Analysis of High Molecular Mass Compounds from the Spider Pamphobeteus verdolaga Venom Gland. A Transcriptomic and MS ID Approach

Abstract

Nowadays, spider venom research focuses on the neurotoxic activity of small peptides. In this study, we investigated high-molecular-mass compounds that have either enzymatic activity or housekeeping functions present in either the venom gland or venom of Pamphobeteus verdolaga. We used proteomic and transcriptomic-assisted approaches to recognize the proteins sequences related to high-molecular-mass compounds present in either venom gland or venom. We report the amino acid sequences (partial or complete) of 45 high-molecular-mass compounds detected by transcriptomics showing similarity to other proteins with either enzymatic activity (i.e., phospholipases A2, kunitz-type, hyaluronidases, and sphingomyelinase D) or housekeeping functions involved in the signaling process, glucanotransferase function, and beta-N-acetylglucosaminidase activity. MS/MS analysis showed fragments exhibiting a resemblance similarity with different sequences detected by transcriptomics corresponding to sphingomyelinase D, hyaluronidase, lycotoxins, cysteine-rich secretory proteins, and kunitz-type serine protease inhibitors, among others. Additionally, we report a probably new protein sequence corresponding to the lycotoxin family detected by transcriptomics. The phylogeny analysis suggested that P. verdolaga includes a basal protein that underwent a duplication event that gave origin to the lycotoxin proteins reported for Lycosa sp. This approach allows proposing an evolutionary relationship of high-molecular-mass proteins among P. verdolaga and other spider species.

Improving the Annotation of the Venom Gland Transcriptome of Pamphobeteus verdolaga, Prospecting Novel Bioactive Peptides

“…Furthermore, two toxins were classified as phospholipase A2 homologs and five as angiotensin-converting enzyme homologs. These two hypothetical phospholipases show homology smaller than 80% when compared to any of the previously reported phospholipase-D-verdolaga [33]. The identification of these hypothetical toxins might indicate that the bite from P. verdolaga could lead to edema, extreme pain, and possible necrosis on vertebrates.…”

“…The 318 ORFs of interest from the HMM analysis were unique; however, 115 ORFs were present in both BLAST and HMM strategies. Finally, a total of 328 ORFs of interest were obtained since 17 and 23 sequences from the BLAST and HMM strategies, respectively, were reported previously [31,33]. As per the HMM classification suggested by Koua and Kuhn-Nentwig [35], 77 families were identified from the peptides/proteins of interest, of which 12% belonged to the α-latrocrustotoxin family, 9% to the leucine-rich peptide family, 9% to the disulfide isomerase family, 8% to the serine protease family, 7% to the tachylectin 5A family, 5% to the α-latroinsectotoxin family, 5% to the α-latrotoxin family, and 3% to the δ-latroinsectotoxin family (Figure S3).…”

“…In total, 15 of the 65 mature sequences presented odd cysteine numbers: between 3 and 19. Previously described P. verdolaga toxins [33], as well as several Selenotypus plumipes toxins [56], have been reported with odd cysteine numbers. Furthermore, odd cysteine numbers have also been described previously in cones [57].…”

“…The main objective of this study is to improve the available transcriptomic resources of the spider Pamphobeteus verdolaga (Araneae: Theraphosidae). Previous works by Estrada and collaborators [18,[31][32][33] allowed the partial characterization of the venom as well as the identification of over 7137 non-redundant (nr) open reading frames (ORFs) (over 100 sequences haven been disclosed so far on the European Nucleotide Archive under accession number HAHO01), 256 disulfide bridged peptide toxins, and over 45 high molecular mass proteins from the venom gland transcriptome of P. verdolaga. Since there is evidence that a single assembly method does not retrieve all genes present within a transcriptome [29,30,34], and there is an expressed need for the generation of transcriptomic data from organisms of the Araneae order, we have improved the amount of information obtained from P. verdolaga's venom gland transcriptome by re-assembling the reads with three different free de novo assembly algorithms.…”