Molecular characterisation of endogenous Vangl2/Vangl1 heteromeric protein complexes.

<h4>Background</h4>Mutations in the Planar Cell Polarity (PCP) core gene Vangl2 cause the most severe neural tube defects (NTD) in mice and humans. Genetic studies show that the Vangl2 gene genetically interacts with a close homologue Vangl1. How precisely Vangl2 and Vangl1 proteins inte...

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Autores principales:	Edwige Belotti, Tania M Puvirajesinghe, Stéphane Audebert, Emilie Baudelet, Luc Camoin, Michel Pierres, Lea Lasvaux, Géraldine Ferracci, Mireille Montcouquiol, Jean-Paul Borg
Formato:	article
Lenguaje:	EN
Publicado:	Public Library of Science (PLoS) 2012
Materias:	Medicine R Science Q
Acceso en línea:	https://doaj.org/article/87709adb080a4559b31b0d6be284f9a3
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spelling	oai:doaj.org-article:87709adb080a4559b31b0d6be284f9a32021-11-18T08:13:44ZMolecular characterisation of endogenous Vangl2/Vangl1 heteromeric protein complexes.1932-620310.1371/journal.pone.0046213https://doaj.org/article/87709adb080a4559b31b0d6be284f9a32012-01-01T00:00:00Zhttps://www.ncbi.nlm.nih.gov/pmc/articles/pmid/23029439/?tool=EBIhttps://doaj.org/toc/1932-6203<h4>Background</h4>Mutations in the Planar Cell Polarity (PCP) core gene Vangl2 cause the most severe neural tube defects (NTD) in mice and humans. Genetic studies show that the Vangl2 gene genetically interacts with a close homologue Vangl1. How precisely Vangl2 and Vangl1 proteins interact and crosstalk has remained a difficult issue to address, with the main obstacle being the accurate discrimination of the two proteins, which share close sequence homology. Experimental evidence previously presented has been sparse and addressed with ectopically expressed proteins or with antibodies unable to biochemically discriminate Vangl1 from Vangl2, therefore giving rise to unclear results.<h4>Methodology and main findings</h4>A highly specific monoclonal anti-Vangl2 antibody was generated and rigorously tested on both recombinant and extracted Vangl2 using surface plasmon resonance (SPR) analysis, western blot, and immunoprecipitation experiments. This antibody efficiently affinity-purified Vangl2 from cell lysates and allowed the unambiguous identification of endogenous Vangl2 by proteomic analysis. Vangl1 was also present in Vangl2 immunoprecipitates, establishing the first biochemical evidence for the existence of Vangl2/Vangl1 heterodimers at an endogenous level. Epitope-tagged Vangl2 and Vangl1 confirmed that both proteins interact and colocalize at the plasma membrane. The Vangl2 antibody is able to acutely assess differential expression levels of Vangl2 protein in culture cell lines, as corroborated with gene expression analysis. We characterised Vangl2 expression in the cochlea of homozygous and heterozygous Lp mutant mice bearing a point mutation within the C-terminal Vangl2 region that leads to profound PCP defects. Our antibody could detect much lower levels of Vangl2(Lp) protein in mutant mice compared to the wild type mice.<h4>Conclusion</h4>Our results provide an in-depth biochemical characterisation of the interaction observed between Vangl paralogues.Edwige BelottiTania M PuvirajesingheStéphane AudebertEmilie BaudeletLuc CamoinMichel PierresLea LasvauxGéraldine FerracciMireille MontcouquiolJean-Paul BorgPublic Library of Science (PLoS)articleMedicineRScienceQENPLoS ONE, Vol 7, Iss 9, p e46213 (2012)
institution	DOAJ
collection	DOAJ
language	EN
topic	Medicine R Science Q
spellingShingle	Medicine R Science Q Edwige Belotti Tania M Puvirajesinghe Stéphane Audebert Emilie Baudelet Luc Camoin Michel Pierres Lea Lasvaux Géraldine Ferracci Mireille Montcouquiol Jean-Paul Borg Molecular characterisation of endogenous Vangl2/Vangl1 heteromeric protein complexes.
description	<h4>Background</h4>Mutations in the Planar Cell Polarity (PCP) core gene Vangl2 cause the most severe neural tube defects (NTD) in mice and humans. Genetic studies show that the Vangl2 gene genetically interacts with a close homologue Vangl1. How precisely Vangl2 and Vangl1 proteins interact and crosstalk has remained a difficult issue to address, with the main obstacle being the accurate discrimination of the two proteins, which share close sequence homology. Experimental evidence previously presented has been sparse and addressed with ectopically expressed proteins or with antibodies unable to biochemically discriminate Vangl1 from Vangl2, therefore giving rise to unclear results.<h4>Methodology and main findings</h4>A highly specific monoclonal anti-Vangl2 antibody was generated and rigorously tested on both recombinant and extracted Vangl2 using surface plasmon resonance (SPR) analysis, western blot, and immunoprecipitation experiments. This antibody efficiently affinity-purified Vangl2 from cell lysates and allowed the unambiguous identification of endogenous Vangl2 by proteomic analysis. Vangl1 was also present in Vangl2 immunoprecipitates, establishing the first biochemical evidence for the existence of Vangl2/Vangl1 heterodimers at an endogenous level. Epitope-tagged Vangl2 and Vangl1 confirmed that both proteins interact and colocalize at the plasma membrane. The Vangl2 antibody is able to acutely assess differential expression levels of Vangl2 protein in culture cell lines, as corroborated with gene expression analysis. We characterised Vangl2 expression in the cochlea of homozygous and heterozygous Lp mutant mice bearing a point mutation within the C-terminal Vangl2 region that leads to profound PCP defects. Our antibody could detect much lower levels of Vangl2(Lp) protein in mutant mice compared to the wild type mice.<h4>Conclusion</h4>Our results provide an in-depth biochemical characterisation of the interaction observed between Vangl paralogues.
format	article
author	Edwige Belotti Tania M Puvirajesinghe Stéphane Audebert Emilie Baudelet Luc Camoin Michel Pierres Lea Lasvaux Géraldine Ferracci Mireille Montcouquiol Jean-Paul Borg
author_facet	Edwige Belotti Tania M Puvirajesinghe Stéphane Audebert Emilie Baudelet Luc Camoin Michel Pierres Lea Lasvaux Géraldine Ferracci Mireille Montcouquiol Jean-Paul Borg
author_sort	Edwige Belotti
title	Molecular characterisation of endogenous Vangl2/Vangl1 heteromeric protein complexes.
title_short	Molecular characterisation of endogenous Vangl2/Vangl1 heteromeric protein complexes.
title_full	Molecular characterisation of endogenous Vangl2/Vangl1 heteromeric protein complexes.
title_fullStr	Molecular characterisation of endogenous Vangl2/Vangl1 heteromeric protein complexes.
title_full_unstemmed	Molecular characterisation of endogenous Vangl2/Vangl1 heteromeric protein complexes.
title_sort	molecular characterisation of endogenous vangl2/vangl1 heteromeric protein complexes.
publisher	Public Library of Science (PLoS)
publishDate	2012
url	https://doaj.org/article/87709adb080a4559b31b0d6be284f9a3
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Molecular characterisation of endogenous Vangl2/Vangl1 heteromeric protein complexes.

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