Venomic, Transcriptomic, and Bioactivity Analyses of Pamphobeteus verdolaga Venom Reveal Complex Disulfide-Rich Peptides That Modulate Calcium Channels

Abstract

Pamphobeteus verdolaga is a recently described Theraphosidae spider from the Andean region of Colombia. Previous reports partially characterized its venom profile. In this study, we conducted a detailed analysis that includes reversed-phase high-performance liquid chromatography (rp-HPLC), calcium influx assays, tandem mass spectrometry analysis (tMS/MS), and venom-gland transcriptome. rp-HPLC fractions of P. verdolaga venom showed activity on CaV2.2, CaV3.2, and NaV1.7 ion channels. Active fractions contained several peptides with molecular masses ranging from 3399.4 to 3839.6 Da. The tMS/MS analysis of active fraction displaying the strongest activity to inhibit calcium channels showed sequence fragments similar to one of the translated transcripts detected in the venom-gland transcriptome. The putative peptide of this translated transcript corresponded to a toxin, here named ω-theraphositoxin-Pv3a, a potential ion channel modulator toxin that is, in addition, very similar to other theraphositoxins affecting calcium channels (i.e., ω-theraphotoxin-Asp1a). Additionally, using this holistic approach, we found that P. verdolaga venom is an important source of disulfide-rich proteins expressing at least eight superfamilies.

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“…In Cupiennius salei, short cationic peptides and ICK peptides together comprise 39% of the venom components, whereas larger proteins only contribute 15% to its diversity . The predominance of ICK peptides has also been reported in venom isolated from Pardosa pseudoannulata, Cyriopagopus hainanus (formerly Haplopelma hainanum), Selenocosmia jiafu, Lycosa singoriensis and Pamphobeteus verdolaga (Zhang et al, 2010;Undheim et al, 2013;Cheng et al, 2016;Huang et al, 2018;Estrada-Gomez et al, 2019;Hu et al, 2019). The general assumption is that ICK peptides are highly diverse components of spider venom and dozens of different peptides may be present per species .…”

Optimization of the de novo assembly of the transcriptome of the venom gland of Pamphobeteus verdolaga, prospecting novel bioactive peptides

“…The transcriptome's raw reads from the venom gland of Pamphobeteus verdolaga were obtained from the European Nucleotide Archive (ENA) under accessions numbers: PRJEB21288/ERS1788422/ERX2067777-ERR2008012. As explained by Estrada and co-workers (25), two female specimens were collected from the province of Antioquia, Colombia under the contract 155 signed by the University of Antioquia and the Environmental Ministry of Colombia, and the venom glands were extirpated. The total RNA was obtained through TRIzol ® reagent (ThermoFisher Scienti c, MA, USA) while the puri cation process of mRNA and the library creation was carried out with the Illumina mRNA TruSeq kit v2, as indicated by the manufacturer.…”

“…When we also included partial transcripts in Trinity analyses, we increased to 33% the percentage of complete genes in relation to the number of contigs (not shown); however, the inclusion of incomplete transcripts might negatively affect subsequent studies as annotation of incomplete genes leads to errors in transcript quanti cation as well as errors in gene expression pro les (40,41). A previous P. verdolaga assembly and annotation made by Estrada and collaborators with Trinity and TransDecoder, reported 78,000 contigs from which 20% (16,030) were identi ed as ORFs and only 45% were identi ed as nonredundant genes, showing a nal 9% (7173 sequences) of the total predicted contigs as genes (25). Our methods classi ed only 0.07 to 25.2% of the ORFs as redundant sequences and functional annotation of these sequences represented an increase of up to 20% more complete genes in the non-redundant merge, when compared to the previous work.…”

“…Theraphosidae venoms contain approximately 40% of proteins related to cellular processes and about 5 to 30% of proteins that evidence the ICK motif (possible toxins), while showing abundance in proteins related to redox activity, histones and proteases that also contribute to toxin diversity (49,50). Alignment of the non-redundant merged transcriptome to the Arachnoserver and EROP databases allowed the identi cation of 171 sequences with biological activities of interest; 36 of these were previously reported (25). All 135 remaining sequences matched to 427 different proteins within the aforementioned databases; however, the best hit was restricted to only 32 proteins at an average e-value of 1x10 -5 and identities smaller than 85% in 98% of the sequences.…”

“…The CD-HIT analysis showed a high level of homology within P. verdolagas' 135 predicted proteins, as 87 clusters were found with global identities higher than 30% (51). All identi ed proteins were clustered and named as per the nomenclature proposed by King and collaborators, were U stands for the unknown function and Pv for Pamphobeteus verdolaga; theraphotoxins-Pv1 to Pv3 were described elsewhere (25,52).…”