Original Paper
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Acta Biochim Biophys
Sin 2008, 40: 391-396
doi:10.1111/j.1745-7270.2008.00414.x
mr1e, a conotoxin from
Conus marmoreus with a novel disulfide pattern
Yanfang Wang1, Xiaoxia Shao1, Min Li1, Sumin Wang2, Chengwu Chi1,3, and Chunguang
Wang1*
1
Institute of Protein
Research, Tongji University, Shanghai 200092, China
2 School of Life Sciences, University of Science
and Technology of China, Hefei 230027, China
3
Institute of
Biochemistry and Cell Biology, Shanghai Institutes for Biological Sciences,
Chinese Academy of Sciences, Shanghai 200031, China
Received: January
18, 2008
Accepted: March 10,
2008
This work was
supported by the grants from the National Basic Research Program of China (No.
2004CB719904), the Chinese Academy of Sciences for Key Topics in Innovation
Engineering (KSCX2-YW-R-104), the Program for young
excellent talents in Tongji University (2006KJ063), and the Dawn
Program of the Shanghai Education Commission (06SG26)
*Corresponding
author: Tel, 86-21-65984347; Fax, 86-21-65988403; E-mail,
Conotoxins
are well known for their highly variable structures and functions. Here we
report the identification of a novel conotoxin named mr1e from Conus
marmoreus. mr1e is composed of 11 amino acid residues cross-linked by two
disulfide bonds (CCHSSWCKHLC). The spacing of intercysteine loops in mr1e is
exactly the same as that in a4/3 conotoxins. However, the native mr1e
peptide co-eluted on reverse-phase HPLC with the regioselectively synthesized
ribbon disulfide linkage isomer (C1-C4, C2-C3) but not the globular linkage isomer (C1-C3, C2-C4). Although this
peptide has the same disulfide connectivity as the c-conotoxins,
their sequences do not share significant homology. Thus, mr1e could be defined
as a novel conotoxin family. By intracranial injection into mice, mr1e showed
an excitatory effect. The characterization of mr1e certainly enriches our
understanding of conotoxins, and also opens an avenue for further structural
and functional investigation.
Keywords conotoxin; Conus marmoreus;
disulfide connectivity; mr1e
Cone snails are a group of predatory marine gastropods that can
produce a mixture of disulfide-rich peptide neurotoxins, namely conotoxins, to
prey and defense. Conotoxins are attracting increasing interest from scientists
because they are highly diversified both structurally and functionally.
Conotoxins can be classified into several families based on the number and
pattern of disulfide bonds and their different targets, including ion channels,
neurotransmitter receptors or transporters, and G-protein-coupled receptors
[1,2]. For instance, a-conotoxins have a CC-C-C pattern and act on nicotinic acetylcholine
receptors [3], whereas w-conotoxins share a C-C-CC-C-C cysteine framework and specifically
target calcium channels [4]. Because of their high specificities, some
conotoxins have been applied as tool agents in neuroscience and developed as
therapeutic and pharmaceutical reagents in clinical applications [5,6]. Conotoxins of several different families have two disulfide bonds. a-conotoxins are
one of the best studied conotoxin families. They have a canonical C1-C3, C2-C4
disulfide bond connectivity. According to the residue numbers in the two
cysteine loops, a-conotoxins are further grouped into different subfamilies, of which
a3/5
and a4/7 are most prominent. In addition, there are a4/6, a4/5, a4/4, and a4/3 conotoxins
[7]. They all act on nicotinic acetylcholine receptors, but with distinct
subtype specificity [8,9]. t-conotoxins have a unique CC-CC motif and also a C1-C3, C2-C4
disulfide bond connectivity, but their molecular target is still elusive [10].
Recently, two other two-disulfide-bond conotoxin families were identified, r-conotoxin TIA
acting on a1-adrenoceptor and c-conotoxin MrIA acting on noradrenaline transporter [11]. r-conotoxin
shares a similar cysteine framework and disulfide bond connectivity with a-conotoxin,
however, c-conotoxins have a unique C1-C4, C2-C3 connectivity and a well-conserved CHOC motif
for the second intercysteine loop, where O is hydroxyproline. Some other
two-disulfide-bond conotoxins were identified by the molecular cloning method.Conotoxins are a natural library of bioactive peptides with a
population of approximately 50,000 of which only a small portion has been
studied [12]. More research is needed to explore the remainder. In this study,
we report the purification and characterization of a novel conotoxin, mr1e,
from the venom of the mollusk-hunting snail Conus marmoreus. This
peptide has a CC-C-C cysteine framework, like a4/3 conotoxins, but adopts
a C1-C4, C2-C3 disulfide
bond connectivity. Thus, mr1e might define a novel conotoxin family.
Materials and Methods
Materials
Sephadex G-15 was purchased from Amersham Biosciences (Uppsala,
Sweden), Zorbax 300SB-C18 analytical HPLC columns (4.6 mm?250 mm, 9.2 mm?250 mm)
were obtained from Agilent Technologies (Santa Clara, USA), and trifluoroacetic
acid and acetonitrile were from Merck (Darmstadt, Germany). Reagents for
amino-terminal sequencing, Fmoc-amino acids, and Fmoc-cys (Trityl)-resin were
purchased from Applied Biosystems (Foster City, USA). Other reagents were of
analytical grade.
