Gurriaran-Rodriguez Uxia, Datzkiw David, Radusky Leandro G, Esper Marie, Xiao Fan, Ming Hong, Fisher Solomon, Rojas Marina A, De Repentigny Yves, Kothary Rashmi, Rojas Adriana L, Serrano Luis, Hierro Aitor, Rudnicki Michael A
bioRxiv. 2023 May 30:2023.05.30.542914. doi: 10.1101/2023.05.30.542914.
Wnt proteins are secreted hydrophobic glycoproteins that act over long distances through poorly understood mechanisms. We discovered that Wnt7a is secreted on extracellular vesicles (EVs) following muscle injury. Structural analysis identified the motif responsible for Wnt7a secretion on EVs that we term the Exosome Binding Peptide (EBP). Addition of the EBP to an unrelated protein directed secretion on EVs. Disruption of palmitoylation, knockdown of WLS, or deletion of the N-terminal signal peptide did not affect Wnt7a secretion on purified EVs. Bio-ID analysis identified Coatomer proteins as candidates responsible for loading Wnt7a onto EVs. The crystal structure of EBP bound to the COPB2 coatomer subunit, the binding thermodynamics, and mutagenesis experiments, together demonstrate that a dilysine motif in the EBP mediates binding to COPB2. Other Wnts contain functionally analogous structural motifs. Mutation of the EBP results in a significant impairment in the ability of Wnt7a to stimulate regeneration, indicating that secretion of Wnt7a on exosomes is critical for normal regeneration . Our studies have defined the structural mechanism that mediates binding of Wnt7a to exosomes and elucidated the singularity of long-range Wnt signalling.
Wnt蛋白是分泌型疏水糖蛋白，通过尚不明确的机制在远距离发挥作用。我们发现，肌肉损伤后Wnt7a会分泌到细胞外囊泡（EVs）上。结构分析确定了负责Wnt7a在EVs上分泌的基序，我们将其称为外泌体结合肽（EBP）。将EBP添加到一种不相关的蛋白质上可引导其在EVs上分泌。棕榈酰化的破坏、WLS的敲低或N端信号肽的缺失均不影响Wnt7a在纯化的EVs上的分泌。生物ID分析确定衣被蛋白是负责将Wnt7a装载到EVs上的候选蛋白。EBP与COPB2衣被蛋白亚基结合的晶体结构、结合热力学和诱变实验共同表明，EBP中的双赖氨酸基序介导了与COPB2的结合。其他Wnt蛋白含有功能类似的结构基序。EBP的突变导致Wnt7a刺激再生的能力显著受损，这表明Wnt7a在外泌体上的分泌对正常再生至关重要。我们的研究确定了介导Wnt7a与外泌体结合的结构机制，并阐明了长距离Wnt信号传导的独特性。
