,
3 150 mm, Waters) developed in the same diphasic gradients as above but at a flow rate of 0.25 mL/min. The eluted fractions with antimicrobial activities were vacuum-dried and used for determination of molecular mass and amino acid sequences. 2.4. Peptide sequencing and modeling The molecular mass of peptide was determined using Matrixassisted laser desorption/ionization-time of flight-mass spectrometry (Voyager-DE STR biospectometry workstation, Applied Biosystems). Purified peptide was submitted to reduction and alkylation, and Edman degradation was performed with a normal automatic cycle program using an Applied Biosystems Model 491 gas-phase sequencer. The 3-Dimensional structure of peptide was predicted from amino acid sequences by SWISS-MODEL platform on line (http:// swissmodel.expasy.org/interactive). 2.5. The full-length cDNA cloning The amino acid sequence detected from Edman sequencing was searched against the dbEST database of Mytilus using tblastn search tool (http://blast.ncbi.nlm.nih.gov/Blast.cgi). The matched EST sequence with high similarity was used as the template for designing specific primer of PCR amplification. Using Rapid Amplification cDNA Ends (RACE) method; the full length of cDNA was obtained by SUPERSWITCH™ RACE cDNA Synthesis Kit (Shanghai, China) according to the manufacturer's instructions. For 3’-RACE, a sense primer (5’-TAAAATGTGGCATGAATGG-3’) and a 3’Adaptor primer were used; Meanwhile, The antisense (5’GGGTCTTGTATGGAATCC-3’) corresponding to the 3’-end of amplified the cDNA fragment, together with 5’-Adaptor primer was used to obtain the cDNA 5’-end. The full-length cDNA was confirmed by specific primers (sense: 5’-CAGACCTACACTGTTCACGATGAT-3’, antisense: 5’- GTTCTCAATCTAATATTCCAAAGTCA-3’) designed in the 5’- and 3’-UTR, respectively, from sequences retrieved through RACE assays. The PCR was performed in 25 mL volume, including of 2.5 mL 10 SUPERSWITCH™ Hot Start Buffer, 0.1 mL of each primer (10 mМ), 2 mL dNTP Mix (2.5 mM), 0.5 mL of the cDNA template, and 0.5 mL SUPERSWITCH™ Hot Start DNA polymerase for 41 cycles on a Thermal Cycler (Bio-Rad, USA). Each cycle consisted of 30 s at 94 C, 30 s at 60 C, and 120 s at 72 C, with a final extension of 5 min. The PCR products were subjected to 1.0% agarose gel electrophoresis, purified, cloned into pMD19-T vector (TaKaRa), and transformed into competent DH5a. The resulting cDNA sequences were analyzed using the software of DNAstar lasergen 7.0 after nucleic acid sequencing. Signal peptides were predicted using the SignalP 4.0 [24] online tool (http:// www.cbs.dtu.dk/services/SignalP/) and conserved domains were predicted using simple modular architecture research tool (SMART) software (http://smart.embl-heidelberg.de/). Following peptide signal removal, the theoretical MW and pI were determined on Expasy-ProtParam online tool (www.expasy.org/tools/protparam. html). Multiple sequence alignments were performed with DNAMAN (version 7.0.2.176). Phylogenetic analyses based on the amino acid sequences were conducted by MEGA 6.0 using Maximum Likelihood algorithm with 1000 bootstrap replicates. 2.6. Quantitative analysis of mytichitin-1 expression The tissue-specific expression and the temporal expression of transcripts in mussels challenged with S. luteus were determined by quantitative real-time PCR. Mussels of 5e6 cm shell length were collected from East China Sea. Total RNA was individually purified
Please cite this article in press as: Qin C-l, et al., Characterization of a novel antimicrobial peptide with chiting-biding domain from Mytilus coruscus, Fish & Shellfish Immunology (2014), http://dx.doi.org/10.1016/j.fsi.2014.09.019
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from previously homogenized hemocytes, digestive gland, mantle, posterior abductor muscle, gill, foot, and gonad. After reverse transcription, the expression of transcripts in tissues was measured by SYBR Green quantitative real-time PCR assay in Applied Biosystem 7500 Real-time PCR System. Two specific primers, mytichitin-RT-F (5’-CCTAAAATGTGGCATGAATGG-3’) and mytichitin-RT-R (5’-GCAAGCGTAGTCACAAAATCC-3’), were used for
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amplifying gene fragment of 158 bp, which was sequenced to verify the PCR specificity. Amplifications were carried out at a final volume of 20 mL, containing 1.0 mL cDNA sample, 10 mL SYBR Green Real-time PCR master mixtures (Takara), 0.4 mL ROX II, 1.0 mL of each primer and 7.6 mL H2O. The following thermal profile was used: an initial step of 5 min denaturation at 95 C, followed by 40 cycles at 95 C for 30 s, 50.3 C for 30 s, and 72 C for 40 s. Amplification
Fig. 1. Purification and identification of mytichitineCB. A, the plasma sample collected from M. coruscus was isolated by Sunfire™ prep C8 column and the target fraction (denoted by “*”) with antibacterial activity was collected for further purification. B, further purification of the target elution by analytical Vydac C18 column and the elusion with antibacterial activity (denoted by “*”) was collected for structural analysis. C, mass spectrometry analysis yielded a single mass of 6297.55 Da; N-terminal sequences (29-AA) determined by Edman degradation are indicated with character abbreviation.
