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Plectreurys tristis venome: A proteomic and transcriptomic analysis.

Zobel-Thropp PA, Thomas EZ, David CL, Breci LA, Binford GJ - J Venom Res (2014)

Bottom Line: With these analyses we found known venom neurotoxins U1-PLTX-Pt1a, U3-PLTX-Pt1a, and we discovered new groups of potential neurotoxins, expanding the U1- and ω-PLTX families and adding U4-through U9-PLTX as six new groups.Other proteins detected in the transcriptome were found to be members of conserved gene families and make up 20% of the transcripts.These include cDNA sequences that match venom proteins from Mesobuthus and Hottentotta scorpions, Loxosceles and Dysdera spiders, and also salivary and secreted peptide sequences from Ixodes, Amblyomma and Rhipicephalus ticks.

View Article: PubMed Central - PubMed

Affiliation: Department of Biology, Lewis & Clark College, Portland, OR 97219, USA.

ABSTRACT
Spider venoms are complex cocktails rich in peptides, proteins and organic molecules that collectively act to immobilize prey. Venoms of the primitive hunting spider, Plectreurys tristis, have numerous neurotoxic peptides called "plectoxins" (PLTX), a unique acylpolyamine called bis(agmatine)oxalamide, and larger unidentified protein components. These spiders also have unconventional multi-lobed venom glands. Inspired by these unusual characteristics and their phylogenetic position as Haplogynes, we have partially characterized the venome of P. tristis using combined transcriptomic and proteomic methods. With these analyses we found known venom neurotoxins U1-PLTX-Pt1a, U3-PLTX-Pt1a, and we discovered new groups of potential neurotoxins, expanding the U1- and ω-PLTX families and adding U4-through U9-PLTX as six new groups. The venom also contains proteins that are homologs of astacin metalloproteases that, combined with venom peptides, make up 94% of components detected in crude venom, while the remaining 6% is a single undescribed protein with unknown function. Other proteins detected in the transcriptome were found to be members of conserved gene families and make up 20% of the transcripts. These include cDNA sequences that match venom proteins from Mesobuthus and Hottentotta scorpions, Loxosceles and Dysdera spiders, and also salivary and secreted peptide sequences from Ixodes, Amblyomma and Rhipicephalus ticks. Finally, we show that crude venom has neurotoxic effects and an effective paralytic dose on crickets of 3.3µg/gm.

No MeSH data available.


Related in: MedlinePlus

Astacin sequence analysis. An alignment of nonredundant Plectreurys pro-enzyme astacins is shown (signal sequence is omitted). Important conserved astacin motifs are boxed. Peptides detected in the proteomic analysis are underlined. The consensus logo (below) was generated using WebLogo (http://weblogo.berkeley.edu) of a ClustalO amino acid alignment (http://www.ebi.ac.uk/Tools/msa/clustalo/), where the tallest letters represent the most conserved amino acids across all seven sequences. Corresponding GenBank accession numbers are listed after the C-terminus of each protein sequence.
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Figure 3: Astacin sequence analysis. An alignment of nonredundant Plectreurys pro-enzyme astacins is shown (signal sequence is omitted). Important conserved astacin motifs are boxed. Peptides detected in the proteomic analysis are underlined. The consensus logo (below) was generated using WebLogo (http://weblogo.berkeley.edu) of a ClustalO amino acid alignment (http://www.ebi.ac.uk/Tools/msa/clustalo/), where the tallest letters represent the most conserved amino acids across all seven sequences. Corresponding GenBank accession numbers are listed after the C-terminus of each protein sequence.

Mentions: Proteomic analysis of Plectreurys venom. A. Protein and peptide gels were used to visualize the venom proteome and to enrich for smaller polypeptides, respectively. Left panel, 4–20% Tris-glycine SDS-PAGE of crude venom (15 µg). Arrows point to regions corresponding to predicted astacin and venom peptide sizes based on transcriptome data. Right panel, 16.5% Tris-tricine SDS-PAGE of crude venom (25µg). Boxed areas indicate excised regions that were digested with trypsin (Princeton Separations) in the presence of ProteaseMAX (Promega) following the manufacturer’s protocol. All extracted peptides were desalted using OMIX C18 tips (Agilent Technologies), combined, and analyzed by LC MS/MS. B. Tryptic peptides that were positive hits against our cDNA database with ≥96% identity are listed and categorized into three groups based on general function. For each peptide, the percent coverage was calculated by dividing the number of amino acids in the peptide detected by the number of amino acids in the open reading frame of the corresponding cDNA sequence (Met through *stop); the superscript “nfl” indicates that the corresponding sequence is not full length. The cDNA sequence for clone 1531 is 77% identical to cDNA 1370, but corresponds only to the C-terminal 100 amino acids of the sequence, so we are not able to calculate size or percent coverage. Alignments of astacin and plectoxin sequences are presented in Figures 3 and 4, respectively.


Plectreurys tristis venome: A proteomic and transcriptomic analysis.

