BVES is a novel interactor of ANO5 and regulates myoblast differentiation

Haiwen Li,Zhiheng Chen,Yandi Gao,Shuliang Guo,Yuanbojiao Zuo,Li Xu,Renzhi Han,Lingling Zhao,Zuocheng Yang

doi:10.1186/s13578-021-00735-w

Abstract

BackgroundAnoctamin 5 (ANO5) is a membrane protein belonging to the TMEM16/Anoctamin family and its deficiency leads to the development of limb girdle muscular dystrophy R12 (LGMDR12). However, little has been known about the interactome of ANO5 and its cellular functions.ResultsIn this study, we exploited a proximal labeling approach to identify the interacting proteins of ANO5 in C2C12 myoblasts stably expressing ANO5 tagged with BioID2. Mass spectrometry identified 41 unique proteins including BVES and POPDC3 specifically from ANO5-BioID2 samples, but not from BioID2 fused with ANO6 or MG53. The interaction between ANO5 and BVES was further confirmed by co-immunoprecipitation (Co-IP), and the N-terminus of ANO5 mediated the interaction with the C-terminus of BVES. ANO5 and BVES were co-localized in muscle cells and enriched at the endoplasmic reticulum (ER) membrane. Genome editing-mediated ANO5 or BVES disruption significantly suppressed C2C12 myoblast differentiation with little impact on proliferation.ConclusionsTaken together, these data suggest that BVES is a novel interacting protein of ANO5, involved in regulation of muscle differentiation.

Highlights

Anoctamin 5 (ANO5) is a member of the TMEM16/Anoctamin family with putative chloride channel function and/or phospholipid scrambling activity [1,2,3]
The biotinylated proteins were readily detectable in the lysates from the stable ANO5-BioID2, ANO6-BioID2 and BioID2-MG53 C2C12 cells with biotin incubation as compared to the cells without biotin treatment, which showed minimal background biotinylation similar to the control C2C12 sample (Fig. 1B, C)
In this study, we identified Blood vessel epicardial substance (BVES) as a new ANO5-interacting protein in skeletal muscle cells using the BioID2 proximity labeling approach and mapped the interacting domains between BVES and ANO5

Summary

Introduction

ANO5 is a member of the TMEM16/Anoctamin family with putative chloride channel function and/or phospholipid scrambling activity [1,2,3]. Ano deficient mice exhibited little pathology in skeletal muscle [5, 6] while others reported a mild muscular dystrophy phenotype in a different strain of Ano5-KO mice, which exhibited defective myoblast fusion and delayed muscle. We employed BioID2 to depict the Ano interactome and identified a novel Ano5-interacting protein, BVES (Blood Vessel Epicardial Substrance, known as POPDC1), which is highly expressed in striated muscles. Anoctamin 5 (ANO5) is a membrane protein belonging to the TMEM16/Anoctamin family and its deficiency leads to the development of limb girdle muscular dystrophy R12 (LGMDR12). Little has been known about the interactome of ANO5 and its cellular functions

Methods

Results

Conclusion