Please cite this article in press as: Qin C-l, et al., Characterization of a novel antimicrobial peptide with chiting-biding domain from Mytilus coruscus, Fish & Shellfish Immunology (2014), http://dx.doi.org/10.1016/j.fsi.2014.09.019
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products were analyzed with a 65 C/95 C melting curve. The expression values were normalized using the b-actin of M. coruscus as housekeeping gene (McActin-F: 5’-ATGAAACCACCTACAACAGT3’; McActin-R: 5’- TAGACCCACCAATCCAGACG-3’). Results are given as the mean with standard deviation of three technical replicates. Comparisons between groups were made by one-way analysis of variance followed by a Duncan test for identification of the statistically distinct groups. 3. Results 3.1. Isolation and identification of new antimicrobial peptide from M. coruscus hemolymph Hemolymph was collected from M. coruscus. After cells and debris were removed by centrifugation, the supernatant was applied on a Sunfire™ prep C8 column and eluted with a gradient of 5e60 % acetonitrile, yielding the chromatogram shown in Fig. 1A. The eluted fractions were assayed for their antibacterial activity against two bacterial strains, S. luteus and E. coli. The fractions containing antibacterial activity (denoted by asterisk in Fig. 1A) were collected, vacuum dried, and subjected to the second C18 reverse-phase column for further purification (Fig. 1B). Finally, the antibacterial peptide (denoted by asterisk in Fig. 1B) was purified to homogeneity by reverse-phase HPLC. The molecular mass of this new antimicrobial peptide determined by MALDI-TOF was 6297.55 Da (Fig. 1C). The alkylated peptide was sequenced by Edman degradation, and a 29-residue Nterminal partial sequence was obtained (Fig. 1C). 3.2. Cloning of cDNA encoding the new antimicrobial peptide When we used the 29-residue N-terminal sequence of the new antibacterial peptide to search against Mytilus EST database, one matched EST sequence (gbjES407161.1j) with 73% identity was obtained. Based on the partial N-terminal amino acids sequence and matched EST sequence, we designed and synthesized specific primer to amplify the cDNA of the new antibacterial peptide from M. coruscus and the complete cDNA was determined using RACE technique. Five distinct variants with high sequence identity (>95%) were obtained (showed in Supplementary Fig. 1), in which, the represent cDNA sequence (gij568218204) is shown in Fig. 2. The complete 1588-bp cDNA obtained revealed an open reading frame of 1341 bp. This open reading frame encodes a protein composed of 446 amino acids with a putative N-terminal 24-residues signal peptide (Fig. 2), and this sequence turns out to be a chitotriosidase-like protein after sequence analysis. A comprehensive search of none-redundant protein sequences data revealed significant homology of this sequence (>35%) with chitotriosidase/chitinases from other species. Furthermore, domain analysis showed that the obtained sequence has one GH18_chitolectin_chitotriosidase domain (cd02872) and one Chitin-binding domain (smart00494). Therefore, we propose the name of mytichitin-1 for this new sequence. The mature peptide of mytichitin-1 consists of a 422-amino acid residue with a calculated molecular mass of 48,005.79 Da and an estimated pI of 9.37. The 29-residues fragment (Thr392-Arg420) of mytichitin-1 perfectly matches the amino acid sequences determined by Edman degradation. Moreover, the 55-residues (containing 6 cysteines) of the carboxyl terminus of mytichitin-1 shows a theoretical molecular mass of 6303.38 Da, which is identical to the experimental molecular mass (6297.55 Da, measured by MALDI-TOF) of the antimicrobial peptide purified in this article, considering the fact that forming of 3 disulfides will lose 6 Da. These results indicate that the isolated antibacterial peptide in present study corresponds to the carboxyl terminus of mytichitin-1.
