Original Paper |
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Acta Biochim Biophys |
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doi:10.1111/j.1745-7270.2006.00192.x |
Isolation of a cDNA Encoding a
Protease from Perinereis aibuhitensis Grube
Rong-Gui LI1,
Dong-Meng QIAN2, Dao-Sen GUO1,
Gui-Cai DU1, Zhi-Yong YAN2,
and Bin WANG2*
1
Department of Biology, Qingdao University, Qingdao 266071, China;
2
Medical School, Qingdao University, Qingdao 266071, China
Received: February
4, 2006
Accepted: May 9,
2006
This work was
supported by a grant from the Special Project of National Grand Fundamental
Research Pre-973 Program of China (2004CCA02400)
*Corresponding
author: Tel, 86-532-82991508; Fax, 86-532-83812439; E-mail, [email protected]
Abstract The cDNA encoding a protease of Perinereis aibuhitensis
Grube (PPA) was cloned. The deduced amino acid sequence analysis showed that the
protein had 49% identity to the C-terminal 169–246
amino acids of serine protease of Heterodera glycines. Northern blotting
analysis indicated that the cDNA could hybridize with mRNA of approximately 260
bases isolated from the marine earthworm. The cDNA was amplified by polymerase
chain reaction and cloned into pMAL-p2 to construct expression vector pMAL-PPA.
pMAL-PPA was introduced into Escherichia coli BL21(DE3) and
overexpression of PPA fused with maltose binding protein was achieved by
isopropyl-b–D-thiogalactopyranoside
induction. The fusion protein was purified by affinity chromatography on an
amylose resin column and ion-exchange chromatography on a
diethylaminoethyl-Sepharose 4B column. Rabbits were immunized with the purified
protein and antiserum was prepared. The antibody could react with a protein of
approximately 9 kDa extracted from the marine earthworm as certified by Western
blotting analysis. The activity analysis of the recombinant PPA suggested that
it was probably a plasminogen activator.
Key words cDNA; protease; Perinereis aibuhitensis Grube
Proteases form a group of enzymes that
specialize in the cleavage of peptide bonds, and are found in diverse organisms
such as prokaryotes, plants, animals and viruses. Some of the proteases are
serine endoproteases that might participate in a number of different
physiological functions, such as coagulation, cellular and humoral immunity,
fibrinolysis, embryonic development and digestion [1]. Those proteases
participating in the digestion process are referred to as trypsins (EC
3.4.21.4) [2].
Perinereis aibuhitensis Grube is a kind
of marine clamworm widely distributed along the seaside in Asia. It has been
used in Chinese traditional herbal medicine for hundreds of years and as fish
bait. Much attention has been focused on the clamworm in recent years because
several bioactive peptides have been isolated from it. Pan et al.
purified a new antimicrobial peptide called perinerin and studied its
biochemical properties [3]. The giant hemoglobin from the marine polychaete P.
aibuhitensis was extensively studied by Tsuneshige et al. [4]. A
fibrinolytic protein consisting of two chains with a molecular weight of 47.4
kDa and a pI of 4.5 was isolated from P. aibuhitensis as reported by Tan
et al. [5]. As several other proteins with fibrinolytic activity have
also been purified from earthworms belonging to the same family as marine
earthworm [6–8], P. aibuhitensis might be a new source of thrombolytic
agents.
Thrombosis is one of the most widely occurring diseases that often
cause disability and death. Thrombolysis is an effective way to treat this kind
of disease. All of the currently used thrombolytic agents are plasminogen
activators, serine proteases that are very efficient in restoring the blood
flow [9]. Despite the widespread use of established thrombolytic agents, such
as streptokinase, tissue-type plasminogen activator and urokinase-type
plasminogen activator, these agents have a number of inadequacies, including
resistance to reperfusion, occurrence of coronary reocclusion and bleeding
complications. The quest continues for new plasminogen activators with higher
potency, more specific thrombolytic activity and fibrin selectivity, and longer
half-life [10,11].
In this article, a cDNA presumed to encode a small protease (PPA)
which might be a new plasminogen activator was cloned from the Perinereis
aibuhitensis Grube clamworm digestive tract and sequenced. The cDNA
amplified by polymerase chain reaction (PCR) was cloned into an expression
plasmid. The recombinant protein PPA fused with maltose-binding protein (MBP)
was expressed in Escherichia coli, and the recombinant protein was
purified to ascertain the biological activities of PPA.
