Full text of DOI:10.1080/152165400306214

IUBMB Life, 49: 207 210, 2000
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Copyright 2000 IUBMB
1521-6543/00 $12.00 + .00
Original Research Article
Cloning and Characterization of a Novel cDNA
Sequence Encoding the Precursor of a Novel Venom
Peptide (BmKbpp) Related to a Bradykinin-Potentiating
Buthus martensii
Peptide from Chinese Scorpion
Karsch
Xian-Chun Zeng, Wen-Xin Li, Fang Peng, and Zhi-Hui Zhu
Department of Virology and Molecular Biology, School of life Science, Wuhan University, Wuhan 430072,
People’s Republic of China
a and b, according to their physiological effects and binding
+
(
)
properties 2, 3 . The toxins that act on the K channel are
Summary
Based on the amino acid sequence of a bradykinin-potentiating
peptide Bpp peptide K-12 from scorpion Buthus occitanus, a
full-lengthcDNAsequenceencoding the precursorof anovel venom
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mainly composed of 30 40 residues stabilized by th¡ree or four
(
) (
)
( )
disulŽ de bonds 4 . Chlorotoxin, speciŽ c for the Cl channel,
( )
has only 36 amino acid residues with four disulŽ de bridges 5 .
In addition, some scorpion venoms contain a different class of
polypeptides with no disulŽ de bridges, which show the activity
of a bradykinin-potentiating peptide Bpp . For example, Nas-
sar et al. 6 isolated Bpps from Buthus occitanus and Leiurus
quinquestriatus scorpion venoms, which showed an activitysim-
ilar to that of the synthetic bradykinin-potentiating factor B and
(
)
peptide named BmKbpp relatedtothisBpp, has been isolated and
analyzed. The cDNA encodes a precursor of 72 amino acid residues,
including a signal peptide of 22 residues and an extra Arg-Arg-
Arg tail at the C-terminal end of the precursor, which have to be
1
(
)
(
)
removed in the processingstep.The C-terminalregion 21 residues
( )
(
)
of the precursor is homologous 57% identical with the sequence
of peptide K-12. Thus, according to the primary structure of the
BmKbpp precursor, there may be a propeptide between the signal
peptide and the putative mature BmKbpp at the C-terminal region
of the precursor.
( )
C; Ferreira et al. 7 isolated a bradykinin-potentiating factor
from scorpion Tityus serrulatus venom. More recently, Meki
IUBMB
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Life, 49: 207 210, 2000
( )
(
)
et al. 8 isolated a Bpp peptide K-12 from the Egyptian scor-
pion B. occitanus, the activity of which may result from the in-
hibition of angiotensin-converting enzyme. However, so far, the
precursor of any venom peptide from scorpion with no disulŽ de
bridges has not been reported yet.
Keywords BmKbpp; bradykinin-potentiating peptide; cDNA; scor-
pion; venom.
(
)
B. martensii Karsch BmK is a representative species in
China. Recently, some toxins from these scorpions, which are
active on Na+ channels or K+ channels, have been described in
some aspects, including their amino acid sequence determina-
tion, molecular cloning, function, and three-dimensional struc-
INTRODUCTION
Venoms from scorpions contain a series of small polypep-
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tides of 31 70 amino acid residues with three or four disulŽ de
«
bridges, which act selec¡tively on different types of ion channels
+
+
(
)
–
tural analysis 9 12 . In this paper, we describe the cloning and
(
)
such as Na , K , or Cl channels and display various degrees
characterization of a cDNA sequence encoding the precursor of
of toxicity towards mammals, insects, or crustaceans, respec-
+
(
)
a novel Bpp-related venom peptide named BmKbpp with no
disulŽ de bridges. To our knowledge, this is the Ž rst report of this
kind.
(
)
–
tively 1 5 . Scorpion toxins that interact with Na channels
–
contain 60 70 residues cross-linked by four disulŽ de bridges.
These long-chain toxins can be divided into two major groups,
1Abbreviations: BmKbpp, Bpp-related venom peptide from Buthus
martensii Karsch; Bpp, bradykinin-potentiating peptide; PCR, polymerase chain
reaction.
