Transcript cb2701 (1) - International Journal of Current Biotechnology

Amrendra kumar, Vaishnavi Ramasubbu, , Lakshminarayanan Kartik, Saravanakumar Ayappan, Cloning, system
analysis and model structural information of PmRab7 protein, Int.J.Curr.Biotechnol., 2014, 2(7):1-6.
International Journal of Current
Biotechnology
ISSN: 2321 - 8371
Journal Homepage : http://ijcb.mainspringer.com
Cloning, system analysis and model structural information of PmRab7 protein
Amrendra kumar1, Vaishnavi Ramasubbu1, Lakshminarayanan Kartik2 Saravanakumar Ayappan1*
1
CAS in Marine biology, Faculty of Marine Sciences, Annamalai University – India.
2
CAS in Crystallography and biophysics, University of Madras – India.
A R T I C L E
I N F O
Article History:
Received 4 July 2014
Received in revised form 10 July 2014
Accepted 18 July 2014
Available online 30 July 2014
Key words:
Rab7, endocytosis, lysosome, vesicular transport and endosomal trafficking.
A B S T R A C T
PmRab7 is membrane bound receptor protein which allows WSSV (White spot syndrome
virus) viral core protein VP28 to attach with PmRab7 receptor surface as well as PmRab7
mediated internalization in cell via endocytosis. In the present study PmRab7 sequence were
cloned by the techniques of polymerase chain reaction (PCR) from shrimp using single set of
primer designed from Penaeus monodon available in NCBI database. The PCR amplicons of
613 bp were consistently observed in P. monodon. The amplicons from these shrimp were
further gel eluted, cloned, sequenced and subjected to homology search, multiple sequence
alignment, phylogenetic tree construction and motif were analyzed. The homology search
confirmed their identity to PmRab7 gene, multiple sequence alignment of the translated
PmRab7 amino acid sequences confirmed the presence of conserved consensus sequences of
205 amino acids, which was uniformly observed across different shrimp and higher eukaryotic cells. Phylogenetic tree constructed based on protein sequences of cloned PmRab7 genes
revealed closed evolutionary relationship between species, six serine, two threonine and two
tyrosine were found as highly phosphorylated sequences, GTP binding site and binding
pocket were observed by 3D modeling.
Introduction
The Penaeus monodon is commonly known as black tiger
shrimp and it is one of the most economically important
crustacean sea food species in Southeast Asian countries,
Viral diseases especially White spot syndrome virus
(WSSV) and yellow head virus (YHV) are two major threat
that cause high mortality in shrimp farm shrimp culture.
WSSV is of the genus Whispovirus, family Nimaviridae.
It is a large enveloped DNA virus with genome size of
nearly 300 kb (Van Hulten et al., 2001). Viral entry into
host cells requires endocytosis machineries of the host,
PmRab7 a small GTPase protein, was investigated for its
function in vesicular transport during viral infection in P.
monodon. The small GTPase Rab7 is one of the most
distinct membrane proteins of Late Endosome (LE), where
Rab7 is able to promote minus-end directed transport of
the LE by interacting with RILP and (oxysterol-binding
protein) related 28 protein (ORP1L), mediating a
connection to the dynein/dynactin motor protein complex
(Johansson, Rocha et al., 2007). Rab7 is capable of
promoting plus-end directed transport of
autophagosomes, likely through the rab7- interacting
protein FYVE and coiled coil domain containing 1
(FYCO1) which in turn is thought to recruit a kinesin
motor (Pankiv, Alemu et al. 2010). Rab7 is also involved
*Corresponding author.
Email address: [email protected]
1
in the recruitment of the retromer to the LE membrane
(Rojas, van Vlijmen et al., 2008). The retromer is essential
for LE to TGN transport of the LE membrane associated
M6PR interacts with Rab7 through its VPS26 domain
(Seaman, 2004; Rojas, van Vlijmen et al., 2008). PmRab7
is a P monodon small GTPase protein possibly involved
in replication of several shrimp viruses. RNA interference
(RNAi) using double-stranded RNA (dsRNA) targeting
PmRab7 gene (dsRNA-PmRab7) was employed to silence
the expression of PmRab7 to investigate the inhibitory
effect on Laem-Singh virus (LSNV) replication. PmRab7
cDNA contains 1357 bp encoding 205 amino acids.
