Download Striped murrel S1 family serine protease: immune

Biologia 69/8: 1065—1078, 2014
Section Cellular and Molecular Biology
DOI: 10.2478/s11756-014-0410-8
Striped murrel S1 family serine protease: immune characterization,
antibacterial property and enzyme activities
Jesu Arockiaraj1*, Rajesh Palanisamy1, Venkatesh Kumaresan1, Prasanth Bhatt1,
Mukesh Kumar Chaurasia1, Marimuthu Kasi2, Mukesh Pasupuleti3 & Annie J. Gnanam4
1
Division of Fisheries Biotechnology & Molecular Biology, Department of Biotechnology, Faculty of Science and Humanities,
SRM University, Kattankulathur – 603 203, Chennai, Tamil Nadu, India; e-mail: [email protected]
2
Department of Biotechnology, Faculty of Applied Sciences, AIMST University, Semeling Bedong, 08100 Bedong, Kedah,
Malaysia
3
Lab PCN 206, Microbiology Division, CSIR-Central Drug Research Institute, B.S. 10/1, Sector 10, Jankipuram Extension,
Sitapur Road, Lucknow – 226 031, Uttar Pradesh, India
4
Institute for Cellular and Molecular Biology, The University of Texas at Austin, 1 University Station A4800, Austin, TX
78712, USA
Abstract: We report a molecular characterization of S1 family serine protease (SP-1) from snakehead murrel (or called
striped murrel) Channa striatus (Cs). CsSP-1 polypeptide contained a catalytic core domain (otherwise known as serine
protease trypsin domain) between H20 and I237 along with a catalytic triad at H61 , D104 and S197 . Phylogenetic analysis
confirmed that CsSP-1 belongs to serine protease S1 family. The tertiary structure showed that CsSP-1 contains 14 βsheets as 2 separate β-barrels (the first β-barrel consists of 8 β-sheets in the N-terminal region and the second β-barrel
consists of 6 β-sheets in the C-terminal region) and 3 α-helical regions. Significantly (P < 0.05) the highest CsSP-1 mRNA
expression was observed in intestine, liver and kidney, moderate expression was seen in spleen, head kidney, skin and
blood, and the lowest one in brain, gill, muscle and heart. Further, the expression was induced in intestine with fungus
Aphanomyces invadans and bacteria Aeromonas hydrophila. The recombinant CsSP-1 protein showed antibacterial activity
against both gram-negative and gram-positive bacteria. The optimum CsSP-1 enzyme activity against the substrate casein
was determined at 8 mM casein concentration. Moreover, the activity was highly influenced by 5 mM phenyl-methylsulfonyl fluoride followed by ethylenediaminetetraacetic acid, 4-(2-aminoethyl)benzenesulfonylfluoride hydrochloride and
calpain inhibitor I. The CsSP-1 enzyme exhibited the highest activity at pH 7.5 and temperature 35 ◦C. The overall results
showed the potential involvement of CsSP-1 in the immune system of murrels. However, further research is necessary to
study the mechanism of implicit trypsin association in the defence process.
Key words: murrel; serine protease; antimicrobial property; enzyme inhibitors; substrate specificity.
Abbreviations: AEBSF HCl, 4-(2-aminoethyl)benzenesulfonylfluoride hydrochloride; ALLN, calpain inhibitor I; CDD,
conserved domain database; CsSP-1, Channa striatus family 1 serine protease; EDTA, ethylenediaminetetraacetic acid; EUS,
epizootic ulcerative syndrome; IPTG, isopropyl-β-thiogalactopyranoside; LB, Luria-Bertani; MBP, maltose-binding protein;
ORF, open reading frame; PBS, phosphate buffer saline; qRT-PCR, quantitative real-time polymerase chain reaction; SP,
serine protease.
Introduction
Striped murrel (or called snakehead fish) Channa striatus, an air breathing freshwater fish, belongs to the
family Channidae (otherwise known as Ophiocephalidae). This species exists as a good food fish due to
its high quality flesh in many parts of India. It procures high market demand due to its low fat content,
a few intramuscular spines and high medicinal properties. It has long been commercially cultured in many
countries from Asia Pacific region. Epizootic ulcerative syndrome (EUS) is a major limiting factor of this
cultivable species. EUS is mainly caused by a fungus Aphanomyces invadans; once the primary infection occurred by the fungus, the secondary infection
colonizes due to other disease causing bacteria including Aeromonas hydrophila, Flavobacterium sp., Bacillus
mycoides, Pseudomonas fluorescens, etc. (Arockiaraj et
al. 2003; Dhanaraj et al. 2008). The syndrome causes
skin lesions in the initial stages, which progress in size
until eventually a circular to oval deep haemorrhagic
ulcer exposing the skeletal musculature is visible. This
disease had a serious impact on C. striatus and also
other commercially important cultivable freshwater and
* Corresponding author
c 2014 Institute of Molecular Biology, Slovak Academy of Sciences
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brackish water fishes all over the world resulting in a
considerable amount of economic loss to the fish farmers (Haniffa et al. 1999, 2002; Marimuthu et al. 1999;
Arthimanju et al. 2011). Hence, it is necessary to investigate the immune system of murrel and characterize
defence genes to regulate the disease at the molecular
level.
Magnadottir (2006), Subramanian et al (2007,
2008) and Uribe et al (2011) reported that the teleosts
innate immune responses include humoral and cellular receptor molecules that are soluble in plasma and
other body fluids. Many protease inhibitors are present
in the serum and other body fluids of teleosts (Bowden
et al. 1997). The exact role of the protease inhibitors is
to regulate the body fluid homeostasis. These molecules
are connected with acute phase reactions and immunity
against pathogenic organisms that produce proteolytic
enzymes (Magnadottir 2010).
Serine proteases (SPs) are proteolytic enzymes,
which rank among the most biologically vital and
widely distributed enzyme. They have been categorized into non-clip domain SPs (or single domain) and
clip domain SPs based on the presence of the domain.