Received 23 November 1999; accepted 25 November 1999.
Address correspondence to Wen-Xin Li. Fax: 86-027-87882661;
E-mail: xianchun zeng@hotmail.com
207

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ZENG ET AL.
MATERIALS AND METHODS
DNA Sequencing
Nucleotide sequence was determined by using the PRISM
Construction of the cDNA Library
Standard recombinant DNA techniques were used 13 . The
B. martensii Karsch scorpions were killed 2 3 days after ex-
traction of their venom to allow the toxin-producing cells of the
venom glands to enter the secretory phase. Total RNA was ex-
tracted from homogenized venom gland tissues by using a mod-
iŽ ed one-step extraction method 14 . Poly A mRNA was pu-
riŽ ed by using a PolyATract mRNA lsolationSystem Promega .
(
)
377 DNA sequencer ABI with a universal M13 forward primer
or T7 promoter primer according to the method of Sanger et al.
(
)
–
(
)
15 .
RESULTS AND DISCUSSION
(
)
( )+
The PCR strategy was prepared for obtaining the full-length
cDNA sequence of BmKbpp as described inFig. 1. The Ž rstPCR
obtained one DNA band of 180 bp. This DNA was inserted into
pGEM-T vector, and DNA sequence analysis demonstrated that
(
)
)
(
The SuperScript plasmid system from GIBCO/BRL was used
to generate the library. Double-stranded cDNA was synthesized
from 5 lg of telson mRNA. The Nucleon QC system Amer-
(
(
it encoded a peptide of 21 residues plus 3 additional residues
)
sham was used to remove the residual adapters and Notl frag-
)
R-R-R , which was homologous with the sequence of peptide
ments from double-stranded cDNA before ligation of cDNA to
K-12. The second PCR obtained a fragment of 350 bp, which
encompassed the 50 untranslated region and the encoding region
of BmKbpp cDNA. The full-length cDNA sequence of BmKbpp
was then completed by overlapping these two PCR fragments.
This combination matched the gene encoding the BmKbpp pre-
cursor of 72 amino acid residues, including a signal peptide of
22 residues in the N-terminal part and an extra Arg-Arg-Arg tail
at the C-terminal end of the precursor that had to be removed in
–
–
the NotI SalI cut plasmid pSPORT 1. Electroporation was used
to introduce ligated cDNA into Escherichia coli 10B. From 1 lg
of cDNA 1.8 £106 E. coli 10B transformants were generated.
«
Screening the cDNA Library by PCR
The polymerase chain reaction PCR was used to screen the
library. The degenerate forward primer was 5 -T TCAG AG-
(
)
0
(
)
0
(
)
the processing step Fig. 2 .
(
)
(
)
(
)
(
)
AG GA TC TA TC GC ACTG AA-3 , corresponding to res-
To investigate whether or not the BmKbpp precursor has ho-
mologs, the partial coding region of BmKbpp cDNA was used
as a probe to screen the same library by speciŽ c probe hybridiza-
tion. The results indicated that 2% of the clones were positive.
Of these, 10 clones were analyzed; all were found to have an
identical sequence and to encode the same protein as BmKbpp.
This suggests BmKbpp may have no any homolog. The nu-
cleotide sequence of BmKbpp cDNA showed that the 50 and 30
untranslated region are 43 and 86 bp long, respectively. The up-
idues 1to6of the N-terminal partialamino acidresidue sequence
( )
of peptide K-12, a Bpp from scorpion B. occitanus 8 ; the re-
verse primer was 50-AGCGGCCGCCCT( )-30, corresponding
15
to a partial sequence of the pSPORT I vector and the 30-terminal
( )
poly A sequence of a cDNA. The PCR was carried out by two
steps. The Ž rst step used only forward primers and consisted
of 10 cycles at 94±C for 1 min, 45±C for 1 min, and 72±C for
1 min. In the second step, reverse primers were added and the
reaction was run for 30 cycles at 94±C for 1 min, 62±C for
1 min, and 72±C for 1 min with a Ž nal extension phase of 5 min.
