Mol Cell Biochem (2014) 387:143–150 DOI 10.1007/s11010-013-1879-0
Molecular cloning, characterization, and expression of hsp60 in caudal fin regeneration of Misgurnus anguillicaudatus Li Li • Ping Nan • Shengna Zhai • Lele Wang Songbo Si • Zhongjie Chang
•
Received: 21 August 2013 / Accepted: 18 October 2013 / Published online: 21 November 2013 Ó Springer Science+Business Media New York 2013
Abstract Urodele amphibians and teleost fish are capable of nearly perfect regeneration of lost appendages. The fin constitutes an important model for studying the molecular basis of tissue regeneration. It has been known that heat shock protein 60 (Hsp60) is a multifunctional protein of the heat shock protein family. The purpose of this study is to investigate the role of hsp60 as a part of a stress response system after fin injury or in fin regeneration. We firstly cloned full-length cDNA of hsp60 from Misgurnus anguillicaudatus (designated as MaHsp60) by RACE method. The cDNA contains a 83-bp 50 UTR, a 1,728-bp open reading frame encoding 492 amino acids and a 542-bp 30 UTR (Accession No.: KF537340). The phylogenetic tree shows that the MaHsp60 fits within the hsp60 clade. Then quantitative RT-PCR detected that MaHsp60 began to increase rapidly its expression at 1 dpa and reached its peak at 2 dpa. Next, spatial distribution analysis of MaHsp60 in fins showed that MaHsp60 located mainly in the deeper layer of regenerated epidermis when MaHsp60 expressed most. After the MaHsp60 had been cloned into the pET-32a vector, SDS-PAGE analysis confirmed that the MaHsp60 protein was efficiently expressed in Escherichia coli BL21 and adjustable with the temperature. These findings have revealed that MaHsp60, a highly conserved gene during vertebrate evolution as well as related to stress response, is involved in the formation of wound epidermis which occurs as the first phase of fin regeneration after fin amputation in caudal fin regeneration.
L. Li P. Nan S. Zhai L. Wang S. Si Z. Chang (&) Molecular and Genetic Laboratory, College of Life Science, Henan Normal University, 46# East of Construction Road, Xinxiang 453007, Henan, People’s Republic of China e-mail: [email protected]; [email protected]
Keywords Hsp60 gene Fin regeneration Phylogenetic analysis Gene expression Immunohistochemistry Misgurnus anguillicaudatus
Introduction All animals have the biological response to injury, but their ability to regenerate are quite different. Mammals are unable to regenerate amputated appendages, such as limbs, yet urodele amphibians, and teleosts exhibit outstanding capability of regenerating. After their appendages are severed, they can complete the structural and functional reconstruction with accurate position relationship through regeneration [1, 2]. Some teleosts are able to regenerate the fin, optic, retina, liver, and even heart [3]. The caudal fin model of tissue regeneration is particularly attractive because the fin is a relatively simple structure, quick to regenerate, and nonessential to the viability of fishes [4]. Mudloach (Misgurnus anguillicaudatus) is a freshwater teleost widely distributed along the eastern coasts of the Asian continent. Furthermore, many recent details regarding the genetic pathways regulating regeneration have been uncovered using mudloach. Although these fins present various and numerous shape, length, and color phenotypes, they are built with the same skeletal elements. And, their regeneration process are basically similar [5]. The teleost fins consist of a protruding visible exoskeleton made of bony rays of dermal origin, which are connected by means of ligaments to the endoskeleton [6]. Fin regeneration process follows a pattern of epithelial migration and accumulation to cover the surface of the cut, formation of a regeneration blastema along the entire width of the cut and ultimately proliferation and cell differentiation leading to replacement of the lost or damaged structure. In addition, these multiple fin structures may
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regenerate very rapidly [5, 7, 8]. It is conceivable and has been proved that many genes are involved [9, 10]. Heat shock proteins are a set of highly conserved proteins and are expressed in response to external stressors, such as thermal stress, tissue trauma, heavy metal toxicity, or inflammation. Also they are involved in a wide range of biochemical processes including folding of nascent polypeptides, intracellular trafficking, modulating signal pathway activity, and regulating immune responses [11]. The multifunctional nature of heat shock proteins and their critical roles in the regulation of tissue healing and cell survival make them good targets in fin regeneration process [12]. In this study, in order to explore the role of heat shock protein 60 gene (hsp60) in fin regeneration, we firstly isolated the full-length cDNA of hsp60 from M. anguillicaudatus and characterized its expression using fluorescent real time RT-PCR and immunohistochemistry. And through construction of expression vector, expression of Hsp60 protein were conducted. These results will help us understand the characterization, expression, and related regeneration functions of the hsp60.
Materials and methods Fish care and fin amputation Adult M. anguillicaudatus were collected from wetlands in the old course of the Yellow River, Yanjin County (Henan, China). The fish were kept in tanks with recirculating fish water which was kept at 26 °C during the whole experiment. Prior to fin amputations the fish were anesthetized in 0.1 % tricaine and then placed on a wet towel. Using a razor blade, approximately 30 % of the caudal fin was amputated.
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using DNAMAN. Degenerate primers P1 and P2 (Table 1) were designed from the conserved regions, after getting the conserved regions sequence of the MaHsp60 by PCR, then based on it, 50 gene-specific primers 50 GSP1 and 50 GSP2 and 30 gene-specific primers 30 GSP1 and 30 GSP2 (Table 1) were designed for the RACE. Reverse transcription and 50 and 30 - RACE PCR reaction conditions were performed according to the manufacturer’s instructions of the RACE Core Set (TaKaRa, Japan). PCR products were gel purified, cloned into pGEM-T vector (Promega, USA). After being transformed into the competent cells of Escherichia coli DH5a, the positive recombinants were identified through anti-Amp selection and sequenced by dideoxy-chain-termination using Sp6 and T7 promoter sequences as primers. Homologous alignment and bioinformatics analysis The full-length cDNA sequence of MaHsp60 was assembled by finding the overlapping region of each fragment. The nucleotide sequence and deduced amino acid sequence were compared with other sequences reported in NCBI’s GenBank using the BLAST program (http://blast.ncbi.nlm.nih. gov/Blast.cgi). The possible ORF was analyzed with ORF Finder tool (http://www.ncbi.nlm.nih.gov/gorf/gorf.html). All Hsp60 protein sequences from ten representative vertebrates were aligned using DNAMAN. A phylogenetic tree of different vertebrate Hsp60 proteins was constructed using neighbor-joining method with the software of Molecular Evolution Genetics Analyses (MEGA) version 4.0 and ClustalW (http://www.ebi.ac.uk/Tools/clustalw2/index). Real-time quantitative RT-PCR analysis In order to further quantify the relative expression of MaHsp60 in different stages of fin regeneration, a real-time
Total RNA isolation and synthesis cDNA For total RNA extraction, approximately 20 mg of regenerated tail tissues of the respective stages, 1 day post amputation (dpa), 2 dpa, 3 dpa, 4 dpa, 5 dpa, and 6 dpa were collected on ice. They were homogenized by using liquid nitrogen grinding, and total RNA was extracted using Trizol in accordance with the manufacturer’s instructions. First-strand cDNA synthesis was carried out with prime script reverse transcriptase (Takara, Japan), using 1 lg of total RNA isolated as described above. The first strand cDNA was obtained and preserved in -20 °C. Rapid amplification of cDNA ends analysis and cloning the full-length cDNA of MaHsp60 Nucleotide sequences of hsp60 genes were retrieved from GenBank (www.ncbi.nlm.nih.gov) and multiple aligned
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Table 1 Primer sequences used in gene cloning and RT-PCR in this study Primer
Oligonucleotide sequences (50 ?30 )
PI
GACTA(C/T)GAGAAGGAGAA(A/G)CT
P2
GTCCTCAC(C/G)ACCTT(T/G)GTG
0
GGGCGATTTCCTCTGGTGTTGTG
5 GSPl 50 GSP2
CTGGTGTTGTGACTGGCTTTGAGG
30 GSPl
GGCTAGCAAAACTTTCTGATGG
30 GSP2
TGACCATTGCTAAGAATGCAGG
FQ-F
CCTCAAAGCCAGTCACAAC
FQ-R
CACGGTCAAACTTCATTCC
GAPDH-F
GCCTCTTGCACGACCAACTG
GAPDH-R
CGGAAGGCCATGCCTGTCAG
P3
CCGGAATTCATGCTGCGTTTACCGAGTG
P4
CCCAAGCTTTTAGAAGCCTCCCATGCCTC
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RT-PCR assay was performed on an ABI 7500 real-time PCR detection system (Applied Bio-systems, USA) with SYBR green fluorescent label. All samples were analyzed in three replicates and all the reactions were independently repeated twice to ensure reproducibility. GAPDH was used to normalize data for differences between samples. Genespecific primers of hsp60 gene (FQ-F and FQ-R) and GAPDH (GAPDH-F and GAPDH-R) are showed in Table 1. Amplifications were performed in 50 ll containing 12.5 ll of 29 SYBR Premix Ex Taq (Takara, Japan), 3.5 pM of each primer, 2 ll of diluted cDNA, and 0.5 ll 509 ROX Reference Dye II (Takara, Japan). Data were analyzed with 7500 system SDS Software v 1.4(Applied Biosystems, USA). The 2-44Ct method was used to analyze the expression levels of MaHsp60 [13]. Statistical calculations were performed using SPSS version 13.0 software. Immunohistochemistry The rat anti-Hsp60 at 1:300 (Boster) were used as primary antibody, and the StreptAvidin-BiotinComplex kit (SA1029, Boster) were used as secondary antibody. Fins were fixed in 4 % paraformaldehyde in PBS, sectioned and immunostained as previously described [14]. Construction of MaHsp60 prokaryotic expression vector DNA fragment of 1,900 bp containing the ORF of MaHsp60 was amplified from caudal-fin cDNA using specific primers P3 and P4 (Table 1). Amplification conditions were as follows: initiation at 95 °C for 5 min; 35 cycles of 94 °C for 30 s, 60 °C for 30 s, and 72 °C for 60 s; and a final extension at 72 °C for 10 min. The PCR product was gel purified, cloned into pGEM-T vector, and sequenced to ensure that no mutations were introduced during PCR amplification. Then the plasmid was digested with XhoII and EcoRI and cloned into pET-32a/His C expression vector (Invitrogen) to obtain the MaHsp60 expression vector, pET-32a/His-Hsp60. The recombinant plasmid was sequenced to confirm that the inserted sequence was in the proper reading frame. Expression and change under different temperature of the M. anguillicaudatus Hsp60 protein (designated as MaHsp60) The pET32a/His-Hsp60 recombinant plasmid was used to transform competent E. coli BL21. A newly transformed single colony was selected and cultured in 5 ml of Luria broth (Kana) culture medium at temperature gradient of 26, 31, and 36 °C, and when the A value (optical density)
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reached 0.6, IPTG was added to a final concentration of 0.1 mmol/l. After incubation for 6 h, 1 ml of the culture media was removed, the bacteria were collected by centrifugation. After re-suspending precipitation by adding 1 % SDS, the bacteria were lysed by sonication. Then the soluble proteins in the supernatant were collected by recentrifugation. The expression of the recombinant Hsp60 protein was analyzed by SDS-PAGE on 10 % gels.