These SPs are multigene encoded protein family sharing a common catalytic mechanism along with structural characteristic features of three conserved amino
acid residues, histidine, aspartic acid and serine, which
constitute the catalytic triad (Perona & Craik 1995;
Piao et al. 2007). Serine protease was named after the
reactive serine residue present in the active site that is
essential for the function of the enzyme. During activation, some SPs act as upstream activating factors, while
others function as downstream factors (Neurath 1984).
SPs exert many physiological functions in mammals, including digestion, blood coagulation, fertilization, embryonic development, fibrinolysis, pro-enzyme,
pro-hormone and complement activation, as well as
they participate in cellular and humoral immune mechanisms (O’Brien & McVey 1993; Rawlings & Barrett
1993; Krem & Di Cera 2002). In invertebrates, it plays
a vital role in signal transduction, embryonic development and immune responses (Anderson 1998; Herrero
et al. 2005; O’Connell et al. 2006).
In the proteolytic cascade, SPs are involved in
many biological processes of aquatic organisms (Simonet et al. 2002; Laskowski & Kato 1980), for example, haemolymph coagulation in horseshoe crab Limulus
polyphemus (Bennett & Ratnoff 1972; Muta & Iwanaga
1996) and activating the prophenoloxidase activating
system in arthropods (Jiang et al. 2003). So far, many
reports have been published on clip domain SPs from
aquatic species (Buda et al. 2005; Jimenez-Vega et al.
2005; Rattanachai et al. 2005; Lin et al. 2006; Amparyup et al. 2007; Charoensapsri et al. 2009; Ren et
al. 2009; Cui et al. 2010; Tassanakajon et al. 2010;
Qin et al. 2010; Vaseeharan et al. 2011) and only a
few on non-clip domain SPs from aquatic species including Penaeus vannamei (Jimenez-Vega et al. 2005),
Panulirus argus (Levine et al. 2001), Fenneropenaeus
chinensis (Ren et al. 2011) and Pacifastacus leniuscu-
lus (Hernandez-Cortes et al. 1999). It is interesting to
note that all the SPs reported in aquatic organisms are
from invertebrates only. There is a serious lack of information on SPs from fish, especially freshwater fish.
Kong et al. (2009) reported a D/adipsin and kallikreinlike serine protease from a marine flatfish (olive flounder) Paralichthys olivaceus. Hence, it is absolutely on
demand to provide sound information on single domain
or non-clip domain SPs from fish, especially freshwater
fish and/or striped murrels.
This study was therefore aimed: (1) to identify C.
striatus serine protease-1 (named as CsSP-1) from the
established C. striatus cDNA library using genome sequencing FLXTM (GS FLXTM ) technology; (2) to study
the bioinformatics characterization including evolutionary analysis of the identified CsSP-1; (3) to investigate the tissue distribution and mRNA expression of
CsSP-1 infected with pathogenic microbes; and (4) to
clone the complete coding sequence of CsSP-1 in an Escherichia coli expression vector system with further purifying the recombinant protein to study the antibacterial, immunological and biochemical properties. These
observations would provide new insights for further research on biological characterization of this potentially
important serine protease-1 in murrel.
Material and methods
C. striatus cDNA library construction and CsSP-1 identification
A full-length cDNA of CsSP-1 was identified from the
established C. striatus cDNA library by the genome sequence FLXTM (GS-FLXTM ) technology. Briefly, total
RNA was isolated using Tri ReagentTM (Life Technologies) from the tissue pool including spleen, liver, kidney, muscle and gills of healthy C. striatus. The mRNA
was then isolated by using mRNA isolation kit (Miltenyi Biotech). The first strand cDNA synthesis and normalization were carried out with CloneMinerTM cDNA library construction kit (Invitrogen) and Trimmer Direct
Kit: cDNA Normalization Kit (BioCat GmbH). Thereafter,
the GS-FLXTM sequencing of C. striatus cDNA was sequenced according to the manufacturer’s protocol (Roche).
The raw data were processed with the Roche quality
control pipeline using the default settings. Seqclean software (http://compbio.dfci.harvard.edu/tgi/software/) was
used to screen for and remove normalization adaptor sequences, homopolymers and reads shorter than 40 bp
prior to assembly. Further, the sequences were subjected
to assembly using MIRA (ver. 3.2.0) software (Chevreux
et al. 2004) into full-length cDNAs. From the established cDNA library of C. striatus sequence database,
a serine protease-1 gene was identified through BLAST
(Altschul et al. 1990) annotation program on the NCBI
(http://www.blast2go.com/b2ghome).
Bioinformatics characterization of CsSP-1
The full-length CsSP-1 sequence was compared with other
sequences available in the NCBI database and the similarities were analyzed. The open reading frame (ORF) and
amino acid sequence of CsSP-1 were obtained by using
DNAssist 2.2 (Patterton & Graves 2000). Characteristic domains or motifs were identified using the PROSITE profile
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Murrel S1 family serine protease
database (De Castro et al. 2006). The N-terminal transmembrane sequence was determined by DAS transmembrane
prediction program (http://www.sbc.su.se/∼miklos/DAS).
Signal peptide analysis was conducted using the SignalP
program (http://www.cbs.dtu.dk). Pair-wise and multiple
sequence alignment was constructed with Clustal-Omega
program (http://www.ebi.ac.uk/Tools/msa/clustalo/) and
edited using BioEdit 7.1.3.0 (Hall et al. 2011). The phylogenetic tree was constructed using the neighbour-joining
method implemented in the MEGA 5 package (Kumar et
al. 2004).
Three-dimensional structure of CsSP-1 protein was
predicted by I-TASSER program (http://zhanglab.ccmb.
med.umich.edu/I-TASSER) that generates the full-length
model of proteins by excising continuous fragments from
threading alignments and then reassembling them using
replica-exchanged Monte Carlo simulations (Zhang 2008;
Roy et al. 2010). To validate the obtained tertiary structures, the Ramachandran plot analysis was also applied
(http://mordred.bioc.cam.ac.uk/∼rapper/rampage.php).