PmRab7 contains conserved motifs involved in
guanosine-5- triphosphate (GTP) binding, GTPase
activity, an effector binding domain and an
isoprenylation site similar to other mammalian Rab
proteins. Knock-down of PmRab7 expression by RNAi
using dsRNA PmRab7 not only inhibited WSSV but also
inhibited RNA viruses including YHV (Ongvarrasopone
et al., 2008). The mechanism underlying this phenomenon
in shrimp is unclear. It is noted that mammalian Rab7
plays important roles in endosomal trafficking processes
in transportation of the cargo from an early to a LE or
from a LE to a lysosome and is involved in lysosome
biogenesis and phagocytosis (Zhang et al., 2007; Dale
et al., 2004; Bucci et al., 2000; Vitelli et al., 1997;Feng et
al., 1995; Meresse et al., 1995; ). Hence, this study was
focused to study cloning, sequencing, functional
Int.J.Curr.Biotechnol.
Volume 2; Issue 7; July, 2014
validation of amino acid involved in PmRab7 and model
structure determination.
Materials and Methods
The post larva of P. monodon was collected from the
shrimp farm of Portonovo region, Tamilnadu, India, and
the post larva was brought to the laboratory and
acclimatized in Fibreglass reinforced plastics (FRP) tank
for experiment.
Total RNA isolation and c-DNA synthesis
Total RNA was extracted from the lymphoid organ using
Trizol reagent (Invitrogen), from the P.monodon post
larva. RNA sample were prepared, concentration and
purity were assessed by Nanodrop technology by
measuring their absorbance at 260 and 280 nm, and stored
at -80 until use. 2 µl RNA was used as the template to
synthesize first strand cDNA. Base on multiple alignment
of highly conserved sequence of PmRab7 (P. monodon)
sequences from various dataset available in the GenBank
database, one set of degenerate primer was designed
corresponding to the conserve site of PmRab7, such as
PmRab7
Forward
F1-5'CGGGATCCCGTGCTGCAGCTCGGATTTC-3', and
reverse
PmRab7-R1
5'GGAATTCCTGTTTAGCCTTGTTGTCATTGG-3'.
PCR amplification of PmRab7 gene
Polymerase chain reaction (PCR) temperature profile was
94°C for 4 min followed by 35 cycles of 94°C for 30 s,
58°C for 30 s, 72°C for 40 s and final extension step at
72°C for 10 min.
T-tail cloning
Amplified product from c-DNA, PmRab 7 product were
purified from agarose gel by using thermo scientific Gene
Jet PCR purification kit, subsequently purified PmRab7
was cloned in pTZ T-tail vector by using T4 DNA ligase,
the ligation mixture was transformed in prepared
competent cell by Thermo scientific manual of E.coli DH5á
cells, transformed cell were then immediately plated on
recently prepared LB agarose plate containing ampiciline,
X-gal and Isopropyl â-D-1-thiogalactopyranoside (IPTG),
plate was incubated on 37°C for overnight, Successfully
transformed white colonies were selected from blue white
selection method, moreover selected transformed
colonies were further confirm by colony PCR.
Plasmid isolation, DNA Sequencing and Bioinformatics
analysis:
Confirmed colonies were subsequently subjected to LB
broth medium containing 50mM ampiciline and incubated
in shaker incubator at 37°C for overnight, further plasmid
(pTZ and PmRab7) were isolated and concentration was
measured by using NanoDrop, isolated plasmid were
sequenced by Sanger method from Applied Biosystem
India, and sequence were submitted to NCBI and
sequenced gene was translated on ExPASy and blasted
by using Blastp, multiple sequence alignment was done
by Clutral W, and the different graphical feature were
studied on NCBI plate form. The various physicochemical parameters were analyzed on ExPASy by using
ProtParam program. Protein domain was found by domain
finder, phosphorylation site were analyzed by NetPhos
2.0 and 3D model structure was made by PyMOL with
the reference to 1vg8 on PDB in the presence of Mg2+
and GTP.
Results
RNA isolation and first strand cDNA synthesis
Extracted RNA samples had very good quality and
integrity based on Nanodrop analysis results. The OD
260/280 ratio for purified RNA was between 0.788 – 1.83,
indicating that preparations were free of any major protein
contamination. NanoDrop results showed that first
strand cDNA synthesis reaction was successful.
PCR amplification
As PCR results showed, synthesized cDNA was
successfully amplified by PCR reaction. The presence of
amplicons is characteristic for the presence of the
P.monodon DNA. Length of PmRab7 specific product
was about 679 bp. The intensity and size of bands was
identical with DNA ladder, which confirmed the accuracy
of performed reactions. Furthermore, no visible bands
can be seen in negative control lanes. PCR products were
used for ligation into pTZ57R/T vector after A-tailing
process.