Fig. 2. cDNA and deduced amino acid sequence of mytichitin-1 (A) and sequence alignment (B) of mytichitineCB with chitinebiding domain (smart00494). The predicted signal peptide is shown in bold. The asterisk (*) indicates the stop codon. A polyadenylation signal (AATAAA, denoted by bold Italic) emerged in the 3’-UTR at position 15 upstream of the poly-(A)-tail. The motif eDXXDXDXE- is boxed. The sequence of mytichitin-CB is underlined. The numbers at right panel indicate the serial number of amino acids and nucleotides.
Additionally, the 55-residues of the carboxyl terminus of mytichitin-1 contains a chitin-biding domain (Fig. 2B), therefore, we named the antimicrobial peptide presented in this article as mytichitin-CB. The tertiary structure modeling by SWISS-MODEL was successful for mytichitin-CB and model with the highest score was shown in Fig. 3A. The predicted tertiary structure of mytichitin-CB is characterized by several bsheets, including Asn25-Ala31, Arg34-Cys40, Val45-Asn47, and Tyr51-Asp53 as determined by Swiss-PdbViewer (v4.0.1). 3.3. Activity spectrum of mytichitin-CB The activity spectrum of mytichitin-CB was investigated against a variety of bacterial strains (Table 1). In liquid growth inhibition assays, the purified mytichitin-CB had marked activity against the Gram-positive strains including B. subtilis, S. aureus, S. luteus; and B. megaterium (MIC5 mM). Additionally, mytichitin-CB had weaker effects on the Gram-negative strains including E. coli, V. Parahaemolyticus, P. aeruginosa, and V. harveyi (MIC > 50 mM). 3.4. Multiple sequences alignment and phylogenetic analysis Blast analysis revealed that mytichitin-1 shared a high homology with chitotriosidase/chitinase from other species. The deduced
Please cite this article in press as: Qin C-l, et al., Characterization of a novel antimicrobial peptide with chiting-biding domain from Mytilus coruscus, Fish & Shellfish Immunology (2014), http://dx.doi.org/10.1016/j.fsi.2014.09.019
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curves for both two genes, indicated that the amplification were specific (Data not shown). Real-time qPCR demonstrated that mytichitin-1 mRNA amount was measured mainly in the gonad (in both male and female, Fig. 6A, C), and to a lesser extent, in the digestive gland. Temporal change of mytichitin-1 mRNA in gonad after microbial challenge was shown in Fig. 6B. Compared to the control group injected with only PBS solution, the expression level of mytichitin-1 mRNA in the challenge group increased and reached the highest level at 12 h (about 9-fold) post-injection after S. luteus challenge (P < 0.01). 4. Discussion Fig. 3. Comparison of the structures of mytichin-CB (A) and tachycitin (B) and 3D structure-based sequence alignment of mytichitin-CB and tachycitin with regard to the region corresponding to Cys-40eAsp53 of tachycitin (C). The putative key residues of tachycitin for chitin-biding are denoted by “*”.
amino acid sequence of mytichitin-1 was aligned with other reported chitotriosidase/chitinase and several conserved features were found in mytichitin-1 (Fig. 4) including two conserved domains, chitin catalytic domain and chitin-biding domain. The motif eDGLDL(P/I/F)DL(W)- regarding chitin degradation were also conserved in mytichitin-1 [25]. In addition, a short link region with abundant Ser/Thr between chitin catalytic domain and chitinbiding domain was observed in most sequences. The sequence similarity and the common structure features suggested that mytichitin-1 was a new counterpart of chitotriosidase/chitinase. The phylogenetic tree of invertebrate and vertebrate chitotriosidase/chitinase (Fig. 5) included six major clades with high bootstrap value: acidic mammalian chitinase from mammal, acidic mammalian chitinase from bird, chitinase1/2 from fish, chitotriosidase from mammal, chitotriosidase from mollusk, and chitinase from arthropod. Despite low bootstrap values, mytichitin-1 was firstly clustered with chitotriosidase-like protein from the bivalve C. gigas, and then grouped with chitotriosidase from mollusk and amphioxus.