The puriŽ ed PCR products were then cloned into a pGEM-T
vector and sequenced. Next, we ampliŽ ed the 50 region of the
BmKbpp cDNA by a second PCR step, using primer03 as the
(
)
stream lateral sequence of the initial code ATG of the cDNA
of BmKbpp precursor is CAAA, which is identical with that
0
(
forward primer 5 -CTCACTATAGGGAAAGCTGC-3 , corres-
)
ponding totheT7promoter of thepSPORT1 vector andprimer 4
0
0
(
as the reverse primer 5 -TCTTCTTCGTTCGGCTGGAGC-3 ,
corresponding to the 30-terminal part of the encoding region of
)
BmKbpp cDNA determined by Ž rst PCR . The second PCR
was carried out as follows: 35 cycles of denaturation at 94±C
for 1 min, annealing at 60±C for 1 min, and extension at 72±C
for 1 min, with a Ž nal extension phase of 5 min. The puriŽ ed
PCR product was cloned and sequenced.
Screening the cDNA Library by SpeciŽc
Probe Hybridization
A speciŽ c cDNA probe used to screen the library was syn-
thesized by PCR using primers 1 and 4 as the forward and re-
verse primers, respectively. Probes were 32P-labeled by nick-
Figure 1. Strategy for screening of BmKbpp full-length
cDNA from the cDNA library by PCR. Primer 1 was comple-
mentary to a portion of the N-terminal part of peptide K-12, a
(
)
Bpp from scorpion B. occitanus 10 . Primer 2 corresponded
0
( )
to a partial sequence of NotI adapter and 3 -poly A sequence
(
)
of the cDNA. Primer 3 corresponded to the T7 promoter se-
translation Boehringer Mannheim, Germany and were used to
screen 20,000 clones from the cDNA library as described else- quence. Primer 4 was synthesized according to the 30 region of
(
)
where 13 .
the BmKbpp cDNA sequence determined by the Ž rst PCR.

209
CDNA FOR PRECURSOR OF SCORPION VENOM PEPTIDE
Figure 2. Nucleotide sequence of a full-length cDNA encoding the BmKbpp precursor. The predicted protein sequence is given
below the nucleotide sequence and is numbered starting from the NH2-terminal amino acid residue of the peptide. The signal
(
)
peptide is underlined; a potential polyadenylation signal AATAAA is underlined twice.
of most other toxin cDNAs from BmK described previously; precursors, a change known to improve the export efŽ ciency of
( )
also, a single AATAAA polyadenylation signal was found 19 most eukaryotic signal peptides. b It also has two negatively
( )
nucleotides upstream of the poly A -tail.
(
)
charged residues at the C-terminal part Asp-Glu versus one
A search for amino acidsequence homology indicatedthat the Asp or Glu residue for most toxin precursors of scorpions.
C-terminal region of BmKbpp precursor showed 57% identity The high sequence identity between the C-terminal region of
with the sequence of peptide K-12 from scorpion B. occitanus; BmKbpp precursor and peptide K-12 suggests that there may
three additional positions showed conservative replacement be- be a propeptide between the signal peptide and putative mature
(
)
tween them, for a total result of 71% homology Fig. 3 . Three BmKbpp at the C-terminal region of the precursor, and that the
extra Arg residues were present at the N-terminus of the pre- BmKbpp may be structurally and functionally related to peptide
cursor, which may be removed in the processing steps as some K12. Although the precursor contains two typical processing
(
)
scorpion toxins do 16, 17 . As with the precursors of most signals, the dibasic pairs Lys-8/Lys-9 and Lys-17/Lys-18, sep-
(
)
BmK mammalian toxins 9, 18 , the signal peptide cleavage of arated from each other by seven residues, the cleavage site of
(
)
the BmKbpp precursor occurred at a small neutral residue Ser ; the propeptide could not be determined because of lack of in-
moreover, the residue at position 3 is a Val. The signal peptide formation about the processing mechanism for propeptides in
¡
of BmKbpp precursor has two properties that are different from scorpions. The similar phenomenon was observed in a precur-
( )
those of most other scorpion toxins: a It has two positively sor of seven Bpps and a C-type natriuretic peptide from the
(
)
(
)
charged amino acid residues Lys-Lys in the 19 and 20 snake Bothrops jararaca 19 . In that precursor, the seven Bpps
¡
¡
positions versus only one Lys residue for other scorpion toxin are aligned tandemly after the signal peptide, followed by a
Figure 3. Sequence analysis of BmKbpp precursor, and sequence comparison of BmKbpp precursor with that of peptide K-12
(
)
from scorpion B. occitanus 10 . Amino acids common to the two peptides are indicated by centered dots; and three conservative
replacement positions are indicated by . The signal peptide is underlined. The dibasic pairs of residues at amino acid positions
+
–
–
8 9 and 17 18 are in boldface type.

