Mass Spectrometric Assessment Bioactive Peptides In Tarantula Venom Biology Essay

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Spiders have some of the oldest known fossil records dating back to 300 million years ago, during the Carboniferous time. In that time period spiders have been the main focus of many integral areas. Such areas include human interest and fascination, modern popular culture and more importantly the basis of scientific research. There are approximately 40,000 known species of spider worldwide, all of which are members of the arachnid order Araneae which is divided into two distinct sub orders. {{2 Rash,Lachlan D. 2002}} The first sub order called Mesothelae includes a small group of primitive spiders where as the second sub order Opisthothelea is further divided into two infra orders named Mygalomorphae and Araneomorphae. Large ground dwelling spiders such as Tarantulas fall into the Mygalomorphe and the larger infra order Araneomorphae consists of nearly 90% of all other spider species. {{23 Gregory,T.R. 2003;}} Some main spiders include; orb weavers, huntsman, wolf and jumping spiders. Spider venoms are recognised as major sources of biologically active molecules that may represent future medicines of the drug market. Their potential as drug sources has been determined through the use of various investigative tools indicating that venoms vary in terms of chemical nature from salts to large multi-protein domains. (Vassilevski, A.A. Kozlov, S.A. and Grishin, E.V. 2009)

Only a few spider species present a real threat to humans and can include the "Widow" spider and the Australian "Funnel web." The Australian funnel web has caused more than 13 fatal deaths before scientists introduced anti-venom onto the drug market in 1981. (Winkel D K, Mirtschin P and Pearn J 2006) Venom constituents from this spider include toxins that act on potassium and sodium channels causing symptoms such as cardiac rhythm, coma then death. Female venoms from this spider are thought to be the most complex to date revealing greater than 600 known peptide components (Vassilevski, A.A. Kozlov, S.A. and Grishin, E.V. 2009

Molecular cloning techniques have made it possible to gain further knowledge of the composition of the Funnel web venom. Other studies that have been performed on spider venoms indicate that there is a predominance of disulfide containing peptide neurotoxins in turn characterised by a primary structure known as a cysteine motif.

Several hundred of these molecules can be present in the same venom consisting of similar structure, stabilised by invariant cysteine residues. The specific action of the molecules is determined by the unique sequence of amino acid residues found in the loop regions between the disulfide bridges and can be referred to as "natural combinatorial libraries." These can also be found in venom from cone snails, snakes, jellyfish, insects and scorpions. {{20 Sollod, Brianna L. 2005;}}

There are approximately 300 species of the tarantula (Theraphosidae) spider and are distributed predominately throughout tropical regions of the USA, Asia and Australia. Their main source of prey for survival is both invertebrates and vertebrates using grasping fangs or by being caught in silk snares. Venom produced from these tarantulas is highly efficient when acting on the central and peripheral nervous systems and have strong capability of paralysing higher vertebrates. Nervous tissue has two important characteristics, the excitability of the cell membrane and the ability to transfer electrical signals across the synapse. Spider toxins therefore use neuronal receptors, ion channels or pre synaptic membrane proteins as targets, thus inhibiting neurotransmitter release. {{2 Rash,Lachlan D. 2002;}} Also due to their distinct size, tarantulas were always thought to be highly dangerous to humans; however recent findings have indicated that they are rather quite harmless. The average human will experience mild to severe pain, and vigorous itching after been bitten. Other severe symptoms may include; muscular spasms and strong cramps that may last several hours. Observing the history of bite reports it is quite evident that some spiders are clearly more neurotoxic than others and include; the Poecilotheria, Stromatopelma and Haplopelma.

However, in saying this there are no documented cases of human death following a tarantula bite. In contrast to this, tarantula venoms given (at various doses) to other animals such as; mice, rats, frogs, birds, dogs and snakes can be extremely lethal. {{1 Escoubas,Pierre 2004}}

The first toxins from spider venoms date back to the late 70's and early 80's. During this time period studies were limited due to the restricted quantities of material available. From this material quantities of venom produced are extremely low in comparison to that from scorpions or snakes for example. {{1 Escoubas,Pierre 2004}}