3.5. Quantitative analysis of mytichitin-1 expression In order to establish the expression patterns of the mytichitin-1 transcripts in tissues, real-time qPCR was employed to quantify the expression of hemocyte, digestive gland, mantle, posterior adductor muscle, gill, foot, and gonad. The amplification specificity for mytichitin-1 and b-actin was determined by analyzing the dissociation curves. Only one peak presented in the dissociation
Table 1 Activity spectrum of mytichitin-CB against bacteria and fungus. Organism Bacteria:gramenegative Escherichia coli Vibrio Parahaemolyticus Pseudomonas aeruginosa Vibrio harveyi Bacteria:gramepositive Bacillus subtilis Staphylococcus aureus Sarcina luteus Bacillus megaterium Fungus Candida albicans Monilia albican
Mytichitin-CB (mmol/L) >100.0 50.0e100.0 50.0e100.0 >100.0 1.56e3.13 3.13e6.25 1.56e3.13 3.13e6.25 6.25e12.5 12.5e25.0
In present study, a novel antimicrobial peptide named mytichitin-CB was purified from M. coruscus plasma. Mytichitin-CB contains 55 residues including 6 cysteines in its sequence, which was further verified by its cDNA analysis. The result of Edman degradation with reduced and alkylated sample, together with the comparison of theoretical and experimental molecular mass, indicates the forming of three disulfides in mytichitin-CB. In present work, mytichitin-CB was active in vitro against Gram-positive bacteria but showed no or limited activities against Gramnegative bacteria, which is similar to the action of other AMPs from mussel, such as mytilins (mytilin A and G1) [12], myticins (myticin A and B) [13], and MGDs [6]. In addition, some defensins from insect [26] and Oyster C. gigas [19] were also found to be specific to Gram-positive bacteria. Unfortunately, the mechanism of selective inhibition against Gram-positive bacteria is unclear. Some researchers suggested that this selective function may results from different composition in cell wall of Gram-positive (lipoteichoic acid) and Gram-negative bacteria (Lipopolysaccharide) [27], the antimicrobial peptide charge [28], and the ratio of Arg/Lys of AMPs [29]. Although mytichitin-CB shows a strong activity against Grampositive bacteria and fungi, there is no sequence homology between mytichitin-CB and those characterized antimicrobial peptides to date. A chitin-biding domain was detected in mytichitin-CB. The antimicrobial activity is initially identified for chitin-binding proteins extracted from plants [30,31], which commonly comprise single or multiple copies of the chitin-binding domain. Some chitin-biding-domain-containing antimicrobial peptides were also found in arthropod. For example, Penaeidins, a family of antimicrobial peptides characterized in the shrimp Penaeus vanname [32], and Tachycitin, a 73-residue polypeptide having antimicrobial activity presented in the hemocyte of horseshoe crab Tachypleus tridentatus [33,34]. For invertebrates, the chitin-binding domain was assumed to comprise about 65 residues involving a high percentage of cysteine and aromatic residues in a similar manner to the plant chitin-binding domain [35]. We compared modeling structure of mytichitin-CD with the structure of tachycitin (1DQC), and the structural similarity between segment Cys40eAsp53 of mytichitin-CB and segment Cys40eAsp54 (proposed as an essential chitin-biding region by Suetake et al. [34]) of tachycitin, both comprising the antiparallel bεta-sheet and a hairpin loop, was examined. As shown in Fig. 3, the residues of Asn47 and Phe51 of mytichitin-CB are located at perfectly corresponding positions to the residues of Asn47 and Val52 (essential chitin-biding residues [34]) of tachycitin. Therefore, one could assume that the region of Cys40eAsp53 from mytichitin-CB serves as an essential chitin-binding site. It is suggested that the chitinbinding ability of Penaeidin and Tachycitin could not only be essential for their antifungal activity but could also be involved in chitin assembly or wound healing [36]. However, there is no significant sequence homology between mytichitin-CB and those chitin-biding-domain-containing antimicrobial peptides from
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Fig. 4. Multiple sequence alignment of mytichitin-1 (gij568218204) with chitotriosidase/chitinase from other species. Conserved residues are shaded in dark gray. Amino acids of the catalytic sites are shown with asterisk (*). The link region and the chitin-biding domain are shown with dash line and solid line, respectively. Gaps are indicated by dot to improve the alignment. The GenBank accession numbers, protein names, and the species are shown in left panel.