Experimental fish
Healthy C. striatus (average body weight of 50 g) were obtained from Tamirabarani river fed systems of Tirunelveli
District, Tamil Nadu, India. They were transported to Division of Fisheries Biotechnology & Molecular Biology (SRM
University) in oxygenated polythene bags. They were maintained in rectangular plastic containers (100 L) with aerated
and filtered freshwater at 29 ± 2 ◦C in the laboratory. The
fishes were acclimatized for a week before being injected
to immune stimulants. A maximum of five C. striatus per
container were maintained during the experiment.
Collection of fish tissues for tissue-specific CsSP-1 mRNA
expression
C. striatus liver, heart, spleen, intestine, kidney, head kidney, muscle, skin, gill and brain tissues were collected from
three healthy fishes to analyze the CsSP-1 mRNA expression by quantitative real time (qRT-PCR). Moreover, blood
(0.5–1 mL/fish) was also collected from the same individuals
from the caudal fin using a sterilized syringe and immediately centrifuged at 4,000×g for 5 min at 4 ◦C. The supernatant was removed and the blood cells were collected. All
the collected tissues were immediately snap-frozen in liquid nitrogen and stored at –80 ◦C until the total RNA was
extracted.
CsSP-1 mRNA expression after infected with fungus and
bacteria
To induce the mRNA expression level in intestine, the fishes
were injected with fungus A. invadans and bacteria A. hydrophila as reported in our earlier studies (Abirami et al.
2013; Arockiaraj et al. 2013a; Kumaresan et al. 2014).
For fungus induced mRNA expression analysis, the fish
were injected with A. invadans (102 spores). A. invadans
were isolated from the EUS infected C. striatus muscle sample. The infected muscle sample was taken from the EUS
infected C. striatus and were placed in a Petri dish of algal boost GP medium with 100 units/mL penicillin G and
100 µg/mL streptomycin. The nutrient medium was incubated at 25 ◦C for 12 h and examined under a binocular microscope (Nikon-Eclipse E400 microscope, Germany). The
fungal species were identified according to the description
of Caster & Cole (1990) using potato dextrose agar and
Czapek Dox agar (Himedia, Mumbai).
For bacterial challenge, the fish were injected intraperitonealy with A. hydrophila (5×106 colony forming units
1067
per mL) suspended in 1X phosphate buffer saline (PBS)
(100 µL/fish). A. hydrophila were also isolated and identified from the muscle sample of EUS infected C. striatus as
described by Dhanaraj et al (2008).
Tissues were collected before (0 h), and after injection
(12, 24, 36, 48 and 72 h) and were immediately snap-frozen
in liquid nitrogen and stored at –80 ◦C until total RNA was
isolated. Fish physiological saline (or called PBS) (1X) was
prepared and served as control (100 µL/fish). All samples
were analyzed in three duplications and the results were
expressed as relative fold of one sample as mean ± standard
deviation as reported by Livak & Schmittgenm (2001).
Isolation of total RNA and conversion of cDNA
Total RNA was isolated from the collected tissues using
Tri ReagentTM (Life Technologies), according to the manufacture’s protocol with slight modifications (Arockiaraj et
al. 2013a,b). Using 2.5 µg of RNA, first strand cDNA was
synthesized by SuperScript VILOTM cDNA Synthesis Kit
(Life Technologies). The resulting cDNA solution was stored
at –20 ◦C for further analysis.
mRNA expression of CsSP-1 analysis using quantitative
real-time PCR (qRT-PCR)
An expression level of CsSP-1 in the liver, heart, spleen,
intestine, kidney, head kidney, muscle, skin, gill, blood and
brain were determined by qRT-PCR. Real-time analysis was
carried out on a BIO-RAD CFX384 Touch Real-Time PCR
Detection System. Reactions were performed in a 20 µL volume containing cDNA 4 µL (10 ng), 10 µL of Fast SYBR
Green Master Mix, 0.5 µL of each primer (20 pmol/µL)
(CsSP-1 F1: 5’-AAG TCC AAG GCC AGC ATA TT-3’
and CsSP-1 R2: 5’-GGG AAT CAG CTC CAA GTA CAA3’) and 5 µL dH2 O. The qRT-PCR had following cycle: 1
cycle of 95 ◦C for 10 s, followed by 35 cycles of 95 ◦C for 5 s,
58 ◦C for 10 s and 72 ◦C for 20 s and finally 1 cycle of 95 ◦C
for 15 s, 60 ◦C for 30 s and 95 ◦C for 15 s. The same qRTPCR cycle profile was used for the internal control gene,
β-actin. The internal control primers (β-actin F3: 5’-TCT
TCC AGC CTT CCT TCC TTG GTA-3’ and β-actin R4:
5’-GAC GTC GCA CTT CAT GAT GCT GTT-3’) were
designed from the β-actin of C. striatus (GenBank Accession No.: EU570219). After the PCR program, data were
analyzed with BIO-RAD software. To maintain consistency,
the baseline was set automatically by the software. The commethod) was used to analyze
parative CT method (2−∆∆C
T
the expression level of CsSP-1 (Livak & Schmittgenm 2001).
Cloning of CsSP-1 coding sequence
All the cloning experiments were carried out according to
our earlier protocol (Arockiaraj et al. 2011, 2012a,b). CsSP1 gene specific primers (F5: 5’-(GA)3 GAATTC ATG GCA
AGA GGC TTT GTA CTT CTG C EcoRI-3’ and R6: 5’(GA)3 AAGCTT GAT TGC ACA AGT CTT AAA CAG
CAA A HindIII-3’) were designed with the corresponding
restriction enzyme sites for EcoRI and HindIII in forward
and reverse, respectively. Using plasmid DNA of CsSP-1
as a template along with Taq DNA polymerase (Invitrogen BioServices India Pvt. Ltd., Bangalore, India), PCR
was carried out to amplify the coding sequence. The PCR
product was purified using the QIAquick Gel Extraction Kit
(Qiagen India Pvt. Ltd., New Delhi, India). After PCR, the
amplified product was purified by using the GeneJETTM
(Thermo Scientific). Further, the cloned products were sequenced using the ABI Prism-Bigdye Terminator Cycle Sequencing Ready Reaction kit and analyzed using an ABI
3730 sequencer. Then, both insert and vector were digested
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with the respective restriction enzymes. The ligated product was transformed into XL1-Blue cells and the correct recombinant product was transformed into competent E. coli
BL21 (DE3) cells for protein expression.
