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FJ
EXPRESS SUMMARY ARTICLE The Full-length version of this article is also available, published online January 22, 2003 as doi:10.1096/fj.02-0635fje. |
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* Department of Pharmacology, Faculty of Medical Sciences, UNICAMP, Campinas (SP), 13081–970, Brazil;
Department of Pharmacology, Institute of Biomedical Sciences, USP, Sao Paulo (SP), 05508–900, Brazil;
Department of Biochemistry and Medical Biotechnology, University of Naples Federico II, Napoli, Italy; and
Department of Toxicological and Pharmaceutical Chemistry, University of Naples Federico II, Napoli, Italy
2Correspondence: Department of Pharmacology, Faculty of Medical Sciences – UNICAMP, PO Box 6111, 13081–970, Campinas, SP, Brazil. E-mail: cleber.teixeira{at}directnet.com.br
SPECIFIC AIM
All known Na+ channel-specific scorpion toxins are composed of a single chain of 60–70 amino acid residues cross-linked by four disulfide bridges. These toxins are modifiers of the channel gating mechanism; according to their principal functional effects and binding properties, they have been divided into two major classes: 
- and ß-toxins. The -toxin class inhibits Na+ current inactivation prolonging the action potential, whereas the ß-toxin class shifts the voltage of activation toward more negative membrane potentials, causing spontaneous and repetitive firing. These toxic polypeptides in scorpion venoms are responsible for a variety of effects seen in scorpion envenomation, including cardiovascular, gastrointestinal, and respiratory disturbances, attributed to a massive neurotransmitter release. Priapism is a common sign of scorpion envenomation, especially in accidents involving children.
Tityus serrulatus venom (TSV) relaxes rabbit and human cavernosal tissue due to NO release from nitrergic nerves. Here we report the isolation, purification, and amino acid sequence of a bioactive component, an -toxin, present in crude TSV. A broad pharmacological characterization of this active toxin mediating the relaxant responses in rabbit corpus cavernosum (RbCC) is described, providing the first documentation of nitrergic actions mediated by a scorpion toxin in erectile tissue. We have attempted to identify short peptide segments in the amino acid sequence that could account for the function of the toxin.
PRINCIPAL FINDINGS
1. Isolation and biochemical characterization of Ts3
Protein elution monitored by UV at 220 nm yielded 11 fractions, I to XI (Fig. 1
a). All fractions were pooled, lyophilized, and tested in the RbCC. Only fraction VII displayed relaxing activity in the cavernosal tissue, which was then submitted to the next purification step. Elution of the biologically active fraction (VII) showed two main subfractions: VIIa and VIIb (Fig. 1b
). The quality and Mr of peaks-containing peptides were obtained by ESI-MS system. The Mr of the toxin present in VIIa was found to be 7427.66 ± 0.15 Da as determined by mass spectrometry analysis, with a purity > 95% (Fig. 1c
). Further experiments were performed with VIIa, since this toxin was 
10-fold more potent than VIIb.
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The Mr of the alkylated toxin measured by ESI-MS was 7911 Da, which confirms the presence of 8.3 cys-CAM modified for protein 7427.66 ± 0.15 Da. A sequence of the first 25 NH2-terminal residues was obtained as follows: KKDGYPVEYDNCAYICWNYDNAYCD. The alignment of this sequence was compared to a data bank of protein sequences (http://www.expasy.ch), and this observation indicated that the VIIa toxin sequence is 100% aligned with SCX1 TITSE (Swiss-Prot Primary Acess Number: P01496) and named Ts3. Proteolytic peptide digestion with trypsin revealed the presence of four peptides of the following Mr: 3056.64, 3070.67, 3184.38, and 3326.85. The results indicate that all four peptides are positively matched with a score of 100% with SCX1 TITSE. Indeed, the Mr of VIIa native toxin (7427.66±0.15 Da) agrees with the calculated Mr for the following sequence reported for Ts3:
KKDGYPVEYDNCAYICWNYDNAYCDKLCKDKKADSGYCYWVHILCYCYGLPDSEPTKTNGKCKS.
