Molecular Characterization and Expression Analysis of Matrix Metalloproteinase 3 in the Asian Yellow Pond Turtle Mauremys mutica

Mi ZHAO， Yan SHI， Jian ZHAO， Xinping ZHU， Kunci CHEN， Debo PAN and Chengqing WEI

Key Laboratory of Tropical and Subtropical Fishery Resource Application and Cultivation， Ministry of Agriculture，Pearl River Fishery Research Institute， Chinese Academic of Fishery Science， Guangzhou 510380， China

Molecular Characterization and Expression Analysis of Matrix Metalloproteinase 3 in the Asian Yellow Pond Turtle Mauremys mutica

Mi ZHAO， Yan SHI， Jian ZHAO， Xinping ZHU*， Kunci CHEN， Debo PAN and Chengqing WEI

Key Laboratory of Tropical and Subtropical Fishery Resource Application and Cultivation， Ministry of Agriculture，Pearl River Fishery Research Institute， Chinese Academic of Fishery Science， Guangzhou 510380， China

Matrix metallopeptidase 3 is a zinc-containing proteinase that participates in tissue remodeling and immune responses. In this study， a cDNA encoding matrix metallopeptidase 3 was isolated and characterized from the Asian yellow pond turtle Mauremys mutica （designated as MaMMP3）. The MaMMP3 cDNA is 1805 bp and consists of a 5′-untranslated region （UTR） of 56 bp， a 3′-UTR of 243 bp， and an open reading frame （ORF） of 1506 bp encoding 481 amino acids. Homology analysis of MaMMP3 revealed that the MaMMP3 shared 25%-63% similarity to other known MMP3 sequences. The genomic sequence covers 6007 bp. Comparative analysis of the cDNA sequence revealed that the Asian yellow pond turtle MMP3 has eight exons and seven introns. The phylogenetic tree showed that the MaMMP3 is closely related to Gallus gallus MMP3 and Taeniopygia guttata MMP3. The mRNA expression of the MaMMP3 in normal group without any bacterial challenge could be detected in all studied tissues including kidney， heart， live and spleen， with the highest level in the spleen. The results of immune challenge showed that the expression level of MaMMP3 was up-regulated in the spleen and liver. These results provided an important information for studying the roles of Asian yellow pond turtle MMP3 in immunity further.

Asian yellow pond turtle， Mauremys mutica， Serratia marcescens， Matrix metallopeptidase 3， Immune responses

1. Introduction

Matrix metalloproteinases 3 （MMP3） is a zinc-containing proteinase， belonging to the MMP family which are capable of cleaving proteinaceous components of the extracellular matrix （ECM） as well as non-ECM proteins，thus affecting cell fate through modifications of a cell's microenvironment （Silence et al.， 1999）. MMP3 has a similar domain structure like other MMPs that include a prepeptide for secretion， a propeptide to maintain latency，and a catalytic domain （Vu et al.， 2010）. It is secreted as latent enzymes and processed to active forms upon the removal of the propeptide domain through various mechanisms （Murphy et al.， 1999）.

The expression and distribution of MMP3 has implicated that MMP3 plays an important role in many physiological processes including embryonic development， angiogenesis， tissue resorption and remodeling， and pathological events such as tumor invasion and arthritis （Uria and Werb， 1998； Sarras et al.，2002； Fu et al.， 2007）. In vitro and cell culture studies showed that MMPs can regulate cell fate and behavior by remodeling the ECM. On the other hand， more evidence has indicated that MMPs are capable of cleaving non-ECM extracellular proteins to regulate varied aspects of inflammation and immunity， such as latent TGF-β （Maeda et al.， 2002）， MCP-1， MCP-2， MCP-3， MCP-4（McQuibban et al.， 2002）， SDF-1/CXCL12 （McQuibban et al.， 2001）， IL-1β （Schonbeck et al.， 1998）， Macrophage chemokine （Haro et al.， 2000）.

After challenge， such as by injury， inflammationor infection， MMP3-deficient mice revealed various phenotypes: decreased neutrophil recruitment in models of acute lung injury （Warner et al.， 2001） and in LPS-induced neuroinflammation （Gurney et al.，2006）； impaired contact hypersensitivity （Wang et al.，1999）； enhanced collagen-induced arthritis （Mudgett et al.， 1998）； reduced accumulation of macrophages in atherosclerotic plaques （Silence et al.， 2001）； reduced macrophage-chemoattractant activity （Haro et al.， 2000）；delayed clearance of bacteria and delayed appearance of CD4+ T lymphocytes into intestinal mucosa （Li et al.，2004）. These phenotypes indicated that the enzymes have specif i c， and at times， essential roles in tissue repair， angiogenesis， host defence and especially in inf l ammation and immunity.

