HUANG Ren-Huai et al.: Purification and Characterization of a Neurotoxic Peptide Huwentoxin-III

　

Purification
and Characterization of a Neurotoxic Peptide Huwentoxin-III and a Natural
Inactive Mutant from the Venom of the Spider Selenocosmia huwena Wang
(Ornithoctonus huwena Wang)

HUANG Ren-Huai,
LIU Zhong-Hua, LIANG Song-Ping*

(College of Life
Sciences, Hunan Normal University, Changsha 410081, China)

 

Abstract novel neurotoxic peptide, named huwentoxin-III,
and a natural mutant have been isolated from the venom of the spider Selenocosmia
huwena Wang (Ornithoctonus huwena Wang). The average molecular weights
of the two peptides were determined as 3853.35 and 3667.40 by mass spectrometry,
respectively. Huwentoxin-III has 33 amino acid residues containing 6 cysteine
residues. Its natural mutant is only truncated a tryptophan residue from C-terminals
of huwentoxin-III. The sequences of the two peptides show 70.5% sequence similarity
with that of lectin-like peptide SHL-I previously isolated from the venom
of the same spider, while they cannot agglutinate human erythrocytes. Huwentoxin-III
can reversibly paralyze cockroaches for several hours with an ED50 of (192.95±120.84)
μg/g (P=0.95) (x±s) and can enhance the muscular contractions
elicited by stimulating the nerve of the isolated rat vas deferens, however,
the mutant of huwentoxin-III has no such effect, which suggests that Trp33
was an important residue related to the biological function of huwentoxin-III..

Key words

huwentoxin-III; natural mutant; spider
venom; neurotoxic peptide; Ornithoctonus huwena

Spider venoms contain a variety
of toxic components, some of which have served as the tools to study the mechanisms
of signal transmission related with ion channels, since most of them can specially
act on ion channels including calcium, sodium and potassium channels［1－3］.
Thus, the studies of spider venoms will contribute to pharmacology, neurobiology,
agriculture and medicine.

Chinese bird spider Selenocosmia
huwena Wang (Ornithoctonus huwena Wang) is distributed in Chinese
Southwestern hilly area and is one of the most venomous spiders in China［4,5］.
In our previous work, we have demonstrated that S. huwena venom contains
a mixture of compounds with different types of biological activities, and
several proteins and polypeptides were isolated and characterized, such as
HWTX-I, -II, -IV and SHL-I［6－8］. HWTX-I, consisting of 33 residues, is an
N-type calcium channel blocker［9－11］. HWTX-II is a peptide with 37 amino acid
residues, which can reversibly paralyze the cockroach with an ED50 value of
(127±54) μg/g (x±s) and can enhance cooperatively the inhibitory action
of huwentoxin-I to the mouse phrenic transmission［12］. HWTX-IV contains 35
amino acid residues and specifically inhibits the neuronal tetrodotoxin-sensitive
(TTX-S) voltage-gated sodium channel with an IC50 value of 30 nmol/L in adult
rat dorsal root ganglion(DRG) neurons through a mechanism quite similar to
that of TTX［13］. SHL-I, which can agglutinate human and mouse erythrocytes
at concentration of 125 mg/L and 31 mg/L respectively, is one of the lowest-molecular
weight lectin-like peptide being specific to mannosamine［14,15］. The three-dimensional
structures of HWTX-I, -II, -IV and SHL-I have been determined using 2D [¹H]-NMR,
which shows that HWTX-I, -IV and SHL-I adopt similar structural motif–inhibitor
cystine knot (ICK) motif, characterized by a triple-stranded, anti-parallel
β-sheet stabilized by a cystine knot containing three disulfide bridges (named
1-4,2-5 and 3-6 pattern)［13, 16－20］. However, the structure of HWTX-II is
different from those of HWTX-I, -IV and SHL-I and adopts a novel scaffold
with an unique disulfide bridge linkage (Cys4-Cys18, Cys8-Cys29 and Cys23-Cys34,
named 1-3, 2-6 and 4-5 pattern)［21－23］.

Here, we report the isolation and purification of a novel neurotoxic peptide,
named huwentoxin-III (HWTX-III), and its natural inactive mutant from the
venom of Chinese bird spider S. huwena, including their amino acid
sequences and the determination of their biological activity. .