other species (data not shown), indicating mytichitin-CB may represent a new class of antimicrobial peptide family with chitinbiding domain. Using RACE technique, the complete cDNA (named mytichitin1) of mytichitin-CB was cloned and a 446-residues precursor including a 24-residues putative signal peptide was detected. The sequence of mytichitin-CB is perfectly matched the carboxyl terminus of mytichitin-1, therefore, we believed that mytichitin-CB is released from the carboxyl-terminal part of mytichitin via protein cleavage. Many antimicrobial peptides are derived from larger precursors, and processing and generation of antibacterial peptides have been reported from several species. For example, in invertebrate, buforin I from the stomach gland cells of the Asian toad Bufo bufo is generated by a pepsin-mediated processing of the cytoplasmic histone H2A [37]. astacidin 1 is released from the carboxylterminal part of crayfish hemocyanin by a cysteine-like proteinase
[38]. In mice, the precursor a-defensin is cleaved by metalloproteinase, a matrilysin to produce a -defensin [39], and human defensin-5 is also processed by paneth cell trypsin [40]. The organization and processing of peptides from one large precursor molecule is an efficient way to synthesize different effector molecules and amplify the antibacterial response. Mytichitin-1 shows a high (about 35e40%) sequence similarity with chitotriosidase/chitinase from other species (Fig. 4). Both chitotriosidase and chitinase is member of the glycosyl_hydrolase_18 superfamily capable of hydrolyzing chitin [41]. In addition, chitotriosidase/chitinase is also regarded as an immune molecule capable of killing chitin-bearing pathogens [42]. It has been demonstrated that recombinant mammalian chitinase is a fungistatic factor capable of inhibiting hyphal growth of chitincontaining fungi, suggesting a physiological role in defense against chitin-bearing pathogens [43,44]. Moreover, previous
Please cite this article in press as: Qin C-l, et al., Characterization of a novel antimicrobial peptide with chiting-biding domain from Mytilus coruscus, Fish & Shellfish Immunology (2014), http://dx.doi.org/10.1016/j.fsi.2014.09.019
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Fig. 5. Phylogenetic tree constructed by maximum likelihood method based on the amino acid sequences of chitotriosidase/chitinase from animals. This tree was generated by MEGA 6.0 using Maximum Likelihood algorithm with 1000 bootstrap replicates. Mytichin-1 from M. couscus is denoted by “*”. Numbers at each branch node represent the percentage values given by bootstrap analysis.
studies have shown that the C-terminal chitin-biding domain of human ChT permitted the enzyme to bind to chitin [45,46], and play a key role in the fungistatic activity of human chitinase [47]. However, little information is available to date regarding the antimicrobial activity of separated chitin-biding domain from chitinase. Phylogenetic analysis showed that mytichtin-1 and reported chitotriosidase/chitinase from other species seem to form different phylogenetic groups. This result, together with the fact that the Cterminal of mytichitin-1 can be released as an antimicrobial peptide, led us to assume that mytichitin-1 may represent a special chitotriosidase-like protein with a different position in evolution from known chitotriosidase/chitinase. This assumption awaits, however, confirmation from the identification of more mytichitinlike proteins. Unlike most of the reported bivalve mollusc AMPs, the expression of mytichitin-1 transcript was predominantly expressed in gonad instead of hemocytes. Considering the fact that mytichitinCB was purified from hemolymph in this study, it is hard to avoid speculating that mytichitin-1 may synthesized in gonad and mytichitin-CB was released from the C-terminal of precursor to hemolymph via proteolytic cleavage after invading of pathogens.
