Two platypus venom gland cDNA libraries were sequenced using the Illumina platform, which produced 19,069,168 reads of 36 nucleotides in length, and the 454 FLX platform, which yielded 239,557 reads (average length 180 nucleotides). These reads were aligned to the platypus Ensembl genebuild (v.42). Of the 239,557 FLX sequences, 50,254 had hits to 8,821 unique cDNA sequences, of which 8,734 had amino acid translations (from the total of 24,981 cDNA sequences, 24,763 of which had amino acid translations) at 85% identity and 10-5. The remaining 189,303 reads that had no hits to cDNA were aligned against the assembly (535,968 sequences from Ensembl v. 42). Of these, 151,313 had hits to the assembly at 10-5 and 85% identity.
A visual representation of Gene Ontology (GO) annotation of 454 read data is shown in Figure S1 in Additional file 1
. The most common GO terms were cellular process, metabolic process, cell and cell part, binding, and catalytic activity; full results are available online [22
]. It should be noted that GO terms such as regulation of transcription and regulation of translation, which would be required to support production and secretion of increased quantities of venom during the breeding season, appear in this list.
We identified platypus venom genes based on homology to known venom proteins. This approach was taken because we have previously found that there are homologues of all three known platypus venom peptides present in the venom of reptiles [5
]. It has previously been speculated by us as well as other groups (for example, [21
]) that there may be specific protein motifs that are preferentially selected for evolution to venom molecules independently in different animals, further supporting the use of our homology approach to identify platypus venom genes. We thus identified novel putative platypus venom genes by using TBLASTN to search the animal toxins contained within the Tox-Prot database [24
] [most toxins contained within the database come from reptilians (1,204 of 2,855; v 57.8 released September 2009)] against the platypus genome, and then looked for Ensembl or GenomeScan gene predictions overlapping with 454 and Illumina reads. Sequences for peptides encoded by these putative venom genes are available online [25
After aligning reads and Tox-Prot proteins to the platypus genome, gene prediction in regions containing both reads and Tox-Prot homologous regions yielded 155 putative genes. Predictions that did not have read support or that were expressed in three or more (of six) non-venom tissues were removed, leaving 83 putative platypus venom genes (see Additional file 1
for further details on toxin classification and Additional file 2
for peptide sequences). A threshold of three non-venom tissues was chosen so as to limit the number of false negatives; we have previously shown that platypus venom OvDLPs, OvNGF and OvCNPs are expressed in some non-venom tissues. Those genes not expressed in any non-venom tissues (33) were classified as probable (likely) platypus venom genes (Table S1 in Additional file 1
BLAST searches of GenBank and the Tox-Prot database using the peptides encoded by these genes allowed classification to toxin family (Figure ; homology was defined using E < 0.0001) and speculation about putative functions (Table ). The 83 putative platypus venom peptides came from 13 different families; it appears that like the venom of many snakes, platypus venom contains a large number of protein toxins from a small number of families [26
], possibly because after the initial emergence of a toxin gene, subsequent duplications will increase expression levels, and thus multigene toxin families are formed [27
]. GO annotation of these predicted peptides is shown in Figure . It can be seen that the GO term 'proteolysis' is highly represented (31 have this annotation), consistent with our analysis showing 33 protease-encoding genes. GO terms, including 'blood coagulation', 'pore complex biogenesis', 'cation transport', 'metallopeptidase activity', 'serine-type endopeptidase activity', and 'peptidase inhibitor activity', also match with the peptides encoded by the classes of venom genes that we discovered. In many cases, it was possible to link the putative functions of these peptides with the symptoms of platypus envenomation and the known pharmacological effects of the venom, which we discuss below.
Representation of the putative platypus venom gene families discovered by homology searching with other toxin sequences. Putative functions are shown in Table 1.
Previously unknown toxins identified in the platypus venom gland transcriptome data
Gene Ontology annotation of putative platypus venom genes. (a) Biological process; (b) cellular component; (c) molecular function. Data can be classified under more than one GO term.
Platypus venom has previously been found to have protease activity [10
], and the largest group of putative platypus venom toxins identified were proteases (33 total; 12 expressed in venom gland alone are probable platypus venom toxins). These included 7 genes that had greater than 500 Illumina reads mapping to them and which therefore appear to be highly expressed. The large number of protease genes and their high expression suggests that proteases are important components of platypus venom. There are a number of hypotheses for the activities of these, discussed in the following paragraphs, but as a group they may act to cleave venom components into active molecules in the secretory cells and lumen of the venom gland or in the tissues of the victim [10
]. The general protease activity could also help to dissolve tissue and facilitate the spread of the venom.