Recombinant CsSP-1 protein: expression and purification
Transformed E. coli BL21 (DE3) cells were incubated in
ampicillin (100 µg/mL) Luria-Bertani (LB) broth overnight.
This culture was then used to inoculate 100 mL of LB broth
in 0.2% glucose-rich medium with ampicillin at 37 ◦C until the cell density reached 0.6 at 600 nm. E. coli BL21
(DE3) harbouring pMAL-c2x-CsSP-1 was induced for overexpression with 1 mM isopropyl-β-thiogalactopyranoside
(IPTG) and incubated at 12 ◦C for 8 h. Cells were harvested
by centrifugation (4,000×g for 20 min at 4 ◦C). Then the
cells were re-suspended in column buffer (Tris-HCl, pH 7.4,
200 mM NaCl) and frozen at –20 ◦C overnight. After thawing
on ice, cells were disrupted by sonication. The crude CsSP1 fusion protein fused with maltose-binding protein (MBP)
was purified using pMALTM protein fusion and purification
system protocol (New England Biolabs UK Ltd., United
Kingdom). Further, DEAE-SepharoseTM ion exchange chromatography method was used to purify the recombinant
CsSP-1 protein away from MBP and the protease, and we
also stipulated an additional purification step for removing
trace contaminants according to the manufacture’s protocol
(New England Biolabs UK Ltd., United Kingdom). Then
the purity of the expressed enzyme was verified by 12%
SDS-PAGE and the molecular weight of target protein was
evaluated using protein molecular weight standards. Proteins were visualized by staining with 0.05% coomassie blue
R-250. The concentrations of purified proteins were determined by the method of Bradford (1976) using bovine serum
albumin as the standard. The purified protein was kept at
–80 ◦C until determination of molecular functional activities.
Antibacterial property of recombinant CsSP-1 protein
The assay was performed as described by Casteels et al
(1993), Rathinakumar et al (2009) and Song et al (2013).
The activity of the purified recombinant CsSP-1 protein was
examined against various gram-negative (A. hydrophila, E.
coli, Edwardsiella tarda, Vibrio parahaemolyticus, Vibrio alginolyticus and Vibrio harveyi) and gram-positive (Bacillus
subtilis, Streptococcus iniae, Staphylococcus aureus, Enterococcus faecium and Lactococcus lactis) bacteria using liquid
growth inhibition assays. In brief, the bacteria were diluted
using 50 mM Tris-HCl buffer (pH 8.0) to obtain the bacterial culture of 103 colony forming units per /mL. In a 96well microplate, 50 µL recombinant CsSP-1 protein along
with equal volume of 50 mM Tris-HCl buffer (pH 8.0) was
taken. Then, 50 µL bacterial cultures were loaded in the
wells. After a gentle mixing, the microplate were incubated
at 37 ◦C for 2 h. Then, 150 µL nutrient medium were added
and further incubated overnight at 37 ◦C. Finally, the optical density was determined at 600 nm using a microplate
reader (Thermo Scientific). The wells with 50 µL Tris-HCl
buffer were used as control. The study was conducted in
three replicate groups.
Protease activity assay
The assay was conducted as explained by Johnvesly & Naik
(2002) with slight changes. The protease activity analysis
was performed by adding various concentrations (0, 2, 4,
6, 8, 10, 12, 14, 16, 18 and 20 mM) of 900 µL casein (in
100 mM sodium phosphate, pH 7.4) into 100 µL of purified
CsSP-1 enzyme (1 µg/µL). The mixture was incubated at
35 ◦C for 20 min. The reaction was stopped by the adding
3 mL 5% trichloroacetic acid. The activity was measured at
400 nm using a spectrophotometer.
Protease inhibitor activity
The inhibitory assay was performed as described by
Hernández-Martínez et al. (2011) with slight modification.
Four different inhibitors, namely 5 mM phenyl-methylsulfonyl fluoride (PMSF, active site inhibitor), 5 mM
ethylenediaminetetraacetic acid (EDTA, metalloprotease
inhibitor), 5 mM 4-(2-aminoethyl)benzenesulfonylfluoride
hydrochloride (AEBSF HCl) and calpain inhibitor I (ALLN)
were used. The inhibitors were individually blended with
recombinant CsSP-1 enzyme and incubated at 35 ◦C for
20 min. Subsequently, the substrate was added, and the solution was further incubated for 40 min. Casein was used
as control. The inhibitory activity was measured at optical
density 400 nm. The inhibitory activity was calculated as
described by Hernández-Martínez et al (2011).
Effect of temperature on the stability of CsSP-1 enzyme
The assay was performed and calculated as described by
Silva-Lopez et al. (2005) with slight modifications. The assay was conducted to find out the temperature tolerance of
recombinant CsSP-1 enzyme. Briefly, the recombinant protein were incubated for 10 min at various (10, 20, 25, 30, 35,
40, 45, 50, 60 and 70) temperatures ( ◦C) in 1 X PBS buffer
(pH 7.5). The assay was performed in three replicates.
pH tolerance of CsSP-1 enzyme
The assay was conducted following the methodology of Cho
et al. (2000) with slight modifications. This assay was conducted to check the tolerance of CsSP-1 enzyme at various
pH (4, 5, 6, 6.5, 7, 7.5, 8, 8.5, 9, 9.5, 10, 11, 12, 13 and 14).
