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J Venom Anim Toxins Incl Trop Dis. 2018 Nov 29;24:36. doi: 10.1186/s40409-018-0173-8. eCollection 2018.

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

Author information

1
1Laboratory of Animal Toxins, School of Pharmaceutical Scienes of Ribeirão Preto, University of São Paulo, Avenida do Café s/n, Ribeirão Preto, SP Brazil.
2
2Laboratory of genetics - LABGEN, Institute of Genetics and Biochemistry, Campus Umuarama, Federal University of Uberlândia, Avenida Pará, Uberlândia, MG 1720 Brazil.
3
3Department of Physics and Chemistry, University of São Paulo, School of Pharmaceutical Sciences of Ribeirão Preto, Av. do Café s/n°, Monte Alegre, Ribeirão Preto, SP 14040-903 Brazil.

Abstract

Background:

Animal 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.

Methods:

Total 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.

Results:

We 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.

Conclusions:

We 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.

KEYWORDS:

Cutaneous secretion; Illumina; RNA-seq; Rhinella schneideri; Skin secretion; Toad protein; Toad secretion; Transcriptome

Conflict of interest statement

Not applicable.Not applicable.The authors declare there are no competing interests.Springer Nature remains neutral with regard to jurisdictional claims in published maps and institutional affiliations.

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