The Asian yellow pond turtle， Mauremys mutica（Cantor）， is a member of the family Bataguridae （Joyce et al.， 2004）. Because of its higher value for food and remedies than other species of turtle， it has become one of the most commonly and highly traded turtles in Asia （Lau and Shi， 2000； Shi and Pharham， 2001）. However， recent reports indicate that the increase in breeding density has caused a high frequency of disease epidemics and the emergence of new diseases in artificial cultivation of Asian yellow turtle. A strain of pathogenic bacteria was isolated from the exterior ulceration of the Asian yellow pond turtle and was identifed as Serratia marcescens by Tan et al.（2007）. S. marcescens， a gram-negative bacillus classified as a member of the Enterobacteriaceae， is widely distributed in nature and mainly infects organisms with weak resistance. So far， many reports have showed that S. marcescens has infected humans and culture animals （Tan et al.， 2007； Jones et al.， 2000）. In order to control disease and enhance the yields and quality of the Asian yellow pond turtle， it is necessary to further study the immune mechanisms of Asian yellow pond turtle（Gui et al.， 2012）. In contrast with detailed knowledge on MMP3 in other organisms， the information of MMP3 in turtle is rare. To study the gene expression regulation and its roles in immune responses， a full-length cDNA of MMP3 was cloned and characterized （designated as MaMMP3） from the Asian yellow pond turtle M. mutica, and its expression patterns in different tissues after infection of S. marcescens were investigated in this study.

2. Materials and Methods

2.1 Animals and immune challengeThe Asian yellow pond turtle M. mutica （body weight 120-160g） was obtained from the turtle culture base of Pearl River Fisheries Research Institute of the Chinese Academy of Fishery Sciences and maintained in tanks for one week before experimentation. The bacterial challenge experiment was performed by injecting intraperitoneally with 600 μl.

S. marcescens resuspended in 6.5% （m/v） saline（1012cfu）. Untreated turtles and turtles which received an injection of 600ul 6.5% （m/v） saline were used as the blank group and the control group， respectively. The turtles were returned to tanks and three turtles were randomly sampled at 12 h， 24 h and 36 h. The tissues from the blank， control and the stimulated groups were collected.

2.2 cDNA library construction and EST analysisA cDNA library was constructed from the liver of an Asian yellow pond turtle challenged by S. marcescens（600 μl， 1012cfu）， using the SMART? PCR cDNA Synthesis Kit （Clontech）（Endege et al.， 1999）. Random sequencing of the library used 5′ PCR Primer II 5′-AAGCAGTGGTATCAACGCAGAGT-3′ and poly（dT） to determine the full length cDNA sequences， then，BLAST analysis of all the sequences.

2.3 RNA extraction and reverse transcriptionTotal RNA samples were extracted from tissues using Trizol Reagent （Invitrogen） and then treated with DNase I（Promega）. Subsequently， the first-strand cDNA was synthesized based on manufacture's instruction of PrimeScript? RT reagent Kit （Perfect Real Time）（TaKaRa）. There were 0.5 μg of total RNAs， 2 μl of PrimeScript Buffer， 25 pmol of Oligo（dT） Primer， 50 pmol of Random6-mers， and 0.5 μl of PrimeScript RT Enzyme Mix I in a 10 μl volume. Reverse transcription was performed at 37°C for 15 min and 85°C for 5 s.

2.4 Real-time PCRReal time quantitative RTPCR was performed on an ABI step one plus Real-Time Detection System （Applied Biosystems）to investigate the expression of MaMMP3. Two MaMMP3 gene-specific primers， MaMMP3-F: 5′-TATGGACCCCCAACCTCT-3′ and MaMMP3-R: 5′-AAATAAGGACTCTTGCGCCAG-3′， were designed to amplify a product of 176 bp. The β-actin was used as an internal control to verify the qRT-PCR reaction and adjust the cDNA templates. Two β-actin gene-specific primers β-actin-F: 5′-GATGTGGATCAGCAAGCA-3′and β-actin-R: 5′-GGGCAAAGTTTACAAGTAA-3′were designed to amplify a fragment of 165 bp. The qRTPCR amplif i cations were carried out in triplicate in a total volume of 20 μl containing 10 μl of 2 × SYBR Green Real-time PCR Master Mix， 1 μl of cDNA， 0.4 μl of eachprimer and 8.2 μl of double-distilled water. The qRT-PCR program consisted of denaturation step at 95°C for 30 s，followed by 40 amplif i cation cycles of 15 s denaturation at 95°C， 15 s annealing at 60°C.

