Evidence for processing enzymes in the abdominal gland of the newt, Cynops pyrrhogaster, that generate sodefrin from its biosynthetic precursor

Tomoaki Nakada, Yoko Ishizuka, Takeo Iwata, Fumiyo Toyoda, Takashi Kato, J. Michael Conlon, Sakae Kikuyama

    Research output: Contribution to journalArticle

    4 Citations (Scopus)

    Abstract

    Sodefrin (Ser-Ile-Pro-Ser-Lys-Asp-Ala-Leu-Leu-Lys) is a female-attracting peptide pheromone secreted by the abdominal gland of the male red-bellied newt, Cynops pyrrhogaster. Sequence analysis of a cDNA encoding sodefrin revealed that the peptide is located in the C-terminal region of its precursor protein (residues 177-186 of preprosodefrin) and extended from its C-terminus by the tripeptide sequence Ile187-Ser188-Ala189 and flanked at its N-terminus by Leu174-Gly175-Arg 176. This suggests that sodefrin is generated by enzymatic cleavage at monobasic (Lys and Arg) sites within the precursor molecule. To demonstrate the presence in the abdominal gland of proteolytic enzymes capable of generating sodefrin, an enzymatic assay was developed using t-butoxycarbonyl (Boc)-Leu-Gly-Arg-4methylcoumaryl-7-amide (MCA) and Boc-Leu-Leu-Lys-MCA as synthetic substrates. A crude extract of the abdominal gland hydrolyzed both substrates to liberate 7-amino-4- methylcoumarin, suggesting that enzymes that generate sodefrin from its precursor molecule are present in the gland. The activity in the extract for cleaving Boc-Leu-Gly-Arg-MCA was optimal at pH 9.0 and 45°C and for Boc-Leu-Leu-Lys-MCA at pH 9.0 and 40°C. The effects of a range of specific inhibitors on activities in the extract suggest an involvement of enzymes belonging to the serine protease family. It was also demonstrated that enzymatic activity in an extract of the abdominal glands of sexually developed males was significantly (three- to six-fold; p<0.01) higher than that of sexually undeveloped males.

    Original languageEnglish
    Pages (from-to)521-524
    Number of pages4
    JournalZoological Science
    Volume24
    Issue number5
    DOIs
    Publication statusPublished - 2007 May

    Fingerprint

    salamanders and newts
    extracts
    enzymes
    peptides
    tripeptides
    serine proteinases
    amides
    pheromones
    sequence analysis
    assays
    proteins

    Keywords

    • Amphibia
    • Newt
    • Proteolytic processing
    • Reproduction
    • Sodefrin

    ASJC Scopus subject areas

    • Animal Science and Zoology

    Cite this

    Evidence for processing enzymes in the abdominal gland of the newt, Cynops pyrrhogaster, that generate sodefrin from its biosynthetic precursor. / Nakada, Tomoaki; Ishizuka, Yoko; Iwata, Takeo; Toyoda, Fumiyo; Kato, Takashi; Conlon, J. Michael; Kikuyama, Sakae.

    In: Zoological Science, Vol. 24, No. 5, 05.2007, p. 521-524.

    Research output: Contribution to journalArticle

    Nakada, Tomoaki ; Ishizuka, Yoko ; Iwata, Takeo ; Toyoda, Fumiyo ; Kato, Takashi ; Conlon, J. Michael ; Kikuyama, Sakae. / Evidence for processing enzymes in the abdominal gland of the newt, Cynops pyrrhogaster, that generate sodefrin from its biosynthetic precursor. In: Zoological Science. 2007 ; Vol. 24, No. 5. pp. 521-524.
    @article{0e6a138038ab4a0ba9bdc7f863465663,
    title = "Evidence for processing enzymes in the abdominal gland of the newt, Cynops pyrrhogaster, that generate sodefrin from its biosynthetic precursor",
    abstract = "Sodefrin (Ser-Ile-Pro-Ser-Lys-Asp-Ala-Leu-Leu-Lys) is a female-attracting peptide pheromone secreted by the abdominal gland of the male red-bellied newt, Cynops pyrrhogaster. Sequence analysis of a cDNA encoding sodefrin revealed that the peptide is located in the C-terminal region of its precursor protein (residues 177-186 of preprosodefrin) and extended from its C-terminus by the tripeptide sequence Ile187-Ser188-Ala189 and flanked at its N-terminus by Leu174-Gly175-Arg 176. This suggests that sodefrin is generated by enzymatic cleavage at monobasic (Lys and Arg) sites within the precursor molecule. To demonstrate the presence in the abdominal gland of proteolytic enzymes capable of generating sodefrin, an enzymatic assay was developed using t-butoxycarbonyl (Boc)-Leu-Gly-Arg-4methylcoumaryl-7-amide (MCA) and Boc-Leu-Leu-Lys-MCA as synthetic substrates. A crude extract of the abdominal gland hydrolyzed both substrates to liberate 7-amino-4- methylcoumarin, suggesting that enzymes that generate sodefrin from its precursor molecule are present in the gland. The activity in the extract for cleaving Boc-Leu-Gly-Arg-MCA was optimal at pH 9.0 and 45°C and for Boc-Leu-Leu-Lys-MCA at pH 9.0 and 40°C. The effects of a range of specific inhibitors on activities in the extract suggest an involvement of enzymes belonging to the serine protease family. It was also demonstrated that enzymatic activity in an extract of the abdominal glands of sexually developed males was significantly (three- to six-fold; p<0.01) higher than that of sexually undeveloped males.",
    keywords = "Amphibia, Newt, Proteolytic processing, Reproduction, Sodefrin",
    author = "Tomoaki Nakada and Yoko Ishizuka and Takeo Iwata and Fumiyo Toyoda and Takashi Kato and Conlon, {J. Michael} and Sakae Kikuyama",
    year = "2007",
    month = "5",
    doi = "10.2108/zsj.24.521",
    language = "English",
    volume = "24",
    pages = "521--524",
    journal = "Zoological Science",
    issn = "0289-0003",
    publisher = "Zoological Society of Japan",
    number = "5",

