FASEB J. Cell Migration Consortium
HOME HELP FEEDBACK SUBSCRIPTIONS ARCHIVE SEARCH TABLE OF CONTENTS
QUICK SEARCH:   [advanced]


     

This Article
Right arrow Abstract Freely available
Right arrow Full Text (PDF)
Right arrow Alert me when this article is cited
Right arrow Alert me if a correction is posted
Services
Right arrow Email this article to a friend
Right arrow Similar articles in this journal
Right arrow Similar articles in PubMed
Right arrow Alert me to new issues of the journal
Right arrow Download to citation manager
Right arrow reprints & permissions
Citing Articles
Right arrow Citing Articles via HighWire
Right arrow Citing Articles via Google Scholar
Google Scholar
Right arrow Articles by Froy, O.
Right arrow Articles by Gurevitz, M.
Right arrow Search for Related Content
PubMed
Right arrow PubMed Citation
Right arrow Articles by Froy, O.
Right arrow Articles by Gurevitz, M.
(The FASEB Journal. 1998;12:1793-1796.)
© 1998 FASEB

HYPOTHESIS

Membrane potential modulators: a thread of scarlet from plants to humans

Oren Froya and Michael Gurevitza,1

a Department of Plant Sciences, George S. Wise Faculty of Life Sciences, Tel Aviv University, Ramat-Aviv 69978, Tel Aviv, Israel


   ABSTRACT
TOP
 ABSTRACT
INTRODUCTION
 BIOACTIVITY
 STRUCTURE
 GENOMIC ORGANIZATION
 REFERENCES
 
The preservation along evolution of specific core motifs in proteins of diverse functions and taxonomic origins pinpoints a possible developmental advantage at the structural level. Such a preservation was observed in a group of membrane potential modulators including plant {gamma}-thionins, scorpion toxins, insect and scorpion defensins, bee venom apamin and MCD peptide, snake sarafotoxins, and human endothelins. These substances are short polypeptides of various lengths and nonhomologous sequences that affect organisms of distant phyla. Despite the structural differences, comparative analysis reveals commonality at three levels: 1) effect on membrane potential; 2) a common cysteine-stabilized {alpha}-helical (CSH) motif; and 3) similar gene organization (except for insect defensins), i.e., an intron that splits a codon toward the end of the leader sequence. We thus propose that these modulators, divided into two groups differing in their CSH motif orientation, have either diverged from two independent ancestors or have evolved by gene diversification via exon shuffling and subsequent modifications. To enforce protein synthesis through the secretory pathway and enable disulfide bond formation and secretion, insertion sites downstream of preexisting leader sequences have been a prerequisite. What seems advantageous for evolution, may also be exploited in attempts to `accelerate evolution' by protein design using the conserved CSH core as a suitable scaffold for reshaping molecular exteriors.—Froy, O., Gurevitz, M. Membrane potential modulators: a thread of scarlet from plants to humans. FASEB J. 12, 1793–1796 (1998)


Key Words: neurotoxins • structural motifs • evolution • MCD peptide


   INTRODUCTION
TOP
 ABSTRACT
 INTRODUCTION
BIOACTIVITY
 STRUCTURE
 GENOMIC ORGANIZATION
 REFERENCES
 
DEFENSE AND PREY MECHANISMS of venomous organisms rely in many instances on glandular `fast-acting' substances injected through a stinging device into the circulatory system and targeted to ion channels in motorneural tissues. However, nature's large arsenal of bioactive polypeptides provides examples of similar modulators able to affect membrane potential of other cell types, including bacteria. All these effectors are usually small, compact molecules whose efficiency may be attributed to their high binding affinity to specific receptors. A variety of such small basic polypeptides, capable of disrupting cell membrane potential, is found in plants and animals.

Plant {gamma}-thionins, scorpion neurotoxins, insect and scorpion defensins, bee venom apamin and mast cell degranulating (MCD)2 peptide, snake sarafotoxins, and human endothelins may be categorized in this functional group albeit each class recognizes a distinct receptor binding site. All these polypeptides display enormous sequence diversity, various lengths, and different overall 3-dimensional structures. Therefore, it has so far been impossible to trace a putative common evolutionary linkage among these substances. Still, all these compounds exhibit commonality at three levels.