The recombinant product was incubated in 50 mM buffer
for 3 h at 25 ◦C. The acetate, phosphate and glycine-NaOH
buffers were used to adjust the pH. The assay was performed
in three replicates.
Statistical analysis
For comparison of CsSP-1 relative gene expression, antibacterial activity, proteolytic activity, proteolytic inhibitory activity, pH and temperature tolerance, statistical analysis
was performed using the one-way ANOVA and mean comparisons were performed by Tukey’s Multiple Range Test
using SPSS 11.5 software at the 5% significant level.
Results
Characterization of CsSP-1 sequence using in silico
tools
CsSP-1 was identified from the constructed cDNA library of C. striatus using GS-FLXTM technology. The
obtained complete sequence of CsSP-1 was submitted to GenBank/EMBL database under the accession
number HG001458. The full length nucleotide sequence
CsSP-1 contains 735 bp with the ORF of 732 bp. The
ORF is capable of encoding a polypeptide of 244 amino
acid residues with an estimated molecular weight of
26 kDa and a predicted theoretical pI of 8.8. The protein consists of a putative signal sequence between M1
and G18 ; a cleavage site being predicted between the
residues G18 and S19 . Moreover, the domain analysis
showed that CsSP-1 polypeptide contains a long single catalytic core domain (otherwise called serine protease trypsin domain) between H20 and I237 (Fig. 1).
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Fig. 1. Nucleotide and deduced amino acid sequences of CsSP-1 gene (GenBank Accession Number HG001458). The nucleotides and
their corresponding amino acid residues are numbered along the right margin. A putative signal peptide region (1-18 amino acid) at the
N-terminal is underlined by a double arrow, and the cleavage site (between 18 and 19 amino acid) is indicated by a red colour vertical
arrow. The catalytic domain (serine protease trypsin domain) between residues 20 and 237 is highlighted in grey shade. Residues
highlighted in red colour circles (H61 , D104 , S197 ) are the catalytic triad of CsSP-1. The boxed sequences represent the potential
attachment sites for the N-linked carbohydrate chain. The asterisk (*) indicates the stop codon.
Further analysis demonstrated that the catalytic core
domain possesses a catalytic triad: H61 , D104 and S197 .
In addition to these, the polypeptide contains 13 high
confidently predicted common motifs including 5 Nmyristoylation sites, 4 N-glycosylation sites, 1 casein
kinase II phosphorylation site, 2 protein kinase C phosphorylation sites and 1 amidation site (data not shown).
Conserved domain database (CDD) analysis showed
that the CsSP-1 also contains a substrate binding site
at G191 , S212 and G215 . Further, the analysis observed 8
cysteine residues (with four pairs of disulphide bonds)
in the serine protease trypsin domain at C46=C62,
C136=C203, C167=C182 and C193=C218.
The bioinformatics analysis showed that the deduced amino acid sequence CsSP-1 shared the highest sequence similarity with SP-1 from olive flounder Paralichthys olivaceus (82%) and the lowest with
subtilisin-like protease of Rattus sp. The other homologous sequences include complement factor D precursor, granzyme, neutrophill elastase precursor, mast cell
protease precursor, serine protease precursor and duodenase precursor, taken for analysis, exhibited not less
than 50% similarity with CsSP-1 (data not shown).
Multiple sequence alignment of CsSP-1 showed
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Fig. 2. Multiple sequence alignment of deduced amino acid sequences of CsSP-1 with other homologues. Sequences for alignment were
taken as follows: PoSP-1, SP-1 from olive flounderParalichthys olivaceus (BAD91574); OmCFDP, complement factor D precursor from
rainbow trout Oncorhynchus mykiss (NP 001233275); OnGZM-1, granzyme-1 from Nile tilapia Oreochromis niloticusis (AAY23199);
HsSP-57P, SP-57 precursor from human Homo sapiens (NP 999875). The gaps (-) were introduced to maximize the similarity. The
conserved catalytic triad (H61 , D104 and S197 ) is indicated by red shade followed by an asterisk. The conserved amino acid residues are
shown in black shade. The number sign (#) denotes the substrate binding sites (G191 , S212 and G215 ). The eight conserved cysteine
residues are highlighted in pink shade and their disulphide linkages are signified by black lines.
that the catalytic triad H61 , D104 and S197 and the 8
cysteine residues are highly conserved among the homologous sequences considered for analysis. Further,
the alignment demonstrated three conserved motifs
named ‘YMAS’ (34–37), ‘CGG’ (46–48) and ‘TAAHC’
(58–62) around the catalytic histidine residue (H61 ) in
the catalytic domain. Similarly, another two conserved
motifs ‘NDI’ (103–105) and ‘MLL’ (106–107) around
the conserved catalytic aspartic acid residue (D104 ).
Moreover, the conserved reactive serine residue (Ser197 )
from the catalytic triad was also found within a motif
called ‘GDSGGPLVCC region’ (195–204) (Fig. 2). All
the above mentioned residues are conserved among fish
and mammals (including human beings).
A bootstrapped phylogenetic tree of CsSP-1 was
constructed using the neighbour-joining method. The
tree revealed that CsSP-1 clustered together with SP1 from P. olivaceus, formed a sister clade with other
serine protease from fish and finally clustered together
with amphibian serine protease. This analysis indicates
that the identified and bioinformatically characterized
CsSP-1 from the cDNA library of C. striatus belongs to
serine protease S1 family. Subtilisin-like protease from
Rattus sp. was set as an outgroup. The genetic distance
of the tree is 0.2 (Fig. 3).