Materials and Methods
Clamworm and bacterial strains
P. aibuhitensis was collected in the
Jiaozhou Bay (Qingdao, China) in June 2004. E. coli strain JM109
(Promega, Madison, USA) was used for subcloning and strain BL21(DE3) (Invitrogen,
Grand Island, USA) was used for protein expression. E. coli was grown in
Luria broth (LB) medium with 100 mg/ml ampicillin.
cDNA cloning and sequencing
mRNA isolation from the epithelial cells of clamworm digestive tract
and reverse transcription were carried out as described previously [12].
According to the amino acid sequence in the conserved domain of serine
proteases published, a cDNA fragment was amplified by PCR using two primers, P1
(5‘-GGTGACTCYGGYGGCCCT-3‘) and P2 (5‘-TTTTTTTTTTTTTTTTTTTTT-3‘),
and the PCR product was cloned into pGEM-T and sequenced. According to the
sequence, the 5‘ upstream sequence was amplified using 5‘ rapid
amplification of cDNA ends according to the manufacturer’s instructions
(Invitrogen) using the primers P3 (5‘-AAAGTCGACTTATTGCATGACACTG-3‘)
and P4 (5‘-CGACTGGAGCACGAGGACACTGA-3‘). The full-length open
reading frame of the cDNA for cloning into the expression plasmid was further
amplified with the two primers P5 (5‘-AAAGAATTCATGTCTGACGCGGAAG-3‘)
and P3 using total cDNA as the template. The cycle program used was as follows:
94 ºC for 45 s, 52 ºC for 45 s and 72 ºC for 1 min. PCR product was separated
by agarose gel electrophoresis, and the corresponding band (approximately 260
bp) was recovered using an Agarose gel DNA fragment recovery kit (TaKaRa,
Dalian, China) and ligated into pGEM-T (Promega) to construct pGEM-TPPA. Those
positive colonies were selected by restriction enzyme digestion with EcoRI
and SalI. The inserted DNA fragment was further confirmed by sequencing
using an ABI PRISM 310 genetic analyzer (Applied Biosystems, Courtaboeuf,
France).
Northern blotting analysis
Total RNA (2 mg) from epithelial cells of clamworm digestive tract was separated in
a denatured agarose gel and transferred to nylon Hybond N+ membranes (Amersham Biosciences, Piscataway, USA). DNA probes for
Northern blotting were 32P-labeled PPA gene amplified by PCR, and hybridization was carried
out at 55 ºC. Other procedures of Northern blotting were according to the
previous method [13].
Construction of expression
vector
The DNA fragment was cut down from pGEM-TPPA with EcoRI and SalI,
and ligated into pMAL-p2 (New England Biolabs, Ipswich, USA) lineared with the
same enzymes to construct recombinant plasmid pMAL-PPA. Positive colonies were
ascertained by digestion with EcoRI and SalI.
Expression and purification
Plasmid pMAL-PPA was introduced into E. coli BL21(DE3). One
transformed colony was inoculated into 50 ml LB medium containing 100 mg/ml ampicillin
and shaken vigorously overnight at 37 ºC. The culture was transferred into a 4
liter flask containing 1 liter of rich LB medium (New England Biolabs)
supplemented with 100 mg/ml ampicillin and cultured for 3 h in the same way. The culture
temperature was then cooled to 30 ºC and isopropyl-b–D-thiogalactopyranoside
was added to a final concentration of 0.5 mM.
The culture was shaken vigorously at 30 ºC for 6 h and the cells
were harvested by centrifugation. Cell pellets were washed with ice-chilled
column buffer (20 mM Tris-HCl, pH 7.4, 200 mM NaCl, 5 mM EDTA, 5 mM b-mercaptoethanol)
and resuspended in the same buffer. The cells were then sonicated at 0 ºC 30
times, 10 s each time, with a 45 s interval. All the following operations were
carried out at 4 ºC. The cell extract was centrifuged at 20,000 g for
30 min, and the recombinant protein in the supernatant was absorbed onto an
amylose resin column (1 cm´10 cm).
After the column was washed with the 100 ml column buffer, the MBP-PPA on the
column was eluted with 20 ml elution buffer (column buffer supplemented with 10
mM maltose). The elution was diluted to 100 ml with TSE buffer (20 mM Tris-HCl,
pH 8.0, 20 mM NaCl, 5 mM EDTA, 5 mM b-mercaptoethanol) and applied to a
diethylaminoethyl (DEAE)-Sepharose 4B column (2 cm´15 cm). The column was eluted with NaCl gradient (from 20 to 1000
mM) in TSE buffer at a flow rate of 0.5 ml/min and 1 ml fraction was collected.