2.5 Genomic DNA isolation and genomic PCRDNA extraction was performed according to the method of Wilson （1990）， with slight modifications. Liver tissue homogenate was diluted in distilled water （f i nal volume 1.5 mL） and centrifuged at 15 000 rpm for 30 min at 4°C. DNA was extracted from the pellet by incubation for 2 h at 37°C in extraction buffer （1 mol/L Tris， 0.5 mol/ L EDTA， proteinase K [20 mg/mL]， and 10% sodium dodecyl sulfate）， purif i ed by a standard phenol/chloroform procedure （Prescott et al.， 1999）， and precipitated with 70% ethanol. According to the cDNA sequence of the MaMMP3， primers were designed to amplify the complete genomic sequences （Table 1）. The full length genomic fragments amplif i ed were cloned into pMD18-T vector and the recombinant plasmids were sequenced and determined. The exons and introns were determined by Splign program （Kapustin et al.， 2008）.

Table 1 Primers used in the experiment for complete genomic cloning.

2.6 Sequence alignments and statistical analysisThe MaMMP3 amino acid sequence was predicted using DNATool v 6.0 software. The position of the putative signal peptide cleavage site was predicted using the SignaIP 3.0 software （Bendtsen and Nielsen， 2004）. The percentage of similarity to and identity with the known MMP3 sequences was calculated by the MatGAT program （Campanella et al.， 2003） with default parameters. The protein domain was predicted with the simple modular architecture research tool （SMART）version 4.0 program （Schultz et al.， 2008； Letunic et al.， 2006） and ScanProsite （Gattiker et al.， 2002）. The hydropathicity index of the amino acids was scaled using ProtScale （http//us.expasy.org/cgi-bin/protscale. pl）. The secondary and 3D structure were predicted using SOPMA （Combet et al.， 2000） and CPHmodels-3.2（Nielsen et al.， 2010）， respectively. The protein sequence of the MaMMP3 was compared to its counterpart sequences currently available in GenBank using the BLAST program （Altschul et al.， 1997）. Position of the prodomain cleavage site and conserved cysteine residues are based on the N-terminal sequence information（Fu et al.， 2009）. Multiple alignment of MaMMP3 was carried out with the Clustal W program （Larkin et al.， 2007）. The phylogenetic tree was constructed with MEGA program version 4.0 （Tamura et al.，2007）. Statistical analysis was carried out with SPSS Statistics 13.0 Software. Data were analyzed by one-way analysis of variance （ANOVA） with default parameters to identify differences between groups. Differences were considered statistically significant when P values were lower than 0.05.

3. Results

3.1 Molecular characterization of MaMMP3BLAST analysis of all the sequences revealed that a sequence of 1806 bp was homologous to the MMP3 of Taeniopygia guttata. The corresponding colony was picked up and resequenced to obtain the complete sequence of the MaMMP3. The full-length of MaMMP3 cDNA consisted of a 5′-untranslated region （UTR） of 55 bp， a 3′-UTR of 243 bp with a polyadenylation signal （AATAAA） at 20 nucleotides upstream of the poly （A） tail， and an open reading frame （ORF） of 1506 bp encoding a polypeptide of 481 amino acids with an estimated molecular mass of 55.15 kDa and a theoretical isoelectric point of 5.86（Figure 1）. The MaMMP3 sequence was deposited in GenBank under accession No. HQ244395.

The preproprotein of MaMMP3 contained a typical signal peptide sequence （Met1-Ala20）， a prodomain（Phe21-Gln103）， a catalytic domain （Phe104-Gly267） and the C-terminal hemopexin-like domain （Cys293-Lys481）. Between Gln103and Phe104was predicted prodomain cleavage site， and the Cys293was the beginning of the C-terminal hemopexin-like domain， based on the N-terminal sequence information from the mature MMP3 sequences of human， rat， frog， chicken and bird（Letunic et al.， 2006）. The prodomain of MaMMP3 contains a highly conserved sequence， PRCGVPD （PRCGVPD， position 94-100）. The catalytic domain is featured by the signature sequence HEFGHSLGLHH （HEFGHSLGLHH， position222-232） and a conserved methionine （Met240）， forming a ‘Met-turn' eight residues downstream. In addition， four conserved catalytic sites were found in the sequence（His222， Glu223， His226， His232）.

