The venom itself is a cocktail of low- and high-molecular-weight components, the toxins constituting a very small proportion of these, often only one compound. They can be classified according to their mode of action and include neurotoxins (acting on the nervous system), cardiotoxins (heart), haemorrhagins (blood vessels), haemotoxins (blood), nephrotoxins (kidney), necrotoxins (tissue) and myotoxins (muscles). These diverse biological properties have led to the discovery of new compounds with useful pharmacological activities.
Modern venom studies are often carried out by proteomics methods, since the targets in these complex mixtures are peptides and/or proteins. However, many of these studies have been limited to the low-molecular-weight peptides of molecular mass 2-9 kDa. The two main reasons for this, according to Songping Liang and colleagues from Hunan Normal University and Peking University, are the relatively small amounts of the higher-molecular weight proteins in the venom and the lack of spider genomes and cDNA libraries.
Liang and his co-workers decided to investigate the venom of the Chinese tarantula, Chilobrachys jingzhao, which is regarded as a good subject since it produces larger amounts of venom. In order to target both the peptide and protein fractions of the venom in the search for toxins and biologically active compounds, the team conducted a two-pronged attack. After an initial gel filtration step into two components with molecular weight above or below 10 kDa, different fractionation and mass spectrometric characterisation processes were instigated to get the best out of each component.
The less highly populated protein fraction was fractionated by 2D gel electrophoresis followed by in-gel digestion of the individual protein spots with trypsin. All of the digests were analysed by matrix-assisted laser desorption ionisation (MALDI) MS/MS with peptide mass fingerprinting. However, this process produced no matches at all, which did not surprise the researchers since there are no proteomic/genomic databases of spider species.
The backup technique for these digests was de novo sequencing using MALDI MS/MS with the LIFT technique, in which high energy collisions were employed, as well as electrospray ionisation LC/MS/MS. In this way, 47 out of 90 protein spots were identified, revealing a collection of enzymes, haemocyanins, toxin-like proteins and many unknown or hypothetical proteins. The enzymes were involved in many different pathways but well-characterised toxic components known to be present in the venom of other tarantula species were not found.
In contrast, the heavily populated peptide fraction of molecular weight lower than 10 kDa was subjected to cation exchange chromatography to give 8 fractions, each of which was fractionated further by reversed-phase HPLC to give a total of 97 fractions. These were analysed individually by MALDI MS and Edman sequencing. About 120 peptides were distinguished with 60 being either fully or partially sequenced. More than 70% of them were in the mass range 3-5 kDa with 10% at 5-8 kDa, consistent with other tarantula venoms.
All of the fully sequenced peptides had either three or four disulphide bonds and about 30% were C-terminally amidated, although the overall amount of post-translational modifications was regarded as quite low. Some of the peptides matched with the known jinzhaotoxins found previously in this tarantula species.
The team carried out a phylogenetic analysis to try and gain insight into the biological functions of the fully sequenced peptides. One group is likely to be insecticidal toxins and a second consists of sodium and potassium channel inhibitors which are the major toxic compounds of this spider.
The results will form a reference database for further studies of the Chinese tarantula and may lead to the discovery of peptides and proteins with unique and useful biological activities as potential drugs.
Sources:
Proteomics 2007, 7, 1892-1907: "Proteomic and peptidomic analysis of the venom from Chinese tarantula Chilobrachys jingzhao"
Proteomics & Genomics
READ MORE - Proteomic of tarantula venom
Modern venom studies are often carried out by proteomics methods, since the targets in these complex mixtures are peptides and/or proteins. However, many of these studies have been limited to the low-molecular-weight peptides of molecular mass 2-9 kDa. The two main reasons for this, according to Songping Liang and colleagues from Hunan Normal University and Peking University, are the relatively small amounts of the higher-molecular weight proteins in the venom and the lack of spider genomes and cDNA libraries.
Liang and his co-workers decided to investigate the venom of the Chinese tarantula, Chilobrachys jingzhao, which is regarded as a good subject since it produces larger amounts of venom. In order to target both the peptide and protein fractions of the venom in the search for toxins and biologically active compounds, the team conducted a two-pronged attack. After an initial gel filtration step into two components with molecular weight above or below 10 kDa, different fractionation and mass spectrometric characterisation processes were instigated to get the best out of each component.
The less highly populated protein fraction was fractionated by 2D gel electrophoresis followed by in-gel digestion of the individual protein spots with trypsin. All of the digests were analysed by matrix-assisted laser desorption ionisation (MALDI) MS/MS with peptide mass fingerprinting. However, this process produced no matches at all, which did not surprise the researchers since there are no proteomic/genomic databases of spider species.
The backup technique for these digests was de novo sequencing using MALDI MS/MS with the LIFT technique, in which high energy collisions were employed, as well as electrospray ionisation LC/MS/MS. In this way, 47 out of 90 protein spots were identified, revealing a collection of enzymes, haemocyanins, toxin-like proteins and many unknown or hypothetical proteins. The enzymes were involved in many different pathways but well-characterised toxic components known to be present in the venom of other tarantula species were not found.
In contrast, the heavily populated peptide fraction of molecular weight lower than 10 kDa was subjected to cation exchange chromatography to give 8 fractions, each of which was fractionated further by reversed-phase HPLC to give a total of 97 fractions. These were analysed individually by MALDI MS and Edman sequencing. About 120 peptides were distinguished with 60 being either fully or partially sequenced. More than 70% of them were in the mass range 3-5 kDa with 10% at 5-8 kDa, consistent with other tarantula venoms.
All of the fully sequenced peptides had either three or four disulphide bonds and about 30% were C-terminally amidated, although the overall amount of post-translational modifications was regarded as quite low. Some of the peptides matched with the known jinzhaotoxins found previously in this tarantula species.
The team carried out a phylogenetic analysis to try and gain insight into the biological functions of the fully sequenced peptides. One group is likely to be insecticidal toxins and a second consists of sodium and potassium channel inhibitors which are the major toxic compounds of this spider.
The results will form a reference database for further studies of the Chinese tarantula and may lead to the discovery of peptides and proteins with unique and useful biological activities as potential drugs.
Sources:
Proteomics 2007, 7, 1892-1907: "Proteomic and peptidomic analysis of the venom from Chinese tarantula Chilobrachys jingzhao"
Proteomics & Genomics