Identification of Crb2 Partner Proteins in Podocytes Using BioID

Background: Crb2 is an essential protein expressed at the slit diaphragm of podocytes and its mutations cause hereditary nephrotic syndrome. We also reported that anti-Crb2 antibodies may be causal in idiopathic nephrotic syndrome. However, how Crb2 functions in podocytes is not fully understood. To obtain insight into the signaling pathways of Crb2, we conducted proximity-dependent biotinylation and proteomics (BioID). Methods: BioID is based on the fusion of a promiscuous E.coli biotin ligase (BirA) to a target protein, Crb2. Intracellular domain of human Crb2 (1223-1285aa) was subcloned downstream of the extracellular/transmembrane domain of IL2R, conjugated with BirA, and expressed in human podocytes. As a control, human podocytes expressing GFP-BirA were used. Biotinylated proteins were captured and analyzed by mass spectrometry. Results (n=3) were analyzed using Scaffold 5. Results: Protein identification by BioID revealed 328 proteins exhibiting significant interaction with Crb2 (compared with GFP-control, p<0.05). Identified proteins were analyzed by enrichment analysis using Reactome pathways and gene ontology annotations. The results showed that Crb2 partner proteins are predominantly involved in Rho, Rac and Cdc42 GTPase signaling pathways, covering 20 out of the 25 most significantly enriched Reactome pathways. Furthermore, Crb2 interactors significantly contribute to actin cytoskeleton organization (GO ID 30036, adjusted p-Value 4.70E-08) and cell adhesion (GO ID 7155, adjusted p-Value 1.91E-06). Of interest, ARHGEF26, an upstream activator of Rho family proteins, was identified as one of the Crb2 interactors. We confirmed the interaction by co-immunoprecipitation and immunostaining showed ARHGEF26 and Crb2 colocalization in both immortalized human podocytes and mouse glomeruli. Analysis of public scRNAseq datasets showed that ARHGEF26 expression in the kidney is highly specific to podocytes. CRISPR/Cas9 KO of ARHGEF26 in podocytes reduced RhoA and Rac1 activation as well as cell projection formation in response to EGF. Conclusion: Crb2 plays a crucial role as a cytoskeletal regulatory protein in podocytes, and alterations in Crb2 signaling will likely lead to cytoskeletal changes and subsequent podocyte injury. We are investigating the identified proteins to elucidate their roles in Crb2 signaling, with the goal to identify novel therapeutic targets.