Figure 1 Full cDNA sequence and predicted amino acid sequence of MaMMP3. The signal peptide is underlined. The MMP3 signature sequences P94RCGVPD100 of the precursor were shown in shadow. The vertical arrow indicates putative cleavage site for prodomain. The catalytic domain signature sequence HEFGHSLGLHH is bold， boxed. The conserved catalytic residues （His222， Glu223， His226， His232）are indicated as pound signs （#） below. The conserved methionine residue of the nearby “Met-turn” （Met240） is boxed and grey shaded. The arrow marked “C” shows the beginning of the C-terminal hemopexin-like domain. The classical polyadenylation signal is double lines. The asterisk marks the stop codon at the end of the open reading frame.

Multiple alignment of the MaMMP3 with other known MMP3 amino acid sequences revealed the strong amino acid conservation in MMP3 domains （Figure 2）. Homology analysis revealed that the MaMMP3 shared 25%-63% similarity to other known MMP3 amino acid sequences （Table 2）.

The hydropathic properties of MaMMP3 amino acid are indicated in Figure 3， in which the hydrophobic aminoacids are below zero， while the hydrophilic amino acids are above zero. The minimum and maximum scores are -3.278 and 2.822 at position 30 and 15. The secondary structure distribution along the sequence and tertiary structure of MaMMP3 are represented in Figure 4 A and B.

Table 2 Homology analysis of MaMMP3 amino acid sequence with other known MMP3 amino acid sequences determined by MatGat software.

Figure 2 Multiple alignment of the predicted amino acid sequence of MaMMP3 with other eukaryote MMP3 amino acid sequences. Numbering begins at the f i rst residue of the proposed mature region of MaMMP3. The predicted cleavage site between the propeptide and the catalytic domain is indicated by an arrow. The conserved sequence in the propeptide involved in the “cysteine-switch” is boxed， and the zinc-binding motif within the bracketed catalytic domain is indicated by a solid line on top. The three conserved histidine residues in the zinc binding motif and the conserved methionine residue of the nearby “Met-turn” are indicated by signs （@） above. The arrow marked “C”shows the beginning of the C-terminal hemopexin-like domain. The MaMMP3 amino acid sequence aligned with other eukaryote MMP3 amino acid sequences are from hummmp3 （Homo sapiens， NP_002413.1）， musmmp3 （Mus musculus， NP_034939.1）， xenmmp3 （Xenopus laevis， NP_001087063.1）， galmmp3 （Gallus gallus， XP_417175.1） and taemmp3（Taeniopygia guttata， XP_002198018.1）.

Table 3 Exon/intron organization of MaMMP3.

Figure 3 The hydropathic properties of MaMMP3 amino acid.

3.2 Genomic organization of MaMMP3Comparison of the cDNA sequence with genomic sequence data revealed that the Asian yellow pond turtle MMP3 covers 6006 bp and has eight exons. The exon/intron junctions correspond to the GT/AG rule （Table 3）. The deduced exon/intron organization is illustrated in Figure 5.

Figure 4 A. The secondary structure distribution of MaMMP3； B. 3-dimensional model of the catalytic domain of MaMMP3 represented as ribbons.

Figure 5 Schematic representation of the structural features of MaMMP3 cDNA and deduced exon/intron organization of MaMMP3. Protein coding region is indicated by f i lled.

3.3 Phylogenetic analysis of MaMMP3The phylogenetic tree was reconstructed by the neighborjoining method based on the multiple alignment built with Clustal W. As shown in Figure 6， a clear clade division was observed between vertebrates and invertebrates MMP3. The MaMMP3 clustered with the vertebrate MMP3 and was closely related to Gallus gallus MMP3 and Taeniopygia guttata MMP3.

3.4 MaMMP3 expression in different tissuesReal-time quantitative RT-PCR analysis was employed to detect the expression pattern of the MaMMP3 mRNA with the β-actin as an internal control. The results showed that the MaMMP3 mRNA was constitutively expressed in all detected tissues including kidney， heart， spleen and liver. As showed in Figure 7 A， in healthy individuals， the expression level of the MaMMP3 in spleen was higher than in other tissues， and the expression in heart and kidney was at a lower level.

3.5 Inductive expression of MaMMP3The temporal expression of the MaMMP3 mRNA in spleen and liver after S. marcescens challenge was measured by real-time quantitative RT-PCR and the results were shown in Figure 7 B. In the spleen， after 12 h of S. marcescens stimulation，the MaMMP3 mRNA expression was significantly upregulated and reached the highest expression （P ＜ 0.01）. At 24 and 36 h post-injection， the MaMMP3 expressionlevel was 13 and 24-fold （P ＜ 0.01） higher than that observed in the control turtle， respectively. The same expression pattern of MaMMP3 was observed in the liver after stimulation.