However, the processing of mytichitin-CB from mytichitin is unknown. Several antimicrobial peptides have been characterized as sex-specific, such as male-specific Andropin and female-specific ceratotoxin from reproductive apparatus of Drosophila melanogaster [48,49]; and male-specific Scygonadin from reproductive tract of Scylla serrata [50]. Additionally, both Andropin and ceratotoxin is unresponsive to bacterial injections but induced by mating. Unlike these reported sex-specific antimicrobial peptides, mytichitin-CB was expressed in both female and male gonad and can be induced by bacterial challenge. In the present study, the expression level of mytichitin-1 mRNA was obviously up-regulated post bacterial challenge, and the peak expression level was detected at 12 h with more than 9-fold increase compared with that of control group. These results indicated that mytichitin-CB was a reproductive-related antimicrobial peptide and served as one of the acute phase proteins involved in the elimination of invasive pathogens. However, much is still left to be explored, including the mechanism and processing of mytichitin-CB from mytichitin, and the roles of mytichitin in Mytilus mating. In conclusion, mytichitin-CB is a new type of cysteine-rich peptides with no homologous sequences available in AMP
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Fig. 6. Quantitative analysis of mytichitin-1 expression. A, Tissue-specific expression of mytichitin-1. Vertical bars represent the mean ± SD of three technical replicates. The Y axis of each graph is scaled based on the highest level of expression. GIL: gills, GON: gonad, FOO: foot, MAN: mantle, ADD: abductor muscle, DIG: digestive gland, HAE: Hemocytes. B, Temporal expression of mytichitin-1 in gonad after challenge. Vertical bars represent the mean ± SD (n ¼ 3). C, The expression of mytichitin-1 in male and female mussel gonad.
database. Mytichitin-CB has a chitin-biding domain and is produced from the C-terminal of mytichitin, a chitotriosidase-like protein of M. coruscus. The antimicrobial activities, and the rather specific and time-dependent expression in gonad, argue in favor of the participation of mytichitin-CB in immune defense. A more detailed study, including recombinant expression mytichitin-1 and mytichitin-CB, the subcellular location, the processing of mytichitin-CB from mytichitin-1, and the antibacterial mechanism need be performed to gain insight into the role of mytichitin in mussel immunity. These data are of great importance to understand how the mussel immune system responds to pathogens in the environment. Acknowledgments We are grateful to Dr. Hu Wei-jun (Hunan Normal University, Changsha, China) for mass spectrometry analysis and protein Nterminal sequencing. We thank Dr. Song xue-mei (Zhejiang academy of agricultural sciences, Hangzhou, China) for advices during cDNA cloning. Appendix A. Supplementary data Supplementary data related to this article can be found at http:// dx.doi.org/10.1016/j.fsi.2014.09.019. References [1] Tincu JA, Taylor SW. Antimicrobial peptides from Marine invertebrates. Antimicrob Agents Chemother 2004;48:3645e54. [2] Mercado L, Schmitt P, Marshall S, Arenas G. Gill tissues of the mussel Mytilus edulis chilensis: a new source for antimicrobial peptides. Electron J Biotechnol 2005;8:284e90. n F, C [3] Arenas G, Guzma ardenas C, Mercado L, Marshall SH. A novel antifungal peptide designed from the primary structure of a natural antimicrobial peptide purified from Argopecten purpuratus. Peptides 2009;30:1405e11. [4] De Zoysa M, Whang I, Youngdeuk L, Sukkyoung L, Lee JS, Lee J. Defensin from disk abalone Haliotis discus discus: molecular cloning, sequence characterization and immune response against bacterial infection. Fish Shellfish Immunol 2010;28:261e6. [5] Kubota Y, Watanabe Y, Otsuka H, Tamiya T, Tsuchiya T, Matsumoto JJ. Purification and characterization of an antibacterial factor from snail mucus. Comp Biochem Physiology C 1985;82:345e8. [6] Hubert F, Noel T, Roch P. A member of the arthropod defensin family from edible Mediterranean mussels (Mytilus galloprovincialis). Eur J Biochem 1996;240:302e6.
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Please cite this article in press as: Qin C-l, et al., Characterization of a novel antimicrobial peptide with chiting-biding domain from Mytilus coruscus, Fish & Shellfish Immunology (2014), http://dx.doi.org/10.1016/j.fsi.2014.09.019
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