Identification of alveolar proteins associated with claudin‐18 using BioID

Abstract

Tight junctions are protein complexes that form at sites of cell contact that function to regulate paracellular flow of small molecules, water, and ions. Distinct protein components contribute to the barrier function of tight junctions including the claudin family of transmembrane proteins and scaffolding proteins such as Zonula Occludens‐1(ZO‐1). Changes in tight junction morphology from the established smooth cobblestone patterning to a spiked junction are associated with an increase in barrier permeability (leak). Tight junction spike formation is associated with changes in claudin‐18 interacting partners, including ZO‐1. This suggests that protein‐claudin‐18 interactions are essential for tight junction morphology and therefore function. Using rat alveolar epithelial cells (rAECs), we used BioID to investigate the proximal claudin‐18 proteome. BioID uses a promiscuous biotinylating enzyme, BirA, which we fused to claudin‐18 to biotinylate proximal and interacting proteins. To determine that BirA‐claudin‐18 localized to tight junctions, rAECs were transduced with adenovirus to express BirA‐claudin‐18 and stained with claudin‐18 and streptavidin‐Cy3. This showed that the assay was functional and our chimera trafficked to tight junctions and colocalized to proximal proteins. To identify biotinylated proteins, we used tandem mass spectrometry (MS/MS) and identified 3771 protein matches within the BirA‐claudin‐18 sample. Proteins of interest were vetted, and a list of protein candidates were compiled based on peptide spectrum matches (PSMs) and known protein localization. Proteins of interest include retinoic acid and palladin, which are of specific interest because they are associated with a spike like structure found in the testis, the tubulobulbar complex, which we hypothesize behaves as a signaling platform in the testis and is comparable to tight junction spikes found in the lung. These findings show that BioID is a valid method for identification of proteins to claudin‐18, providing a discovery‐based approach to identify novel proteins that play a role in barrier function and tight junction regulation.