Peptide purification
The venom apparatus of C. marmoreus was dissected out, cut
into segments, and homogenized. The venom was extracted with 1.1% (v/v) acetic acid for 30 min at 0 ?C. The homogenate was
centrifuged at 10,000 g for 10 min at 4 ?C, and the supernatant was
collected. This procedure was repeated twice, and the supernatants were pooled,
lyophilized, and stored at –20 ?C. For conotoxin purification, the lyophilized crude venom was
dissolved with 1.1% acetic acid and briefly centrifuged, the supernatant was
loaded onto a Sephadex G-15 column (2.6 cm?100 cm) and eluted with 1.1% acetic acid at a flow rate of 0.5
ml/min. The eluted fractions were pooled and further fractionated on a Zorbax
300SB-C18 semipreparative column (9.2 mm?250 mm; Agilent 1100 reverse-phase HPLC). Further purification of
peptide mr1e was carried out on a Zorbax 300SB-C18 analytical column (4.6 mm?250 mm). The purity of the prepared peptide was determined by mass
spectrometry.
Peptide sequencing
The purified peptide mr1e was dissolved in 0.1 M Tris-HCl, 6 M
guanidine-HCl (pH 8.5), and 0.01 M EDTA, and reduced with 100? overdose of dithiothreitol at 37 ?C for 2 h. Then 4 ml 4-vinylpyridine
was added. After being mixed and flushed with N2, the
mixture was incubated at room temperature for 3 h in darkness. The alkylated
peptide was then purified by HPLC. The amino acid sequence of the
pyridylethylated mr1e was determined by automated Edman degradation on an ABI
491A Procise protein sequencing system (Applied Biosystems).
Molecular mass determination
Molecular mass determination
The molecular mass of all the purified and synthetic peptides was
analyzed by a QTrap mass spectrometer using the Enhanced MS scan type (Applied
Biosystems). The mass spectrometer equipped with a TurboIonSpray Source was
operated in positive ionization mode.
Peptide synthesis and
refolding
The linear peptide of mr1e was synthesized by standard Fmoc
chemistry. The protected peptide was independently grown on a Wang resin, using
the
2-(1H-benzotriazol-1-yl)-1,1,3,3-tetramethyluroniumhexafluorophosphate(HBTU)/N-hydroxybenzotriazole
(HOBt) amino acid activation method. Solid phase peptide synthesis was carried
out on a 433A peptide synthesizer (Applied Biosystems).Two isomers of mr1e with different protecting groups on Cys pairs
[Trt-C1,3 Acm-C2,4 (isomer A C1-C3, C2-C4) and Trt-C1,4, Acm-C2,3 (isomer B C1-C4, C2-C3)] were synthesized.
During the cleavage of synthesized peptides from the resin, the residue side
chains were simultaneously deprotected except Cys (Acm). The linear peptides
were oxidized at room temperature with 50 mM NH4AC
buffer (pH 8.0) overnight to form the disulfide bond between free Cys residues.
The purified monocyclic peptides were then dissolved in 10% CH3CN, 4% trifluoroacetic acid. Then 0.5 mg/ml iodine was added to form
the second disulfide bond between Cys residues protected by Acm. The final
fully disulfide bonded peptides were then purified and co-eluted with the
natural mr1e.
Bioassay
The biological activity of mr1e was studied by intracranial
injection into mice. The toxin mr1e was dissolved in normal saline solution at
different concentrations. Male Kunming mice (4 weeks old, 25–30 g body
weight) were injected intracranially with 20 ml mr1e solution (n=4
for each dose). The control animals were injected with the same volume of
normal saline solution (n=4). The symptoms of injected mice were
observed for 2 h.
Results
mr1e purification and
sequencing
The crude venom extract from C. marmoreus was first
size-fractionated on a Sephadex G-15 column. The major peak was further
separated by reverse-phase HPLC [Fig. 1(A)]. Each peak was repurified on
the same C18 semipreparative column for molecular mass determination and
sequencing.The peak indicated by an arrow in Fig. 1 gave an 11 residue
sequence after being reduced and pyridylethylated, CCHSSWCKHLC. The determined
molecular mass of this toxin was 1301.7 Da, consistent with the calculated mass
(1301.8 Da) assuming that two disulfide bonds are formed. This toxin shares the
same cysteine framework with a4/3 conotoxins, with four and three residues in two cysteine loops,
respectively. However, the disulfide bond connectivity of this toxin is not
like that of a-conotoxins (C1-C3, C2-C4) but similar to the connectivity of c-conotoxins (C1-C4, C2-C3).