Figure 6 Phylogenetic analysis of the MaMMP3 amino acid sequence with other known MMP3 sequences. The branches were validated by bootstrap analysis from 1000 replications， which were represented by percentage in branch nodes. The MMP3 amino acid sequences used in this analysis are listed in Table 2.

4. Discussion

So far， there have been no reptile MMP3 cloned and characterized， and the function of turtle MMP3 in the immune response remains unknown. To further understand the characteristics and function of turtle MMP3 in the immune responses， a MMP3 was cloned and characterized from Asian yellow pond turtle in the present study.

Sequence analysis suggested that the MaMMP3 contained typical signal peptide sequence （Met1-Ala20），a prodomain （Phe21-Gln103）， a catalytic domain （Phe104-Gly267） and the C-terminal hemopexin-like domain（Cys293-Lys481）. The perfect PRCGXPD （PRCGVPD）motif is discovered in the prodomain activity （Pharham and Shi， 2001）， the assumped “cysteine switch”， within which the cysteine residue （Cys96） interacts with the catalytic Zn2+to maintain enzyme latency （van Wart and Birkedal， 1990）. The catalytic domain of a typical MMP contains a zinc ion （Zn2+） in the active site that is ligated to three conserved histidine residues in the sequence HEXXHXXGXXH. The glutamic acid residue（E） in this catalytic motif provides the nucleophile that severs peptide bonds. The backbone structures of the MMP3 catalytic domain， including a characteristic MET TURN that is caused by a conserved methionine residue downstream of the zinc-binding site. MaMMP3 has a carboxy （C）-terminal HEMOPEXIN-LIKE DOMAIN，which functions in substrate recognition. MMP3 is secreted as latent enzymes and processed to the active forms upon the removal of the propeptide domain throughvarious mechanisms like many other MMPs.

Figure 7 A. Expression level of the MaMMP3 mRNA in different tissues. Real-time quantitative RT-PCR was done with RNA samples from kidney， heart， spleen and liver from Asian yellow pond turtle. The Asian yellow pond turtle β-actin gene was used as an internal control to calibrate the cDNA template for all the samples. All the data were analyzed from 3 individuals； B. Expression level of the MaMMP3 mRNA in heart， kidney， spleen and liver after S. marcescens stimulation. The MaMMP3 mRNA expression in S. marcescens treated samples was normalized to that in the relevant control. In this experiment， the Asian yellow pond turtle β-actin gene was used as an internal control to calibrate the cDNA template for all the samples. All the data were analyzed from 3 individuals. Signif i cant differences were indicated with the asterisk （*represented P ＜ 0.05，**represented P ＜ 0.01）.

To further understand the possible biological function of the MaMMP3， its mRNA expression was examined at different time points after S. marcescens challenge in spleen and liver， the spleen was thought to be an important immune organ in the reptiles and the liver is the very issue from which we constructed the cDNA library and from which we detected the MMP3. So we selected the spleen and liver to research the temporal expression pattern of MaMMP3 after bacteria simulation. The expression level of the MaMMP3 was significantly up-regulated at all time points studied both in spleen and liver.

These results seem to be similar to data reported by others. Pagensteche et al. showed enhancement of MMP3 expression in rat in response to LPS both in the spleen and liver （Pagenstecher et al.， 2000）. Handley and Miller demonstrated that during Salmonella enterica and Yersinia enterocolitica infection， the mRNA of MMP3 in Peyer's patches and mesenteric lymph nodes are both updated （Handley and Miller， 2007）. In addition， MMP3 is also upregulated during LPS-induced neuroinf l ammation （Mun-Bryce et al.， 2002）. These results suggest the MMP3 could participate in the immune responses in Asian yellow pond turtle. Understanding these interactions might help to shed light on the immune mechanisms of Asian yellow pond turtle.

Acknowledgements This research was supported by the Ministry of Science and Technology funds（No. 2011GB23260021）； Major State Basic Research Development Program of China （No. 2004CB117401）；Special Funds of Science and Technology New Star from Guangzhou City （No. 2012089） and Fundamental Research Funds from CAFS （No. 2012A0403）.

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10.3724/SP.J.1245.2014.00038

*Corresponding author: Dr. Xinping Zhu， from Pearl River Fishery Research Institute， Chinese Academic of Fishery Science， Guangzhou，China， with his research focusing on genetics and breeding of aquatic animals.

E-mail: zhuxinping_1964@163.com

25 September 2013 Accepted: 21 February 2014

Asian Herpetological Research 2014， 5（1）: 38-48