The proteins in each fraction were analyzed by sodium
dodecylsulfate-polyacrylamide gel electrophoresis (SDS-PAGE).
Assay of caseinolytic activity
Caseinolytic activity has been widely used to measure the activity
of plasminogen activators [14]. For the determination of caseinolytic activity
of the purified recombinant protein, 9 ml of TS buffer (50 mM Tris-HCl, pH 8.0,
150 mM NaCl) containing 100 mg agarose and 100 mg skim milk was boiled for 2
min and cooled to 42 ºC, then 50 ml human plasminogen solution (2 mg/ml in TS
buffer) was added and poured down to a plate. In solidified agarose gel in a
plastic dish, 3 mm apertures were holed out and various different samples were
placed in them. After incubation for at least 6 h at 37 ºC, the clear zone
around the small aperture was estimated for the caseinolytic activity.
To determine the substrate specificity of PPA toward plasminogen, an
agarose gel plate was prepared as described above without plasminogen. Samples
of MBP-PPA, plasminogen, Factor Xa (Sigma, St. Louis, USA), MBP-PPA and
plasminogen, and a mixture of MBP-PPA, Factor Xa and plasminogen were placed
into the small apertures. The plate was then incubated at 37 ºC for 6 h, and
the clear zone around the small apertures was estimated for the caseinolytic
activity.
Preparation of antiserum
against MBP-PPA
One Japanese adult male rabbit was immunized with a mixture of 150 mg MBP-PPA and the
same volume of complete Freund’s adjuvant. The rabbit was again immunized with
the same mixture but with only 100 mg MBP-PPA after three weeks. Ten days later,
the blood serum was collected from the rabbit’s carotid artery.
Western blotting analysis
One gram of P. aibuhitensis was mixed with 10 ml TE buffer
(20 mM Tris-HCl, pH 8.0, 100 mM NaCl, 5 mM EDTA, and 1 mM dithiothreitol) and
homogenized with a glass pestle on ice. The homogenate was then centrifuged at
15,000 g for 30 min at 4 ºC, and 5 ml of the supernatant was
mixed with the same volume of 2´SDS-PAGE
loading buffer and boiled for 5 min. After the sample was separated by SDS-PAGE
with 18% separating gel, the proteins in the gel were transferred onto
polyvinyldifluoridine membranes (Pall, Ann Arbor, USA). The following operation
was processed according to the standard protocol using antisera against MBP
(New England Biolabs) and MBP-PPA, respectively. Horseradish peroxidase-labeled
sheep anti-rabbit immunoglobulin G was used as the secondary antibody [15].
Results
Cloning of the open reading
frame encoding a serine protease
A DNA band of approximately 260 bp was amplified by reverse
transcription-PCR [Fig. 1(A)]. PCR product was directly cloned into pGEM-T,
confirmed by restriction endonuclease digestion and sequencing. The nucleotide
and deduced amino acid sequence are shown in Fig. 2. The inserted DNA
cut down from pGEM-TPPA was cloned into expression vector pMAL-p2 and those
positive clones were also confirmed by endonuclease digestion [Fig. 1(B)].
Protein sequence alignment of
PPA and serine protease of Heterodera glycines
Protein sequence alignment of PPA indicated that this protein was homologous
to the C-terminal domains of several serine proteases. This peptide possessed
49% identity to the C-terminal amino acid 169–246 of serine protease of H.
glycines (SPHG) (Fig. 3) [16]. For the positive amino acids, the
similarity was up to 65%. There were four cysteine residues encoded by the
cloned cDNA, and each had its counterpart in the C-terminal domain of the above
serine protease.
Northern blotting analysis
In order to confirm that the cloned cDNA was indeed an intact gene transcript
and not a degraded cDNA fragment, total RNA from the epithelial cells of
clamworm digestive tract was analyzed by Northern blotting. The results showed
that the probe could hybridize with RNA of 260 bases, and no larger RNA
molecules could be detected (Fig. 4). It could be inferred from these
results that the small cDNA was indeed reverse-transcripted from an intact mRNA
molecule.
