Original Paper |
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Acta Biochim Biophys |
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doi:10.1111/j.1745-7270.2008.00385.x |
Cloning and isolation of a conus
cysteine-rich protein homologous to Tex31 but without proteolytic activity
Jing Qian1,
Zhan-yun Guo1*, and Cheng-wu Chi1,2*
1 Institute of
Protein Research, Tongji University, Shanghai 200092, China
2 Institute of Biochemistry
and Cell Biology, Shanghai Institutes for Biological Sciences, Chinese Academy
of Sciences, Shanghai 200031, China
Received: August
15, 2007
Accepted: October
16, 2007
This work was
supported by grants from the National Basic Research Program of China
(2004CB719900) and Initiation Funding of Tongji University (2000144005)
*Corresponding
authors:
Cheng-wu Chi: Tel, 86-21-54921165;
Fax, 86-21-65988403; E-mail, [email protected]
Zhan-yun
Guo: Tel, 86-21-65988634; Fax, 86-21-65988403; E-mail,
[email protected]
We cloned and
isolated a cysteine-rich protein, designated Mr30, from Conus marmoreus.
Mr30 belongs to the cysteine-rich secretory protein family that is highly homologous
to Tex31 previously obtained from Conus textile and reported as a
protease responsible for processing of pro-conotoxins. Mr30, purified by a
procedure similar to that of Tex31, indeed showed low proteolytic activity.
However, further investigations revealed that the detected protease activity
actually resulted from a trace amount of protease(s) contamination rather
than from Mr30 itself. This finding led us to rethink the role of conus
cysteine-rich secretory proteins: they were probably not responsible for the
processing of pro-conotoxins as previously deduced, but their real biological
functions remained to be clarified.
Keywords cone snail;
cysteine-rich secretory protein; protease; protease inhibitor; covalent
modification
The cysteine-rich secretory protein (CRISP) family is highly
conserved during evolution, but their functions have not been well defined. The
mammalian CRISPs are mainly found in saliva and male reproductive tracts, as
well as in human granulocytes and plasma. Their functions are related to
sperm-egg fusion and the innate immune system [1–7]. The CRISPs are also
present in the venom of snakes and lizards, and some of them could function as
ion channel blockers [8–13].
Recently, a novel CRISP, namely Tex31, was isolated from the venom
of cone snails, Conus textile [14]. It was reported that Tex31 had
protease activity and was responsible for pro-conotoxin processing. To our
knowledge, of the CRISPs, only Tex31 was reported as a protease. To confirm
this interesting finding, we cloned and isolated a Tex31 homolog, designated
Mr30, from Conus marmoreus that were collected from the South China Sea
near Sanya City, China. The Mr30 fraction, purified from the venom of C.
marmoreus by a procedure similar to that for Tex31, indeed had a low
proteolytic activity, but further investigations revealed that the detected
activity was actually a result of a trace amount of protease(s) contamination.
Materials and Methods
Materials
The cone snails (C. marmoreus) were collected from the South
China Sea near Sanya City. The TRIzol RNA isolation agents and a rapid
amplification of cDNA ends (3‘-RACE)
kit were purchased from Invitrogen (Carlsbad, USA). The pGEM-T Easy Vector was
the product of Promega (Madison, USA). The Dec-RVKR-chloromethylketone
(RVKR-CMK) was from Biomol (Plymouth Meeting, USA). Sepharose 4B and DEAE
Sepharose Fast Flow were purchased from Amersham Biosciences (Peapack, USA).
Bovine trypsin and aprotinin were purchased from Sigma (St Louis, USA).