   BIOACTIVITY
TOP
 ABSTRACT
 INTRODUCTION
 BIOACTIVITY
STRUCTURE
 GENOMIC ORGANIZATION
 REFERENCES
 
Plant {gamma}-thionins are composed of 47 amino acid residues constrained by four disulfide bridges and are implicated in defense against pathogens (1, 2) by increasing the permeability of cell membranes (3). The Pyrularia thionin, for example, depolarizes cell membranes and opens Ca2+ channels (3, 4; Fig. 1). Scorpion neurotoxins modulate ion channel conductance in excitable tissues by depolarizing membrane potential. They are divided into two major groups: 1) `long' toxins (60–70 amino acid residues), which are constrained by four disulfide bridges and capable of binding to sodium channels thereby disrupting their function (5; Fig. 1); and 2) `short' toxins (less than 40 amino acid residues), which are divided into potassium and chloride channel blockers ( Fig. 1) bearing three and four disulfide bridges, respectively (6, 7). Insect (14) and scorpion (15, 16) defensins are 37–40 amino acid-long polypeptides constrained by three disulfide bridges. The former were shown to disrupt the permeability barrier of the cytoplasmic membrane and generate a partial depolarization of the inner membrane in the bacterium Micrococcus luteus (8; Fig. 1). Apamin and MCD peptide, two constituents of the honey bee venom, are 18 and 22 amino acids long, respectively, contain two disulfide bridges (17), and affect potassium channel conductance (9; Fig. 1). Human endothelins and snake sarafotoxins are 21 amino acids long. Their binding to the endothelin receptor generates transient and sustained Ca2+ waveforms, which result from mobilization of intracellular stores and influx through a calcium channel (10, 18; Fig. 1).



View larger version (16K):
[in this window]
[in a new window]
  		Figure 1. Schematic gene organization and site or mode of action of selected membrane potential modulators. The coding region for the mature polypeptide appears in black. The leader sequence and pro-region are crosshatched. Introns are designated by the triangles. The genomic organization of scorpion defensins and snake sarafotoxins are unknown. The asterisk indicates unpublished results.


   STRUCTURE
TOP
 ABSTRACT
 INTRODUCTION
 BIOACTIVITY
 STRUCTURE
GENOMIC ORGANIZATION
 REFERENCES
 
The determined structures of the aforementioned polypeptides (19–27; structure of scorpion defensins has not been determined) reveal that, regardless of their length and number of S-S bonds, they share a similar cysteine-stabilized {alpha}-helical (CSH) motif, which involves a Cys-X-X-X-Cys stretch of the {alpha}-helix bonded through two disulfide bridges to a Cys-X-Cys triplet of a ß-strand (17, 25, 26). Intriguingly, the orientation of both Cys stretches, relative to the amino terminus, in apamin, MCD peptide, snake sarafotoxins, and human endothelins is inverted in comparison with that of scorpion neurotoxins, scorpion and insect defensins, and plant {gamma}-thionins ( Fig. 2).



View larger version (22K):
[in this window]
[in a new window]
  		Figure 2. Sequence comparison and a putative evolutionary scheme for the various membrane potential modulators: plant {gamma}-thionin (11); scorpion `long' toxin (28); scorpion chlorotoxin and charybdotoxin (6, 7); scorpion defensin (15); insect defensin (12); bee apamin (29); snake sarafotoxin (30); human endothelin (31). Dashed line between the insect defensin and the bee apamin suggests that either both groups evolved independently from two ancestors or that all these molecules evolved from one ancestor through exon shuffling. The CSH motif is boxed in the sequences and is also depicted by the endothelin 1 structure [PDB accession number 1EDP (27)] in the inset. The blue {alpha}-helix and ß-strand are connected by two yellow disulfide bridges.


