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


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Article Info

DATE

2018-11-29

AUTHORS

Priscila Yumi Tanaka Shibao, Camila Takeno Cologna, Romualdo Morandi-Filho, Gisele Adriano Wiezel, Patricia Tiemi Fujimura, Carlos Ueira-Vieira, Eliane Candiani Arantes

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. More... »

PAGES

36

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    21 schema:description 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.
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    27 schema:keywords AMP
    28 Brazilian territory
    29 ConclusionsWe
    30 Edman degradation
    31 HiSeq2500
    32 Illumina HiSeq2500
    33 MethodsTotal RNA
    34 NCBI database
    35 PLA2
    36 RNA
    37 RNA-seq analysis
    38 ResultsWe
    39 Rhinella
    40 TRIzol reagent
    41 Trinity
    42 activity test
    43 addition
    44 analysis
    45 annotation
    46 antimicrobial peptides
    47 attention
    48 biochemical characterization
    49 biochemical tests
    50 biomolecules
    51 brevinins
    52 buforin
    53 characterization
    54 chromatography
    55 class
    56 column
    57 cutaneous secretions
    58 database
    59 de novo
    60 deep sequencing analysis
    61 defense peptides
    62 degradation
    63 depth characterization
    64 duplicates
    65 end analysis
    66 evidence
    67 fast liquid protein chromatography
    68 first time
    69 fraction
    70 frog skin
    71 functional annotation
    72 galectins
    73 gland
    74 gland secretion
    75 greater number
    76 host defense peptides
    77 lectin
    78 metalloproteases
    79 molecules
    80 mucous glands
    81 non-redundant NCBI database
    82 novel AMPs
    83 novel toxins
    84 novo
    85 number
    86 partial biochemical characterization
    87 peptides
    88 pioneer study
    89 poison gland
    90 poisons
    91 presence
    92 presence of serine
    93 protease
    94 protein
    95 protein chromatography
    96 protein molecules
    97 protein sequencing
    98 raw reads
    99 reads
    100 reagents
    101 results
    102 reversed-phase column
    103 richness
    104 secretion
    105 sequence
    106 sequencing
    107 sequencing analysis
    108 serine
    109 serine proteases
    110 skin
    111 skin glands
    112 skin secretions
    113 source
    114 source of biomolecules
    115 study
    116 territory
    117 test
    118 time
    119 toad Rhinella
    120 toad secretion
    121 toad skin
    122 toads
    123 toxin
    124 venom
    125 whole Brazilian territory
    126 schema:name Deep sequencing analysis of toad Rhinella schneideri skin glands and partial biochemical characterization of its cutaneous secretion
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