Results Molecular cloning and structural analysis of MaHsp60 In an attempt to isolate the MaHsp60 gene from caudal fins, we have first cloned conserved fragment by using a degenerate RT-PCR strategy. Based on the DNA sequence information, we designed primers for cloning full-length MaHsp60 by RACE-PCR. By overlapping the 30 - and 50 RACE fragments, a 2,357 bp transcript was obtained. It includes a 50 -terminal untranslated region of 83 bp, a 30 terminal untranslated region of 542bp with a short polyA tail (GenBank accession no. KF537340), and an ORF of 1,728 bp encoding a polypeptide of 576 amino acids with a predicted molecular mass of 61.05 kDa and theoretical isoelectric point of 4.75. The nucleotide and predicted amino acid sequence of the ORF of MaHsp60 are shown in Fig. 1. Homology analysis and evolutionary relationships of MaHsp60 The BLAST analysis revealed that MaHsp60 has high homology to the Hsp60 of other species. The homology of deduced amino acid sequences of Hsp60 from ten species are shown in Table 2 and Fig. 1. The deduced amino acid sequence of MaHsp60 was 95.0, 93.6, 90.3, 87.0, 85.0, 86.5, 86.1, and 71.7 % identical to that of Danio rerio Hsp60, Carassius auratu Hsp60, Paralichthys olivaceus Hsp60, Gallus gallus Hsp60, Xenopus laevis Hsp60, Sus scrofa Hsp60, Homo sapiens Hsp60, and Drosophila melanogaster Hsp60, respectively. To evaluate the evolutionary relationships of MaHsp60 with other species, we constructed a phylogenetic tree using software MEGA 4.0 on the basis of the Hsp60 amino acids sequences (Fig. 1). Amino acids sequences from 20 species were used for the rooted phylogenetic tree constructed by the neighbor-joining method. And the phylogenetic tree shows that The Hsp60 proteins from different vertebrates were divided into two subgroups. The birds, mammals, and amphibians were grouped into one cluster, and the teleosts were separated and formed another independent group. The results support a monophyletic origin of fish again. As far as the evolution distance was
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Mol Cell Biochem (2014) 387:143–150
(A) GAAACTCTCGTGCTTATATCATT CAGTCTGCTGTACAGACGGACACACCCATCCCTTCATTCACCTCCAGAACACTTTACAAA 1 1 61 21 121 41 181 61 241 81 301 101 361 121 421 141 481 161 541 181 601 201 661 221 721 241 781 261 841 281 901 301 961 321 1021 341 1081 361 1141 381
1201 401 1261 421 1321 441 1381 461 1441
123
ATGCTGCGTTTACCGAGTGTGATGAAACAGATGAGGCCAGTGTGCAGGGCGCTGGCCCCA M
L
R
L
P
S
V
M
K
Q
M
R
P
V
C
R
A
L
A
P
CATTTGACCCGCGCGTACGCAAAGGATGTCAAGTTTGGAGCCGATGCCCGGGCCCTCATG H
L
T
R
A
Y
A
K
D
V
K
F
G
A
D
A
R
A
L
M
CTCCAGGGCGTTGACCTGTTGGCTGATGCTGTGGCAGTCACAATAGGACCAAAGGGTCGC L
Q
G
V
D
L
L
A
D
A
V
A
V
T
I
G
P
K
G
R
ACTGTCATCATTGAGCAGAGCTGGGGAAGCCCCAAGGTCACCAAAGATGGCGTCACGGTG T
V
I
I
E
Q
S
W
G
S
P
K
V
T
K
D
G
V
T
V
GCCAAAAGTATTGATCTAAAGGATAGGTATAAGAACATCGGGGCCAGGCTTGTACAAGAC A
K
S
I
D
L
K
D
R
Y
K
N
I
G
A
R
L
V
Q
D
GTAGCCAACAACACTAATGAGGAGGCTGGAGACGGCACCACAACCGCCACAGTGCTGGCT V
A
N
N
T
N
E
E
A
G
D
G
T
T
T
A
T
V
A
L
CGGGCTATTGCCAAGGAGGGATTTGACACCATCAGCAAAGGCGCCAACCCTGTGGAGATC R
A
I
A
K
E
G
F
D
T
I
S
K
G
A
N
P
V
E
I
CGTAGAGGAGTTATGACTGCAGTGGAGACTGTCATCAATGAACTTAAAAAAACCTCAAAG R
R
G
V
M
T
A
V
E
T
V
I
N
E
L
K
K
T
S
K
CCAGTCACAACACCAGAGGAAATCGCACAGGTGGCCACAATTTCTGCCAATGGAGACACT P
V
T
T
P
E
E
I
A
Q
V
A
T
I
S
A
N
G
D
T
GAAGTTGGTGAGATCATCTCCAATGCCATGAAGAAAGTTGGACGTAAAGGTGTTATTACT E
V
G
E
I
I
S
N
A
M
K
K
V
G
R
K
G
V
I
T
GTAAAGGATGGTAAAACTCTGCACGATGAGCTTGAGATCATTGAGGGAATGAAGTTTGAC V
K
D
G
K
T
L
H
D
E
L
E
I
I
E
G
M
K
F
D
CGTGGACACATTTCTCCTTACTTCATCAACACTGCTAAAGGTCAAAAATGTGAGTTCCAG R
G
H
I
S
P
Y
F
I
N
T
A
K
G
Q
K
C
E
F
Q
GATGCTTATCTGCTTCTGAGTGAGAAGAAGATCTCCAGTGTACAGAGCATCGTGCCAGCA D
A
Y
L
L
L
S
E
K
K
I
S
S
V
Q
S
I
V
P
A
CTGGAACTTGCCAACCAGCACCGCAAGCCTCTGATCATTGTGGCTGAAGATGTGGATGGA L
E
L
A
N
Q
H
R
K
P
L
I
I
V
A
E
D
V
D
G
GAAGCACTCAGTACTCTGGTCCTCAACAGGTTAAAGGTTGGACTGCAAGTTGTTGCAGTC E
A
L
S
T
L
V
L
N
R
L
K
V
G
L
Q
V
V
A
V
AAGGCTCCAGGATTTGGGGACAACCGAAAGAACCAGCTGCAGGATATGGCAGTGGCCACT K
A
P
G
F
G
D
N
R
K
N
Q
L
Q
D
M
A
V
A
T
GGAGGCACAGTGTTTGGTGATGATGCTGTGGGTCTGGCCCTTGAGGATATCCAGGCACAT G
G
T
V
F
G
D
D
A
V
G
L
A
L
E
D
I
Q
A
H
GACTTTGGCAAGGTTGGCGAGGTCATCGTGACCAAGGATGACACGATGCTCCTCAAAGGC D
F
G
K
V
G
E
V
I
V
T
K
D
D
T
M
L
L
K
G
CGTGGTGATCCAGCAGCCGTTGAGAAACGTGCGAACGAGATCGCCGAACAGCTGGAGAGC R
G
D
P
A
A
V
E
K
R
A
N
E
I
A
E
Q
L
E
S
ACAAACAGCGACTATGAGAAGGAGAAACTCAACGAGCGGCTGGCAAAACTCTCTGATGGA T
N
S
D
Y
E
K
E
K
L
N
E
R
L
A
K
L
S
D
G
GTCGCTGTAATTAAGGTTGGAGGAACAAGTGACGTTGAAGTAAACGAGAAGAAAGACCGT V
A
V
I
K
V
G
G
T
S
D
V
E
V
N
E
K
K
D
R
GTCACCGATGCACTGAACGCCACGCGAGCTGCTGTGGAGGAGGGAATTGTGCCTGGAGGA V
T
D
A
L
N
A
T
R
A
A
V
E
E
G
I
V
P
G
G
GGCTGTGCCCTGTTGCGCTGCATCCCAGCCTTGGATACCATCAAGCCTCTAAATGAGGAT G
C
A
L
L
R
C
I
P
A
L
D
T
I
K
P
L
N
E
D
CAGAAAATAGGTATTGACATTATTCGCAGATCATTGCGTATTCCTGCCATGACCATTGCT Q
K
I
G
I
D
I
I
R
R
S
L
R
I
P
A
M
T
I
A
AAGAATGCAGGAGTTGAAGGATCTCTGGTGGTGGAGAAGATTTTACAGAGTGCTCCAGAA
Mol Cell Biochem (2014) 387:143–150
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b Fig. 1 Homologous alignment and bioinformatics analysis of
Expression changes of MaHsp60 during caudal fins regeneration
