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

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.

Expression of Cell-Adhesion Molecules in E. coli: A High Throughput Screening to Identify Paracellular Modulators

“…A recent article suggested that Barrier and Fence functions are independent of each other, indicating that in vivo ZO-1 is dispensable for the Barrier function [ 47 ]. The Barrier function can be impacted by the use of CLDN extracellular loop-peptides or other protein engineering strategies employing CLDNs [ 39 , 48 , 49 ]. We thus conclude that disrupting PPIs of CLDNs equates to disrupting cell–cell adhesion.…”

Chimeric Claudins Provide a Novel Method to Research Neural Tube Defects

“…The goal of the chCLDN is to bind to native CLDN proteins and disrupt their ability to bind naturally with other CLDNs (see Figure 1a). To create this protein, we utilized the method outlined by Taylor et al, 2021. After collecting the DNA sequences of our CLDN of interest, the sequences corresponding with the transmembrane regions of the protein were substituted for a synthetic protein (referred to hereafter as 836), allowing it to maintain the general CLDN structure while preventing binding within the cell membrane. By doing so, the chCLDN has the ability to bind to specific types of CLDNs and interrupt their native interactions across the TJ.…”

“…The human genome contains 23 different types of CLDNs which play an important role in creating a structure called the tight junction (TJ). This structure enables neighboring cells to adhere to one another (Gamero-Estevez et al, 2018;Taylor et al, 2021) and is essential for numerous important steps in development including proper NT folding (Baumholtz, Gupta, et al, 2017;El Andalousi et al, 2020).…”

“…To overcome these limitations and better understand the individual contributions of various CLDN proteins, we utilized a chCLDN, a labsynthesized protein that blocks specific types of CLDN interactions (Taylor et al, 2021). Using this method, we inhibited CLDN3 within chicken embryos and observed the results.…”

"Cations as Molecular Switches for Junctional Adhesion Molecule-A"

“…In this study we recombinantly expressed the two extracellular immunoglobulin domains of JAM-A. We used Maltose Binding Protein (MBP) as a fusion partner to allow for the generation of high yield of proteins, and to maintain consistency with our previous work [14,47,[70][71][72]. The pET28-MBP-JAM-A (Figure S3) plasmid was expressed in the SHuffle T7 bacterial strain to allow for the proper folding, disulfide formation and cytoplasmic expression of the JAM-A extracellular protein [45].…”