JF M SE, 28(4), pp. 903~912, 2016. www.ksfme.or.kr 수산해양교육연구 제 권 제 호 통권 호, 28 4 , 82 , 2016. http://dx.doi.org/10.13000/JFMSE.2016.28.4.903
. Introduction
Ⅰ
Cathepsin L superfamily is a multifunctional cysteine protease enzyme and widely distributed in most animals. Approximately 11 cysteine proteases (cathepsins B, C, F, H, K, L, O, S, V, X(Z), and W), 2 aspartic proteases (D and E), and 2 serine
protease (A and G) have been recognized (Zavašnik Bergant & Turk, 2006). In the field of
‐
classification, although most mature enzymes share highly homologous amino acid sequences, their motifs significantly differ on the basis of the sequence analysis in the proregion. Two distinct subfamily, namely, cathepsin L subfamily
Cloning, Expression Analysis and Enzymatic Characterization of Cathepsin L from the Inshore Hagfish (Eptatretus burgeri)
Jin-Hyeon JANG So-Hee SON Hyeon-Kyeong JO Joon-Ki CHUNG Hyung-Ho LEEㆍ ㆍ ㆍ ㆍ (Pukyong National University)
먹장어
Cathepsin L
의 분자생물학적 클로닝 발현 및,
효소학적 특성 분석장진현 손소희 조현경 정준기 이형호
부경대학교
( )
Abstract
Hagfish which belongs to the chordate contact cyclostomata, is important phylogenetic relationship between vertebrate and invertebrate. Cathepsins of the cysteine protease family have traditionally been thought to play a major role in intracellular protein degradation and turnover in lysosomes.
In this study, Catepsin L was cloned from Inshore hagfish (Eptatretus burgeri), the cDNA encoding ORF of the Eptatretus burgeri Cathepsin L (EbCtL) is 978 bp. The cDNA encoding proEbCtL was expressed in Escherichia coli strain BL21(DE3) using the pGEX-4T-1 expression vector system. The recombinant proEbCtL protein was overexpressed as a approximately 55 kDa fusion protein. The overproduced soluble GST-fusion protein was then applied to glutathione-Sepharose 4B column chromatography; the sample harboring the fusion protein evidenced a high degree of purity when analyzed via SDS-PAGE and Western blot analysis. Its activity was quantied by cleaving the synthetic peptide Z-FR-AMC, Z-LLE-AMC, and Suc-AAF-AMC, and the optimal pH for the protease activity was 8, 9.5, and 9, respectively.
Key words : Cathepsin L, Cysteine protease, Eptatretus burgeri.
Corresponding author : 051-629-5864, [email protected]
(2014 ) .
(cathepsins L, V, F, H, S, K and W) and cathepsin B subfamily (cathepsins B, C and X(Z)), have been classified. The two groups differ in proregion and mature peptide sequence (Barrett et al., 1998).
In cathepsin L subfamily, the propeptide comprises 100 residues and 2 conserved motifs, namely, ERFNIN and GNFD. In cathepsin B subfamily, the propeptide is approximately 60 residues in length and contains the GNFD motif only (Dacks, Kuru, Liapounova, & Gedamu, 2008;
Guo et al., 2000; McDonald, 1985). Most proteomic research and related studies on cysteine cathepsins have focused on vertebrates, particularly mammals, including primates and rodents (Fonović
& Turk, 2014; Shahinian, Tholen, & Schilling, 2013). Currently, cysteine cathepsins should not be solely considered as lysosomal proteases because they are also found in other cellular compartments.
These cathepsins participate in many biological processes in addition to protein turnover. The isoforms of cathepsin L are detected in the nucleus and function as a regulator of cell cycle by cutting the histone H3’s N-terminus tail (Duncan et al., 2008). In zebrafish, a cathepsin L variant is involved in developing fish embryos (Tingaud-Sequeira & Cerdà, 2007). A series of cathepsin L-like proteases is also discovered in rodents; these proteases perform specific roles in gestation (Song et al., 2010). Cysteine cathepsins are significant signaling molecules and vital regulators in physiological events, as indicated by the experimental evidence accumulated. Their nonendosomal functions also become highly fascinating (Zhou, Zhang, Li, & Cai, 2015).