The three-dimensional protein model of CsSP-1
was constructed by I-TASSER online structure prediction tool. The obtained tertiary structure was validated using the Ramachandran plot analysis (data not
shown). The protein model seems to be unstructured
due to high proportion of random coils (61.8%). The
high proportion of random coil regions in CsSP-1 protein may be due to presence of high quantity of small
amino acid residues including glycine (10%), leucine
(9%), valine (9%) and alanine (7%). The tertiary protein structure of CsSP-1 consists of 14 β-sheets as 2
separate β-barrels (the first β-barrel consists of 8 βsheets in the N-terminal region and the second β-barrel
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Fig. 3. Phylogenetic analysis of CsSP-1 protein using neighbour-joining method. The tree is based on the alignment corresponding
full-length amino acid sequences. The sequences were aligned using the Clustal-Omega program and the tree was constructed in MEGA
(ver. 5). The numbers at the branches denote the bootstrap majority consensus values on 1,000 replicates. The details for abbreviation, species name, gene description and GenBank accession numbers are as follows: PoSP-1, Paralichthys olivaceus serine protease-1
(BAD91574); ElCFDP, Esox lucius complement factor D precursor (ACO14314); OmCFDP, Oncorhynchus mykiss complement factor D precursor (NP 001233275); SsCFDP, Salmo salar complement factor D precursor (ACI69308); XlLOC733303P, Xenopus laevis
LOC733303 protein (AAH99292); XtNEP, Xenopus tropcalis neutrophill elastase precursor (NP 001006847); OaMCP-3P, Ovis aries
mast cell protease-3 precursor (NP 001009411); OaMCP-1AP, Ovis aries mast cell protease-1A precursor (NP 001009472); BtDDN-1,
Bos taurus duodenase-1 precursor (DAA17498); BtGZMB, Bos taurus granzyme B (ACI15823); TcSP-57, Tupaia chinensis serine
protease-57 (ELV09314); MdSP-57, Myotis davidii serine protease-57 (ELK29413); MmSP-57P, Mus musculus serine protease-57 precursor (NP 001036175); HsSP-57P, Homo sapiens serine protease-57 precursor (NP 999875); RSLP, Rattus sp. subtilisin like protease
(AAB22663).
is formed by 6 β-sheets in the C-terminal region) and
3 α-helical regions. The catalytic active sites are situated in a helix (His61 ) and the coil region (Asp104 and
Ser197 ) (Fig. 4).
mRNA expression of CsSP-1
The tissue distribution of CsSP-1 mRNA was analyzed
in qRT-PCR using gene specific primers. cDNA synthesized from different tissues including liver, heart, spleen,
intestine, kidney, head kidney, muscle, skin, gill, blood
and brain were used as a template for the analysis. Significantly (P<0.05) the highest CsSP-1 mRNA expression was observed in intestine, liver and kidney, moderate expression was seen in spleen, head kidney, skin
and blood, while the lowest one in brain, gill, muscle
and heart (Fig. 5a).
Based on the tissue specific mRNA expression results, CsSP-1 gene expression in murrel was inducted
in intestine following challenge by fungus A. invadans
and bacteria A. hydrophila. In A. invadans injected C.
striatus, the CsSP-1 mRNA expression was significantly
(P < 0.05) increased until 48 h post injection (24-fold),
when compared to the control group (Fig. 5b), and the
expression was dropped at 72 h post injection. In A.
hydrophila challenged individuals the mRNA expression
was significantly (P < 0.05) the highest at 24 h post injection (6-fold), compared to the control (Fig. 5c), and
again there was a slight increase at 48 h post injection
and then the expression dropped at 72 h post injection.
Recombinant CsSP-1 protein purification and activity
analysis
CsSP-1 coding sequence was cloned into pMAL-c2x vector. Further, the expression of CsSP-1 was induced with
IPTG in an E. coli BL 21 (DE3) cells. The recombinant
CsSP-1 protein was isolated from the IPTG-induced E.
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Fig. 4. The cartoon representation of CsSP-1 tertiary structure using I-TASSER program. The random coils, β-sheets and α-helices
are showed as blue, green and red cartoon models, respectively. The catalytic triad and the substrate-binding site in the serine protease
trypsin domain are indicated by stick and ball models, respectively.
coli cells. The SDS-PAGE analysis showed the recombinant CsSP-1 protein along with the MBP. The molecular size of the recombinant CsSP-1 protein along with
the MBP was 68.5 kDa (42.5 kDa for MBP and 26 kDa
for CsSP-1). Further, the recombinant CsSP-1 protein
was isolated from the maltose-binding fusion protein
using DEAE-Sepharose ion exchange chromatography.
After the removal of the fusion protein, the purified
recombinant protein (molecular weight 26 kDa) gave
a major single band on 12% SDS-PAGE (data not
shown).
The antibacterial property of the recombinant
CsSP-1 against various gram-negative and gram-positive bacteria was examined by liquid growth inhibition method. The recombinant CsSP-1 showed antibacterial activity against both gram-negative and grampositive bacteria (Fig. 6). The strong inhibition was
noticed against A. hydrophila followed by E. coli, V.
alginolyticus, E. tarda, V. parahaemolyticus, V. harveyi, S. aureus, B. subtilis, S. iniae, L. lactis and E.
faecium. It is interesting to note that the recombinant CsSP-1 protein significantly (P < 0.05) inhibited the growth of gram-negative bacteria in comparison with Gram-positive bacteria. The protease activity
of purified CsSP-1 enzyme was measured using various concentrations of casein as a substrate. The optimum CsSP-1 enzyme activity (64 units) was observed in
8 mM substrate used. Further, the usage of higher substrate concentration against CsSP-1 yielded no higher
enzyme activity (Fig. 7). The protease activity of the
purified CsSP-1 enzyme was highly influenced by PMSF
followed by EDTA, AEBSF HCl and ALLN (Fig. 8).
The thermal stability of the purified CsSP-1 enzyme
was determined against various temperatures. The enzyme had an optimum activity at 35 ◦C, and after then
the activity decreased (Fig. 9a). The enzyme activity
reached zero at 70 ◦C. Similarly, the pH tolerance of
purified CsSP-1 enzyme was measured against various
acidic and alkaline values of pH. The highest enzyme
activity was observed at pH 7.5. The enzyme activity
reached zero at pH 13 (Fig. 9b).