   GENOMIC ORGANIZATION
TOP
 ABSTRACT
 INTRODUCTION
 BIOACTIVITY
 STRUCTURE
 GENOMIC ORGANIZATION
REFERENCES
 
We isolated genomic sequences and found similarity in gene arrangement among all distinct classes of scorpion `long' and `short' toxins in that they all consist of two exons and a phase I intron splitting a codon toward the end of the leader sequence (O. Froy et al. unpublished observations). A similar gene organization ( Fig. 1) was also found in {gamma}-thionin genes (11). Apamin and MCD peptide are arranged in tandem and the first intron in both genes ( Fig. 1) occurs in homologous positions, between the penultimate and the ultimate codon of the pro-protein (9). Surprisingly, insect defensin genes are intronless, i.e., a single exon consists of the coding and noncoding regions (12; Fig. 1). Human endothelin genes appear to contain five exons (13). The first intron has features similar to those of the scorpion toxin and {gamma}-thionin introns ( Fig. 1). The second exon contains the endothelin sequence and the third exon encodes an endothelin-like peptide, a product likely to be formed via exon duplication of the endothelin coding region (13). Snake sarafotoxins show high sequence similarity to human endothelins and possess remarkable similarity in structure and target receptor (32, 33). There is little information as to the sarafotoxin gene organization. Landan and colleagues (32) suggested that the sarafotoxin lineage diverged from the ancestral gene of endothelin prior to its first duplication event. In all the above-mentioned genes, except for those encoding insect defensins, it apparently has been found that the first exon encodes the 5'-untranslated region and most, if not all, of the leader sequence, and that this exon is followed by a phase I intron.

Evidently, all these polypeptides share similar characteristics at three levels of comparison: 1) effect on membrane potential; 2) a common CSH motif; and 3) similar gene organization (except for insect defensins), i.e., an intron that splits a codon toward the end of the leader sequence. These common features may suggest an evolutionary linkage among these membrane potential modulators. The exception of the intronless insect defensin genes and the inverse orientation of the CSH motif in the different polypeptides may be explained by the following possible mechanisms: 1) Independent evolution of the group including plant {gamma}-thionins, scorpion toxins, and insect and scorpion defensins vs. the group of bee venom apamin and MCD peptide, snake sarafotoxins, and human endothelins, in which the CSH motif is inversely oriented ( Fig. 2). In the first group, a primordial gene diverged to plants and scorpions and was further transferred to as ancient an insect order as Odonata to generate the insect defensin gene, which might have lost its intron. In the other group, another gene, developed to the state represented in the honey bee, was transferred to snakes and humans. 2) Exon shuffling. The exon bearing the CSH motif was excised and reintegrated in various orientations downstream of leader sequences preceding an intron (plant {gamma}-thionins and scorpion toxins), a pro-region (insect defensins), or both (apamin, MCD peptide, and endothelins). Since appropriate functioning of these polypeptides necessitates oxidation of half cysteines, only such integration sites could assure mobilization into the secretory apparatus enabling disulfide bridge formation. 3) Independent evolution, followed by exon shuffling within both groups.

Regardless of the evolutionary pathway, it seems that the overall length of these membrane potential modulators has diminished to practically the rudimentary size bearing the CSH motif. This process occurred in parallel to the generation of new active surfaces, e.g., the putative active sites of two scorpion neurotoxins: the `long' sodium channel toxin, Lqh{alpha}IT (28), and the `short' potassium channel modulator, charybdotoxin (7). Hence, the evolutionary competence of the CSH scaffold to adopt versatile molecular faces, each displaying a distinct, specific function, may be of practical use and lead to the design of novel bioactive effectors of cell membranes and excitable tissues.


   ACKNOWLEDGMENTS
 
This research was supported by grant no. IS-2486–94C from BARD, The United States-Israel Binational Agricultural Research & Development Fund, grant no. 891–0112–95 from the Israeli Ministry of Agriculture, and grant 466/97 from The Israel Academy of Sciences and Humanities.