Molecular phylogenetic data announced that extant two vertebrates, which are consist of the living jawless vertebrates (cyclostomes) and jawed the vertebrates (Kuraku, Hoshiyama, Katoh, Suga,
& Miyata, 1999; Mallatt & Sullivan, 1998). Hagfish which belong to cyclostomata typify the most basal lineage of vertebrates (Oisi, Ota, Kuraku, Fujimoto,
& Kuratani, 2013).
In this study, we demonstrated sequence analysis, expression analysis, and enzymatic assay of the EbCtL.
. Materials and methods
Ⅱ
1. Full-cDNA Cloning of
Eptatretus burgeri
Cathepsin LTo identify the full-length of E. burgeri cathepsin L gene, oligonucleotides were designed for the fish cathepsin L highly in conserved region.
Total RNA was isolated from hagfish using the Hybrid-R kit (GeneAll). cDNA was synthesized from isolated mRNA using the Transcriptor First Strand cDNA Synthesis Kit (Roche) and SuperScriptTM RT (Invitrogen). Authors constructed 5' and 3' RACE-PCR using the hagfish specific and Universal primers for 5' and 3' RACE from a cDNA mixture which was prepared from total RNA of hagfish using the SMART RACE cDNA amplification kit (Clontech). The primer sequences used in this study are listed in <Table 1>.
The amplified RACE products were subcloned into a pGEM T-Easy vector (Promega) and transformed into E. coli DH5 competent cells. The α E. coli clones containing the recombinants were overlaid with 100g/ml of ampicillin, 40g/ml 5-bromo-4-chloro-3-indoly D-galactoranoside — (X-Gal) and 0.4 mM isopropyl-thiogalactopyranoside (IPTG) was added to agar gel in Luria-Bertani broth.
By selecting at random white colonies cultivated and then plasmid DNA was extracted. Plasmid
DNA was extracted from E. coli DH5α using the PureHelix Fast-n-Pure Plasmid Kit (NANOHELIX).
DNA sequencing carry into effect using M13(-20)F/M13(-20)R primers in the COSMO co, Ltd. (Seoul, Korea).
Primer
name 5'-3' sequence Information
EbCtL-F GCG ACT TCA CCT GCT
GAT A Primers for
cDNA library screening EbCtL-R AAG GAG GAT TCA TAT
TTA TTT CCA C 5'pGEX GGG CTG GCA AGC CAC
GTT TGG TG Sequencing primers 3'pGEX CCG GGA GCT GCA TGT
GTC AGA GG BamHI-
EbCtL-pro CGG GAT CCA TGG GCC
TGA CCA TCT CC Primers for constructionthe of expression
vector BamHI-
EbCtL-mat CGG GAT CCC TCC CAG CCG CCA TA EcoRI-
EbCtL-R CGG AAT TCT CAG ACG AGT GGG TAG CTG GC
<Table 1> Oligonucleotide primers used for EbCtL cDNA library screening and expression
2. Sequence and Phylogenetic Analysis The EbCtL amino acid sequences homology and identity were analyzed using, BioEdit (Hall, 2004).
The signal peptides of EbCtL was identifed using
the SignalP 4.0 server
(http://www.cbs.dtu.dk/services/SignalP/). Multiple alignments of EbCtL amino acid sequences were analyzed with the GENETYX Version 7.0.3 (Genetyx Co., Tokyo, Japan). The phylogenetic tree used neighbor-joining methods in MEGA 6 (Kumar, Nei, Dudley, & Tamura, 2008).
