Deep sequencing analysis of toad Rhinella schneideri skin glands and partial biochemical characterization of its cutaneous secretion

Abstract

BackgroundAnimal poisons and venoms are sources of biomolecules naturally selected. Rhinella schneideri toads are widespread in the whole Brazilian territory and they have poison glands and mucous gland. Recently, protein from toads’ secretion has gaining attention. Frog skin is widely known to present great number of host defense peptides and we hypothesize toads present them as well. In this study, we used a RNA-seq analysis from R. schneideri skin and biochemical tests with the gland secretion to unravel its protein molecules.MethodsTotal RNA from the toad skin was extracted using TRizol reagent, sequenced in duplicate using Illumina Hiseq2500 in paired end analysis. The raw reads were trimmed and de novo assembled using Trinity. The resulting sequences were submitted to functional annotation against non-redundant NCBI database and Database of Anuran Defense Peptide. Furthermore, we performed caseinolytic activity test to assess the presence of serine and metalloproteases in skin secretion and it was fractionated by fast liquid protein chromatography using a reverse-phase column. The fractions were partially sequenced by Edman’s degradation.ResultsWe were able to identify several classes of antimicrobial peptides, such as buforins, peroniins and brevinins, as well as PLA2, lectins and galectins, combining protein sequencing and RNA-seq analysis for the first time. In addition, we could isolate a PLA2 from the skin secretion and infer the presence of serine proteases in cutaneous secretion.ConclusionsWe identified novel toxins and proteins from R. schneideri mucous glands. Besides, this is a pioneer study that presented the in depth characterization of protein molecules richness from this toad secretion. The results obtained herein showed evidence of novel AMP and enzymes that need to be further explored.Electronic supplementary materialThe online version of this article (10.1186/s40409-018-0173-8) contains supplementary material, which is available to authorized users.

Protein identification from the parotoid macrogland secretion of Duttaphrynus melanostictus

“…The only protein that we would call the attention is the antimicrobial peptide precursor (Additional file 1: fractions 3 and 4; Additional file 2: fraction 5). Recently, Shibao et al [41] identified several classes of antimicrobial peptides in Rhinella schneideri skin glands by transcriptomic analysis. However, complementary studies are still necessary to confirm the actual existence of them.…”

Action of heparin and acetylcholine modulators on the neurotoxicity of the toad Rhinella schneideri (Anura: Bufonidae) in Brazil

“…The lower temperature condition is known to attenuate the neuromuscular blockade by Bothrops PLA 2 neurotoxins (Cogo et al, 1998;Ponce-Soto et al, 2009;Floriano et al, 2013;Sucasaca-Monzón et al, 2015). Although there is evidence of the presence of PLA 2 enzymes in the mucous gland secretions of R. schneideri toad (Shibao et al, 2018), the same was not observed in the poison of this species. However, incubation at lower temperatures abolished the neuromuscular blockade in BC preparations while a mild and progressive neuromuscular facilitation was observed in 60 min exposure time.…”

From Animal Poisons and Venoms to Medicines: Achievements, Challenges and Perspectives in Drug Discovery

“…Mucus-rich samples, such as toad and frog poisons, is also another issue, which may hinder the use of omic approaches (Shibao et al, 2018). In this context, studies comprising animal toxins are not a simple task since many challenges must be addressed.…”