Mahsp60 and deduced amino acid sequence. a Full-length cDNA and deduced amino acid sequence of Mahsp60. The deduced amino acid is shown below the nucleotide sequence. Nucleotide and amino acid sequences are numbered on the left. Amino acid sequence is represented with one-letter codes below the cDNA sequence. ATG shows the initiation codon. An asterisk indicates the stop codon. ATP/ ADP and Mg2? binding segments appear as double lined. A signature motif appears as dashed line. Conserved GGM repeats at the C-terminal appear as single- lined. This sequence has been submitted to GenBank (Accession no.: KF537340). b Multiple alignment of amino acid sequences of Hsp60. Protein sequences were obtained from GenBank: Ma Misgurnus anguillicaudatus (this study); Dr Danio rerio (NP851847); Ca Carassius auratus (ABI26641); Po Paralichthys olivaceus(ABB76384); Hs Homo sapiens (NP955472); Ss Sus scrofa (XP001928634); Mm Mus musculus (NP071565); Gg Gallus gallus (NM001012916); Xl Xenopus laevis (NP001083970); Dm Drosophila melanogaster (NP727489); and Lv Litopenaeus vannanmei (ACN30235). c Phylogenetic tree of hsp60 proteins from various species. The tree was obtained by bootstrap analysis with the neighbor-joining method and was bootstrapped 1,000 times. Filled diamond indicates Hsp60 from Misgurnus anguillicaudatus. Accession numbers for these sequences are: Tanichthys albonubes, ADK27679; Cten opharyngodon idella, ADU34083; Kryptolebias marmoratus, AEM65177; Epinephelus akaara, HQ141338; Salmo salar, ACI33148; Bos Taurus, NP001160080; Oryctolagus cuniculus, XP002712414; Pan troglodytes, XP001169156; Strogylocentrots purpuratus, XP795205; and Branchiostoma floridae, XP002595084. The other organisms with GenBank reported sequences see Fig. 1b
The fluorescent real-time RT-PCR were conducted to determine the expression of MaHsp60 in different periods of caudal fins regeneration. It revealed that expression of MaHsp60 rapidly increased in response to fin amputation (Fig. 2). By 2 dpa, the expression of gene reached to the levels that were approximately 18-fold higher than the expression measured in the uninjured caudal fin (0 dpa). Thereafter, the expression began to decrease. By 3 dpa, the expression of MaHsp60 decreased to the levels that were approximately half of the peak expression levels. Then the expression of MaHsp60 remained low expression levels during late regeneration. Spatial distribution of MaHsp60 in regenerated caudal fins We next asked how this Hsp60 distributes in the regenerated fin, then immunostaining was performed in tissue sections from fins when the expression was at peak. Longitudinal sections show that wound healing has been finished at this time, that is, at 2 dpa as Akimenko reported [5]. It can be seen that positive cells are mainly involved in the composition of deeper epidermis, while there are nearly no distribution in flat epithelial cells of superficial layers of the epidermis and blastema inside the caudal fin (Fig. 3).
concerned, the MaHsp60 has a closer genetic relationship with that of D. rerio, which agrees with the comparability result of deduced amino acid sequence.
Fig. 1 continued
481 1501 501 1561 521 1621 541
K
N
A
G
V
E
G
S
L
V
V
E
K
I
L
Q
S
A
P
E
ATTGGATATGATGCTATGAATGGAGAATATGTCAACATGGTTGAAAAAGGAATTATTGAC I
G
Y
D
A
M
N
G
E
Y
V
N
M
V
E
K
G
I
I
D
CCCACAAAGGTTGTGAGGACAGCATTACTTGATGCTGCAGGTGTTGCGTCTCTGCTGTCC P
T
K
V
V
R
T
A
L
L
D
A
A
G
V
A
S
L
L
S
ACTGCTGAAGCCGTCGTCACCGAGATCCCCAAGGAGGAGAAGGACATGCCAGGTGGAGGG T
A
E
A
V
V
T
E
I
P
K
E
E
K
D
M
P
G
G
G
1681
ATGGGAGGCATGGGAGGTATGGGTGGTATGGGAGGCATGGGAGGATTCTAA
561
M G G M G G M G G M G G M G G F * ACCACACTTCACTGACTTTAGAGAGAAGGGTTGTGGGCAGGAGACATGATTCGCCCCTTT CTTTCAACTCGGATAAACCTGCCGATACAGAGTGCTGGTCTTGACACGGAGCAAAGATAT GGACCTTACTATTCCCCCATCTTCCACCACTGTCCATCCCATCTGATCTCATCTCTCTGC TCATGACCGAAGACATGACTCTATTGTTTCAAAGACAGGCTCCAGAATGTCTATTTGCTT GGATATTTAGCATTGTGCTTATTGTTTCCATCCAACCATTCAAATGGGAATGTGCATATT GAAAACTGCTTCATGCTGTTGTATTGTTTGAAAGCATTTATCATTTTGCATAAAGCTTGT TCTGAAAGACCATCTGAAGGGGCTTACAAAGCCAGACATTCAGGTTGAAATCTGTAAGAA AGCAGATACTTTGACTAGTTGTATCTAAAAAATTCCTTCTCATTGATCAGTTCTTTTTTT GTACACTGTTCATCATTTCATATAAAAATAAAACTCATTTTGGGATTACAAAAAAAAAAA AA
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Mol Cell Biochem (2014) 387:143–150
Fig. 1 continued
Table 2 Comparability of deduced amino acid sequences of Hsp60 from various species Gene
Mahsp60 (%)
Mahsp60
100
Drhsp60 (%)
Cahsp60 (%)
Pohsp60 (%)
Hshsp60 (%)
Sshsp60 (%)
Mmhsp60 (%)
Gghsp60 (%)
100
Xlhsp60 (%)
Drhsp60
95.0
Cahsp60
93.6
94.8
Pohsp60
90.3
90.2
88.5
Hshsp60
86.1
87.3
86.5
85.6
Sshsp60
86.5
87.5
87.2
86.3
97.6
Mmhsp60 Gghsp60
87.0 87.0
88.2 88.6
87.7 87.2
86.8 87.0
97.6 94.1
97.2 94.4
100 94.2
Xlhsp60
85.0
85.7
85.2
83.8
90.4
89.9
90.1
90.4
Dmhsp60
71.7
70.7
71.9
72.0
72.8
73.1
73.3
72.6
72.4
Lvhsp60
68.7
69.2
69.5
68.6
72.2
72.1
71.7
71.4
70.8
Lvhsp60 (%)
100 100 100 100
Expression and temperature-associated change of MaHsp60 According to the SDS-PAGE analysis, after IPTG induction, 78.9 kDa band (Fig. 4a, lane 2, 3) corresponding to the expectant size of the fusion protein was observed. And the constructed plasmid pET32a/His-Hsp60 exhibited a higher trend of the His-Hsp60 fusion protein expression levels in BL21 over time during 6 h (Fig. 4a, lane 2) to 8 h (Fig. 4a, lane 3) when the concentration of IPTG was 0.1 mmol/l. There was basal low expression levels of the protein without IPTG induction (Fig. 4a, lane 1). Furthermore, this protein had the maximum expression amount at 36 °C (Fig. 4b, lane 3) among temperature gradients of 26, 31, and 36 °C after incubation for 6 h, while at the latter two temperature expression seemed to have no significant change (Fig. 4b, lane1, 2).