Zobel-Thropp PA, Thomas EZ, David CL, Breci LA, Binford GJ - J Venom Res (2014)

Astacin sequence analysis. An alignment of nonredundant Plectreurys pro-enzyme astacins is shown (signal sequence is omitted). Important conserved astacin motifs are boxed. Peptides detected in the proteomic analysis are underlined. The consensus logo (below) was generated using WebLogo (http://weblogo.berkeley.edu) of a ClustalO amino acid alignment (http://www.ebi.ac.uk/Tools/msa/clustalo/), where the tallest letters represent the most conserved amino acids across all seven sequences. Corresponding GenBank accession numbers are listed after the C-terminus of each protein sequence.
© Copyright Policy - open-access
Related In: Results  -  Collection

License
Show All Figures
getmorefigures.php?uid=PMC4231235&req=5

Figure 3: Astacin sequence analysis. An alignment of nonredundant Plectreurys pro-enzyme astacins is shown (signal sequence is omitted). Important conserved astacin motifs are boxed. Peptides detected in the proteomic analysis are underlined. The consensus logo (below) was generated using WebLogo (http://weblogo.berkeley.edu) of a ClustalO amino acid alignment (http://www.ebi.ac.uk/Tools/msa/clustalo/), where the tallest letters represent the most conserved amino acids across all seven sequences. Corresponding GenBank accession numbers are listed after the C-terminus of each protein sequence.
Mentions: Proteomic analysis of Plectreurys venom. A. Protein and peptide gels were used to visualize the venom proteome and to enrich for smaller polypeptides, respectively. Left panel, 4–20% Tris-glycine SDS-PAGE of crude venom (15 µg). Arrows point to regions corresponding to predicted astacin and venom peptide sizes based on transcriptome data. Right panel, 16.5% Tris-tricine SDS-PAGE of crude venom (25µg). Boxed areas indicate excised regions that were digested with trypsin (Princeton Separations) in the presence of ProteaseMAX (Promega) following the manufacturer’s protocol. All extracted peptides were desalted using OMIX C18 tips (Agilent Technologies), combined, and analyzed by LC MS/MS. B. Tryptic peptides that were positive hits against our cDNA database with ≥96% identity are listed and categorized into three groups based on general function. For each peptide, the percent coverage was calculated by dividing the number of amino acids in the peptide detected by the number of amino acids in the open reading frame of the corresponding cDNA sequence (Met through *stop); the superscript “nfl” indicates that the corresponding sequence is not full length. The cDNA sequence for clone 1531 is 77% identical to cDNA 1370, but corresponds only to the C-terminal 100 amino acids of the sequence, so we are not able to calculate size or percent coverage. Alignments of astacin and plectoxin sequences are presented in Figures 3 and 4, respectively.

Bottom Line: With these analyses we found known venom neurotoxins U1-PLTX-Pt1a, U3-PLTX-Pt1a, and we discovered new groups of potential neurotoxins, expanding the U1- and ω-PLTX families and adding U4-through U9-PLTX as six new groups.Other proteins detected in the transcriptome were found to be members of conserved gene families and make up 20% of the transcripts.These include cDNA sequences that match venom proteins from Mesobuthus and Hottentotta scorpions, Loxosceles and Dysdera spiders, and also salivary and secreted peptide sequences from Ixodes, Amblyomma and Rhipicephalus ticks.

View Article: PubMed Central - PubMed

Affiliation: Department of Biology, Lewis & Clark College, Portland, OR 97219, USA.

ABSTRACT
Spider venoms are complex cocktails rich in peptides, proteins and organic molecules that collectively act to immobilize prey. Venoms of the primitive hunting spider, Plectreurys tristis, have numerous neurotoxic peptides called "plectoxins" (PLTX), a unique acylpolyamine called bis(agmatine)oxalamide, and larger unidentified protein components. These spiders also have unconventional multi-lobed venom glands. Inspired by these unusual characteristics and their phylogenetic position as Haplogynes, we have partially characterized the venome of P. tristis using combined transcriptomic and proteomic methods. With these analyses we found known venom neurotoxins U1-PLTX-Pt1a, U3-PLTX-Pt1a, and we discovered new groups of potential neurotoxins, expanding the U1- and ω-PLTX families and adding U4-through U9-PLTX as six new groups. The venom also contains proteins that are homologs of astacin metalloproteases that, combined with venom peptides, make up 94% of components detected in crude venom, while the remaining 6% is a single undescribed protein with unknown function. Other proteins detected in the transcriptome were found to be members of conserved gene families and make up 20% of the transcripts. These include cDNA sequences that match venom proteins from Mesobuthus and Hottentotta scorpions, Loxosceles and Dysdera spiders, and also salivary and secreted peptide sequences from Ixodes, Amblyomma and Rhipicephalus ticks. Finally, we show that crude venom has neurotoxic effects and an effective paralytic dose on crickets of 3.3µg/gm.

No MeSH data available.


Related in: MedlinePlus