Twenty-six peptides were predicted from platypus venom gland cDNA to have homology to serine proteases of several types, which are found in the venom of most snakes [28
]. Nine of these are expressed in venom gland alone and are classified as probable venom toxins. A phylogenetic tree of platypus serine protease sequences is shown in Figure S2 in Additional file 1
. The kallikrein-type serine proteases encoded by five genes found in the platypus venom transcriptome may have effects including vasodilation, smooth muscle contraction, inflammation and nociperception (pain) (reviewed in [29
]). Kallikrein-like proteases are also present in shrew [30
], lizard [32
] and some snake venoms [28
]. Venom kallikreins generally possess a catalytic triad and 10 to 12 conserved cysteine residues [31
]. Not all of the identified platypus peptides contain this catalytic triad (Figure ), possibly due to problems with gene prediction, which is error-prone. However, the shrew peptides have rare non-homologous insertions near Asp of this triad [31
], and non-homologous insertions are also found in lizard gilatoxin [32
], indicating that some sequence variation is possible whilst still maintaining the kallikrein-like activity of the peptide.
Figure 3 Partial MUSCLE alignment of putative platypus venom kallikrein serine protease sequences, showing the most conserved regions. The full alignment can be seen in Figure S5 in Additional file 1. Gilatoxin (P43685), blarina toxin (BAD18893), blarinasin (Q5FBW2), (more ...)
Six of the putative platypus venom serine proteases were found to have homology to endogenous coagulation factors (for example, Factor X), which are involved in the blood coagulation cascade, and snake venom group D prothrombin activators such as trocarin D, which cause coagulation and inflammation [35
]. Many other proteins encoded by genes identified in the platypus venom transcriptome also appear to have hemostatic effects (Table ), as do many snake venoms [36
]. At first glance, the symptoms of platypus envenomation do not point to hemostatic effects, but several studies have shown that the venom does in fact affect blood characteristics. Fenner et al.
] recorded that an envenomated patient had a high erythrocyte sedimentation value, meaning that there were increased levels of pro-clotting factors present in the blood, which can be indicative of inflammation. The patient himself also noted that the spur wounds, despite being deep, bled little even though the platypus had to be forcibly removed. In vitro
experiments have shown the venom to be a coagulant, and it also causes hemorrhagic edema [16
]. We hypothesize that the putative venom serine proteases are responsible for some of these effects.
Seven genes encoding PIII zinc metalloproteinases, which contain the zinc binding motif HEXXHXXGXXH [28
], were found in the platypus venom transcriptome. Three of these were found to be expressed in venom gland alone and are classified as probable venom toxins. Zinc metalloproteinases are a second group of protease enzymes present in snake venom, which cause bleeding in the victim through fibrin(ogen)olytic activity (reviewed in [28
]). This is not a known symptom of platypus envenomation. However, some snake venom metalloproteinases (including PIIIs) do not cause bleeding, and have instead been shown to cause inflammation (reviewed in [37
]). We thus hypothesize that the seven metalloproteinases in platypus venom have inflammatory effects. The platypus venom peptides follow the same structure as snake venom PIII metalloproteinases, containing preprosequence, metalloproteinase, disintegrin, and cysteine-rich domains [28
] (Figure ). This conservation of domain and domain order across such widely divergent species as the platypus and reptiles again suggests the selection of certain peptide motifs for evolution to venom molecules.
Figure 4 Representation of domain order in the platypus venom metalloproteinases for which we appear to have complete sequence. Lowercase h denotes that the residue is not found in all platypus sequences. This arrangement mirrors that of the snake venom PIII metalloproteinases (more ...)
Ten putative platypus venom genes encode proteins with homology to kunitz-type protease inhibitors, many of which are involved in controlling the blood coagulation cascade [38
]. Six of these are expressed in venom gland alone and are classified as probable platypus venom toxins. A neighbor-joining tree of putative platypus venom kunitz-type protease inhibitors plus non-venom homologues is shown in Figure . It can be seen that the putative platypus venom peptides cluster together into a single clade, displaying the duplications that have given rise to this putative toxin family.
Unrooted neighbor-joining phylogenetic tree of the kunitz domain-containing putative platypus venom peptides (boxed). Bootstrap values less than 50 have been omitted. ENSOANT represents platypus homologues not expressed in venom gland.
