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Relaxin-like factor (RLF)/insulin-like peptide 3 (INSL3) is secreted from testicular Leydig cells as a monomeric protein comprising three domains B–C–A with full biological activity in boars

RLF (relaxin-like factor), also known as INSL3 (insulin-like peptide 3), is a novel member of the relaxin/insulin gene family that is expressed in testicular Leydig cells. Despite the implicated role of RLF/INSL3 in testis development, its native conformation remains unknown. In the present paper we...

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Autores principales: Minagawa, Itaru, Fukuda, Masafumi, Ishige, Hisako, Kohriki, Hiroshi, Shibata, Masatoshi, Park, Enoch Y., Kawarasaki, Tatsuo, Kohsaka, Tetsuya
Formato: Online Artículo Texto
Lenguaje:English
Publicado: Portland Press Ltd. 2011
Materias:
Acceso en línea:https://www.ncbi.nlm.nih.gov/pmc/articles/PMC3242508/
https://www.ncbi.nlm.nih.gov/pubmed/21899516
http://dx.doi.org/10.1042/BJ20111107
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author Minagawa, Itaru
Fukuda, Masafumi
Ishige, Hisako
Kohriki, Hiroshi
Shibata, Masatoshi
Park, Enoch Y.
Kawarasaki, Tatsuo
Kohsaka, Tetsuya
author_facet Minagawa, Itaru
Fukuda, Masafumi
Ishige, Hisako
Kohriki, Hiroshi
Shibata, Masatoshi
Park, Enoch Y.
Kawarasaki, Tatsuo
Kohsaka, Tetsuya
author_sort Minagawa, Itaru
collection PubMed
description RLF (relaxin-like factor), also known as INSL3 (insulin-like peptide 3), is a novel member of the relaxin/insulin gene family that is expressed in testicular Leydig cells. Despite the implicated role of RLF/INSL3 in testis development, its native conformation remains unknown. In the present paper we demonstrate for the first time that boar testicular RLF/INSL3 is isolated as a monomeric structure with full biological activity. Using a series of chromatography steps, the native RLF/INSL3 was highly purified as a single peak in reverse-phase HPLC. MS/MS (tandem MS) analysis of the trypsinized sample provided 66% sequence coverage and revealed a distinct monomeric structure consisting of the B-, C- and A-domains deduced previously from the RLF/INSL3 cDNA. Moreover, the N-terminal peptide was four amino acid residues longer than predicted previously. MS analysis of the intact molecule and PMF (peptide mass fingerprinting) analysis at 100% sequence coverage confirmed this structure and indicated the existence of three site-specific disulfide bonds. RLF/INSL3 retained full bioactivity in HEK (human embryonic kidney)-293 cells expressing RXFP2 (relaxin/insulin-like family peptide receptor 2), the receptor for RLF/INSL3. Furthermore, RLF/INSL3 was found to be secreted from Leydig cells into testicular venous blood. Collectively, these results indicate that boar RLF/INSL3 is secreted from testicular Leydig cells as a B–C–A monomeric structure with full biological activity.
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spelling pubmed-32425082011-12-20 Relaxin-like factor (RLF)/insulin-like peptide 3 (INSL3) is secreted from testicular Leydig cells as a monomeric protein comprising three domains B–C–A with full biological activity in boars Minagawa, Itaru Fukuda, Masafumi Ishige, Hisako Kohriki, Hiroshi Shibata, Masatoshi Park, Enoch Y. Kawarasaki, Tatsuo Kohsaka, Tetsuya Biochem J Research Article RLF (relaxin-like factor), also known as INSL3 (insulin-like peptide 3), is a novel member of the relaxin/insulin gene family that is expressed in testicular Leydig cells. Despite the implicated role of RLF/INSL3 in testis development, its native conformation remains unknown. In the present paper we demonstrate for the first time that boar testicular RLF/INSL3 is isolated as a monomeric structure with full biological activity. Using a series of chromatography steps, the native RLF/INSL3 was highly purified as a single peak in reverse-phase HPLC. MS/MS (tandem MS) analysis of the trypsinized sample provided 66% sequence coverage and revealed a distinct monomeric structure consisting of the B-, C- and A-domains deduced previously from the RLF/INSL3 cDNA. Moreover, the N-terminal peptide was four amino acid residues longer than predicted previously. MS analysis of the intact molecule and PMF (peptide mass fingerprinting) analysis at 100% sequence coverage confirmed this structure and indicated the existence of three site-specific disulfide bonds. RLF/INSL3 retained full bioactivity in HEK (human embryonic kidney)-293 cells expressing RXFP2 (relaxin/insulin-like family peptide receptor 2), the receptor for RLF/INSL3. Furthermore, RLF/INSL3 was found to be secreted from Leydig cells into testicular venous blood. Collectively, these results indicate that boar RLF/INSL3 is secreted from testicular Leydig cells as a B–C–A monomeric structure with full biological activity. Portland Press Ltd. 2011-12-14 2012-01-01 /pmc/articles/PMC3242508/ /pubmed/21899516 http://dx.doi.org/10.1042/BJ20111107 Text en © 2012 The Author(s) The author(s) has paid for this article to be freely available under the terms of the Creative Commons Attribution Non-Commercial Licence (http://creativecommons.org/licenses/by-nc/2.5/) which permits unrestricted non-commercial use, distribution and reproduction in any medium, provided the original work is properly cited. http://creativecommons.org/licenses/by-nc/2.5/ This is an open-access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted non-commercial use, distribution, and reproduction in any medium, provided the original work is properly cited.
spellingShingle Research Article
Minagawa, Itaru
Fukuda, Masafumi
Ishige, Hisako
Kohriki, Hiroshi
Shibata, Masatoshi
Park, Enoch Y.
Kawarasaki, Tatsuo
Kohsaka, Tetsuya
Relaxin-like factor (RLF)/insulin-like peptide 3 (INSL3) is secreted from testicular Leydig cells as a monomeric protein comprising three domains B–C–A with full biological activity in boars
title Relaxin-like factor (RLF)/insulin-like peptide 3 (INSL3) is secreted from testicular Leydig cells as a monomeric protein comprising three domains B–C–A with full biological activity in boars
title_full Relaxin-like factor (RLF)/insulin-like peptide 3 (INSL3) is secreted from testicular Leydig cells as a monomeric protein comprising three domains B–C–A with full biological activity in boars
title_fullStr Relaxin-like factor (RLF)/insulin-like peptide 3 (INSL3) is secreted from testicular Leydig cells as a monomeric protein comprising three domains B–C–A with full biological activity in boars
title_full_unstemmed Relaxin-like factor (RLF)/insulin-like peptide 3 (INSL3) is secreted from testicular Leydig cells as a monomeric protein comprising three domains B–C–A with full biological activity in boars
title_short Relaxin-like factor (RLF)/insulin-like peptide 3 (INSL3) is secreted from testicular Leydig cells as a monomeric protein comprising three domains B–C–A with full biological activity in boars
title_sort relaxin-like factor (rlf)/insulin-like peptide 3 (insl3) is secreted from testicular leydig cells as a monomeric protein comprising three domains b–c–a with full biological activity in boars
topic Research Article
url https://www.ncbi.nlm.nih.gov/pmc/articles/PMC3242508/
https://www.ncbi.nlm.nih.gov/pubmed/21899516
http://dx.doi.org/10.1042/BJ20111107
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