Gene cloning
The total RNA was isolated from the venom ducts of C. marmoreus using
TRIzol agents according to the manufacturer’s manual. The single-strand cDNA
was then synthesized from the total RNA templates using the reverse
transcriptase and a 3‘-end primer, 5‘-GGCCACGCGCGTCGACTAGTAC(dT)17-3‘. Based on the
published sequence of Tex31, a 5‘-end
primer was designed, 5‘-ATGTTGTCTACGATGCAGACTGTTGG-3‘. Using the designed 5‘-end primer and the 3‘-end primer included in the 3‘-RACE kit (5‘-GGCCACGCGCGTCGACTAGTAC-3‘),
we amplified an approximately 1.0 kb DNA fragment that was subsequently cloned
into pGEM-T Easy Vector and sequenced.
Isolation and purification of
Mr30
Six venom ducts from C. marmoreus were homogenized in cold 20
mM Tris-HCl buffer (pH 7.5). After centrifugation (10,000 g for 10 min),
the supernatant was loaded onto a DEAE-Sepharose fast flow column (8 mm´55 mm) pre-equilibrated with 20 mM Tris-HCl buffer (pH 7.5). The
column was then eluted with step-wise increases of NaCl concentration (150 mM,
300 mM, and 500 mM) in the initial buffer. Fractions were collected and
analyzed by SDS-PAGE. The fractions containing Mr30 were pooled and
concentrated by ultrafiltration (cut-off molecular weight 10 kDa) then applied
to a gel filtration column (Sephacryl S-100 HR, 16 mm´600 mm; Amersham Biosciences, Peapack, USA) pre-equilibrated with 20
mM Tris-HCl buffer (pH 7.5). The column was eluted with 20 mM Tris-HCl buffer
(pH 7.5) at a flow rate of 0.4 ml/min, and fractions were automatically
collected (1.4 ml/tube). After analysis using SDS-PAGE, the fractions
containing Mr30 were pooled and concentrated by ultrafiltration (cut-off
molecular weight 10 kDa). The concentration of the purified Mr30 was
quantified by the Bradford method [15]. The purity of the Mr30 was analyzed by
SDS-PAGE and HPLC. For SDS-PAGE analysis, 2 mg purified Mr30 was loaded
onto a 12% SDS gel, and the gel was stained with Coomassie Brilliant Blue R 250
after electrophoresis.
N-terminal sequencing and mass
spectrometry
For N-terminal sequencing, the purified Mr30 (approximately 50 mg) was
transferred to a PVDF membrane and its N-terminal amino acid sequence was
determined by Edman degradation on a protein sequencer (Model 491; Applied Biosystems,
Foster City, USA). For mass spectrometry, Mr30 and the RVKR-modified Mr30 were
further purified by a C3 reverse-phase column (Zorbax 9.4 mm´250 mm; Agilent Technologies, Santa Clara, USA) and eluted by an
acetonitrile gradient. The unmodified Mr30 and modified Mr30 (approximately 100
mg)
were lyophilized and their molecular masses were measured on a Q-trap mass
spectrometer (Applied Biosystems).
Proteolytic activity assay
The protease activity was measured using the fluorescent substrate
(pGlu-Arg-Thr-Lys-Arg- methylcoumarinamide). The reaction was carried out in 20 mM Tris-HCl
buffer (pH 7.5) at 37 ºC for 1 min. The pre-warmed fluorescent substrate (at
the final concentration of 1 mM) and an appropriate amount of protease were added to the pre-warmed
assay solution (total volume of 1 ml), and the increase of fluorescence at 460
nm (excited at 370 nm) was measured by a fluorometer (F-2500; Hitachi, Tokyo,
Japan). For the inhibition assay, the inhibitors (final concentrations:
RVKR-CMK, 1 mM; aprotinin, 200 mg/ml) were pre-incubated with 10 mg purified Mr30 at 37 ºC
for 30 min in 20 mM Tris-HCl buffer (pH 7.5) (total 20 ml), then the protease
activity was measured as described above.
Affinity absorption
Aprotinin was
covalently immobilized onto Sepharose 4B resin using 2-chloromethyl-oxirane as
a cross-linker. Approximately 1 mg aprotinin was covalently bound to 1 ml
Sepharose 4B resin. To absorb the protease(s), 10 mg Mr30 was incubated
with 100 ml affinity
resin in 20 mM Tris-HCl (pH 7.5) buffer (total volume 1.1 ml) at 4 ºC for 2 h.