   FOOTNOTES
 
1 Correspondence: E-mail: mamgur{at}post.tau.ac.il

2 Abbreviations: CSH, cysteine-stabilized {alpha}-helical; MCD, mast cell degranulating.


   REFERENCES
TOP
 ABSTRACT
 INTRODUCTION
 BIOACTIVITY
 STRUCTURE
 GENOMIC ORGANIZATION
 REFERENCES
 

  1. Garcia-Olmedo, F., Rodriguez-Palenzuela, P., Hernandez-Lucas, C., Ponz, F., Marana, C., Carmona, M. J., Lopez-Fando, J., Fernandez, J. A., and Carbonero, P. (1989) The thionins: a protein family that includes purothionins, viscotoxins and crambins. Oxford Surv. Plant Mol. Cell Biol. 6, 31–60
  2. Mendez, E., Moreno, A., Colilla, F., Pelaez, F., Limas, G. G., Mendez, R., Soriano, F., Salinas, M., and de Haro, C. (1990) Primary structure and inhibition of protein synthesis in eukaryotic cell-free system of a novel thionin, {gamma}-hordothionin, from barley endosperm. Eur. J. Biochem. 194, 533–539[Medline]
  3. Vernon, L. P. (1992) Pyrularia thionin: physical properties, biological responses and comparison to other thionins and cardiotoxin. J. Toxicol. Toxin Rev. 11, 169–191
  4. Evans, J., Wang, Y., Shaw, K., and Vernon, L. P. (1989) Cellular responses to Pyrularia thionin are mediated by Ca2+ influx and phospholipase A2 activation and are inhibited by thionin tyrosine iodination. Proc. Natl. Acad. Sci. USA 86, 5849–5853[Abstract/Free Full Text]
  5. Catterall, W. A. (1980) Neurotoxins that act on voltage-sensitive sodium channels in excitable membranes. Am. Rev. Pharmacol. Toxicol. 20, 15–43[Medline]
  6. DeBin, J., Maggio, J. E., and Strichartz, G. R. (1993) Purification and characterization of chlorotoxin, a chloride channel ligand from the venom of the scorpion. Am. J. Physiol. 264, C361–C369[Abstract/Free Full Text]
  7. Miller, C. (1995) The charybdotoxin family of K+ channel-blocking peptides. Neuron 15, 5–10[Medline]
  8. Cociancich, S., Ghazi, A., Hetru, C., Hoffmann, J. A., and Letellier, L. (1993) Insect defensin, an inducible antibacterial peptide, forms voltage-dependent channels in Micrococcus luteus. J. Biol. Chem. 268, 19239–19245[Abstract/Free Full Text]
  9. Gmachl, M., and Kreil, G. (1995) The precursors of the bee venom constituents apamin and MCD peptide are encoded by two genes in tandem which share the same 3'-exon. J. Biol. Chem. 270, 12704–12708[Abstract/Free Full Text]
  10. Simonson, M. S., Osanai, T., and Dunn, M. J. (1990) Endothelin isopeptides evoke Ca2+ signaling and oscillations of cytosolic free [Ca2+] in human mesangial cells. Biochim. Biophys. Acta 1055, 63–68[Medline]
  11. Karunanandaa, B., Singh, A., and Kao, T. (1994) Characterization of a predominantly pistil-expressed gene encoding a {gamma}-thionin-like protein of Petunia inflata. Plant Mol. Biol. 26, 459–464[Medline]
  12. Dimarcq, J. L., Hoffmann, D., Meister, M., Bulet, P., Lanot, R., Reichhart, J. M., and Hoffmann, J. (1994) Characterization and transcriptional profiles of a Drosophila gene encoding an insect defensin: a study in insect immunity. Eur. J. Biochem. 221, 201–209[Medline]
  13. Bloch, K. D., Friedrich, S. P., Lee, M. E., Eddy, R. L., Shows, T. B., and Quertermous, T. (1989) Structural organization and chromosomal assignment of the gene encoding endothelin. J. Biol. Chem. 264, 10851–10857[Abstract/Free Full Text]
  14. Hoffmann, J. A., and Hetru, C. (1992) Insect defensins: inducible anti-bacterial peptides. Immunol. Today 13, 411–415[Medline]
  15. Cociancich, S., Goyffon, M., Bontems, F., Bulet, P., Bouet, F., Menez, A., and Hoffmann, J. (1993) Purification and characterization of a scorpion defensin, a 4 kDa antibacterial peptide presenting structural similarities with insect defensins and scorpion toxins. Biochem. Biophys. Res. Commun. 194, 17–22[Medline]
  16. Ehret-Sabatier, L., Loew, D., Goyffon, M., Fehlbaum, P., Hoffmann, J. A., van Dorsselaer, A., and Bulet, P. (1996) Characterization of novel cysteine-rich antimicrobial peptides from scorpion blood. J. Biol. Chem. 271, 29537–29544[Abstract/Free Full Text]