3. Expression and Purification of Recombinant proEbCtL protein
In order to construct an expression vector for recombinant EbCtL in E. coli strain BL21(DE3), the coding sequence contained in the E. burgeri procathepsin L was amplified by PCR using the
p r i m e r s ( B a m H I - p r o E b C t L - F , 5'-CGGGATCCATGGGCCTGACCATCTCC-3';
E c o R I - E b C t L - R ,
5'-CGGAATTCTCAGACGAGTGGGTAGCTGGC-3') which harbor BamHI/EcoRI restriction sites (underlined). The resulting fragment (978 bp) was cloned into the pGEX-4T-1 (Amersham Pharmacia Biotech). The recombinant plasmid (proEbCtL/pGEX-4T-1) was transformed into E. coli strain BL21(DE3). Transformed cells were
grown in LB broth containing ampicillin (50g/ml) at 37 ℃ for about 3 hr. Next, Isopropyl-D-thiogalactopyranoside (IPTG) was added to a final concentration of 0.4 mM, and incubation was shifted to 20 ℃ for 6hr. The induced cells were harvested, resuspended in phosphate buffered saline (PBS), sonicated by using a sonicator (Vibra cell, Sonics & materials Inc, USA), and centrifuged at 20,000g × 20 min at 4 ℃. After, The glutathione S-transferase (GST) fused proEbCtL protein was affinity purified at 4 ℃ by a glutathione-Sepharose 4B column (Pharmacia Biotech Co., USA), and protein concentrated using Centrifugal Filters Centriprep 30K devices (Merrck Millipore).
4. SDS-PAGE and Western Blotting Analysis Electrophoresis procedures were performed as described by Laemmli (Laemmli, 1970). Purified proEbCtL protein was analyzed by 12%
SDS-PAGE.
Western blotting analysis was perfomed using mouse anti-GST antibody (1:2000, Santa Cruz Biotechnology).
5. Enzymatic Assays
The proEbCtL protein activity was assayed
according to the modified method of Barret and Kirschke (Barrett & Kirschke, 1981).
Z-Phe-Arg-AMC (Z-FR-AMC), Z-Leu-Leu-Glu-AMC (Z-LLE-AMC), and Suc-Ala-Ala-Phe-AMC (Suc-AAF-AMC), and the optimal pH for the proEbCtL activity was 8, 9.5, and 9, respectively.
The 7-amido-4-methylcoumarin (AMC) was
measured at an excitation wavelength of 380 nm and an emission wavelength of 460 nm using a Microplate Fluorometer (Packard Co. USA).
The effects of enzyme inhibitors on protease activity were measured using Z-FR-AMC, Z-LLE-AMC and suc-AAF-AMC. The proEbCtL inhibitor assay was used as shown in <Table 3>.
[Fig. 1] E. burgeri cathepsin L (EbCtL) nucleotide and amino acid sequences. The shaded box sequence indicate the signal peptide and thick line box indicate the proregion. The three catalytic site triad residues Cys133, His159 and Asp292 (based on mature EbCtL numbering) are marked with a shaded box and underlined.
The effect of various metal ions on proEbCtL assay was using ZnSO4, CuSO4, MgSO4, HgCl2, CaCl2, KCl, and CoCl2. Also, the effects of detergent (Triton X-100, Tween 20, SDS and Brij
35) on enzyme stability were examined. The effects of inhibitors, metal ions and detergents on the activity of proEbCtL protein were studied at pH 8, pH 9.5, pH 10 at 37 ℃.
[Fig. 2] Multiple sequence alignment analysis of Cathepsin L family in various species. The GenBank accession numbers are as follows : MgCtL Myxine glutinosa cathepsin L (AAF19631.1), PmCtL Petromyzon marinus cathepsin L (AAQ01146.1), BlCtL Branchiostoma lanceolatum cathepsin L (AAQ01144.1), DrCtL Danio rerio cathepsin L (CAA69623.1), PoCtL Paralichthys olivaceus cathepsin L (ACN43674.1).
