Abstract
BackgroundMutations 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.Methodology and Main FindingsA 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 Vangl2Lp protein in mutant mice compared to the wild type mice.ConclusionOur results provide an in-depth biochemical characterisation of the interaction observed between Vangl paralogues.
Highlights
Planar cell polarity (PCP) is crucial for the regulation of tissue morphogenesis and embryonic development
Vangl2 and Vangl1 proteins belong to the Vangl protein family and are key members in mechanisms regulating Planar Cell Polarity (PCP) in vertebrates and invertebrates
The corresponding region from Vangl1 was fused to the GST as a control (GST-NVangl1). Both recombinant GST fusion proteins were produced in E. coli and were correctly expressed and purified as analysed by SDS-PAGE and Coomassie staining (Figure 1A)
Summary
Planar cell polarity (PCP) is crucial for the regulation of tissue morphogenesis and embryonic development. Vangl/ Strabismus is one of these core PCP genes and was originally identified in Drosophila where its function is mandatory for the correct development and function of eye, wing, and bristles [7,8]. Lethal missense mutations in Vangl as well as in Vangl have been identified in human embryos affected with severe neural tube defects [11,12]. Mice carrying spontaneous mutant alleles of Vangl such as the Looptail mutation (Lp) have clear PCP defects at homozygous state, including the most severe failure in neural tube closure (craniorachischisis), whereas Lp heterozygotes show only a mild phenotype [2,13]. Mutations in the Planar Cell Polarity (PCP) core gene Vangl cause the most severe neural tube defects (NTD) in mice and humans. Experimental evidence previously presented has been sparse and addressed with ectopically expressed proteins or with antibodies unable to biochemically discriminate Vangl from Vangl, giving rise to unclear results
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