  17. Kobayashi, Y., Takashima, H., Tamaoki, H., Kyogoku, Y., Lambert, P., Kuroda, H., Chino, N., Watanabe, T., Kimura, T., Sakakibara, S., and Moroder, L. (1991) The cysteine-stabilized {alpha}-helix: a common structural motif of ion-channel blocking neurotoxic peptides. Biopolymers 31, 1213–1220[Medline]
  18. Yang, C. M., Ong, R., Hsieh, J. T., and Yo, Y. L. (1994) Sarafotoxin-induced calcium mobilization in cultured dog tracheal smooth muscle cells. J. Recept. Res. 14, 423–445[Medline]
  19. Lippens, G., Najib, J., Wodak, S. J., and Tartar, A. (1995) NMR sequential assignments and solution structure of chlorotoxin, a small scorpion toxin that blocks chloride channels. Biochemistry 34, 13–21[Medline]
  20. Bontems, F., Roumestand, C., Gilquin, B., Menez, A., and Toma, F. (1991) Refined structure of charybdotoxin: common motifs in scorpion toxins and insect defensins. Science 254, 1521–1523[Abstract/Free Full Text]
  21. Tugarinov, V., Kustanovitz, I., Zilberberg, N., Gurevitz, M., and Anglister, Y. (1997) Solution structures of a highly insecticidal recombinant scorpion {alpha}-toxin and a mutant with increased activity. Biochemistry 36, 2414–2424[Medline]
  22. Pease, J. H., and Wemmer, D. E. (1988) Solution structure of apamin determined by nuclear magnetic resonance and distance geometry. Biochemistry 27, 8491–8498[Medline]
  23. Kumar, N. V., Wemmer, D. E., and Kallenbach, N. R. (1988) Structure of P401 (mast cell degranulating peptide) in solution. Biophys. Chem. 31, 113–119[Medline]
  24. Mills, R. G., Ralston, G. B., and King, G. F. (1994) The solution structure of sarafotoxin-c. Implications for ligand recognition by endothelin receptors. J. Biol. Chem. 269, 23413–23419[Abstract/Free Full Text]
  25. Bruix, M., Jimenez, M. A., Santoro, J., Gonzalez, C., Colilla, F. J., Mendez, E., and Rico, M. (1993) Solution structure of gamma 1-H and gamma 1-P thionins from barley and wheat endosperm determined by 1H-NMR: a structural motif common to toxic arthropod proteins. Biochemistry 32, 715–724[Medline]
  26. Bonmatin, J. M., Bonnat, J. L., Gallet, X., Vovelle, F., Ptak, M., Reichhart, J. M., Hoffmann, J. A., Keppi, E., Legrain, M., and Achstetter, T. (1992) Two-dimensional 1H NMR study of recombinant insect defensin A in water: resonance assignments, secondary structure and global folding. J. Biomol. NMR 2, 235–256[Medline]
  27. Andersen, N. H., Chen, C. P., Marschner, T. M., Krystek, S. R., and Bassolino, D. A. (1992) Conformational isomerism of endothelin in acidic aqueous media: a quantitative NOESY analysis. Biochemistry 31, 1280–1295[Medline]
  28. Zilberberg, N., Froy, O., Cestele, S., Loret, E., Arad, D., Gordon, D., and Gurevitz, M. (1997) Identification of structural elements of a scorpion alpha neurotoxin important for receptor-site recognition. J. Biol. Chem. 272, 14810–14816[Abstract/Free Full Text]
  29. Gualdie, J., Hanson, J. M., Shipolini, R. A., and Vernon, C. A. (1978) The structures of some peptides from bee venom. Eur. J. Biochem. 83, 405–410[Medline]
  30. Takasaki, C., Tamiya, N., Bdolah, A., Wolberg, Z., and Kochva, E. (1988) Sarafotoxins S6: several isotoxins from Atractaspis engaddensis (burrowing asp) venom that affect the heart. Toxicon 26, 543–548[Medline]
  31. Yanagisawa, M., Kurihara, H., Kimura, S., Tomobe, Y., Kobayashi, M., Mitsui, Y., Yazaki, Y., Goto, K., and Masaki, T. (1988) A novel potent vasoconstrictor peptide produced by vascular endothelial cells. Nature (London) 332, 411–415[Medline]
  32. Landan, G., Bdolah, A., Wollberg, Z., Kochva, E., and Graur, D. (1991) Evolution of the sarafotoxin/endothelin superfamily of proteins. Toxicon 29, 237–244[Medline]
  33. Lin, W. W., Lee, C. Y., and Chuang, D. M. (1991) Endothelin- and sarafotoxin-induced phosphoinositide hydrolysis in cultured cerebellar granule cells: biochemical and pharmacological characterization. J. Pharmacol. Exp. Ther. 257, 1053–1061[Abstract/Free Full Text]