[Fig. 3] Neighbor-joining phylogenetic tree analysis of EbCtL with other cathepsins. Each brace point was decided by 1000 replicates bootstrap analysis. The red box indicates EbCtL. The GenBank accession numbers are as follows : MgCtL Myxine glutinosa cathepsin L (AAF19631.1), PoCtL Paralichthys olivaceus cathepsin L (ACN43674.1), AcCtL Anolis carolinensis cathepsin L (XP_003216502.1), GgCtL Gallus gallus cathepsin L (NP_001161481.1), Xenopus laevis cathepsin L (NP_001087489.1), DrCtL Danio rerio cathepsin L (CAA69623.1), Mus musculus cathepsin L (AAD32137.1), HsCtL Homo sapiens cathepsin L (NP_001903.1), HsCt S Homo sapiens cathepsin S (AAC37592.1), HsCtC Homo sapiens cathepsin C (AAQ08887.1), HsCtF Homo sapiens cathepsin F (NP_003784.2), HsCtK Homo sapiens cathepsin K (NP_000387.1), HsCtH Homo sapiens cathepsin H (UniProtKB : P09668.4), HsCtV Homo sapiens cathepsin V (BAA25909.1), HsCtO Homo sapiens cathepsin O (NP_001325.1), HsCtD Homo sapiens cathepsin D (AAP35556.1), HsCtZ Homo sapiens cathepsin Z (NP_001327.2), HsCtB Homo sapiens cathepsin B (AAH10240.1), HsCtE Homo sapiens cathepsin E (NP_001901.1), HsCtW Homo sapiens cathepsin W (NP_001326.2), HsCtA Homo sapriens cathepsin A (UniProtKB/Swiss-Prot: P10619.2), HsCtG Homo sapiens cathepsin G (NP_001902.1).
. Results and discussion
Ⅲ
1. Cloning of Cathepsin L cDNA from hagfish Hagfish cathepsin L (EbCtL) sequence was obtained from cDNA library screening. EbCtL
accession number is KU744007. The 1,127 bp EbCtL cDNA was comprised open reading frame of 978 bp and the 44 bp 5'-UTR and the 105 bp 3'-UTR. The EbCtL cDNA encode 325 amino acids, which comprised a signal peptide of 16 amino acids analyzed with SignalP 4.1 Server
(Petersen, Brunak, von Heijne, & Nielsen, 2011), 93 residues propeptide, and 217 residues mature protein [Fig. 1].
The EbCtL amino acid sequence included an ERFNIN motif, which is conserved in the cathepsin L-like subfamily (Turk, Turk, & Turk, 2000). Also, EbCtL included the GNFD motif, which is conserved in the papain superfamily (Turk et al., 2012; Vernet et al., 1995) [Fig. 2]. Generally, cysteine proteases conserved catalytic site consisting of cysteine (C), histidine (H), and asparagine (N)
residue. The EbCtL mature protein sequence has included cysteine residue (Cys25), histidine residue (His159) and asparagine (Asn175). EbCtL proteins have included all of these features, like all members of cysteine protease [Fig. 2].
2. Phylogenic and Sequence Analysis of hagfish Cathepsin L
By comparing the amino acid sequence of EbCtL with that of MgCtL in the closest relations, it showed 70% of identity.
(C) [Fig. 4] SDS-PAGE and western bolt analysis of
proEbCtL. (A) SDS-PAGE analysis using 12%
SDS PAGE gel and coomassie R-250 blue. (B) – Western blot analysis using anti-GST antibody. Lane M, marker; lane 1, lysate from BL21(DE3) with pGEX-4T-1-Self; lane 2, lysate from IPTG-not induced EbCtL- expressing BL21(DE3); lane 3, lysate from 0.4mM IPTG-induced EbCtL-expressing BL21(DE3); lane 4, purified and concentrated proEbCtL protein. The asterisk (*) indicates the purified and concentrated EbCtL protein (55 kDa). (C) pH dependency of recombinant proEbCtL. The proEbCtL activity was assayed against Z-FR-AMC, Z-LLE-AMC, sub-AAF- AMC. The points show the average values of 3 independent experiments.
