| HOME | HELP | FEEDBACK | SUBSCRIPTIONS | ARCHIVE | SEARCH | TABLE OF CONTENTS |
|
|
|
|||||||
|
|||||||||
|
FJ
EXPRESS SUMMARY ARTICLE The Full-length version of this article is also available, published online July 3, 2003 as doi:10.1096/fj.02-1044fje. |
| ||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||
* Laboratory of Toxicology, University of Leuven, 3000 Leuven, Belgium;
AFMB, CNRS UMR 6098 et Universités d’Aix-Marseille I and II, 13402 Marseille Cedex 20, France;
Department of Physiology, University of Potchefstroom, Potchefstroom 2520, South Africa; and
Interdisciplinary Research Centre, University of Leuven Campus Kortrijk, 8500 Kortrijk, Belgium
2Correspondence: Laboratory of Toxicology, University of Leuven, E. Van Evenstraat 4, 3000 Leuven, Belgium. E-mail: Jan.Tytgat{at}pharm.kuleuven.ac.be
SPECIFIC AIMS
The inhibitor cystine knot (ICK) fold is an evolutionarily conserved structural motif shared by a large group of polypeptides with diverse sequences and functions. Although found in different phyla (animal, plant, and fungus), ICK peptides appear to be most prominent in venoms of cone snail and spider. Recently, two scorpion toxins activating a calcium release channel were also characterized to adopt an ICK fold. The evolutionary relationship among these peptides, however, has not been elucidated to date. Structural similarity often provides evidence to support a divergent evolution mechanism, but structural convergence is not completely ruled out in this case. We isolated and identified full-length cDNAs encoding two new members of the scorpion venom ICK toxin family and determined the first complete gene structure of this family. Together, our data provide compelling evidence to trace the evolutionary process of ICK peptides. By analyzing the precursor organization and gene structure combined with the 3-dimensional fold and functional data, we suggest that divergent as well as convergent evolution might have taken place in the ICK superfamily.
PRINCIPAL FINDINGS
1. Determination of the first complete gene structure of a scorpion ICK peptide
We describe the first complete gene structure of one scorpion ICK peptide (Opicalcine1). We isolated full-length cDNAs encoding precursors of scorpion ICK peptides (Maurocalcine and IpTx A homologues) from the scorpion Opistophthalmus carinatus, a close relative of Scorpio maurus and Pandinus imperator, all belonging to the family of Scorpionidae. The cDNA sequences code for a precursor of 66 residues that is composed of three domains: an amino-terminal signal peptide of 22 residues followed by a small propeptide of 11-amino acid sequence rich in negatively charged residues (2 Asp and 3 or 4 Glu) terminated by a typical prohormone processing signal Lys-Arg or Arg, and a carboxyl-terminal part comprising the mature peptide of 33 residues (named Opicalcine). Opicalcines share a high degree of sequence homology to Maurocalcine and IpTx A (91% and 88%, respectively). To determine the gene structure of Opicalcines, we designed and synthesized primers based on the cDNA sequence and performed a PCR amplification of genomic DNA. A comparison of the Opicalcine genomic sequence with the corresponding cDNA sequence revealed that the gene contains three exons (5` exon, internal exon, and 3` exon) interrupted by one phase-1 intron of 487 bp and one phase-2 intron of 544 bp. Two introns have a consensus GT-AG splice junction. A BLAST search revealed that the Opicalcine1 gene and an unrelated EqtIV (a pore-forming protein) gene from the sea anemone Actinia equina code for two highly homologous presequences but completely different mature peptide sequences. The highly homologous presequence region corresponds exactly to the first two exons of Opicalcine1 together with their conserved precursor organization, indicating that an ancient ICK motif may have accepted the presequence of the sea anemone peptide precursor to generate a secreting function by the exon shuffling mechanism (see Fig. 3
).
|
2. Comparative analysis suggests that animal and plant ICK peptides might share different ancestors
Given a selective pressure (functional constraints) on gene structure (efficient splicing), precursor organization (efficient post-translational processing, peptide maturation, and sorting), and 3-dimensional fold (function borne by tertiary structure) during the course of evolution, conservation between two proteins or peptides at these three levels certainly provides convincing evidence for a common evolutionary origin. These common features have been found in ICK toxins from animals (snail, scorpion, and spider) (Fig. 1
). 1) Conserved precursor organization. Their precursors are composed of three segments that include a signal peptide, a propeptide, and a mature peptide. Despite the absence of a detectable sequence similarity, conserved residues were found at cleavage sites (Ala/Asp-Asp/Glu between the signal peptide and the propeptide; Arg or Lys-Arg at the terminus of the propeptide). 2) Conserved gene structure. Despite the larger size of its two introns, the global structure of the snail 
-TxVIA gene is very similar to that of the scorpion Opicalcine1. The three domain regions (signal peptide, propeptide, and mature peptide) in their precursor are separated by two introns. The location of the second intron of -TxVIA is moved slightly from the boundary of the mature peptide to the boundary of the propeptide compared with Opicalcine1. A similar intron sliding case is also observed in other homologous genes. Given a close evolutionary relation between scorpion and spiders (both belonging to the Arachnida), we expect that a similar gene structure should be present in the ICK toxins from spiders. 3) Conserved 3-dimensional fold. Superposition of the backbone heavy atoms (C, C
, and N) of scorpion Maurocalcine and snail -TxVIA reveals a good fit with an RMSD of 0.75 Å at the structurally conserved regions (three ß-strands) of the ICK motif. A close structural similarity was observed previously between spider 
-agatoxin IVA/IVB and snail -conotoxin GVIA.