This article has been cited by other articles:


Home page
 J. Biol. Chem.Home page
L. Cohen, N. Lipstein, I. Karbat, N. Ilan, N. Gilles, R. Kahn, D. Gordon, and M. Gurevitz
Miniaturization of Scorpion {beta}-Toxins Uncovers a Putative Ancestral Surface of Interaction with Voltage-gated Sodium Channels
J. Biol. Chem., May 30, 2008; 283(22): 15169 - 15176.
[Abstract] [Full Text] [PDF]

Home page
 Plant Physiol.Home page
P. Mergaert, K. Nikovics, Z. Kelemen, N. Maunoury, D. Vaubert, A. Kondorosi, and E. Kondorosi
A Novel Family in Medicago truncatula Consisting of More Than 300 Nodule-Specific Genes Coding for Small, Secreted Polypeptides with Conserved Cysteine Motifs
Plant Physiology, May 1, 2003; 132(1): 161 - 173.
[Abstract] [Full Text] [PDF]

Home page
 FASEB J.Home page
M. GUREVITZ, D. GORDON, S. BEN-NATAN, M. TURKOV, and O. FROY
Diversification of neurotoxins by C-tail wiggling': a scorpion recipe for survival
FASEB J, May 1, 2001; 15(7): 1201 - 1205.
[Abstract] [Full Text] [PDF]

This Article
Right arrow Abstract Freely available
Right arrow Full Text (PDF)
Right arrow Alert me when this article is cited
Right arrow Alert me if a correction is posted
Services
Right arrow Email this article to a friend
Right arrow Similar articles in this journal
Right arrow Similar articles in PubMed
Right arrow Alert me to new issues of the journal
Right arrow Download to citation manager
Right arrow reprints & permissions
Citing Articles
Right arrow Citing Articles via HighWire
Right arrow Citing Articles via Google Scholar
Google Scholar
Right arrow Articles by Froy, O.
Right arrow Articles by Gurevitz, M.
Right arrow Search for Related Content
PubMed
Right arrow PubMed Citation
Right arrow Articles by Froy, O.
Right arrow Articles by Gurevitz, M.

HOME HELP FEEDBACK SUBSCRIPTIONS ARCHIVE SEARCH TABLE OF CONTENTS