Classification of ESTs from
1. Introduction
In the last decades, poisonous animals have gained notoriety since their venoms (secreted or injected) contain several of potentially useful bioactive substances (polypeptide toxins), which are mostly codified by a single gene or, in the case of venom organic compounds, by a given enzymatic route presented in a specialized tissue where the biosynthesis occur – the venom gland.
In this context, in the age of genomic sciences, sequencing the entire genome or portion of it, can be thought as the straightforward step to understand a given venom composition. Particularly because, in many cases, the venom is produced in so small quantities, requiring great challenge (natural and bureaucratic) to obtain biological material for its investigation or the necessity of sacrifice the animal to get samples for analysis by conventional biochemical methods. Genome sequencing allows us the identification of mRNAs, as well as prediction of protein structure and function. In addition, the construction of cDNA libraries is useful to clone, catalog and identify genes, and subsequently express the proteins of interest from these libraries. By this approach, we can have adequate amounts of polypeptide toxins for functional analysis and application, by which otherwise would be difficult to isolate.
According to [1], venoms’ complexity in terms of peptide and protein contents, together with the number of venomous species indicate that only a small proportion (less than 1%) of the all bioactive molecules has been identified and characterized to date, and little is known about the genomic background of the venomous organisms. Consequently, if we take into account that nature, operated by evolutionary processes, is the most efficient source of new functional molecules and drug candidates, the study of all species of venomous animals, including small insects, such as those belonging to the order Hymenoptera [2] will be crucial and timely for basic and applied research.
2. Ants biology: Subfamily Ponerinae
Ants (Vespoidea: Formicidae) belong to the insect order Hymenoptera, which includes other important families like Apidae (bees) and Vespidae (wasps) [3]. The family Formicidae consists of approximately 13.000 species of ants, most of them exibiting an advanced and sophisticated social life. With colonies ranging from tens to millions of individuals, a high diversity as well as numerical and biomass dominance in almost every habitat throughout the world, ants form an important component of terrestrial biodiversity, especially in the Neotropical Region, where about 30% of all known ant species are found [4,5]. All ant species possess eusocial habits, the most conspicuous one being the reproductive division of labor, with one to many queens specialized in reproduction, while the more and less sterile, and nonreproductive workers, help the queen(s) reproduction, tending the brood and dealing with all other tasks of the colony like food collection, nest repair, nest and/territory defense [6].
With more than 1000 species distributed in 28 genera, like
Like all Aculeata hymenopterans (Chrysidoidea, Apoidea, Vespoidea),
In solitary Aculeata hymenopterans, and in social bees and wasps, the venom has two main functions: prey capture and defense, respectively [13,16]. In ants, the products from the venom exhibit much higher diversity of biological roles. Particularly In stinging ants, particularly in primitive groups like Ponerinae, the primary function of venom gland products is to serve as injectable offensive or/and defensive agents (to capture prey, fight with competitors or against predators, for example) [13,16]. In more derived functions, the venom gland products are used as defensive (toxic and/or repellent) agents by non-stinging ants that topically apply them on the cuticle of enemies, as in
3. Clinical aspects of ants’ stings
Many insect stings are associated with local pathophysiological events, characterized by pain, swelling and redness at the sting site for about 1-2 days [18]. The most severe reactions are associated with allergic disorders, presenting neutrophilic and eosinophilic infiltration and specific IgE production [19]. These manifestations are common in accidents with Hymenoptera insects. Most studies that describe the clinical aspects of ant stings reported accidents with ants of the genus
Accidents with ants of the Ponerinae subfamily are rare or rarely reported. In fact, several concomitant or sequential stings are necessary in order to produce significant clinical symptoms of envenomation, in giant ants, multiple attacks are less probable, since workers have a solitary foraging behavior. However, some of the accidents with giants ants may have medical importance, such as the ones produced by the genus
4. Venom composition and pharmacological properties
The ant’s venoms have been investigated in a relatively small number of species. In the group of stinging ants, the most investigated species belong to the Myrmeciinae, Ponerinae, Pseudomyrmecinae and Myrmicinae subfamilies. They produce aqueous solutions of proteinaceous venoms containing enzymatic and non-enzymatic proteins, free amino-acids and small biologically active compounds like histamine, 5-hydroxytryptamine, acetylcholine, norepinephrine, and dopamine [16,17]. Venoms with proteinaceous components are considered as most primitive and are consequently found in other aculeate hymenopterans like wasps and bees [4,16]. A notable exception to this proteinaceous nature of the venom in ants with sting is found in ants of the genera
As a member of a group of predatory ants (Ponerinae), it is expected that
5. Pharmacology and therapeutic uses of venom form ants
The first reported case about the therapeutic use of venoms from ants were to treat rheumatoid arthritis. In fact, insects might have components that justify its use in traditional medicine in countries of East Asia, Africa and South America [36]. Lately, several studies of ant venom aimed to demonstrate their beneficial intrinsic properties such as reduction of inflammation, pain relief, improved function of the immune system and liver [37,38].