Early fractionation techniques included gel filtration and paper/gel electrophoresis yielding very small amounts of product. Introduction of modern venom purification techniques such as High Performance Liquid Chromatography (HPLC) and automated Edman sequencing have enabled efficient characterisation to be carried out. HPLC operates in such a way.... Utilisation of several different approaches has enabled isolation of various different tarantula toxins. One of the most abundant methods used is injection assays using mice or insects. Advances in Mass Spectrometry techniques have also enabled the composition of spider venoms to be identified and characterised whether pure or in a mixture. (Auvin-Guette,C 2002) In the case of identifying peptides, this method can also provide information regarding amino acid sequence under certain conditions. In the process of Mass Spectrometry, neutral molecules are ionized and their positively charged products are directed through an electric field. These products are then separated according to their mass to charge ratio. There are two complementary methods in Mass Spectrometry used in determining the mass of biological molecules named; Matrix-assisted laser desorption ionisation (MALDI) coupled to a mass analyser of the time of flight (TOF). This method is regularly used in the direct study of a mixture of products however it is also used in the ionization of non volatile or biological macromolecules. (Graham LJ R, Graham C and Mc Mullan G 2007) For example; it is commonly used for arthropod venom characterisation due to its ability to resolve large numbers of analytes in one defined step. {{1 Escoubas,Pierre 2004;}}

venom. During this technique, the macromolecule (sample) is placed in a matrix consisting of small organic molecules and ionized by a laser beam. The mass of the positively charged ions that are obtained are then analysed using a mass spectrometer. MALDI-TOF MS analysis is also applied to crude tarantula venoms to enable fingerprinting of venoms and identification of a particular species. {{1 Escoubas,Pierre 2004;}} This technique can also be used in combination with genetic and morphological identification methods. Another technique that has substantial use in the field of Mass Spectrometry is Electro-spray Ionization. This method enables proteins to be ionized without any fragmentation occurring and thus analysed as a whole. During this process a solution of the proteins is sprayed from a needle normally following separation using a chromatography technique. The molecules then become ionized due to the charge of the nozzle's tip, the solvent then evaporates and the ions are directed into the spectrometer to be analysed. Tandem Mass Spectrometry (MS/MS) can be used to determine further structural characterisation of the sample where a spectrometer is used to separate ions based on the mass of the sample. Specific tandem MS equipment normally includes Electro-Spray Ionization or MALDI materials working in combination with various types of mass analyzers for example ion trap, Q-TOF, QqQ etc to fragment the peptide. De novo sequencing must be performed in order to determine partial or primary sequences for unknown genomes where a set of observed mass to charge ratio signals is compared to a computerised database. {{4 Favreau,Philippe 2006;}} Separation methods that have been highly successful have indicated that three main groups of peptides exist in terms of molecular mass; low molecular weight substances of various structure, peptides and high molecular weight substances. Low molecular weight substances have less than 1KDa and can include biogenic amines, free inorganic acids, amino acids and nucleotides. {{13 Escoubas,Pierre 2000;}} An amine called 5-hydroxytryptamine was discovered by Welsh and Betty in 1963 in the venoms of several species of Brazilian tarantula.

Other low molecular components such as adenine nucleotides have been found within the venom of three types of tarantula called; D. Hentzi, Aphonopelma sp and E Californicum. {{2 Rash,Lachlan D. 2002;}} Peptides have 1-10 KDa and consist of disulfide containing neurotoxins and linear peptides with cytolytic properties. High molecular weight substances also exist within venoms and are greater than 10 KDa. Research has proved that spider venoms consist of approximately 25% polypeptides by weight. (Vassilevski, A.A. Kozlov, S.A. and Grishin, E.V. 2009)

There are approximately 300 species of the tarantula (Theraphosidae) spider and are distributed predominately throughout tropical regions of the USA, Asia and Australia. A total of 33 peptide toxin sequences from tarantula venoms have been discovered. {{1 Escoubas,Pierre 2004;}} The peptide GrTx1 isolated from the venom of the "Rosean" tarantula, Grammostola Rosea proved to be one of 15 identified components obtained. High Performance Liquid Chromatography was used as a method of separation. This proved that the GrTx1 peptide has 29 amino acids residues compactly folded by 3 disulfide bridges with a molecular weight of 3697 Da. The GrTx1 peptide from the Chilean rose tarantula has proven to affect various distinct ion channel functions. {{17 Clement,Herlinda 2007;}} Another peptide isolated from venom of the same tarantula called GsMTx4 has a possible clinical use in treating some aspects of mechanical pain. Sensory neurones within the body contain mechanosensitive channels thought to act as molecular sensors for pressure, pain or touch. In order to determine the potential use of GsMTx4 as a blocker for pain treatment a recombinant form of the peptide was constructed and various testing was performed. It was found that when the peptide was injected into rat's significant analgesic responses to inflammation-induced mechanical hyperalgesia were obtained. {{9 Park,Seung Pyo 2008;}} Another tarantula found in areas of Southern China known as the Chinese tarantula; Chilobrachys jingzhao is extremely venomous and is composed of a mixture of compounds. Using proteomic and peptidomic analysis methods it was found that there are approximately 120 peptides, 60 being fully or partially sequenced.

