Chimeric Claudins: A New Tool to Study Tight Junction Structure and Function.

Abigail Taylor,Julie Keller,Calvin Memmott,Sara Bailey,Christopher Mendoza,Dario Mizrachi,Colter Christensen,Tom Lecheminant,Arminda Suli,Mark Warner

doi:10.3390/ijms22094947

Abigail Taylor, Julie Keller + Show 8 more

Open Access

https://doi.org/10.3390/ijms22094947

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Abstract

The tight junction (TJ) is a structure composed of multiple proteins, both cytosolic and membranal, responsible for cell–cell adhesion in polarized endothelium and epithelium. The TJ is intimately connected to the cytoskeleton and plays a role in development and homeostasis. Among the TJ’s membrane proteins, claudins (CLDNs) are key to establishing blood–tissue barriers that protect organismal physiology. Recently, several crystal structures have been reported for detergent extracted recombinant CLDNs. These structural advances lack direct evidence to support quaternary structure of CLDNs. In this article, we have employed protein-engineering principles to create detergent-independent chimeric CLDNs, a combination of a 4-helix bundle soluble monomeric protein (PDB ID: 2jua) and the apical—50% of human CLDN1, the extracellular domain that is responsible for cell–cell adhesion. Maltose-binding protein-fused chimeric CLDNs (MBP-CCs) used in this study are soluble proteins that retain structural and functional aspects of native CLDNs. Here, we report the biophysical characterization of the structure and function of MBP-CCs. MBP-fused epithelial cadherin (MBP-eCAD) is used as a control and point of comparison of a well-characterized cell-adhesion molecule. Our synthetic strategy may benefit other families of 4-α-helix membrane proteins, including tetraspanins, connexins, pannexins, innexins, and more.

Highlights

The tight junction (TJ) is the most apical cell adhesion complex, found in epithelial and endothelial tissue [1]
The TJ is involved in cell signaling pathways that control cell motility, apicobasal polarity determination, and vesicular trafficking [3], and it has been found to reside in diverse compartments during the cell cycle, playing key roles regulating transcription and proliferation [4]
The cytoplasmic counterpart consists of zonula occludens proteins (ZO-1, ZO-2, ZO-3) and protein complexes related to cell polarity determination [5,6]

Summary

Introduction

The tight junction (TJ) is the most apical cell adhesion complex, found in epithelial and endothelial tissue [1]. It serves primarily as a barrier, selectively regulating the paracellular passage of solutes based on size and charge [2,3]. The TJ is involved in cell signaling pathways that control cell motility, apicobasal polarity determination, and vesicular trafficking [3], and it has been found to reside in diverse compartments during the cell cycle, playing key roles regulating transcription and proliferation [4]. The cytoplasmic counterpart consists of zonula occludens proteins (ZO-1, ZO-2, ZO-3) and protein complexes related to cell polarity determination [5,6]

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