    }

    TY - JOUR

    T1 - Evidence for processing enzymes in the abdominal gland of the newt, Cynops pyrrhogaster, that generate sodefrin from its biosynthetic precursor

    AU - Nakada, Tomoaki

    AU - Ishizuka, Yoko

    AU - Iwata, Takeo

    AU - Toyoda, Fumiyo

    AU - Kato, Takashi

    AU - Conlon, J. Michael

    AU - Kikuyama, Sakae

    PY - 2007/5

    Y1 - 2007/5

    N2 - Sodefrin (Ser-Ile-Pro-Ser-Lys-Asp-Ala-Leu-Leu-Lys) is a female-attracting peptide pheromone secreted by the abdominal gland of the male red-bellied newt, Cynops pyrrhogaster. Sequence analysis of a cDNA encoding sodefrin revealed that the peptide is located in the C-terminal region of its precursor protein (residues 177-186 of preprosodefrin) and extended from its C-terminus by the tripeptide sequence Ile187-Ser188-Ala189 and flanked at its N-terminus by Leu174-Gly175-Arg 176. This suggests that sodefrin is generated by enzymatic cleavage at monobasic (Lys and Arg) sites within the precursor molecule. To demonstrate the presence in the abdominal gland of proteolytic enzymes capable of generating sodefrin, an enzymatic assay was developed using t-butoxycarbonyl (Boc)-Leu-Gly-Arg-4methylcoumaryl-7-amide (MCA) and Boc-Leu-Leu-Lys-MCA as synthetic substrates. A crude extract of the abdominal gland hydrolyzed both substrates to liberate 7-amino-4- methylcoumarin, suggesting that enzymes that generate sodefrin from its precursor molecule are present in the gland. The activity in the extract for cleaving Boc-Leu-Gly-Arg-MCA was optimal at pH 9.0 and 45°C and for Boc-Leu-Leu-Lys-MCA at pH 9.0 and 40°C. The effects of a range of specific inhibitors on activities in the extract suggest an involvement of enzymes belonging to the serine protease family. It was also demonstrated that enzymatic activity in an extract of the abdominal glands of sexually developed males was significantly (three- to six-fold; p<0.01) higher than that of sexually undeveloped males.

    AB - Sodefrin (Ser-Ile-Pro-Ser-Lys-Asp-Ala-Leu-Leu-Lys) is a female-attracting peptide pheromone secreted by the abdominal gland of the male red-bellied newt, Cynops pyrrhogaster. Sequence analysis of a cDNA encoding sodefrin revealed that the peptide is located in the C-terminal region of its precursor protein (residues 177-186 of preprosodefrin) and extended from its C-terminus by the tripeptide sequence Ile187-Ser188-Ala189 and flanked at its N-terminus by Leu174-Gly175-Arg 176. This suggests that sodefrin is generated by enzymatic cleavage at monobasic (Lys and Arg) sites within the precursor molecule. To demonstrate the presence in the abdominal gland of proteolytic enzymes capable of generating sodefrin, an enzymatic assay was developed using t-butoxycarbonyl (Boc)-Leu-Gly-Arg-4methylcoumaryl-7-amide (MCA) and Boc-Leu-Leu-Lys-MCA as synthetic substrates. A crude extract of the abdominal gland hydrolyzed both substrates to liberate 7-amino-4- methylcoumarin, suggesting that enzymes that generate sodefrin from its precursor molecule are present in the gland. The activity in the extract for cleaving Boc-Leu-Gly-Arg-MCA was optimal at pH 9.0 and 45°C and for Boc-Leu-Leu-Lys-MCA at pH 9.0 and 40°C. The effects of a range of specific inhibitors on activities in the extract suggest an involvement of enzymes belonging to the serine protease family. It was also demonstrated that enzymatic activity in an extract of the abdominal glands of sexually developed males was significantly (three- to six-fold; p<0.01) higher than that of sexually undeveloped males.

    KW - Amphibia

    KW - Newt

    KW - Proteolytic processing

    KW - Reproduction

    KW - Sodefrin

    UR - http://www.scopus.com/inward/record.url?scp=34547297195&partnerID=8YFLogxK

    UR - http://www.scopus.com/inward/citedby.url?scp=34547297195&partnerID=8YFLogxK

    U2 - 10.2108/zsj.24.521

    DO - 10.2108/zsj.24.521

    M3 - Article

    C2 - 17867852

    AN - SCOPUS:34547297195

    VL - 24

    SP - 521

    EP - 524

    JO - Zoological Science

    JF - Zoological Science

    SN - 0289-0003

    IS - 5

    ER -