Thereafter, the protease activity of the supernatant was measured as described
above and the Mr30 amount in the supernatant was determined by SDS-PAGE and
densitometry. For the control experiment, Sepharose 4B was used instead of the
affinity resin.
RVKR-CMK modification
RVKR-CMK was dissolved in DMSO as a stock solution (20 mM) and
stored at –80 ºC. The purified Mr30 (1 mg/ml) was first treated with RVKR-CMK
(at the final concentration of 1 mM) at 37 ºC for 30 min in 20 mM Tris-HCl
buffer (pH 7.5). For the control experiment, the same amount of DMSO was used
to treat Mr30. Thereafter, part of the modified Mr30 was used for the protease
activity assay; the remaining was further purified by C3 reverse-phase HPLC
and its molecular mass was measured by mass spectrometry. In the control
experiment, trypsin (0.5 mg/ml) was modified by RVKR-CMK, and its molecular
mass (before and after modification) and proteolytic activity were measured.
Results
Mr30 cloning from C.
marmoreus
Based on the published cDNA sequence of Tex31, we designed several
5‘-end primers to amplify the cDNA of
Tex31 homolog from the RNA templates isolated from the venom ducts of C.
marmoreus by 3‘-RACE, and finally
obtained an approximately 1.0 kb fragment. After T-vector cloning, the
amplified fragment was sequenced (from eight individual clones). Its open
reading frame encoded a polypeptide chain with 289 residues including an
N-terminal signal peptide as well as a dibasic cleavage site [Fig. 1(A)].
The cloned polypeptide contained a polymorphism at position 186. In one
isoform (Mr30-1; GenBank accession No. EF493183), this position is a Glu
residue (found in four clones), and in the other isoform (Mr30-2; GenBank accession
No. 493184) it is a Lys residue (found in four clones). The protein was named
Mr30 because it was cloned from C. marmoreus with a molecular weight of
approximately 30 kDa. The sequence alignment showed that Mr30 was highly
homologous to Tex31 cloned from C. textile, their amino acid sequence
similarity was as high as 65% [Fig. 1(B)]. The high sequence homology
implied that Mr30 and Tex31 probably had similar biological functions.
Purification of Mr30 from
venom of C. marmoreus
In addition to the cDNA cloning of Mr30, we also isolated and
purified the mature Mr30 protein from the conus venom in sequential steps that
were similar to the procedure used for Tex31 purification.
The material extracted from the venom ducts was first applied to a
DEAE-Sepharose column and eluted in a step-wise manner (data not shown). The
eluted fractions were analyzed by SDS-PAGE [Fig. 2(A)]. The major
fraction of Mr30 was eluted by 300 mM NaCl, and the minor fraction was eluted
by 500 mM NaCl. The two Mr30 fractions were combined, concentrated by
ultrafiltration, and applied to a gel filtration column [Fig. 2(B)].
Each eluted fraction from the gel filtration column was analyzed by SDS-PAGE [Fig.
2(C)]. Mr30 was found in tube numbers 36–40, corresponding to the
second elution peak in Fig. 2(B). These Mr30 fractions were pooled
together, concentrated by ultrafiltration, and used for purity analysis, mass
spectrometry, N-terminal sequencing, and proteolytic activity assay. As shown
in Fig. 2(D,E), the purified Mr30 showed a major peak on C3
reverse-phase HPLC, and a single band on SDS-PAGE. The purity of the Mr30
fraction was over 90%.
Characterization of Mr30
The N-terminal amino acid sequencing of the purified Mr30,
determined by Edman degradation, yielded a single N-terminal sequence,
HAXDSKYSDVTPTHT, where X, usually being a cysteine residue, could not be
determined experimentally. The measured N-terminal sequence of the mature Mr30
was identical to that deduced from its cDNA.