The most abundant peptides are those with a molecular weight lower than 10KDa. In order for these results to be obtained gel filtration, SDS-PAGE- (early techniques), MALDI-TOF, LC/ESI-Q TOF MS, HPLC and Edman degradation sequencing were used and it is because of these techniques that accurate results have been yielded. (Liao Z, Find all citations by this author (default).Cao J, Li S, Yan X, Hu W, He Q, Chen J, Tang J, Xie J, Liang S 2007) Or filter your current search Find all citations by this author (default)LiLOr filter your current searchFind all citations by this author (default)Or filter your current search Find all citations by this author (default).Or filter your current searchFind all citations by this author (default).Or filter your current search

Analysis of venom components from the African Citharischius crawshayi spider (Theraphosidae) prove that fractions contain more than 600 molecular masses having a toxic influence on crickets and altered sodium currents in channels expressed in xenopus oocytes. (Diego-Garcia E, Peigneur S, Waelkens E, Debaveye S, Tytgat J 2010) Peptides from various other spider venoms have been discovered and include; the B. Smithi, P. Cambridgei, C. Validus and S. Hainana species. The Brachypelma Smithi tarantula originates from Mexico, using reverse phase and cation exchange chromatography methods its venom was separated. The peptide obtained is called "Bs toxin 1" and consists of 41 amino acids, linked by 3 disulfide bridges. (Corzo G, Diego-García E, Clement H, Peigneur S, Odell G, Tytgat J, Possani LD, Alagón A. 2008) The sequence of this peptide is similar to that of the Ornithoctonus Huwena spider from China and the Macrothele Gigas from Japan, all having insecticidal properties. Peptides purified from the venom of the Coremiocnemis Validus spider known as "Cvtx- ll" is also insect specific and contains a total of 31 amino acids. (Balaji RA, Sasaki T, Gopalakrishnakone P, Sato K, Kini RM, Bay BH. 2000) In relation to other spiders the sequence of this spider has less than 40% identity with toxins of other spiders. The novel peptides called: "Psalmopeotoxin 1" and "Psalmopettoxin 2" from the Psalmopoeus Cambridgei spider native to Trinidad have been identified. The first peptide is made up of 33 amino acid residues whereas the second consists of 28; however the sequences are both joined by 3 disulfide bridges. These peptides are thought to have use in the development of anti-malarial drugs. {{5 Choi,Soo-Jin 2004;}} Five peptides with various functions have been isolated from the venom of the Selenocosmia huwena now known as Ornithoctonus huwena.

The first peptide to be isolated from this spider is known as "HwTxl" where it is thought to inhibit pre-synaptic N-type Ca2þ channels. This inhibition will therefore block neurotransmitter release. "SHL-1" is a peptide without neurotoxic abilities, found in subjects such as; mice, insects and mouse phrenic nerve diaphragm assays. The second peptide is an insecticidal peptide and is called "HwTxll." Three other important toxins originating from the S.Huwena and from the Selenocosmia hainana spider act as selective inhibitors of TTX-S sodium currents in dorsal root ganglion neurons of rats. Each are known as; "HwTxIV, HnTxlV and HnTxV." These last two peptides; "HnTxlV and HnTxV" are made up of 35 amino acids and have high homology with one other. The last peptide isolated from the venom of the Selenocosmia hainana spider is thought to have no toxic effect on insects and vertebrates and is commonly known as: "HnTxl" {{14 Liang,Songping 2004;}}

Through the discovery of complex molecular mechanisms spiders have been able to produce a diverse range of neurotoxins from their venom and therefore can be seen as "busy drug chemists." Their great structural differences allow them to carryout various physiological roles thus allowing us to gain a better knowledge of ligand-receptor binding and interactions. The clinical use of the "GsMTx4" peptide as an orphan drug to treat Duchenne Muscular Dystrophy supports the concept that other tarantula venoms may be an open source for novel new toxins to treat disease. Mass Spectrometric techniques have been the fundamental success to our current knowledge of these bioactive peptides. However, a lack of genomic work on the toxins and venoms has caused a gap to emerge, not only this but our own personal understanding of toxin selectivity and relationships is restricted due to the availability of venom. {{1 Escoubas,Pierre 2004;}} This gap could possibly be sealed through further molecular biology work undertaken on the structure of genes and the identification of conserved pre-pro peptide areas. This will therefore allow all potential peptides within venoms to be recognized. Combined efforts of Mass Spectrometry and library viewings of cDNA may develop a new era in toxin research via highly specific De Novo sequencing. As demonstrated in this review the majority of toxins from different species of tarantula have the ability to act on sodium, calcium and potassium channels in the nervous system blocking post synaptic cholinergic receptors.

Through the use of this current information further work on tarantula venoms may help discover new peptides with therapeutic potential. In our own laboratory we will be undertaking work on the Aphonopelma chalcodes spider otherwise known as the western desert, Arizona blond or Mexican blond tarantula. Using some of the techniques already mentioned earlier we will hopefully be able to isolate and discover the composition of this spider