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

 

Priscila Yumi Tanaka Shibao¹, Camila Takeno Cologna¹, Romualdo Morandi-Filho¹, Gisele Adriano Wiezel¹, Patricia Tiemi Fujimura², Carlos Ueira-Vieira², Eliane Candiani Arantes^2 3

 

1 Laboratory of Animal Toxins, School of Pharmaceutical Scienes of Ribeirão Preto, University of São Paulo, Avenida do Café s/n, Ribeirão Preto, SP, Brazil

2 Laboratory of genetics - LABGEN, Institute of Genetics and Biochemistry, Campus Umuarama, Federal University of Uberlândia, Avenida Pará, Uberlândia, MG 1720, Brazil

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

 

Received: July 04, 2018.

Accepted: November 07, 2018.

Published: November 29, 2018.

 

Correspondence: ecabraga@fcfrp.usp.br

 

Authors’ contributions

PYTS was responsible for carrying out the transcriptome data analysis, toad's poison biochemical analysis and was a major contributor in writing the manuscript. CTC also was responsible for data analysis, design experiment and writing the manuscript. RMF performed the de novo assembling and FPM calculations. GAW performed part of the functional annotation and deposited the data. CUV surpervisioned the in silico analysis and assisted in the project design. ECA coordinated the whole team, assisted in the experiments design and advised PYTS. All authors read and approved the final manuscript.

 

Competing interests

The authors declare there are no competing interests.