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Dmhsp60 (%)
100
100 100 77.4
100
Discussion Fin regeneration has emerged as an ideal model for our further understanding of vertebrate regeneration due to the simpler anatomical structure compared to the urodele limb [15]. As we think, numerous factors or genes have been implicated in the processes of limb/fin bud initiation and outgrowth in vertebrates [16, 17]. Nevertheless, fin regeneration is firstly a process of wound repair which is subject to mechanical stress caused by a severe injury. Therefore, it is interesting to speculate the role of some heat shock proteins in fin regeneration. We successfully cloned the MaHsp60 cDNA sequence via RACE method. The gene encodes a 576 amino acid neutral protein with a molecular weight of 61.05 kD and has a high similarity with other known hsp60s sequences. The MaHsp60 is conserved in evolution and its encoded
Mol Cell Biochem (2014) 387:143–150
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20
(A)
18
1
(B) 2
3
M
KD
M
1
2
3
97.2
16 14
66.4
12 10
44.3
8
n.s.
6 4
29.0
2 0
0 dpa
1 dpa
2 dpa
3 dpa
4 dpa
5 dpa
6 dpa
Fig. 2 Expression analysis of MaHsp60 in different periods of caudal fins regeneration by real-time fluorescent quantitative RT-PCR. GAPDH was the internal control. Vertical bars the mean ± SD (N = 3). Asterisk indicates a significant difference with the control (P \ 0.05) and n.s. no significant, respectively
we
sc bl be
chr
fe
Fig. 3 Spatial distribution of MaHsp60 in regenerated caudal fins. A distal part of a regenerated caudal fin containing Hsp60-positive cells was shown at 2 dpa. bl blastema; be basal epidermis; chr chromatophores; fe flat epithelial cells; sc secretory cells; and we wound epidermis. Scale bars 50 lm
protein contains conserved structural domains, including two highly conserved motifs corresponding to the ATP/ ADP and Mg2? binding segments 76-DGVTVAK-82 and 109-AGDGTTTATVL-119, a classical HSP60 signature motif 427-ATRAAVEEGIVPGGG-441, a conserved GGM repeat at the C-terminal end [18, 19]. There is no report about expression of Hsp60 gene during fin regeneration. In this report, quantitative expression studies of the gene MaHsp60 were performed during fin regeneration. Basal levels were observed at o dpa, a mature non-regenerating fin. An obvious upregulation of the expression was observed at 1 dpa when the
Fig. 4 SDS-PAGE analysis of His-Hsp60 fusion protein. a Molecular weight marker is in the right lane. Lane 1 Cell lysates of bacteria transformed with pET32a/His-Hsp60 without IPTG induction. Lane 2 and lane 3 represent cell lysates of bacteria transformed with pET32a/ His-Hsp60 under IPTG (0.1 mM) induction after incubation for 6 and 8 h, respectively. b Molecular weight marker is in the left lane. Lane 2, lane 3, and lane 4 represent cell lysates of bacteria transformed with pET32a/His-Hsp60 under IPTG (0.1 mM) induction after incubation 6 h at 26, 31, and 36 °C, respectively
wound is firstly healed by the rapid migration and rearrangement of the epithelial cells of the stump [20]. Then the expression peaked on the following day, which is of no difficulty to understand because the formation of the wound epidermis is under way at this stage, an inevitable stress process after lesion. From 3 dpa, the gene expression decreased gradually until reaching the lowest level which was still above the basal, homeostatic levels were at 6 dpa when stress response and regeneration is finishing [21]. These results suggest that MaHsp60 are strongly upregulated during the wound epidermis formation stage and it begins to decrease when wound repair will complete. The fact might indicate that MaHsp60 has made contribution to the fin formation. In order to clarify how MaHsp60 participates in the morphogenesis of fin regeneration or how MaHsp60 distributes in regenerated fin immunohistochemical method was used. We confirmed the distribution of its protein when expression of MaHsp60 reached the maximum amount. The results of positive cells located mainly in the deep epidermis, but absence of mesenchymal cells inside the fin proves that Hsp60 do participate in the first phase of fin regeneration—wound healing. In this process, the injured stump is repaired by rapid migration of epidermal cells over the amputation surface. The tissues located in the vicinity of the resection site undergo disorganization and display enhanced cell proliferation. It is the proliferation of the cells forming this transitory structure that is responsible for the regrowth of the epidermis. Then following second phase is the formation of the blastema, cells located in the blastema differentiate and participate in the re-establishment of the missing structures [22, 23]. So, the distribution of Hsp60 in fin quite match with its feature as stress
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proteins because the first phase is essentially a kind of stress response to injury. To determine the activity of the MaHsp60, we constructed prokaryotic expression vector, detected the relationship between the MaHsp60 expression and temperature. The expression of protein is efficient and adjustable with the temperature. The 60 kD heat shock protein is an important molecular chaperone, a stress protein constitutively synthesized in all cells analyzed so far, and located on cell membrane [19, 24]. In the last few years, the study of hsp60 focus on expression in the case of tumor, bacterial infection, different temperature, salinity, and other environmental changes [25–27]. Vertebrate limb/fin bud regeneration is considered an excellent and interesting process for studying morphogenesis. The study shows MaHsp60 may play an important role in the recovery of wound epidermis after fin amputation during caudal fin in M. anguillicaudatus. The information gathered from the study provides resources for further investigations into the function of hsp60 in fin regeneration. Acknowledgments This work is supported by Grants from the Natural Science Foundation of Henan Province (No. 112300410275), Scientific Research Project Fund of the Education department of Henan Province (No. 2011B180030), and Youth Foundation of Henan Normal University (No. 2012QK17).
