NLGN3-tagging system for Evaluating Membrane Localization Applied to Drug Screening

Abstract

Aberrant membrane localization of proteins often results in functional impairment, contributing to the development of pathological conditions. Neuroligin-3 (NLGN3) is a rare but recurrent genetic risk factor for autism spectrum disorder (ASD). Comparing surface localization across NLGN3 variants requires that surface signal be referenced to expression level in the same cell. Using NLGN3 as a model, we developed I-CLiQ, a live-cell method in which the target coding sequence is introduced at a single site of a fixed vector backbone and DsRed is co-encoded as an internal expression reference, based on a 2A peptide of Perina nuda virus modified to abolish ribosomal skipping. Combining I-CLiQ with AlphaFold/AlphaMissense analysis, we evaluated ASD-associated NLGN3 variants. Structural modeling predicted steric clashes for p.R451C, p.P514S, and p.R597W. I-CLiQ-based flow cytometry confirmed that these variants significantly decrease surface expression. Furthermore, as a proof-of-concept application, we used I-CLiQ to evaluate two compounds, 4-phenylbutyrate (4-PBA) and 3-amino-1-propanesulfonic acid (3-APS); 4-PBA partially restored the surface expression of p.R451C and p.R597W. These findings establish I-CLiQ as a quantitative tool for analyzing membrane trafficking defects and demonstrate its applicability to compound screening for variants associated with conformational diseases.