Protein expression and
purification
E. coli BL21(DE3) cells harboring the
pMAL-PPA induced by isopropyl-b–D-thiogalactopyranoside could overexpress the MBP-PPA. Gel
scanner analysis indicated that the amount of recombinant protein could be up
to 68% of the total proteins in cells [Fig. 5(A)]. Further research
results showed that most of the recombinant protein appeared in soluble form
and only a very small part in inclusion bodies (data not shown). After breakage
of the cells and removal of insoluble materials of the cell extracts by
high-speed centrifugation, the recombinant MBP-PPA in the supernatant was
purified through chromatography on an amylose resin column followed by a
DEAE-Sepharose 4B column. The protein eluted from the ion-exchange
chromatography was homogeneous in SDS-PAGE [Fig. 5(B), lane 3] and had
an apparent molecular weight of approximately 51 kDa [Fig. 5(B)]. In
order to confirm the eluted protein contained MBP, the protein was analyzed by
Western blotting using antiserum against recombinant MBP. Fig. 5(C)
shows that the purified protein was indeed an MBP fused protein and its
molecular weight was much larger than MBP alone (42 kDa).
Western blotting analysis of
proteins in P. aibuhitensis
To ascertain the actual molecular weight of PPA in vivo,
total proteins of the clamworm were analyzed by Western blotting. The results showed
that two positive bands were detected (Fig. 6). One band was very weak
with a molecular weight of approximately 30 kDa, whereas the other was very
clear with an apparent molecular weight of approximately 9 kDa (Fig. 6).
The molecular weight of the small band was the same as that of the peptide
deduced from the cloned cDNA.
Caseinolytic activity of the
purified protein
The caseinolytic activities of MBP-PPA and the digested product by Factor
Xa were assayed with skim milk plates, which were incubated at 37 ºC for at
least 6 h. The activities could be detected by the formation of transparent
plaques around the wells filled with active proteins. The results showed that
both MBP-PPA [Fig. 7(A), sample 3] and MBP-PPA digested by Factor Xa [Fig.
7(A), sample 2] could produce transparent plaques, and the
plasminogen activating activity of digested MBP-PPA using Factor Xa was larger
than that of intact MBP-PPA. Comparing the activities of digested MBP-PPA with
recombinant streptokinase, we could see that the former showed a much lower
activity.
To determine the substrate specificity of PPA to plasminogen, we
checked the caseinolytic activities of various samples with or without
plasminogen. Neither plasminogen alone [Fig. 7(B), sample 2] nor MBP-PPA
alone [Fig. 7(B), sample 3] had caseinolytic activity, but the mixture
of the two proteins did [Fig. 7(B), sample 4]. As a protease of MBP-PPA,
Factor Xa alone could not degrade casein [Fig. 7(B), sample 1], but
could promote the caseinolytic activity of the mixture of MBP-PPA and
plasminogen [Fig. 7(B), sample 5]. From these results, we suspected that
PPA could specifically activate plasminogen and degrade casein in the agarose
gel.
Discussion
To date, a large number of serine proteases with fibrinolytic
activity have been found. Many of them have been used in the prevention and
treatment of cardiac and cerebrovascular diseases. However, some fibrinolytic
proteases are restricted to narrow medicinal uses because of their
immunoreactions and short half-life. Usually, the peptides with a smaller
apparent molecular weight are less immunogenic than larger proteins. Therefore,
the cloning of genes encoding fibrinolytic peptides with low molecular weight
would offer some clues to finding new fibrinolytic agents.
In this paper, a short cDNA was cloned and expressed as an MBP-fused
protein. The fusion protein showed fibrinolytic activity, although its activity
was much lower compared with that of recombinant streptokinase. In our previous
study, the gene was expressed in E. coli with or without a short
His-tag, but both of the expressed products appeared in inclusion bodies (data
not shown). In order to get soluble and active recombinant proteins, various refolding
measures were taken, but none of them really worked (data not shown).
Therefore, it is suspected that the natural form of PPA in P. aibuhitensis
might be glycosylated.
PPA deduced from the cloned cDNA lacks an N-terminal domain of most
serine proteases from other organisms [17]. We initially suspected that we had
only cloned a partial cDNA sequence encoding the C-terminal domain of a
protease, but the results of Western blotting analysis from this study
indicated that there indeed existed a small peptide of 9 kDa in vivo (Fig.
6). At present, the exact physiological functions in P. aibuhitensis
are not clear. This study will offer some clues to find peptides of small
molecular weights, which might act as new plasminogen activators, and a basis
for further investigation to identify new thrombolytic agents with high
biological activity by site-directed mutagenesis.
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