References 1. Zhang J, Wagh P, Guay D, Sanchez-Pulido L, Akimenko MA (2010) Loss of fish actinotrichia proteins and the fin-to-limb transition. Nature 466:234–237 2. Azevedo AS, Grotek B, Jacinto A, Weidinger G, Saude L (2011) The regenerative capacity of the zebrafish caudal fin is not affected by repeated amputations. PLoS ONE 6:1–8 3. Iovine M (2007) Conserved mechanisms regulate outgrowth in zebrafish fins. Nat Chem Biol 10:613–618 4. Iovine MK, Johnson SL (2000) Genetic analysis of isometric growth control mechanisms in the zebrafish caudal fin. Genetics 155:1321–1329 5. Akimenko MA, Manuel MB, Jose B, Jacqueline G (2003) Old questions, new tools, and some answers to the mystery of fin regeneration. Dev Dyn 226:190–201 6. Becerra J, Montes GS, Bexiga SRR, Junqueira LCU (1983) Structure of the tail fin in teleosts. Cell Tissue Res 230:127–137 7. Santos-Ruiz L, Santamaria JA, Ruiz-Sanchez J, Becerra J (2002) Cell proliferation during blastema formation in the regenerating teleost fin. Dev Dyn 223:262–272 8. Shao J, Chen D, Ye Q, Cui J, Li Y, Li L (2011) Tissue regeneration after injury in adult zebrafish: the regenerative potential of the caudal fin. Dev Dyn 240:1271–1277
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Mol Cell Biochem (2014) 387:143–150 9. Bhaja KP, Lucille J, Patricia T, Amanda S, Marie-Andree A (2004) Screen for genes differentially expressed during regeneration of the zebrafish caudal fin. Dev Dyn 231:527–541 10. Nozomi Y, Takashi I, Akira K, Atsushi K (2009) Gene expression and functional analysis of zebrafish larval fin fold regeneration. Dev Biol 325:71–81 11. Feder ME, Hofmann GE (1999) Heat-shock proteins, molecular chaperones, and the stress response: evolutionary and ecological physiology. Annu Rev Physiol 61:243–282 12. Marcel T, Catherine J, Sophie V (2000) Zebrafish Hsp40 and Hsc70 genes are both induced during caudal fin regeneration. Mech Dev 99:183–186 13. Livak KJ, Schmittgen TD (2001) Analysis of relative gene expression data using real-time quantitative PCR and the 2 (-delta delta C (T)) method. Methods 25:402–408 14. Poss KD, Shen J, Nechiporuk A, McMahon G, Thisse B, Thisse C, Keating MT (2000) Roles for Fgf signaling during zebrafish fin regeneration. Dev Biol 222:347–358 15. Poss KD, Keating MT, Nechiporuk A (2003) Tales of regeneration in zebrafish. Dev Dyn 226:202–210 16. Manuel MB, Carmen M (2010) Dermoskeleton morphogenesis in zebrafish fins. Dev Dyn 239:2779–2794 17. Tamara LT, Jill AF, Robert LT (2010) Molecular signaling networks that choreograph epimorphic fin regeneration in zebrafish—a mini-review. Gerontology 56:231–240 18. Ranford JC, Coates ARM, Henderson B (2000) Chaperonins are cell-signalling proteins: the unfolding biology of molecular chaperones. Expert Rev Mol Med 2:1–17 19. Brocchieri L, Karlin S (2000) Conservation among HSP60 sequences in relation to structure, function, and evolution. Protein Sci 9:476–486 20. Duran I, Mari-Beffa M, Santamaria JA, Becerra J, Santos-Ruiz L (2011) Actinotrichia collagens and their role in fin formation. Dev Biol 354:160–172 21. Chablais F, Jazwinska A (2010) IGF signaling between blastema and wound epidermis is required for fin regeneration. Development 137:871–879 22. Wood A, Thorogood P (1984) An analysis of in vivo cell migration during teleost fin morphogenesis. J Cell Sci 66: 205–222 23. Mercader N (2007) Early steps of paired fin development in zebrafish compared with tetrapod limb development. Dev Growth Differ 49:421–437 24. Rosella C, Andrea D, Pierluca P, Mauro C, Daniela V (2004) Expression of 60 kDa heat shock protein (Hsp60) on plasma membrane of Daudi cells. Mol Cell Biochem 259:1–7 25. Cha IS, Kwon J, Park SB, Jang HB, Nho SW, Kim YK, Hikima J, Aoki T, Jung TS (2013) Heat shock protein profiles on the protein and gene expression levels in olive flounder kidney infected with Streptococcus parauberis. Fish Shellfish Immunol 34:1455–1462 26. Anju B, Piyush KP, Sarada SKS, Mustoori S (2010) Effect of adjuvants on immune response and protective immunity elicited by recombinant Hsp60 (GroEL) of Salmonella typhi against S. typhi infection. Mol Cell Biochem 337:213–221 27. Chow AM, Beraud E, Tang DW, Ferrier-Page`s C, Brown IR (2012) Hsp60 protein pattern in coral is altered by environmental changes in light and temperature. Comp Biochem Physiol A 161:349–353
Molecular cloning, characterization, and expression of hsp60 in caudal fin regeneration of Misgurnus anguillicaudatus Li Li • Ping Nan • Shengna Zhai • Lele Wang Songbo Si • Zhongjie Chang
•
Received: 21 August 2013 / Accepted: 18 October 2013 / Published online: 21 November 2013 Ó Springer Science+Business Media New York 2013
Abstract Urodele amphibians and teleost fish are capable of nearly perfect regeneration of lost appendages. The fin constitutes an important model for studying the molecular basis of tissue regeneration. It has been known that heat shock protein 60 (Hsp60) is a multifunctional protein of the heat shock protein family. The purpose of this study is to investigate the role of hsp60 as a part of a stress response system after fin injury or in fin regeneration. We firstly cloned full-length cDNA of hsp60 from Misgurnus anguillicaudatus (designated as MaHsp60) by RACE method. The cDNA contains a 83-bp 50 UTR, a 1,728-bp open reading frame encoding 492 amino acids and a 542-bp 30 UTR (Accession No.: KF537340). The phylogenetic tree shows that the MaHsp60 fits within the hsp60 clade. Then quantitative RT-PCR detected that MaHsp60 began to increase rapidly its expression at 1 dpa and reached its peak at 2 dpa. Next, spatial distribution analysis of MaHsp60 in fins showed that MaHsp60 located mainly in the deeper layer of regenerated epidermis when MaHsp60 expressed most. After the MaHsp60 had been cloned into the pET-32a vector, SDS-PAGE analysis confirmed that the MaHsp60 protein was efficiently expressed in Escherichia coli BL21 and adjustable with the temperature. These findings have revealed that MaHsp60, a highly conserved gene during vertebrate evolution as well as related to stress response, is involved in the formation of wound epidermis which occurs as the first phase of fin regeneration after fin amputation in caudal fin regeneration.
L. Li P. Nan S. Zhai L. Wang S. Si Z. Chang (&) Molecular and Genetic Laboratory, College of Life Science, Henan Normal University, 46# East of Construction Road, Xinxiang 453007, Henan, People’s Republic of China e-mail: [email protected]; [email protected]
Keywords Hsp60 gene Fin regeneration Phylogenetic analysis Gene expression Immunohistochemistry Misgurnus anguillicaudatus
Introduction All animals have the biological response to injury, but their ability to regenerate are quite different. Mammals are unable to regenerate amputated appendages, such as limbs, yet urodele amphibians, and teleosts exhibit outstanding capability of regenerating. After their appendages are severed, they can complete the structural and functional reconstruction with accurate position relationship through regeneration [1, 2]. Some teleosts are able to regenerate the fin, optic, retina, liver, and even heart [3]. The caudal fin model of tissue regeneration is particularly attractive because the fin is a relatively simple structure, quick to regenerate, and nonessential to the viability of fishes [4]. Mudloach (Misgurnus anguillicaudatus) is a freshwater teleost widely distributed along the eastern coasts of the Asian continent. Furthermore, many recent details regarding the genetic pathways regulating regeneration have been uncovered using mudloach. Although these fins present various and numerous shape, length, and color phenotypes, they are built with the same skeletal elements. And, their regeneration process are basically similar [5]. The teleost fins consist of a protruding visible exoskeleton made of bony rays of dermal origin, which are connected by means of ligaments to the endoskeleton [6]. Fin regeneration process follows a pattern of epithelial migration and accumulation to cover the surface of the cut, formation of a regeneration blastema along the entire width of the cut and ultimately proliferation and cell differentiation leading to replacement of the lost or damaged structure. In addition, these multiple fin structures may
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regenerate very rapidly [5, 7, 8]. It is conceivable and has been proved that many genes are involved [9, 10]. Heat shock proteins are a set of highly conserved proteins and are expressed in response to external stressors, such as thermal stress, tissue trauma, heavy metal toxicity, or inflammation. Also they are involved in a wide range of biochemical processes including folding of nascent polypeptides, intracellular trafficking, modulating signal pathway activity, and regulating immune responses [11]. The multifunctional nature of heat shock proteins and their critical roles in the regulation of tissue healing and cell survival make them good targets in fin regeneration process [12]. In this study, in order to explore the role of heat shock protein 60 gene (hsp60) in fin regeneration, we firstly isolated the full-length cDNA of hsp60 from M. anguillicaudatus and characterized its expression using fluorescent real time RT-PCR and immunohistochemistry. And through construction of expression vector, expression of Hsp60 protein were conducted. These results will help us understand the characterization, expression, and related regeneration functions of the hsp60.
Materials and methods Fish care and fin amputation Adult M. anguillicaudatus were collected from wetlands in the old course of the Yellow River, Yanjin County (Henan, China). The fish were kept in tanks with recirculating fish water which was kept at 26 °C during the whole experiment. Prior to fin amputations the fish were anesthetized in 0.1 % tricaine and then placed on a wet towel. Using a razor blade, approximately 30 % of the caudal fin was amputated.