Therefore, this conotoxin was designated mr1e. According to the classical
nomenclature of Conus peptides [1] , the two small letters ?r represent the Conus species
from which the conotoxin was identified, number 1 indicates the disulfide
framework, and the letter ? indicates the order of discovery. Before the identification of this
peptide, four conotoxins with a CC-C-C framework had been purified from C.
marmoreus, namely c-MrIA (also termed CMrVIB or mr10e in different publications), c-MrIB, CMrVIA,
and CMrX [11,13,14]. However, the nomenclature of these peptides is rather
confused. Following the suggestion of Jimenez et al [15], these four
conotoxins should all use number 1 for the cysteine framework and could be
named sequentially as MrIA, MrIB, MrIC and MrID.
Assignment of disulfide
pattern of mr1e
Although mr1e has a typical a-conotoxin cysteine framework,
its sequence does not share the conserved Pro residue at the third position in
the first intercysteine loop of a-conotoxins. To clarify the disulfide bond
connectivity of mr1e, two isomers of mr1e with different disulfide pairing were
chemically synthesized and selectively oxidized. We did not synthesize the
isomer with C1-C2, C3-C4 pairing, because vicinal disulfide bonds are
extremely rare. Successful peptide synthesis and the formation of each
disulfide bond were confirmed by mass spectrometry. To our surprise, the native
mr1e co-eluted with the isomer with ribbon pairing (C1-C4, C2-C3) but not the isomer with
globular pairing (C1-C3, C2-C4) (Fig. 2), which clearly indicates that
mr1e has the same topological fold as c-conotoxins.
BioassayIntracranial injection into mice showed that mr1e has an excitatory
effect. When 25 mg mr1e were injected, the mice looked agitated which lasted for
about 30 min without obvious behavior symptom. But injection of 100 mg mr1e
immediately elicited stiffening of the body which lasted for about 10 min.
Until 30 min after injection, the mice could not move due to the rigid
paralysis of the rear legs. All the injected mice recovered after 30 min (data
not shown).
Discussion
In this paper, we report the purification and characterization of a
novel conotoxin, mr1e, from the venom of C. marmoreus. This toxin might
present a new family of conotoxins because it shares the same cysteine
framework as a4/3 conotoxins but adopts distinct disulfide connectivity.mr1e is composed of only 11 residues, four of which are cysteines in
a CC-C-C pattern. The spacing of the intercysteine loops is exactly the same as
a4/3
conotoxins, but mr1e does not show any sequence homology with other a4/3 conotoxins.
In particular, mr1e does not have Pro at the third position in the first
intercysteine loop, which is highly conserved in all the published a-conotoxins.
This Pro residue proved to be critical for the globular disulfide pairing of a-conotoxins (C1-C3, C2-C4).
Mutation of this Pro into either bulky charged side chain residue Lys or small
side chain residue Ala switched the original globular conformation into ribbon
conformation (C1-C4, C2-C3) [13]. Thus, the clarification of the
disulfide pairing of mr1e became the primary interest. Indeed, mr1e has a ribbon disulfide connectivity (C1-C4, C2-C3), like
previously reported c-conotoxins (Table 1). c-Conotoxins constitute a
unique family of conotoxins and act on a special target noradrenaline
transporter [11]. Interestingly, all the c-conotoxins identified so
far, as well as mr1e, are from the same species, C. marmoreus. Sequence
comparison clearly showed that mr1e has distinct features compared with c-conotoxins.
First, the spacing of the second intercysteine loop is different. There are
three residues in this loop of mr1e, but only two residues exist in the
corresponding loop of c-conotoxins, and the second residue hydroxyproline is highly
conserved. Second, the sequence of the first intercysteine loop is also
significantly different between mr1e and other c-conotoxins. Based on the
Ala-scanning study of c-MrIA, the residues in this loop are all of great importance for the
activity or structure of this toxin [14]. It would thus be plausible to
speculate that mr1e might have a different biological function. The bioactivity of mr1e was preliminarily explored by intracranial
injection into mice, a method popularly used for conotoxin studies
[20,21,22,23]. The descriptive behavior symptoms induced by different
conotoxins are often taken as qualitative and suggestive information for
further study [21,23]. The injection bioassay showed that mr1e could induce an
excitatory effect on mice. The symptoms produced by mr1e are similar to those
induced by CMrVIA [16], but their potencies are certainly at different levels.
CMrVIA is lethal at a dosage of 40 ng/g body weight, but mice can survive mr1e
even up to approximately 5 mg/g. Their effective pathways might be totally different. It would
be very interesting to identify the molecular target of mr1e and explore the
relationships between the biological activity and the unique structure of mr1e.
Conotoxins are well known for their extremely diversified structures
and functions, easily seen by comparing the two-disulfide-bond conotoxins
described in Table 1. These toxins possess typically 10–19 residues
including four cysteines. However, they show highly divergent cysteine
frameworks, disulfide pairing, non-cysteine sequences, and biological
functions. Furthermore, the same cysteine framework could form different
disulfide connectivities, such as the contrast between mr1e and a4/3
conotoxin. Toxins with the same disulfide linkage might have different targets
and functions. For instance, r-TIA has the same topological fold as a4/7 conotoxin, but it
targets adrenoreceptors instead of acetylcholine receptors. The mechanism of
the diversity of conotoxins is still unclear. The identification of mr1e
certainly enriches our understanding of conotoxins, and also opens the way for
further structural and functional investigation.
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