|
In contrast, plant and fungus ICK peptides possess a different gene structure, where an intron is found to be located within the signal peptide coding region. Despite similarity in precursor organization, an obvious difference can be found at propeptide cleavage sites. Instead of basic residue(s), plants and fungi appear to select two hydrophobic residues as their cleavage signals (Fig. 2
). Inconsistency in the gene structure between animal and plant/fungus ICK peptides is further strengthened by their deviation at the structurally conserved regions of the ICK motif. Superposition of the backbone heavy atoms (C, C, and N) of scorpion Maurocalcine and plant PotCPI reveals a larger deviation with a RMSD of 1.92 Å.
|
Different gene structures and precursor processing signals together with a more deviated 3-dimensional structure between animal and plant ICK peptides make their descent from a common ancestor extremely unlikely. If we consider related functions as indicators of common ancestry, our hypothesis is further supported. Thus, it seems most likely that the structural similarity observed between animal and plant ICK peptides might be a result of convergent evolution.
3. Several polypeptides from animal viruses are structurally homologous to animal ICK peptides
By searching sequence and structure databases using PROSITE, we found that several virus genomes contain ORFs encoding polypeptides that may adopt an ICK fold. These ORF products are composed of a precursor of 50–52 residues with a putative signal peptide of 21–23 residues. A cysteine pattern (CX6CX5CCX3CX6C) found in the predicted mature peptide sequences nicely matches the consensus sequence of the ICK motif and is more like the animal ICK signature. However, it is distinct from those of plants and fungi, which suggests that these virus ICK sequences may be more closely related genetically to animal toxins than plant inhibitors. Structure simulation allows us to consider them as new members of ICK superfamily. Given that all these viruses belong to the Baculoviridae and only infect invertebrates, along with their ICK structure, which is related more to animal ICK peptides, we hypothesize that a gene transfer event might have occurred between an ancestor virus and its host, whereby the virus acquired an ICK gene from the genome of the infected host cells and subsequently lost the two introns and the exon encoding propeptide in the course of evolution (Fig. 3
).
CONCLUSION AND SIGNIFICANCE
Although distantly related venomous animals (e.g., scorpion and snail) have extremely different habitats and diets, an evolutionary linkage between their ICK toxins can be established based on conserved gene structure, precursor organization, and 3-dimensional fold together with a related function. It is estimated that 1500 scorpion species in the world have produced at least 100,000 unique peptides in their venoms while 500 cone snail species have developed at least 50,000 venom peptides. Given a large number of components unidentified at present, further research that integrates gene structure with fold recognition and functional analysis (structural genomics) may discover a wide evolutionary link that might be useful for a better understanding of the venomous animal evolution process and for the design of new peptidyl drugs.
FOOTNOTES
1 To read the full text of this article, go to http://www.fasebj.org/cgi/doi/10.1096/fj.02-1044fje; doi: 10.1096/fj.02-1044fje 
This article has been cited by other articles:
|
|
 |
|
  M. Nishizawa and K. Nishizawa Molecular Dynamics Simulations of a Stretch-Activated Channel Inhibitor GsMTx4 with Lipid Membranes: Two Binding Modes and Effects of Lipid Structure Biophys. J., June 15, 2007; 92(12): 4233 - 4243. [Abstract] [Full Text] [PDF]
|
|
|
|
 |
|
 J. P. Mulvenna, F. M. Foley, and D. J. Craik Discovery, Structural Determination, and Putative Processing of the Precursor Protein That Produces the Cyclic Trypsin Inhibitor Sunflower Trypsin Inhibitor 1 J. Biol. Chem., September 16, 2005; 280(37): 32245 - 32253. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
Y. Xiao, J. Tang, W. Hu, J. Xie, C. Maertens, J. Tytgat, and S. Liang Jingzhaotoxin-I, a Novel Spider Neurotoxin Preferentially Inhibiting Cardiac Sodium Channel Inactivation J. Biol. Chem., April 1, 2005; 280(13): 12069 - 12076. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
Y. Xiao, J. Tang, Y. Yang, M. Wang, W. Hu, J. Xie, X. Zeng, and S. Liang Jingzhaotoxin-III, a Novel Spider Toxin Inhibiting Activation of Voltage-gated Sodium Channel in Rat Cardiac Myocytes J. Biol. Chem., June 18, 2004; 279(25): 26220 - 26226. [Abstract] [Full Text] [PDF]
|
|
| ||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||
| HOME | HELP | FEEDBACK | SUBSCRIPTIONS | ARCHIVE | SEARCH | TABLE OF CONTENTS |
-PVIIA; spiders: Tx3-2 and Pn3A; viruses: McCTL, BsCTL, XcCTL, OpCTL1, and OpCTL2. b) Conserved gene structure. Signal peptide, propeptide, and mature peptide coding regions are shown by the blocks in different colors. Introns are designated by triangles.