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using DNAMAN. Degenerate primers P1 and P2 (Table 1) were designed from the conserved regions, after getting the conserved regions sequence of the MaHsp60 by PCR, then based on it, 50 gene-specific primers 50 GSP1 and 50 GSP2 and 30 gene-specific primers 30 GSP1 and 30 GSP2 (Table 1) were designed for the RACE. Reverse transcription and 50 and 30 - RACE PCR reaction conditions were performed according to the manufacturer’s instructions of the RACE Core Set (TaKaRa, Japan). PCR products were gel purified, cloned into pGEM-T vector (Promega, USA). After being transformed into the competent cells of Escherichia coli DH5a, the positive recombinants were identified through anti-Amp selection and sequenced by dideoxy-chain-termination using Sp6 and T7 promoter sequences as primers. Homologous alignment and bioinformatics analysis The full-length cDNA sequence of MaHsp60 was assembled by finding the overlapping region of each fragment. The nucleotide sequence and deduced amino acid sequence were compared with other sequences reported in NCBI’s GenBank using the BLAST program (http://blast.ncbi.nlm.nih. gov/Blast.cgi). The possible ORF was analyzed with ORF Finder tool (http://www.ncbi.nlm.nih.gov/gorf/gorf.html). All Hsp60 protein sequences from ten representative vertebrates were aligned using DNAMAN. A phylogenetic tree of different vertebrate Hsp60 proteins was constructed using neighbor-joining method with the software of Molecular Evolution Genetics Analyses (MEGA) version 4.0 and ClustalW (http://www.ebi.ac.uk/Tools/clustalw2/index). Real-time quantitative RT-PCR analysis In order to further quantify the relative expression of MaHsp60 in different stages of fin regeneration, a real-time
Total RNA isolation and synthesis cDNA For total RNA extraction, approximately 20 mg of regenerated tail tissues of the respective stages, 1 day post amputation (dpa), 2 dpa, 3 dpa, 4 dpa, 5 dpa, and 6 dpa were collected on ice. They were homogenized by using liquid nitrogen grinding, and total RNA was extracted using Trizol in accordance with the manufacturer’s instructions. First-strand cDNA synthesis was carried out with prime script reverse transcriptase (Takara, Japan), using 1 lg of total RNA isolated as described above. The first strand cDNA was obtained and preserved in -20 °C. Rapid amplification of cDNA ends analysis and cloning the full-length cDNA of MaHsp60 Nucleotide sequences of hsp60 genes were retrieved from GenBank (www.ncbi.nlm.nih.gov) and multiple aligned
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Table 1 Primer sequences used in gene cloning and RT-PCR in this study Primer
Oligonucleotide sequences (50 ?30 )
PI
GACTA(C/T)GAGAAGGAGAA(A/G)CT
P2
GTCCTCAC(C/G)ACCTT(T/G)GTG
0
GGGCGATTTCCTCTGGTGTTGTG
5 GSPl 50 GSP2
CTGGTGTTGTGACTGGCTTTGAGG
30 GSPl
GGCTAGCAAAACTTTCTGATGG
30 GSP2
TGACCATTGCTAAGAATGCAGG
FQ-F
CCTCAAAGCCAGTCACAAC
FQ-R
CACGGTCAAACTTCATTCC
GAPDH-F
GCCTCTTGCACGACCAACTG
GAPDH-R
CGGAAGGCCATGCCTGTCAG
P3
CCGGAATTCATGCTGCGTTTACCGAGTG
P4
CCCAAGCTTTTAGAAGCCTCCCATGCCTC
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RT-PCR assay was performed on an ABI 7500 real-time PCR detection system (Applied Bio-systems, USA) with SYBR green fluorescent label. All samples were analyzed in three replicates and all the reactions were independently repeated twice to ensure reproducibility. GAPDH was used to normalize data for differences between samples. Genespecific primers of hsp60 gene (FQ-F and FQ-R) and GAPDH (GAPDH-F and GAPDH-R) are showed in Table 1. Amplifications were performed in 50 ll containing 12.5 ll of 29 SYBR Premix Ex Taq (Takara, Japan), 3.5 pM of each primer, 2 ll of diluted cDNA, and 0.5 ll 509 ROX Reference Dye II (Takara, Japan). Data were analyzed with 7500 system SDS Software v 1.4(Applied Biosystems, USA). The 2-44Ct method was used to analyze the expression levels of MaHsp60 [13]. Statistical calculations were performed using SPSS version 13.0 software. Immunohistochemistry The rat anti-Hsp60 at 1:300 (Boster) were used as primary antibody, and the StreptAvidin-BiotinComplex kit (SA1029, Boster) were used as secondary antibody. Fins were fixed in 4 % paraformaldehyde in PBS, sectioned and immunostained as previously described [14]. Construction of MaHsp60 prokaryotic expression vector DNA fragment of 1,900 bp containing the ORF of MaHsp60 was amplified from caudal-fin cDNA using specific primers P3 and P4 (Table 1). Amplification conditions were as follows: initiation at 95 °C for 5 min; 35 cycles of 94 °C for 30 s, 60 °C for 30 s, and 72 °C for 60 s; and a final extension at 72 °C for 10 min. The PCR product was gel purified, cloned into pGEM-T vector, and sequenced to ensure that no mutations were introduced during PCR amplification. Then the plasmid was digested with XhoII and EcoRI and cloned into pET-32a/His C expression vector (Invitrogen) to obtain the MaHsp60 expression vector, pET-32a/His-Hsp60. The recombinant plasmid was sequenced to confirm that the inserted sequence was in the proper reading frame. Expression and change under different temperature of the M. anguillicaudatus Hsp60 protein (designated as MaHsp60) The pET32a/His-Hsp60 recombinant plasmid was used to transform competent E. coli BL21. A newly transformed single colony was selected and cultured in 5 ml of Luria broth (Kana) culture medium at temperature gradient of 26, 31, and 36 °C, and when the A value (optical density)
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reached 0.6, IPTG was added to a final concentration of 0.1 mmol/l. After incubation for 6 h, 1 ml of the culture media was removed, the bacteria were collected by centrifugation. After re-suspending precipitation by adding 1 % SDS, the bacteria were lysed by sonication. Then the soluble proteins in the supernatant were collected by recentrifugation. The expression of the recombinant Hsp60 protein was analyzed by SDS-PAGE on 10 % gels.
Results Molecular cloning and structural analysis of MaHsp60 In an attempt to isolate the MaHsp60 gene from caudal fins, we have first cloned conserved fragment by using a degenerate RT-PCR strategy. Based on the DNA sequence information, we designed primers for cloning full-length MaHsp60 by RACE-PCR. By overlapping the 30 - and 50 RACE fragments, a 2,357 bp transcript was obtained. It includes a 50 -terminal untranslated region of 83 bp, a 30 terminal untranslated region of 542bp with a short polyA tail (GenBank accession no. KF537340), and an ORF of 1,728 bp encoding a polypeptide of 576 amino acids with a predicted molecular mass of 61.05 kDa and theoretical isoelectric point of 4.75. The nucleotide and predicted amino acid sequence of the ORF of MaHsp60 are shown in Fig. 1. Homology analysis and evolutionary relationships of MaHsp60 The BLAST analysis revealed that MaHsp60 has high homology to the Hsp60 of other species. The homology of deduced amino acid sequences of Hsp60 from ten species are shown in Table 2 and Fig. 1. The deduced amino acid sequence of MaHsp60 was 95.0, 93.6, 90.3, 87.0, 85.0, 86.5, 86.1, and 71.7 % identical to that of Danio rerio Hsp60, Carassius auratu Hsp60, Paralichthys olivaceus Hsp60, Gallus gallus Hsp60, Xenopus laevis Hsp60, Sus scrofa Hsp60, Homo sapiens Hsp60, and Drosophila melanogaster Hsp60, respectively. To evaluate the evolutionary relationships of MaHsp60 with other species, we constructed a phylogenetic tree using software MEGA 4.0 on the basis of the Hsp60 amino acids sequences (Fig. 1). Amino acids sequences from 20 species were used for the rooted phylogenetic tree constructed by the neighbor-joining method. And the phylogenetic tree shows that The Hsp60 proteins from different vertebrates were divided into two subgroups. The birds, mammals, and amphibians were grouped into one cluster, and the teleosts were separated and formed another independent group. The results support a monophyletic origin of fish again. As far as the evolution distance was