The molecular mass of the purified Mr30 was measured and is shown in
Fig. 3(A). The measured values (a major peak, 29,858 Da, and several
small heavier peaks) were slightly heavier than its theoretical value, 29,666
Da (186E isoform) and 29,665 Da (186K isoform) (assuming 11 disulfide bonds
were formed in the mature Mr30). This phenomenon was probably a result of
heterogeneous post-translational modifications that were also observed in
Tex31 [14] and are very common in cone snails [16]. After the protease
activity was removed from the Mr30 fraction by affinity absorption, little
change occurred in the mass spectrum of Mr30 (data not shown). Thus the small
peaks observed in the mass spectrum were not caused by protease(s)
contamination.
Protease activity assay
As it was reported that Tex31 had proteolytic activity, we
previously deduced Mr30 to also be a protease because of high sequence homology
shared with Tex31. As shown in Fig. 4(A), the purified Mr30 indeed
showed proteolytic activity. However, the measured activity of Mr30 was
extremely low compared with that of trypsin; the specific activity of trypsin
was approximately 5000-fold that of Mr30. We tried adding Ca2+ into the assay buffer or increasing the pH of the buffer to 8.5 (optimal
reaction conditions for Tex31), but no significant activity increase was
observed (data not shown). As shown in Fig. 4(B), the activity of Mr30
could be almost completely inhibited by aprotinin (a reversible serine protease
inhibitor) and RVKR-CMK (an irreversible serine protease inhibitor). However, N-ethylmaleimide
(a cysteine-specific modification reagent) had no effect on the proteolytic
activity of Mr30 (data not shown). Reduced glutathione (5 mM) had no effect on
its proteolytic activity (data not shown). Although the proteolytic activity
was detected in the purified Mr30 fraction, its extremely low specific
activity raised the question whether the detected activity really resulted from
Mr30 itself or from a trace amount of protease(s) contamination. Although the
previous analyses showed the purified Mr30 was homogeneous enough [Fig.
2(D,E)], we could not exclude the possibility that it might still contain
a trace amount of protease(s) contamination.
Affinity absorption
As aprotinin could completely inhibit the activity of Mr30, as shown
in Fig. 4(B), we cross-linked aprotinin onto the Sepharose resin and
used the aprotinin resin to remove the possible contaminated protease(s). As
shown in Fig. 5(A), after absorbed by the affinity resin, the protease
activity of the supernatant became undetectable, whereas the treatment by
Sepharose alone had little effect. This suggested that the protease(s) in the
Mr30 fraction had been absorbed by the affinity resin. However, as shown in Fig.
5(B), affinity absorption had little effect on the Mr30 concentration in
the supernatant. Therefore, the detected proteolytic activity of the Mr30
fraction actually resulted from a trace amount of protease(s) contamination
rather than from Mr30 itself.
RVKR-CMK modification
As shown in Fig. 4(B), RVKR-CMK could completely inhibit the
proteolytic activity of the Mr30 preparation. It is known that RVKR-CMK exerted
its inhibitory effect by covalent modification of the His residue of the
catalysis triad (composed of Ser, His, and Asp) of serine proteases. If the
detected proteolytic activity of the Mr30 preparation was really a result of
Mr30 itself, RVKR-CMK could be covalently bound to Mr30 and cause a molecular
weight increase (708 Da). Otherwise, the molecular weight of Mr30 would not be
changed after the RVKR-CMK treatment. To test the validity of this method,
bovine trypsin (a typical serine protease) was used as a model protease. The
RVKR-CMK treatment completely inhibited the proteolytic activity of trypsin
(data not shown). It also caused a 706 Da increase in the molecular weight of
trypsin as revealed by mass spectrometry (for untreated trypsin: measured
value, 23,309 Da; theoretical value, 23,311 Da; for RVKR-CMK-treated trypsin:
measured value, 24,015 Da; theoretical value, 24,019 Da). The measured
molecular weight increase (706 Da) of trypsin was quite consistent with the
molecular weight of the inhibitor moiety (708 Da). Therefore, RVKR-CMK was
covalently bound to the trypsin molecule. The results of trypsin modification
suggested that the method could be used to test whether Mr30 was a protease or
not. Although the proteolytic activity of the Mr30 preparation could be
completely inhibited by the RVKR-CMK treatment [Fig. 4(B)], the
molecular weight of Mr30 itself was not at all increased after the RVKR-CMK
treatment, as revealed by mass spectrometry [Fig. 3(B)], implying that
RVKR-CMK did not covalently bind to Mr30 itself. Therefore, the RVKR-CMK
modification experiment also suggested that the detected protease activity of
the Mr30 fraction was actually caused by a trace amount of protease(s)
contamination.