Comparison of native and recombinant plasmids
A-tailed PCR products were successfully ligated into
pTZ57R/T vector by TA cloning scheme. Resultant
recombinant plasmid (pTZ-PmRab7) was compared with
native pTZ57R/T by electrophoresis on 1% agarose gel.
As expected, pTZ-PmRab7 (651 bp length) was longer
than native pTZ57R/T (2886 bp). Different bands revealed
in each plasmid lane can be attributed to different forms
of extracted plasmid DNA (linear, open circular and
supercoil).
Blue white selection, Colony PCR and Enzymatic
digestion
White colonies were selected as a preliminary clone then
Colony PCR was used to confirm recombination with
PmRab7 gene specific primers. All PCR reaction
conditions were as before. Selected white colonies
generated strong bands after PCR that showed
recombination process was done as expected. To further
confirm presence and size of insert in pTZ-NcGRA7,
recombinant plasmid was simultaneously digested with
two enzymes (EcoRI/BamHI). After electrophoresis of
digestion reaction on 1% agarose gel, 2 bands were
detected in each lane that can be attributed to pTZ57R/T
band (2886 bp) and insert band (654 bp for NcGRA7). As
shown in Fig. 1. An insert with expected length was
detected.
Sequencing of PMRB7
PCR generated PmRab7 gene was successfully cloned
and sequenced. Sequence data reported in this paper is
available in the Gene Bank database under the accession
number KF199870 Based on the in silico estimates using
CLC main workbench software package (CLC bio), protein
encoded by PmRab7 gene had length of 205 amino acids
with the calculated molecular mass of 24 kDa (Fig. 3)
which was similar to the PmRab7 protein sequences
obtained from the NCBI database.
Bioinformatics Study
Blast analysis of PmRab7 gene revealed 67 to 100%
identity with other recorded PmRab7 genes in NCBI Gene
bank. Similarity of this protein with other shrimp genes
varied between 1-5% (LvRab7 99%), moreover human
Rab7 and rat Rab7 showed 86% (HsRab7 and RnRab7),
where as in plant Rice (PgRab7) shows maximum variation
67% (Fig. 2).
Different protein features were showing in PmRab7
sequence like GTP /Mg2+ has been found in between 17
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Int.J.Curr.Biotechnol.
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Table – 1: Various physico-chemical properties of studied protein computed using ProtParam program.
Number of amino acid
205
Molecular weight
23219.3
Theoretical pI
5.77
Negatively charged residues (Asp+Glu)
27
Positively charged residues (Arg+Lys)
25
Formula
C1030H1615N279O314S9
Total number of atoms
3247
Estimated half-life period
Instability index
>30 hours (mammalian reticulocytes, in vitro).
>20 hours (yeast, in vivo).
>10 hours (E-coli, in vivo).
30.84
Aliphatic index
80.39
GRAVY
-0.348
Table – 2: Protein Domain linker
Rank
SVM-ALL
SVM-LONG
SVM-SHORT
1
1
1
Peak
Value
1.641
1.287
1.589
SVM-JOINT
1
1.287
Peak
Position
114
116
113
Region
Sequence
109 - 120
109 - 123
110 - 118
QASPRDPDHFPF
QASPRDPDHFPFVVL
ASPRDPDHF
116
109 - 123
QASPRDPDHFPFVVL
to 157 amino acid at different four places, GDI interaction
site were found in between 17 to 79 amino acid, PmRab7
conserve domain conation five G box, two molecular
switch regions, five Rab subfamily motif and two putative
GEF interaction sites (Fig. 3.). The results of different
physico-chemical parameter were shown in Table 1. And
the role of this parameter in PmRab7 has discussed in
discussion part, the best protein domain were found in
114 peak region with maximum 1.641 peak value (Table
2.).
Predicted Protein phosphorylation site
Six serine, two threonine, and two tyrosine were found
during protein phosphorylation site analysis, out of six
possible prediction two serine position shows high score,
serine position 101 shows 0.998 and position 135 shows
0.998 where as serine 178 shows minimum score value
0.858 (Table .3.) (Fig 4.).
3D Model structure prediction and GTP binding site
Given sequence structure in PDB shows 90% similar to
1Vg8 reference, further more the supplemented Mg2+
and GTP were attached on define binding site in induce
fit docking by Glydoc. Best Mg2+ and GTP binding site
were chosen on high score and high energy value (Fig 5
A.B.C D).