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(A) GAAACTCTCGTGCTTATATCATT CAGTCTGCTGTACAGACGGACACACCCATCCCTTCATTCACCTCCAGAACACTTTACAAA 1 1 61 21 121 41 181 61 241 81 301 101 361 121 421 141 481 161 541 181 601 201 661 221 721 241 781 261 841 281 901 301 961 321 1021 341 1081 361 1141 381
1201 401 1261 421 1321 441 1381 461 1441
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ATGCTGCGTTTACCGAGTGTGATGAAACAGATGAGGCCAGTGTGCAGGGCGCTGGCCCCA M
L
R
L
P
S
V
M
K
Q
M
R
P
V
C
R
A
L
A
P
CATTTGACCCGCGCGTACGCAAAGGATGTCAAGTTTGGAGCCGATGCCCGGGCCCTCATG H
L
T
R
A
Y
A
K
D
V
K
F
G
A
D
A
R
A
L
M
CTCCAGGGCGTTGACCTGTTGGCTGATGCTGTGGCAGTCACAATAGGACCAAAGGGTCGC L
Q
G
V
D
L
L
A
D
A
V
A
V
T
I
G
P
K
G
R
ACTGTCATCATTGAGCAGAGCTGGGGAAGCCCCAAGGTCACCAAAGATGGCGTCACGGTG T
V
I
I
E
Q
S
W
G
S
P
K
V
T
K
D
G
V
T
V
GCCAAAAGTATTGATCTAAAGGATAGGTATAAGAACATCGGGGCCAGGCTTGTACAAGAC A
K
S
I
D
L
K
D
R
Y
K
N
I
G
A
R
L
V
Q
D
GTAGCCAACAACACTAATGAGGAGGCTGGAGACGGCACCACAACCGCCACAGTGCTGGCT V
A
N
N
T
N
E
E
A
G
D
G
T
T
T
A
T
V
A
L
CGGGCTATTGCCAAGGAGGGATTTGACACCATCAGCAAAGGCGCCAACCCTGTGGAGATC R
A
I
A
K
E
G
F
D
T
I
S
K
G
A
N
P
V
E
I
CGTAGAGGAGTTATGACTGCAGTGGAGACTGTCATCAATGAACTTAAAAAAACCTCAAAG R
R
G
V
M
T
A
V
E
T
V
I
N
E
L
K
K
T
S
K
CCAGTCACAACACCAGAGGAAATCGCACAGGTGGCCACAATTTCTGCCAATGGAGACACT P
V
T
T
P
E
E
I
A
Q
V
A
T
I
S
A
N
G
D
T
GAAGTTGGTGAGATCATCTCCAATGCCATGAAGAAAGTTGGACGTAAAGGTGTTATTACT E
V
G
E
I
I
S
N
A
M
K
K
V
G
R
K
G
V
I
T
GTAAAGGATGGTAAAACTCTGCACGATGAGCTTGAGATCATTGAGGGAATGAAGTTTGAC V
K
D
G
K
T
L
H
D
E
L
E
I
I
E
G
M
K
F
D
CGTGGACACATTTCTCCTTACTTCATCAACACTGCTAAAGGTCAAAAATGTGAGTTCCAG R
G
H
I
S
P
Y
F
I
N
T
A
K
G
Q
K
C
E
F
Q
GATGCTTATCTGCTTCTGAGTGAGAAGAAGATCTCCAGTGTACAGAGCATCGTGCCAGCA D
A
Y
L
L
L
S
E
K
K
I
S
S
V
Q
S
I
V
P
A
CTGGAACTTGCCAACCAGCACCGCAAGCCTCTGATCATTGTGGCTGAAGATGTGGATGGA L
E
L
A
N
Q
H
R
K
P
L
I
I
V
A
E
D
V
D
G
GAAGCACTCAGTACTCTGGTCCTCAACAGGTTAAAGGTTGGACTGCAAGTTGTTGCAGTC E
A
L
S
T
L
V
L
N
R
L
K
V
G
L
Q
V
V
A
V
AAGGCTCCAGGATTTGGGGACAACCGAAAGAACCAGCTGCAGGATATGGCAGTGGCCACT K
A
P
G
F
G
D
N
R
K
N
Q
L
Q
D
M
A
V
A
T
GGAGGCACAGTGTTTGGTGATGATGCTGTGGGTCTGGCCCTTGAGGATATCCAGGCACAT G
G
T
V
F
G
D
D
A
V
G
L
A
L
E
D
I
Q
A
H
GACTTTGGCAAGGTTGGCGAGGTCATCGTGACCAAGGATGACACGATGCTCCTCAAAGGC D
F
G
K
V
G
E
V
I
V
T
K
D
D
T
M
L
L
K
G
CGTGGTGATCCAGCAGCCGTTGAGAAACGTGCGAACGAGATCGCCGAACAGCTGGAGAGC R
G
D
P
A
A
V
E
K
R
A
N
E
I
A
E
Q
L
E
S
ACAAACAGCGACTATGAGAAGGAGAAACTCAACGAGCGGCTGGCAAAACTCTCTGATGGA T
N
S
D
Y
E
K
E
K
L
N
E
R
L
A
K
L
S
D
G
GTCGCTGTAATTAAGGTTGGAGGAACAAGTGACGTTGAAGTAAACGAGAAGAAAGACCGT V
A
V
I
K
V
G
G
T
S
D
V
E
V
N
E
K
K
D
R
GTCACCGATGCACTGAACGCCACGCGAGCTGCTGTGGAGGAGGGAATTGTGCCTGGAGGA V
T
D
A
L
N
A
T
R
A
A
V
E
E
G
I
V
P
G
G
GGCTGTGCCCTGTTGCGCTGCATCCCAGCCTTGGATACCATCAAGCCTCTAAATGAGGAT G
C
A
L
L
R
C
I
P
A
L
D
T
I
K
P
L
N
E
D
CAGAAAATAGGTATTGACATTATTCGCAGATCATTGCGTATTCCTGCCATGACCATTGCT Q
K
I
G
I
D
I
I
R
R
S
L
R
I
P
A
M
T
I
A
AAGAATGCAGGAGTTGAAGGATCTCTGGTGGTGGAGAAGATTTTACAGAGTGCTCCAGAA
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b Fig. 1 Homologous alignment and bioinformatics analysis of
Expression changes of MaHsp60 during caudal fins regeneration
Mahsp60 and deduced amino acid sequence. a Full-length cDNA and deduced amino acid sequence of Mahsp60. The deduced amino acid is shown below the nucleotide sequence. Nucleotide and amino acid sequences are numbered on the left. Amino acid sequence is represented with one-letter codes below the cDNA sequence. ATG shows the initiation codon. An asterisk indicates the stop codon. ATP/ ADP and Mg2? binding segments appear as double lined. A signature motif appears as dashed line. Conserved GGM repeats at the C-terminal appear as single- lined. This sequence has been submitted to GenBank (Accession no.: KF537340). b Multiple alignment of amino acid sequences of Hsp60. Protein sequences were obtained from GenBank: Ma Misgurnus anguillicaudatus (this study); Dr Danio rerio (NP851847); Ca Carassius auratus (ABI26641); Po Paralichthys olivaceus(ABB76384); Hs Homo sapiens (NP955472); Ss Sus scrofa (XP001928634); Mm Mus musculus (NP071565); Gg Gallus gallus (NM001012916); Xl Xenopus laevis (NP001083970); Dm Drosophila melanogaster (NP727489); and Lv Litopenaeus vannanmei (ACN30235). c Phylogenetic tree of hsp60 proteins from various species. The tree was obtained by bootstrap analysis with the neighbor-joining method and was bootstrapped 1,000 times. Filled diamond indicates Hsp60 from Misgurnus anguillicaudatus. Accession numbers for these sequences are: Tanichthys albonubes, ADK27679; Cten opharyngodon idella, ADU34083; Kryptolebias marmoratus, AEM65177; Epinephelus akaara, HQ141338; Salmo salar, ACI33148; Bos Taurus, NP001160080; Oryctolagus cuniculus, XP002712414; Pan troglodytes, XP001169156; Strogylocentrots purpuratus, XP795205; and Branchiostoma floridae, XP002595084. The other organisms with GenBank reported sequences see Fig. 1b
The fluorescent real-time RT-PCR were conducted to determine the expression of MaHsp60 in different periods of caudal fins regeneration. It revealed that expression of MaHsp60 rapidly increased in response to fin amputation (Fig. 2). By 2 dpa, the expression of gene reached to the levels that were approximately 18-fold higher than the expression measured in the uninjured caudal fin (0 dpa). Thereafter, the expression began to decrease. By 3 dpa, the expression of MaHsp60 decreased to the levels that were approximately half of the peak expression levels. Then the expression of MaHsp60 remained low expression levels during late regeneration. Spatial distribution of MaHsp60 in regenerated caudal fins We next asked how this Hsp60 distributes in the regenerated fin, then immunostaining was performed in tissue sections from fins when the expression was at peak. Longitudinal sections show that wound healing has been finished at this time, that is, at 2 dpa as Akimenko reported [5]. It can be seen that positive cells are mainly involved in the composition of deeper epidermis, while there are nearly no distribution in flat epithelial cells of superficial layers of the epidermis and blastema inside the caudal fin (Fig. 3).
concerned, the MaHsp60 has a closer genetic relationship with that of D. rerio, which agrees with the comparability result of deduced amino acid sequence.