Discussion
In the present study, the cloning and isolation of a cysteine-rich
protein from C. marmoreus was reported. The newly obtained Mr30 was
highly homologous to Tex31, reported to be inherent in proteolytic activity and
concluded to be responsible for pro-conotoxin processing. Mr30 purified by a
procedure similar to that of Tex31 also had low proteolytic activity. However,
using two independent experiments we showed that the detected activity
actually resulted from a trace amount of protease(s) contamination rather than
from Mr30 itself.
While we were working on this project, another laboratory also cloned
and isolated Mr30 (they named it GlaCrisp) [17]. They found GlaCrisp by chance
during their isolation of g-carboxyglutamate-containing proteins, and they also could not
detect any proteolytic activity in their purified GlaCrisp fraction. The
purification of GlaCrisp involved an affinity purification step (anti-Gla
antibody) that probably removed the protease(s) contamination (without g-carboxyglutamate
modifications) from the GlaCrisp fraction. The fact that Mr30/GlaCrisp had no
proteolytic activity led us to rethink the possible role of the conus CRISPs:
they are probably not involved in the processing of pro-conotoxins, despite the
low protease activity detected in the purified Tex31 fraction. We thought more
work (such as affinity absorption and covalent modification) was needed to
confirm that the detected proteolytic activity really resulted from Tex31
itself.
Although the purified Mr30 was essentially homogeneous, as analyzed
by SDS-PAGE and reverse-phase HPLC [Fig. 2(D,E)], molecular weight heterogeneity
was observed in mass spectrometry, and all of the measured molecular weight
values were slightly heavier than the theoretical value deduced from its amino
acid sequence (Fig. 3). This phenomenon also occurred in Tex31 [14] in
that several related isoforms corresponding to different post-translational
modifications were present in the mass spectrometry. The molecular weight
heterogeneity of Tex31 and Mr30 was probably caused by heterogeneous
post-translational modifications. It is known that the conus peptides/proteins
contain rich post-translational modifications [16]. In GlaCrisp an N-terminal g-carboxyglutamate
residue had been identified experimentally [17], and other possible
modifications, such as hydroxylation, might also occur in GlaCrisp/Mr30 and
Tex31.
For both Tex31 and Mr30/GlaCrisp, the mature protein was cleaved
after a conserved dibasic site [Fig. 1(B)]. The processing probably
involved two sequential steps, the signal peptidase removed the signal peptide
(20 or 21 residues), then the furin-like protease recognized the dibasic site
and removed the extremely short “pro” sequence (three or four
residues).
GlaCrisp/Mr30 is not a protease, but its real biological functions
remain unknown. Considering that the CRISPs found in the venom of snakes could
function as ion channel blockers [8,11–13], the conus CRISPs might
also be ion channel blockers that target the ion channels of prey.
Acknowledgements
We thank Xiaoxia Shao for mass spectrometry analysis, and thank Rui
Bao for N-terminal amino acid sequence analysis. We thank Carrie Baron
(Dartmouth College, Hanover, USA) for careful editing of the manuscript.
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