Discussion
Although white spot syndrome remains a serious global
problem in sea food production (shrimp) due to high
mortality and less define immune system, till the date
several group are working to develop a potential drug
(herbal, antibody, gene silencing and suppression of
receptor gene (PmRab7) expression by dsRNA
(Chalermporn et al., 2007). There is concurrent
agreement that new antiviral drug are needed to shorten
or simplify treatment by focusing the structural
orientation, active site and week interaction of amino acid
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of PmRab7, this work is one of the step. In this article one
gene encoding PmRab7 was cloned from P.monodon
containing 650 bp and similarly this was code for 205
amino acid sequences.
The Rab7 domain Of PmRab7 from P.monodon has one
highly conserve region, block, five G box, two switch
regions and one GTP binding site similar to all Rab7
monomeric from plant to animal, with one G box and GPT
binding rich amino acids that will participate to bind up
with Mg2+ and GTP. In agreement with this, the PmRab7
gene sequence codes for a deduced polypeptide
containing five extremely conserved motifs (G boxes)
involved in GTP-binding or GTPase activity and an
isoprenylation site (last both C), therefore PmRab7
belonging from GTPase family which will activate after
GTP attachment, P-loop was maximum similar to Homo
sapiens Rab7 A. Suggesting that PmRab7 is an active
GTPase that is able to cycle between GDP and GTP bound
states (Kallaya et al., 2006).The value of extinction
coefficient produced by ProtParam shown that all cystine
residues appear as half cystine, (Pace et al., 1995)
tyrosine, tryptophan and the cystine residues covalently
joined by disulphide bond. Predicted half life showing
that half of the amount of protein in a cell to disappear
after its synthesis in the cell, according to “N-end rule”
the N terminus amino acid of PmRab7 protein is
Methionine, so based on N-end rule, it will be stable
more than 10 hours in E. coli cells (Bachmair et al., 1986).
The instability index provides an estimate of the stability
of protein of interest in a test tube. Values greater than 40
indicate that the protein may be unstable in vitro
(Guruprasad et al., 1990). GRAVY score is the virtual
important for the hydrophobic residues of the protein
positive GRAVY protein indicates hydrophobic while
negative GRAVY protein indicates hydrophilic, integral
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Table – 3: Potential serine, threonine and tyrosine as phosphotylation site
Figure - 1: Agarose Gel electrophoresis of restriction enzyme digested recombinant plasmid lane 1- 1 kb DNA size marker, lane 2 purified insert (651 bp), lane 3- pRSET-B undigested, lane 4 -EcoRI/
BamHI digested pTZ with insert (PmRab7 651), lane 5- pTZ without
insert as a control digested
Figure – 2: Multiple sequence alignment of PmRab7 with various species of Rab7 amino acid sequence, five
conserve motif G boxes, three Different GTP binding site and effector binding site (LTKE).
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Figure – 3: Unique features of PmRab7 protein including different GTP binding site, GDI interaction site, Motifs
and switch regions
Figure – 4: Predicted phosphorylation site of PmRab7
Figure – 5: Showing 3D model of PmRab7 in different mode with Mg2+ and GTP binding site
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Volume 2; Issue 7; July, 2014
membrane proteins typically have higher GRAVY scores
than do globular proteins (Kyte and Doolittle, 1982).
During protein phophorylation prediction serine 101,
threonine 136 and tyrosine 151 shows maximum score, In
a recently published systematic comparative and
structural analysis of protein phosphorylation, Jiménez
et al. (2007) reported that serine and threonine
phosphorylation sites exhibit only a marginal tendency
to occur preferentially in structurally more flexible loops
with approximately 35% actually being located in helices
or b-strands, which can be assumed as relatively rigid
secondary structural elements. And for tyrosine sites,
no tendency to occur more frequently in loops was
detectable at all. In this study we also presented 3D model
of PmRab7 for Mg2+ and GTP binding site analysis, the
same model was shown by Arunima et al., 2012. also, but
here first time we are reporting the inactive PmRab7 (GDP)
to active PmRab7 (GTP) on the basis of various hydrogen
bonding G 18-20, T 46 and K 126 were involved in PmRab7
activation.
Acknowledgement
Authors are thankful to authority of CAS in Marine
Biology, Annamalai University for providing necessary
facility for completion of work and INCOIS, MoES for
providing financial support throughout the work. We are
grateful to Dr.K.V.Rajendran (CIFE Mumbai) for providing
the lab for cloning, and we also thank Dr.K.Gunasekaran
(Department of Crystallography and Biophysics,
University of Madras) for his kind support.
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