Fig. 1 continued
481 1501 501 1561 521 1621 541
K
N
A
G
V
E
G
S
L
V
V
E
K
I
L
Q
S
A
P
E
ATTGGATATGATGCTATGAATGGAGAATATGTCAACATGGTTGAAAAAGGAATTATTGAC I
G
Y
D
A
M
N
G
E
Y
V
N
M
V
E
K
G
I
I
D
CCCACAAAGGTTGTGAGGACAGCATTACTTGATGCTGCAGGTGTTGCGTCTCTGCTGTCC P
T
K
V
V
R
T
A
L
L
D
A
A
G
V
A
S
L
L
S
ACTGCTGAAGCCGTCGTCACCGAGATCCCCAAGGAGGAGAAGGACATGCCAGGTGGAGGG T
A
E
A
V
V
T
E
I
P
K
E
E
K
D
M
P
G
G
G
1681
ATGGGAGGCATGGGAGGTATGGGTGGTATGGGAGGCATGGGAGGATTCTAA
561
M G G M G G M G G M G G M G G F * ACCACACTTCACTGACTTTAGAGAGAAGGGTTGTGGGCAGGAGACATGATTCGCCCCTTT CTTTCAACTCGGATAAACCTGCCGATACAGAGTGCTGGTCTTGACACGGAGCAAAGATAT GGACCTTACTATTCCCCCATCTTCCACCACTGTCCATCCCATCTGATCTCATCTCTCTGC TCATGACCGAAGACATGACTCTATTGTTTCAAAGACAGGCTCCAGAATGTCTATTTGCTT GGATATTTAGCATTGTGCTTATTGTTTCCATCCAACCATTCAAATGGGAATGTGCATATT GAAAACTGCTTCATGCTGTTGTATTGTTTGAAAGCATTTATCATTTTGCATAAAGCTTGT TCTGAAAGACCATCTGAAGGGGCTTACAAAGCCAGACATTCAGGTTGAAATCTGTAAGAA AGCAGATACTTTGACTAGTTGTATCTAAAAAATTCCTTCTCATTGATCAGTTCTTTTTTT GTACACTGTTCATCATTTCATATAAAAATAAAACTCATTTTGGGATTACAAAAAAAAAAA AA
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Mol Cell Biochem (2014) 387:143–150
Fig. 1 continued
Table 2 Comparability of deduced amino acid sequences of Hsp60 from various species Gene
Mahsp60 (%)
Mahsp60
100
Drhsp60 (%)
Cahsp60 (%)
Pohsp60 (%)
Hshsp60 (%)
Sshsp60 (%)
Mmhsp60 (%)
Gghsp60 (%)
100
Xlhsp60 (%)
Drhsp60
95.0
Cahsp60
93.6
94.8
Pohsp60
90.3
90.2
88.5
Hshsp60
86.1
87.3
86.5
85.6
Sshsp60
86.5
87.5
87.2
86.3
97.6
Mmhsp60 Gghsp60
87.0 87.0
88.2 88.6
87.7 87.2
86.8 87.0
97.6 94.1
97.2 94.4
100 94.2
Xlhsp60
85.0
85.7
85.2
83.8
90.4
89.9
90.1
90.4
Dmhsp60
71.7
70.7
71.9
72.0
72.8
73.1
73.3
72.6
72.4
Lvhsp60
68.7
69.2
69.5
68.6
72.2
72.1
71.7
71.4
70.8
Lvhsp60 (%)
100 100 100 100
Expression and temperature-associated change of MaHsp60 According to the SDS-PAGE analysis, after IPTG induction, 78.9 kDa band (Fig. 4a, lane 2, 3) corresponding to the expectant size of the fusion protein was observed. And the constructed plasmid pET32a/His-Hsp60 exhibited a higher trend of the His-Hsp60 fusion protein expression levels in BL21 over time during 6 h (Fig. 4a, lane 2) to 8 h (Fig. 4a, lane 3) when the concentration of IPTG was 0.1 mmol/l. There was basal low expression levels of the protein without IPTG induction (Fig. 4a, lane 1). Furthermore, this protein had the maximum expression amount at 36 °C (Fig. 4b, lane 3) among temperature gradients of 26, 31, and 36 °C after incubation for 6 h, while at the latter two temperature expression seemed to have no significant change (Fig. 4b, lane1, 2).
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Dmhsp60 (%)
100
100 100 77.4
100
Discussion Fin regeneration has emerged as an ideal model for our further understanding of vertebrate regeneration due to the simpler anatomical structure compared to the urodele limb [15]. As we think, numerous factors or genes have been implicated in the processes of limb/fin bud initiation and outgrowth in vertebrates [16, 17]. Nevertheless, fin regeneration is firstly a process of wound repair which is subject to mechanical stress caused by a severe injury. Therefore, it is interesting to speculate the role of some heat shock proteins in fin regeneration. We successfully cloned the MaHsp60 cDNA sequence via RACE method. The gene encodes a 576 amino acid neutral protein with a molecular weight of 61.05 kD and has a high similarity with other known hsp60s sequences. The MaHsp60 is conserved in evolution and its encoded
Mol Cell Biochem (2014) 387:143–150
149
20
(A)
18
1
(B) 2
3
M
KD
M
1
2
3
97.2
16 14
66.4
12 10
44.3
8
n.s.
6 4
29.0
2 0
0 dpa
1 dpa
2 dpa
3 dpa
4 dpa
5 dpa
6 dpa
Fig. 2 Expression analysis of MaHsp60 in different periods of caudal fins regeneration by real-time fluorescent quantitative RT-PCR. GAPDH was the internal control. Vertical bars the mean ± SD (N = 3). Asterisk indicates a significant difference with the control (P \ 0.05) and n.s. no significant, respectively
we
sc bl be
chr
fe
Fig. 3 Spatial distribution of MaHsp60 in regenerated caudal fins. A distal part of a regenerated caudal fin containing Hsp60-positive cells was shown at 2 dpa. bl blastema; be basal epidermis; chr chromatophores; fe flat epithelial cells; sc secretory cells; and we wound epidermis. Scale bars 50 lm
protein contains conserved structural domains, including two highly conserved motifs corresponding to the ATP/ ADP and Mg2? binding segments 76-DGVTVAK-82 and 109-AGDGTTTATVL-119, a classical HSP60 signature motif 427-ATRAAVEEGIVPGGG-441, a conserved GGM repeat at the C-terminal end [18, 19]. There is no report about expression of Hsp60 gene during fin regeneration. In this report, quantitative expression studies of the gene MaHsp60 were performed during fin regeneration. Basal levels were observed at o dpa, a mature non-regenerating fin. An obvious upregulation of the expression was observed at 1 dpa when the
Fig. 4 SDS-PAGE analysis of His-Hsp60 fusion protein. a Molecular weight marker is in the right lane. Lane 1 Cell lysates of bacteria transformed with pET32a/His-Hsp60 without IPTG induction. Lane 2 and lane 3 represent cell lysates of bacteria transformed with pET32a/ His-Hsp60 under IPTG (0.1 mM) induction after incubation for 6 and 8 h, respectively. b Molecular weight marker is in the left lane. Lane 2, lane 3, and lane 4 represent cell lysates of bacteria transformed with pET32a/His-Hsp60 under IPTG (0.1 mM) induction after incubation 6 h at 26, 31, and 36 °C, respectively
wound is firstly healed by the rapid migration and rearrangement of the epithelial cells of the stump [20]. Then the expression peaked on the following day, which is of no difficulty to understand because the formation of the wound epidermis is under way at this stage, an inevitable stress process after lesion. From 3 dpa, the gene expression decreased gradually until reaching the lowest level which was still above the basal, homeostatic levels were at 6 dpa when stress response and regeneration is finishing [21]. These results suggest that MaHsp60 are strongly upregulated during the wound epidermis formation stage and it begins to decrease when wound repair will complete. The fact might indicate that MaHsp60 has made contribution to the fin formation. In order to clarify how MaHsp60 participates in the morphogenesis of fin regeneration or how MaHsp60 distributes in regenerated fin immunohistochemical method was used. We confirmed the distribution of its protein when expression of MaHsp60 reached the maximum amount. The results of positive cells located mainly in the deep epidermis, but absence of mesenchymal cells inside the fin proves that Hsp60 do participate in the first phase of fin regeneration—wound healing. In this process, the injured stump is repaired by rapid migration of epidermal cells over the amputation surface. The tissues located in the vicinity of the resection site undergo disorganization and display enhanced cell proliferation. It is the proliferation of the cells forming this transitory structure that is responsible for the regrowth of the epidermis. Then following second phase is the formation of the blastema, cells located in the blastema differentiate and participate in the re-establishment of the missing structures [22, 23]. So, the distribution of Hsp60 in fin quite match with its feature as stress
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proteins because the first phase is essentially a kind of stress response to injury. To determine the activity of the MaHsp60, we constructed prokaryotic expression vector, detected the relationship between the MaHsp60 expression and temperature. The expression of protein is efficient and adjustable with the temperature. The 60 kD heat shock protein is an important molecular chaperone, a stress protein constitutively synthesized in all cells analyzed so far, and located on cell membrane [19, 24]. In the last few years, the study of hsp60 focus on expression in the case of tumor, bacterial infection, different temperature, salinity, and other environmental changes [25–27]. Vertebrate limb/fin bud regeneration is considered an excellent and interesting process for studying morphogenesis. The study shows MaHsp60 may play an important role in the recovery of wound epidermis after fin amputation during caudal fin in M. anguillicaudatus. The information gathered from the study provides resources for further investigations into the function of hsp60 in fin regeneration. Acknowledgments This work is supported by Grants from the Natural Science Foundation of Henan Province (No. 112300410275), Scientific Research Project Fund of the Education department of Henan Province (No. 2011B180030), and Youth Foundation of Henan Normal University (No. 2012QK17).
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