Discussion
Serine protease families are widely present in animals
and are involved in dietary protein digestion, antimicrobial peptide, haemolymph coagulation, melanin synthesis and activation of immune system in response
to pathogen detection (Iwanaga et al. 1998; Hoffmann
et al. 1999; Levashina et al. 1999; Gorman & Paskewitz 2001; Dana et al. 2005; Tang et al. 2006). In this
study, we reported a full-length CsSP-1 cDNA identified from the established C. striatus cDNA library using
GS-FLXTM technology. The bioinformatics analysis revealed that CsSP-1 contains a serine protease trypsin
domain along with a highly conserved catalytic triad
at H61 , D104 and S197 , which belongs to single domain
serine protease; hence it is predicted as a trypsin like
serine protease. Moreover, according to the available reports, the size of the single domain SPs are less than 300
amino acids; similarly, in this study also the reported
single domain CsSP-1 is 244 amino acids in length. Furthermore, the data from the results of protease activity
and inhibitor assays also confirmed that the reported
sequence from C. striatus is a trypsin like serine protease. Gao & Zhang (2009) reported that the serine
protease trypsin domain exhibits antimicrobial property, for example, trypsin like serine protease from amphioxus Branchiostoma belcheri scrubs the growth of
E. coli and V. parahaemolyticus.
The catalytic domain of CsSP-1 contains eight cysteine residues which are involved in the formation of
disulphide bridges. The numbers of cysteine residues
in the catalytic domain vary from species to species.
For example, six cysteine residues have been reported
from an invertebrate organism, crayfish (Titani et al.
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Murrel S1 family serine protease
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(a)
(b)
(c)
Fig. 5. Gene expression of CsSP-1 by qRT-PCR. (a) Tissue specific mRNA expression of CsSP-1 from different tissues C. striatus.
Data are given as a ratio to CsSP-1 mRNA expression in heart. (b,c) The time course of CsSP-1 mRNA expression in intestine at 0,
12, 24, 36, 48 and 72 h post-injection with fungus (A. invadans) and bacteria (A. hydrophila), respectively.
Fig. 6. Antimicrobial property of the recombinant CsSP-1 protein. The recombinant CsSP-1 protein was examined against various
gram-negative and gram-positive bacteria. The results are presented as minimal inhibition concentrations (MIC) of the recombinant
proteins.
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J. Arockiaraj et al.
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(a)
(b)
Fig. 7. Protease activity of purified CsSP-1 enzyme towards the
substrate casein. The purified enzyme was incubated with various
concentration of substrate at 35 ◦C for 20 min. The activity was
measured at 400 nm in spectrophotometer.
Fig. 9. The proteolytic activity of purified recombinant CsSP-1
enzyme at various temperature and pH. (a) The thermal stability of CsSP-1 enzyme incubated at various temperatures. (b) The
tolerance of CsSP-1 enzyme incubated at various pH. Data expressed here represent an average of three replicates ± standard
deviation.
Fig. 8. Protease activity of purified CsSP-1 enzyme against inhibitor. The substrate casein was used as the control. Protease activity of CsSP-1 enzyme was analysed against various inhibitors,
namely phenyl-methyl-sulfonyl fluoride (PMSF), ethylenediaminetetraacetic acid (EDTA), 4-(2-aminoethyl)benzenesulfonylfluoride hydrochloride (AEBSF HCl) and calpain inhibitor I
(ALLN). The bar shows the average values of three replicates.
Data given here mean the ratio of inhibitors to the substrate
(control) at absorbance measured at 400 nm.
1983) and twelve from human (Emi et al. 1986). Among
the four disulphide bonds from CsSP-1, the third disulfide bridge (Cys136 –Cys201 ) is essential for the structural features of serine proteases (Emi et al. 1986).
Similar results were observed in SPs from abalone
(Groppe & Morse 1986), shrimp (Klein et al. 1996), centipedes (Yang et al. 2012; Junli et al. 2013) and human
(Emi et al. 1986). But in some trypsin like SPs from
aquatic invertebrates the third disulfide bridge is missing, e.g., Pacifastacus leniusculus (Hernandez-Cortes et
al. 1999) and Astacus fluviatilis (Titani et al. 1983). Interestingly, two additional disulphide bridges at Cys22 –
Cys157 and Cys127 –Cys232 were reported in vertebrates
(Rawlings & Barrett 1994) to anchor the pro-peptide
to the protein core (Botos et al. 2000). These variations indicate the evolutionary importance of SPs, but
the exact role and pairing mechanism of these cysteine
residues are still unclear (Zhu et al. 2005).
Phylogenetic analysis showed that CsSP-1 has high
degree of similarity to trypsin like SP-1 from P. oli-
vaceus; hence it is possible to suggest that CsSP-1 belongs to the same serine protease S1 family. In addition,
these two sequences share 82% sequence similarity, several typical structural features including catalytic triad
and conserved eight cysteine residues. The phylogenetic analysis and multiple sequence alignment revealed
that CsSP-1 exhibited a significant similarity with various types of SPs including complementary factor, neutrophil elastase, granzymes and mast cell protease. It
is thus possible to suggest that SPs may be evolved
from a common ancestral gene which shared structural
similarities. After continuous and several gene replications each protease might have originated with different
functions.
The tertiary structure of CsSP-1 predicted using
I-TASSER tool contains 2 β-barrels: one at the Nterminal and another at the C-terminal end. Among
the three catalytic active-site residues, two (His61 and
Asp104 ) are located within the N-terminal β-barrel,
whereas the third residue (Ser197 ) is present at the Cterminal β-barrel by making an oxyanion hole with its
nucleophilic substitution in nature as reported by Page
et al. (2005), who pointed out that the two β-barrels
in the S1 family of serine proteases align asymmetrically in a classical Greek key formation, bringing the
catalytic residues together at their interface.