1 Materials and Methods
1.1 Materials
The venom was obtained by electrical stimulation of female spiders and the
freeze-dried crude venom was stored at －20 ℃ prior to analysis. Adult male
Sprague-Dawley rat (200－300 g) and adult male cockroaches (Periplaneta
americna) were from Peking University. All sequencing reagents were from
Applied Biosystems Division of Perkin Elmer. α-cyano-4-hydroxycinnamic acid
(CCA) and trifluoroacetic acid (TFA) were purchased from Sigma. All the other
reagents were of analytical grade.
1.2 Isolation and purification of HWTX-III and its natural mutant
HWTX-III and its natural mutant were isolated by a combination of ion exchange
and reverse phase high performance liquid chromatogram (RP-HPLC) as described
in our previous work［12］. 20 mg lyophilized crude venom was dissolved in 1.0
ml H2O and centrifuged at 12 000 r/min for 10 min.
The supernatant was filtered through 0.22 μm filters (MilliGen Corp.) and
fractionated on a Waters Protein-Pak CM 8HR column (10 mm×100 mm) connected
with Waters 650E pump and Waters 486 detector. The pooled effluent was further
desalted and purified on a Waters YWG-Pak C18 column (7.9 mm×250 mm) connected
with Waters 510 pump and 486 detector.
1.3 MALDI-TOF mass spectra analyses
The molecular masses of peptides were determined by MALDI-TOF (Bruker Proflex
III). 2 μl of peptide solution in 0.1% TFA/H2O, were
mixed with 20 μl of saturated solution of CCA dissolved in 50% ACN/H2O
containing 0.1%TFA. 2 μl of this mixture was deposited on the target and dried.
Prior to each analysis, the masses were calibrated internally using angiotention-II
(Mr 1296.49).

1.4 Amino acid sequence analyses

he native and alkylated peptides
were submitted to automatic N-terminal sequencing on an Applied Biosystems
491 pulse-liquid-phase sequencer. The phenythiohydation(PTH) amino acids were
identified using on-line RP-HPLC with PTH C18 column on an Applied Biosystems
140C analyzer.

1.5 Bioassay
Haemagglutination assays were performed as the procedure described previously［14］.
The insect toxicity of HWTX-III and its mutant were qualitatively assayed
by intra-abdominal injection of 20 μl toxin dissolved in insect saline into
adult male cockroaches (body weight of 0.35 g). Contrasted experiments were
received by injection saline into insects. Observation of paralysis was made
at 5 min, 15 min, 60 min, 24 h, 48 h, and 72 h, respectively. The assay end-point
for calculation of ED50 was 30 min and ED50 was calculated using probit analysis
with SoftTox program (softLabWare Inc). Physiological studies were carried
out using the isolated rat vas deferens (VD) from the adult male Sprague-Dawley
rats［24］. In brief, the dissected preparations were bathed in a 15 ml glass
organ bath filled with oxygenated (95%O2 and 5%CO2)
in Krebs solution at (36±1) °C and were stretched by a passive force of about
1 gram. The twitch contractions of these muscles were elicited by a field
stimulation of the nerves with single rectangular pulse of 30 V with 0.2 ms
duration every 14 s. The muscular contractions were transformed into electric
signals by a mechanical-electric transducer made of semiconductor stain gauge.
The signals were amplified and recorded with a pen recorder. 50 μl of sample
was added into the bath solution after muscular contraction was stable for
30 min.

2 Results and Discussions
2.1 Purification and mass spectra analyses
The ion exchange chromatography of the crude venom of the spider S. huwena
was shown as Fig. 1(A). The fractions labeled 1, 4, 5 and 6 mainly contain
SHL-I, huwentoxin-I, -II and -IV, respectively. The fraction, labeled 2, was
applied to RP-HPLC［Fig. 1(B)］. The peak with retention time of 36.70 min,
which can enhance the muscular contraction of isolated rat vas deferens, is
named huwentoxin-III. The peak with retention time of 30.60 min is its natural
mutant. The average molecular weights of the two peptides were determined
as 3853.35 and 3667.40, respectively (Fig. 2).

Fig.1 The isolation of HWTX-III
and it natural mutant
(A) Ion exchange HPLC of crude female S. huwena venom. Lyophilized
venom (20 mg in 1 ml) was applied to a Waters Protein-Pak CM 8HR column (10
mm×100 mm) initially equilibrated with buffer A (0.02 mol/L of sodium phosphate
buffer, pH 6.5). The column was eluted with a linear gradient of 0%－35% of
buffer B (1 mol/L of sodium chloride in buffer A) for 40 min at a flow rate
of 2.5 ml/min. (B) Further RP-HPLC chromatography of the fraction labeled
2 in panel A on YWG C18 column (7.9 mm×250 mm). Elution was performed with
a linear gradient of 5%－40% of 0.1% TFA/ACN for 40 min at a flow rate of 2.5
ml/min.