In order to determine the evolutionary relationship of EbCtL with other cathepsins, a comprehensive phylogenetic tree was constructed with the other cathepsins amino acid sequences [Fig. 3]. Based on a comprehensive phylogenetic analysis, cathepsins classified into three evolutionary brace, A, B and C. Brace A includes cysteine protease divided into two subgroup including cathepsin L subfamily and cathepsin B subfamily. Brace B includes Aspartic protease.
Brace C includes serine protease. EbCtL have more to do with the cathepsin L subfamily than cathepsin B subfamily.
3. Enzymatic characterization of recombinant proEbCtL
The recombinant proEbCtL was overexpressed in E. coli strain BL21(DE3) as a 55 kDa fusion protein by using glutathione S-ransferase (GST) tag in pGEX-4T-1 vector. The overproduced soluble GST-tag fusion protein (proEbCtL) was then applied to glutathione-Sepharose 4B column chromatography, and the proEbCtL fusion protein band had a high purity. but It seems the processing various band [Fig. 4A and B].
Due to cut GST-tag from proEbCtL fusion protein during GST purification, we can see processing various band. But its may not reflect in vivo activity.
A fluorogenic substrate Z-FR-AMC, Z-LLE-AMC and Suc-AAF-AMC were used for assessing the recombinant proEbCtL protein activity.
The optimum pH that make proEbCtL has high activity for respective substrates was determined to be 8 (Z-FR-AMC), 9 (Suc-AAF-AMC), 9.5 (Z-LLE-AMC) [Fig. 4C]. On the other hand, Z-RR-AMC was efficiently hydrolyzed by only
cathepsin B (Barrett & Kirschke, 1981), both cathepsin B and L were shown to efficiently hydrolyze Z-FR-AMC derivatives in this study.
Extraordinarily, the high activity was showed in Z-LLE-AMC and Suc-AAF-AMC. Z-LLE-AMC is
fluorogenic substrate for the
peptidylglutamyl-peptide hydrolysing (caspase-like) activity of the proteasome (Wang et al., 2015). And Suc-AAF-AMC is a highly sensitive fluorogenic chymotrypsin substrate (Kisselev, Akopian, Castillo,
& Goldberg, 1999).
Substrate specificity assays showed that proEbCtL protein was low activated on Bz-RGFFP-4MeOHβ NA and MeOSuC-AAPV-AMC. On the other hand, Z-LLE-AMC and Suc-AAF-AMC showed a higher activity than Z-FR-AMC <Table 2>.
<Table 2> Substrate specificity of Eptatretus burgeri cathepsin L
The proEbCtL activity was reduced by the antipain (50% or less compared to Z-FR-AMC, and 70% or less compared to Suc-AAF-AMC), chymostain (46% or less compared to Z-FR-AMC and Z-LLE-AMC, and 72% or less compared to Suc-AAF-AMC), leupeptin (52% or less compared to Z-LLE-AMC and 78% or less compared to Suc-AAF-AMC) <Table 3>.
<Table 3> Effect of various inhibitors on proEbCtL activity
But the specificity of influence among these substrates, dependence of pH, and enzyme inhibition with GST-fused recombinant enzyme that we analyzed in vitromay not reflect that in vivo (Xu, Nakajima, Suzuki, & Yamaguchi, 2002).
In addition, The enzymatic activity of proEbCtL was inactivated by all used metal ions (ZnSO4, CuSO4, MgSO4, HgCl2, CaCl2, KCl and CoCl2), and some detergents <Table 4>. And the enzyme activity using an substrate Suc-AAF-AMC was inactivated by all detergents, but that using Z-LLE-AMC was activated in detergents except for SDS, and that using Z-FR-AMC was activated 1mM TritonX-100 and 1mM Brij35 <Table 4>.
The results presented in this study provides valuable information about the Inshore hagfish cathepsin L.
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Received : 21 March, 2016 Revised : 17 May, 2016 Accepted : 30 May, 2016