As the venom from Ponerinae subfamily is composed of a complex mixtures of proteins and neurotoxins [39] we would expected to have several pharmacological properties. Small peptides isolated from
Several distinctive pharmacological activities were demonstrated with peptides isolated from
6. Genomic study of ant venom composition
Since the description of DNA double helix by Francis Crick and James Watson (1953), recombinant DNA technology and genomics revolutionized numerous areas of life science. The comprehension of the biochemical and molecular basis of inheritance had been improved our knowledge about the complexity of all forms of life and the manner how genes and proteins interact to create diversity. The genomic revolution was additionally expanded with the advent of bioinformatic, the ‘omic’ science (transcriptomic, proteomic, peptidome, metabolomic, glycome) and, presently, system biology.
Collective efforts have been joined to annotate the gene composition of insects. The first complete sequenced genome of insect was from the fruit fly
Up to now, at least 10 ant species had their genomes analyzed and published. The ants whose genomes were sequenced include: the fire ant
Apart of a detailed genome analysis, the construction of cDNA libraries from ants’ venom glands is an important tool in order to analyze venom composition and discover new molecules that could have biological and pharmacological properties. But an important question arises: why hymenopteran venoms? As we pointed at the beginning of this chapter, there are several reports that hymenopteran venom could have biological properties useful for medical purposes. In this scope, from traditional and modern medicine reports, description can be found not only about clinical manifestation caused by hymenopterans venom, as allergic response, but also the benefits of ant venom to treat disease like rheumatoid arthritis and pain [36].
Genomic and transcriptomic studies of hymenopteran cDNA libraries would provide useful information about their protein constituents. Some of these informations would include signal peptide sequences and the presence of post-translational modifications, which cannot be predicted by the studies of mature proteins. Ants genomic studies have shown a number of substances involved in the biology of these insects, such as: vittelogenins, gustatory and odorant receptors, molecules involved in immune response, as well as metabolic and structural proteins like cytochrome P450.
7. Molecular pharmacology and toxinology of D. quadriceps venom
Recently, we have initiated a research project dedicated to investigate the composition, the pharmacological properties, and the transcripts from the venom gland components of
Using one-dimensional (SDS-PAGE) electrophoresis (1-DE) to resolve
The peptide mass fingerprint (PMF), as well as other proteomic analysis is being conducted and a report will be published elsewhere.
Pharmacological studies have been realized with
Recently we also demonstrated the neuroprotective activity of
A part of proteomic and pharmacological studies, we prepared a
|
|
|
No hit | Typical ORF with no hits | 40.8 |
DnTx | Mast cell degranulation | 28.8 |
Hypothetical protein | Unknown function | 12.0 |
Antigen like | Allergenic | 9.6 |
Cytocrome c oxidase | Metabolism | 1.6 |
Cytocrome b | Metabolism | 1.6 |
Transferase | Metabolism | 2.4 |
Ionic channel blocker | Toxin | 1.6 |
Ribossomal protein | Structural protein | 1.6 |
Chymotripsin inhibitor | Metabolism | 0.8 |
Dehydrogenase | Metabolism | 0.8 |
ATP synthase | Metabolism | 0.8 |
Phospholipase A1 | Enzyme/Toxin | 0.8 |
Bacterial ESTs | Symbionts (?) | 4.0 |
Mitocondrial protein | Metabiolism | 0.8 |
As a matter of example, the most abundant toxin was dinoponera toxin (DnTx). The dinoponeratoxin whole sequence (accounting for 27% of the total clones analysed) was identified in this cDNA library. Deduced aminoacid sequences (DnTx01 and DnTx02), corresponding to two cDNA isoform precursos, from
8. Conclusion
Taking into account the information presented in this chapter, a second question arises and should be answered in the near future: “Is there any hymenopteran venom component that could be used as a biotechnological tool?” The majority of works done to discovery new biotechnological tools from hymenopteran venoms were performed using proteomic science analysis, probably because ants apparatus venom is so hard to identify and dissect. Nevertheless, the size of some poneromorph primitive ants may permit subdue these difficulties allowing us to construct a cDNA library and thus opening new perspectives to better understand the biology of ants as well as to analyze the properties of the venom in the search for new molecules with pharmacological and / or biotechnological potential.
Thus, its clear that further work is necessary to understand ant venom, as well venoms from hymenopteran, since several precursors comprises hypothetical and predicted toxins/polypeptides with unknown function. Moreover, a deep functional analysis in the coming period will be made to comprehend the effects presented by total venom and peptides isolated from it.
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