Fig.2MALDI-TOF mass spectra
of HWTX-III (A) and its natural inactive mutant (B)

2.2 Amino acid sequence analysis

The amino acid sequence of native HWTX-III and its natural mutant were determined
end to end by automated Edman degradation. All positions were unequivocally
assigned except for positions 2, 9, 14, 15, 20 and 27, which showed no new
peak. Then the two peptides was reduced, alkylated and resequenced to determine
the positions of cysteine residues, respectively. Carboxymethylated-cysteines
were identified at each of the previously unidentified positions. All the
results revealed that HWTX-III is a 33 amino acid polypeptide, while the sequence
of the natural mutant is only truncated a tryptophan residue from C-terminal
of huwentoxin-III (Fig.3). The average molecular weight of HWTX-III calculated
from the sequence is 3859.69 Da, which is 6 Da higher than the experimental
value, indicating that 6 cysteine residues of HWTX-III formed three pairs
of disulfide brigdes. So does the natural mutant of HWTX-III.

Fig.3 Comparison of amino acid
sequence of HWTX-III with those of its natural mutant, SHL-I and TXP5
Identical and conservative residues are shaded in black and gray, respectively.
The disulfide bridge pattern (named 1-4, 2-5 and 3-6) is indicated below the
sequences. Secondary structure for SHL-I is shown at the top of the panel,
where black squares and shaded arrows represent turns and β-sheet, respectively.

The amino acid sequence of HWTX-III
was used to search the protein database for possible homologues online using
BLAST search (www. ncbi.nlm.nih.gov/blast). Amino acid sequence alignment
(using Bioedit software) showed that HWTX-III has 70.5% and 61% sequence similarity
with SHL-I［14］ from the same spider and TXP5［25］ purified from Mexican red
knee tarantula (Brachypoelma smithii), respectively (Fig.3). The three-dimensional
structure of SHL-I was determined by 2D-NMR studies as inhibitor cystine knot
(ICK) structural motif containing a triple-stranded β-sheet (Asp5-Cys7, Tyr17-Ser20
and Trp25-Leu28, respectively) and three β-turns (Gys2-Asp5, Cys14-Tyr17 and
Arg21-Lys24, respectively) (Protein Data Bank code: 1QK7)［19］. ICK structural
motif is proved to be a common structural motif, adopted by a number of ion
channel toxins and related polypeptides from diverse sources, including spiders,
coneshells, plants and fungi. This structural motif is characterized by a
triple-stranded, anti-parallel β-sheet stabilized by a cystine knot containing
three disulfide bridges (named 1-4, 2-5 and 3-6 pattern). To date, all the
peptides with the ICK motif share a well-known consensus sequence: CX3-7CX3-9CX0-5CX1-5CX4-20C,
where X can be any amino acid residue［26－28］. It is obvious that the sequences
of HWTX-III and its native mutant are in accord with that consensus sequence.
In addition, the two peptides have significant sequence identity with that
of SHL-I. So we speculated that HWTX-III and its mutant should share similar
disulfide linkage pattern (named 1-4, 2-5 and 3-6 pattern) and homologous
three-dimensional structure with that of SHL-I.
2.3 Biological activity of huwentoxin-III and its natural mutant
Spiders use their venoms to paralyze or kill the preys. The well-studied components
in the venom of spiders have been broadly classified in two types: polyamines
and polypeptides. Since we have found that the crude venom of this spider
was toxic to cockroach with a LD50 value of 300 μg/g［6］, preliminary studies
were carried out to determine the biological activity of HWTX-III on insects,
which shows that, although HWTX-III cannot kill the cockroaches during 72
h after being injected, it can reversibly paralyze the cockroaches within
30 min in a dose-dependent manner with an ED50 value of (192.95±120.84) μg/g
(body weight) (P=0.95) (x±s). In addition, HWTX-III can significantly
enhance the muscular contraction of isolated rat vas deferens even at a lower
dose of 1.3 μmol/L［Fig. 4(A)］. However, the natural mutant of HWT-III cannot
affect the cockroaches within 24 h at the concentration of 400 μg/g, and has
no effect on the muscular contraction of isolated rat vas deferens［Fig. 4(B)］,
which suggests that Trp33 in the sequence of HWTX-III should be an important
residue related to the biological function of HWTX-III.

Fig.4 Effect of HWTX-III (A)
and it natural inactive mutant (B) on the neuromuscular transmission in the
rat vas deferens nerve muscle tissues

HWTX-III and its natural mutant
share high sequence identity and homologous structure with that of SHL-I,
however, they cannot agglutinate human A, B and O-type erythrocytes (data
not shown) at a concentration of 2×10³. mg/L, while SHL-I does
at a minimum concentration of 125 mg/L［14］, indicating that the different
residues in these peptides should be related to their different biological
functions. The further structural and functional studies of HWTX-III will
help us to insight into the interaction between the native peptides and their
receptors and the relationship of structure and function.

---

Received: June 23, 2003Accepted:
August 10, 2003
This work was supported by a grant from the National Natural Science Foundation
of China ( No. 30170193)
*Corresponding author: Tel, 86-731-8872556; Fax, 86-731-8861304; e-mail,
[email protected]

Updated at: 12-18-2003