Many snake venoms also contain serine protease inhibitors, which affect hemostasis and produce inflammation [40
]; toxin kunitz-type protease inhibitors called kalicludines are also found in sea anemones [41
]. The presence of these potential anticoagulant molecules may seem at odds with the proposed coagulation effects of some of the putative platypus venom serine proteases identified above, but there are examples in snakes where one venom contains multiple proteases with coagulant and anticoagulant effects, or where one protease has both effects; it is thought that in these cases the concentration of toxins determines the type of effect on the victim (reviewed in [28
]). The function of protease inhibitors in platypus venom gland is unclear, but it is suggested that perhaps these act to inhibit the catalytic activity of proteases [29
] in the venom gland, so that their effects are only released once the venom is injected into the victim. Alternatively, these inhibitors may act as neurotoxins or pro-inflammatory agents, as is the case for some of the snake venom analogues (reviewed in [42
]). It should also be noted that in other species the non-venom protease inhibitor bikunin inhibits proteolysis and inflammation [44
]. The platypus protease inhibitors thus may be expressed in the venom gland in a protective capacity to prevent inflammation in the host tissue and thus allow storage of the venom.
Proteins homologous to invertebrate venom components: alpha-latrotoxin, CRiSPs, cytolytic toxin
Genes encoding proteins with homology to invertebrate venom toxins were also found. For example, we identified seven genes encoding peptides with homology to spider venom alpha-latrotoxin, a neurotoxin also containing ankyrin repeats, which causes a massive release of neurotransmitters on contact with vertebrate neurones (reviewed in [45
]). Three of these are expressed in venom gland alone and are classified as probable platypus venom toxins. However, searches of alpha-latrotoxins against the GenBank database do reveal ankyrin repeat-containing proteins from non-venomous species at similar identities, raising the possibility that this peptide family plays a non-toxin role in the platypus venom gland. It is also possible that the homologous platypus peptides may act, like the alpha-latrotoxins, as potent neurotoxins responsible for the production of pain. Functional studies will be required to determine which hypothesis is correct.
Six genes encoding proteins with homology to CRiSPs (cysteine rich secretory proteins), which are present in a diverse range of vertebrate and invertebrate organisms, were also found. All putative platypus venom CRiSP genes were found expressed in one or more non-venom tissues, raising the possibility that they may have non-venom function. However, CRiSPs have been found in cone snail venom acting as proteases, and in snake and lizard venom acting as ion channel blockers, blockers of smooth muscle contraction (reviewed in [46
]), and myotoxins [47
]. The platypus CRiSPs may thus act as ion channel blockers to produce the muscle wasting observed in envenomated patients [9
] and the in vitro
effect of smooth muscle relaxation [10
]. An analysis of the domains contained within the putative platypus venom CRiSPs is shown in Figure S3 in Additional file 1
One protein with homology to sea anemone cytolytic toxins (for example, actinoporin) was also found. This was not found expressed in tissues other than the venom gland and on this basis is classified as a probable platypus venom toxin. This peptide has a sea anemone cytotoxic protein domain, is homologous to peptides such as hemolytic toxin and actinoporin Or-A, and does not show significant homology along its length to any proteins from other species in the National Center for Biotechnology Information (NCBI) database. Sea anemone cytotoxic proteins bind to cell membranes and have cation-selective pore-forming activity [48
]; we thus suggest that the platypus homologue could cause the weak hemolysis (breaking open of red blood cells) [17
] as well as pain [9
] that have been observed in envenomated victims. However, actinoporin homologues have also recently been discovered in some vertebrates and plants (for example, [49
]), again raising the possibility that this peptide is not a venom toxin and plays some other role in the venom gland. Functional studies will be required to confirm or refute the role of the platypus homologue in toxicity.
Another large group of putative platypus venom genes (18; 8 expressed in venom gland alone) were found to encode proteins with homology to stonustoxin, verrucotoxin and neoverrucotoxin (related peptides from the venom of the stonefish Synanceja
]), and snake venom ohanins. Previously, no overall sequence homology between the stonefish toxins and other proteins had been found [51
]. The alpha- and beta-subunits of stonustoxin are partially homologous and share a domain (B.30.2, also known as PRY-SPRY) with other proteins that may be involved in ligand binding or protein folding [52
], as well as with snake venom ohanin. All of the platypus peptides also possess SPRY, PRY, or both domains, in combination with other domains (Figure S4 in Additional file 1
Ohanin affects the central nervous system and is proposed to cause pain and reduce locomotion for both offence and defense [53
]. This effect is strikingly similar to what has been proposed as the mechanism of action for platypus venom on other platypuses [20
]. Stonustoxin and neoverrucotoxin produce hypertension (high blood pressure), hemolysis, edema, and increased vascular permeability (reviewed in [51
]), some of which are symptoms of platypus envenomation. The edema produced by stonefish envenomation is persistent (reviewed in [55
]), and it is thus possible that the platypus homologues are responsible for the persistent edema that is characteristic of platypus envenomation. The fact that B.30.2-domain-containing peptides have been found in the venom of fish, reptiles, and putatively the platypus is strong support for the hypothesis that certain protein motifs have been independently selected for evolution to venom function multiple times in different lineages.