Tissue specific mRNA expression showed that the
CsSP-1 was highly expressed in intestine among the
eleven tissues tested. Maxwell & Enrico (2002) reported
that the single domain SPs like CsSP-1 is involved
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Murrel S1 family serine protease
in many physiological processes, especially in digestive
and degradative processes; this could be a possible reason why CsSP-1 is highly expressed in intestine. Rothman (1977) reported that the activation of some SPs
inhibits the degradation of pancreas, but it allows activity in the small intestine, where it gets activation by
entero-peptidase. Most of the physiological processes including digestion are regulated by proteases – produced
as inactive version activated by the upstream protease
(Shi et al. 2008). The mRNA expression after viral and
bacterial infection indicated that CsSP-1 is predominantly involved in the acute phase response of inflammation (Hwang et al. 2007). In the host organism, the
pathogenic microbes are recognized as foreign particles,
when pattern recognition receptors bind to the sugar
compound of the microbial cell wall, thus commencing
the activation of SPs for killing the pathogens. These
SPs are controlled by their inhibitors, so they are not
causing damage to the host organisms (Ashida & Sasaki
1994; Dieguez-Uribeondo & Cerenius 1998). Hence it is
possible to suggest that CsSP-1 may be participated in
the defence process against pathogenic infection including fungi and bacteria in murrels.
The CsSP-1 enzyme over-expression and purification was carried out using a pMAL-c2x expression vector system to study the antibacterial activity, protease
activity, protease inhibitor activity, thermal stability
and pH tolerance. Antibacterial activity studies showed
a wide range of bactericidal activity of CsSP-1 against
all the bacteria that were included in the analysis which
clearly explains the role of CsSP-1 as an antimicrobial
protein. Song et al. (2013) tested the minimal inhibition
concentrations of the bacteria using the recombinant
SP from swimming crab Portunus trituberculatus and
found its antibacterial activity only against the gramnegative bacteria such as V. alginolyticus and Pseudomonas aeruginosa, but not against the gram-positive
bacteria (e.g., Micrococcus luteus and S. aureus) and
fungus Pichia pastoris. Similar results were observed in
SP homologue from horseshoe crab by Kawabata et al
(1996). But Yu et al (2003) observed that the SP from
tobacco hornworm, Manduca sexta, showed the antibacterial activity against both gram-negative and grampositive bacteria. Further researchers (Lee & Söderhäll
2001; Wang et al. 2001; Lin et al. 2006; Jitvaropas et al.
2009) also observed that the SP homologues from various crustacean species have the antibacterial activity
against both gram-negative and gram-positive bacteria. Tsuji et al. (1998) found that a 26 kDa SP from
flesh fly Sarcophaga peregrina was toxic to the following bacteria and yeasts: S. aureus, Corynebacterium bovis, B. subtilis, E. coli, Salmonella typhi, Candida albicans and Candida pseudotropicalis. Another SP isolated from haemocytes of the mud crab, Scylla paramamosain, could bind to a number of bacteria and bacterial cell wall components (Zhang et al. 2013). The
studies on the antimicrobial activity of SP and its homologue showed that the defence responses are mediated by the recognition and binding of pattern recognition receptors on the surface of the molecules. Thus,
1075
the immune molecules are sensing the pathogen and
killing it. Overall, the results indicated that the SP and
its homologues recognize any kind of bacteria; after its
recognition the molecules activate the immune signals
and defend themselves against the pathogens. Based on
the results obtained from this study and from the earlier findings, it is possible to suggest that the bacterial
recognition triggered by SP and its homologues might
be species specific. It may be due to different molecular
structures of the bacterial cell wall components present
on their surfaces. However, further research is necessary
focused on the bacteria specific immunity triggered by
SP and its homologues in vertebrates and invertebrates.
The purified recombinant CsSP-1 showed activity
against the substrate casein. Hartley (1964) reported
that the substrate specificity of SPs varies, for example, chymotrypsin like SP from Chinese shrimp Fenneropenaeus chinensis exhibited activity against SucAAPF-pNA. These variations are due to the differences
in their binding pockets (Shi et al. 2008). For instance,
in the substrate binding site serine (in trypsin-like SPs)
is replaced by aspartic acid (in chymotrypsin-like SPs),
hence the activity differs. Single domain trypsin like
SPs possess a common catalytic mechanism, i.e., protease relies on the coordination of catalytic triad (His61 ,
Asp104 and Ser197 ) for hydroxylation of peptide bonds
at the C-terminal region. This hydroxylation breaks the
peptide bonds and converts them into simple amino
acid, so that a transient acyl-enzyme intermediate is
formed between the substrate and the protease (Rawlings & Barrett 1994; Chen et al. 2003), thus the activation occurred.
Further, the protease activity of CsSP-1 was determined against various protease inhibitors. Among
the inhibitors used, PMSF significantly inhibited the
enzyme activity followed by EDTA, AEBSF HCl and
ALLN. The inhibitory effect of CsSP-1 against PMSF
confirms that the purified CsSP-1 enzyme was a serine
protease. Similar results were reported from F. chinensis (Shi et al. 2008), Aspergillus clavatus ES1 (Hajji
et al. 2007) and Haliotis discus discus (Nikapitiya et
al. 2010). The enzyme activity in various pH buffer solutions and at various temperatures showed that the
CsSP-1 enzyme had the highest activity at pH 7.5 and
35 ◦C, respectively. The optimum pH and temperature
for the protease activity was also found to vary among
SPs originating from various organisms, for details, see
Hajji et al. (2007) and Choudhury et al (2009).
Conclusion
The bioinformatics analysis, tissue specific mRNA expression and its pattern after fungal and bacterial infection, antibacterial properties and enzyme activities
of the first reported trypsin like serine protease from
C. striatus provide a basic knowledge on the importance of this novel gene. The overall results showed its
potential involvement in the immune system of murrels. However, further research is necessary to study the
mechanism implicit trypsin association in the defence
process.
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Acknowledgements
This research was supported by DBT’s Prestigious Ramalingaswami Re-entry Fellowship (D.O.NO.BT/HRD/35/02/
2006) funded by Department of Biotechnology, Ministry of
Science and Technology, Government of India, New Delhi.
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Received January 22, 2014
Accepted April 16, 2014
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