210
ZENG ET AL.
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propeptide linker sequence with unknown function and a CNP
at the C terminus.
7. Ferreira, L. A., Alves, E. W., and Henriques, O. B. 1993 Peptide T, a novel
bradykinin potentiator isolated from Tityus serrulatus scorpion venom.
Toxicon 31, 941 947.
8. Meki, A. R.,Nassar, A. Y.,and Rochat, I. I. 1995 A bradykinin-potentiating
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In conclusion, we have described the precursor of a novel
venom peptide related to a Bpp. We believe this is the Ž rst re-
port of its kind. The discovery of a putative propeptide in the
precursor sequence suggests that the activation and functioning
of BmKbpp may be dependent on other factors. Our result may
provide a new way studying the mechanism of BmKbpp and its
pharmacological effects.
(
)
(
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peptide peptide K12 isolated from the venom of Egyptian scorpion Buthus
–
occitanus. Peptides 16, 1359 1365.
9. Xiong, Y. M., Ling, M. H., Lan, Z. D., Wang, D. C., and Chi, C. W. 1999
The cDNA sequence of an excitatory insect selective neurotoxin from the
scorpion Buthus martensii Karsch. Toxicon 37, 335 341.
10. Gong, J., Kini, R. M., Guee, M. C., Gopalakrishnakone, P., and Chung,
(
)
–
(
)
M. C. 1997 Makatoxin I, a novel toxin isolated from the venom of the
scorpion Buthus martensii Karsch, exhibits nitrergic actions. J. Biol. Chem.
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272, 8320 8324.
ACKNOWLEDGEMENTS
This work has been sponsored by the National Medicine Cre-
ation Doctor-Foundation of China to X.-C. Z. and W.-X. L.
11. Romi-Lebrun, R., Lebrun, B., Martin-Eauclaire, M. F., Ishiguro, M.,
(
)
Escoubas, P., Wu, F. Q., Hisada, M., Pongs, O., and Nakajima, T. 1997
PuriŽ cation, characterization and synthesis of three novel toxins from the
Chinese scorpion Buthus martensii Karsch, which act on K+ channels.
(
)
no. 96-901-033 , by the Wuhan Chenguang Project of Sci-
–
Biochemistry 36, 13473 13482.
(
)
ence and Technology to X.-C. Z. no. 965001037-24 , and by
the National Natural Science Foundation of China to W.-X. L.
(
)
12. Li, H. M., Wang, D. C., Zeng, A. H., Jin, L., and Hu, R. Q. 1996 Crystal
structure of an acidic neurotoxin from scorpion Buthus martensii Karsch at
1.85 A resolution. J. Mol. Biol. 261, 415 431.
(
)
no. 39970897 . The nucleotide sequence data presented here
Ê
–
(
)
were submitted to the GenBank database; the accession number
is AF 145952.
13. Sambrook, J., Fritsch, E. F., and Maniatis, T. 1989 Molecular Cloning:
A Laboratory Manual. Cold Spring Harbor Laboratory, Cold Spring Harbor,
NY.
(
)
14. Chomezynski, P., and Sacchi, N. 1987 Single step method of RNA iso-
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