Molecular Characterisation of Endogenous Vangl2/Vangl1 Heteromeric Protein Complexes

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.

背景 平面细胞极性（Planar Cell Polarity，PCP）核心基因Vangl2的突变会在小鼠与人类中引发最严重的神经管缺陷（neural tube defects，NTD）。遗传学研究表明，Vangl2基因与其同源近缘基因Vangl1存在遗传互作。然而，Vangl2与Vangl1蛋白具体的相互作用与串扰机制始终是难以攻克的科学难题，核心阻碍在于二者序列同源性极高，难以实现精准区分。此前已发表的实验证据较为匮乏，且多基于异位表达的蛋白或无法生化区分Vangl1与Vangl2的抗体开展研究，因此所得结果往往模糊不清。
方法学与主要发现 本研究制备了一种高特异性抗Vangl2单克隆抗体，并通过表面等离子体共振（surface plasmon resonance，SPR）分析、蛋白质免疫印迹（western blot）与免疫沉淀实验，对重组提取的Vangl2蛋白进行了严格验证。该抗体可高效地从细胞裂解液中亲和纯化Vangl2，并能通过蛋白质组学分析明确鉴定内源性Vangl2。研究人员在Vangl2免疫沉淀产物中同时检测到Vangl1，这首次为内源性Vangl2/Vangl1异二聚体的存在提供了生化证据。通过表位标签标记的Vangl2与Vangl1实验证实，两种蛋白可相互作用并共定位于细胞膜。该抗Vangl2抗体可灵敏评估培养细胞系中Vangl2蛋白的差异表达水平，这一结论得到了基因表达分析的佐证。本研究还对携带Vangl2基因C端区域点突变（可引发严重PCP缺陷）的纯合与杂合Lp突变小鼠的耳蜗组织中Vangl2的表达特征进行了表征。相较于野生型小鼠，该抗体可在突变小鼠中检测到更低水平的Vangl2Lp蛋白。
结论 本研究结果对Vangl旁系同源蛋白之间的相互作用进行了深入的生化表征与解析。