Differences in a Single Extracellular Residue Underlie Adhesive Functions of Two Zebrafish Aqp0s.

Irene Vorontsova,Noriaki Nagai,James E Hall,Thomas F Schilling,Yosuke Nakazawa

doi:10.3390/cells10082005

Irene Vorontsova, Noriaki Nagai + Show 3 more

Open Access

https://doi.org/10.3390/cells10082005

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Abstract

Aquaporin 0 (AQP0) is the most abundant lens membrane protein, and loss of function in human and animal models leads to cataract formation. AQP0 has several functions in the lens including water transport and adhesion. Since lens optics rely on strict tissue architecture achieved by compact cell-to-cell adhesion between lens fiber cells, understanding how AQP0 contributes to adhesion would shed light on normal lens physiology and pathophysiology. We show in an in vitro adhesion assay that one of two closely related zebrafish Aqp0s, Aqp0b, has strong auto-adhesive properties while Aqp0a does not. The difference appears to be largely due to a single amino acid difference at residue 110 in the extracellular C-loop, which is T in Aqp0a and N in Aqp0b. Similarly, P110 is the key residue required for adhesion in mammalian AQP0, highlighting the importance of residue 110 in AQP0 cell-to-cell adhesion in vertebrate lenses as well as the divergence of adhesive and water permeability functions in zebrafish duplicates.

Highlights

Membrane intrinsic protein (MIP), known as aquaporin 0 (AQP0), is the most abundant lens membrane protein
We first tested the ability of WT zebrafish Aqp0a and Aqp0b to adhere to themselves when stably expressed in L-type mouse fibroblast cell lines
We showed a functional difference in adhesive properties between zebrafish Aqp0a and Aqp0b in an in vitro adhesion assay

Summary

Introduction

Membrane intrinsic protein (MIP), known as aquaporin 0 (AQP0), is the most abundant lens membrane protein. Self-adhesive properties of human AQP0 were first demonstrated in vitro by expressing AQP0 in adhesion-deficient mouse fibroblast L cells [8]. Mouse AQP0 was shown to adhere to untransfected adhesion-deficient mouse fibroblast L cells, negatively charged L-α-phosphatidylserine large unilamellar vesicles, and lens vesicles derived from the WT or AQP0-KO mouse lens cortex [11]. Electron crystallography of the sheep lens suggested that AQP0 connects through 109Pro, 110Pro, 113Arg, and 123Pro in the C-loop between two AQP0 molecules on adjoining membranes [12]. By mutating these residues in rat AQP0 and transfecting them in vitro, we confirmed their importance utilizing the aforementioned adhesion assay [13]. P109A/P110A significantly reduced adhesion between homotypic pairs of cells, while AQP0 V107I and R113G mutations did not alter adhesion [13]

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