2. Release of NO from nitrergic fibers induced by Ts3
Application of the NO synthesis inhibitor L-NAME (100 µM; n=4) caused a significant increase in smooth muscle tone and markedly reduced the relaxations induced by Ts3 (30 nM; P<0.01). Prior addition of L-Arg (1 mM; n=4) prevented this inhibitory effect. Addition of the neuronal NO synthesis inhibitor 7-NI (100 µM; n=3) to the bathing medium reduced Ts3-evoked relaxations (30 nM; 54±6% in the absence and 11±2% in the presence of 7-NI; P<0.01). The selective inhibitor of NO-stimulated soluble guanylyl cyclase ODQ (10 µM; n=3) significantly increased the tone of the preparations and virtually abolished relaxations induced by Ts3 (30 nM; 98±1% inhibition; P<0.01), whereas addition of the PDE5 inhibitor sildenafil (100 nM) significantly enhanced relaxations induced by Ts3 at concentrations of 3 nM (P<0.05), 10 nM (P<0.01), and 30 nM (P<0.05).
Figure 2
shows that the addition of the neuronal Na+ channel blockers tetrodotoxin (100 nM) and saxitoxin (100 nM) to the bathing medium fully prevented the relaxant response elicited by Ts3 (30 nM; 98±1% and 95±3% inhibition in the presence of tetrodotoxin and saxitoxin, respectively; P<0.01; n=3 each). Tetrodotoxin and saxitoxin promptly reversed the response to the toxin when applied during the established relaxation (n=3, each; Fig. 2
).
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3. Biological effects of synthetic peptides
Based on a recent study published by Srinivasan et al. (2001), four distinct peptides were initially synthesized by organic synthesis on solid phase from the known sequence of Ts3: P1–16, P17–32,P33–48, and P49–64. The addition of these peptides (up to 30 µM) alone or in combination had no appreciable effect when assayed in the RbCC (mean relaxation 9–13%; n=4, each). We have also carried out the synthesis of peptides HORSE-1, HORSE-2, HORSE-3, HORSE-4, PDKVP, PDSEP, Buka11, and Buka11-B. Addition of either peptide to the bathing medium slightly relaxed the RbCC in concentrations of up to 30 µM (mean relaxation 7–22%; n=4, each). However, neither tetrodotoxin (100 nM) nor L-NAME (100 µM) blocked the relaxations induced by these peptides.
CONCLUSIONS AND SIGNIFICANCE
Based on functional data, our results demonstrate that TSV contains an -toxin (namely Ts3) that presents nitrergic activity when assayed in cavernosal tissue. The Ts3-induced RbCC relaxations were markedly reduced in the presence of nonselective (L-NAME) or neuronal NO synthesis inhibitor (7-NI), indicating that Ts3 releases NO in the RbCC. That the relaxations mediated by Ts3 are blocked by soluble guanylyl cyclase inhibitor (ODQ) and potentiated by selective PDE5 inhibitor (sildenafil) further demonstrates the involvement of the NO-cGMP pathway in these responses. The relaxant responses to Ts3 were susceptible to full blockade by the Na+ channel blockers tetrodotoxin and saxitoxin, indicating that NO is generated within and released from nitrergic nerve fibers (Fig. 3
).
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Fragmentation of Ts3 into four sequential 16 amino acid peptides (P1–16, P17–32, P33–48, and P49–64) resulted in no appreciable relaxant effect when assayed in the cavernosal tissue either alone or in combination. These results indicate the requirement for a 3-D space conformation rather than linear sequences for a scorpion toxin (or toxin-derived short peptides) to bind to its respective Na+ channel receptor site. Moreover, the assessment of activity of peptides PDKVP, PDSEP, Buka11, Buka11-B, as well as HORSE peptides (P9–24, P25–40, P41–56) and a modified peptide (YGLPDKVPTKT) synthesized based on Ts3 structure, revealed that these peptides slightly relaxed the RbCC by a mechanism independent on either Na+ channel activation or NO release, since the relaxations evoked were not sensitive to inhibition by tetrodotoxin or L-NAME.
The assessment of biological activity of synthetic peptides in this work revealed the crucial importance of the Ts3 structure 3-D conformation maintenance for biological activity, since linear peptide sequences neither presented an appreciable relaxing effect when tested in the RbCC nor was this effect related to Na+ channel activation and NO release, which contradicts the previous observation that the PDKVP sequence is involved in receptor site recognition by scorpion -toxins. Further biochemical/pharmacological studies and mutational analysis combined with 3-D model assembly may be required in order to better characterize the residues making up Ts3 surface that interacts with the channel.
FOOTNOTES
1 To read the full text of this article, go to http://www.fasebj.org/cgi/doi/10.1096/fj.02-0635fje; to cite this article, use FASEB J. (January 22, 2003) 10.1096/fj.02-0635fje 
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