Functional Characterization of Rare Genetic Variants in the LOXL1 Gene Associated with Pseudoexfoliation Syndrome/Glaucoma

Abstract

Purpose : Pseudoexfoliation (PEX) syndrome/glaucoma has been linked to variants in LOXL1, coding for the cross-linking matrix enzyme lysyl oxidase-like 1. More than 60 rare mutations in LOXL1 were previously reported to confer a protective effect against PEX syndrome and glaucoma, but their contribution to disease etiology remains largely unknown. This study aims at analyzing the functional effects of three prioritized rare variants, leading to amino acid substitutions at p.407, p.425 and p.537 in the catalytic domain of LOXL1, on enzyme function and extracellular matrix synthesis in vitro.

Methods : Vector constructs containing full-length LOXL1 were subjected to site-directed mutagenesis to generate six naturally occurring haplotype combinations of rare (p.Y407F, p.R425H, p.V537M) and common variants (p.R141L, p.G153D). His-tagged LOXL1 protein variants were purified from stably transfected HEK293-EBNA cells using Ni2+-affinity chromatography and subjected to appropriate in vitro assays to analyze enzymatic activity, protein aggregation, proteolytic processing and protein interaction. Furthermore, human Tenon’s capsule fibroblasts (hTCF) were stably transfected with Flag-tagged LOXL1 protein variants to analyze subcellular localization of LOXL1, aggregation and matrix assembly based on immunofluorescence staining.

Results : LOXL1 variants carrying one of the three rare protective alleles exhibited significantly decreased enzymatic activity using synthetic (cadaverine) and natural substrates (tropoelastin) compared to variants carrying the major alleles. The presence of rare variants also enhanced binding of LOXL1 to matrix proteins involved in elastogenesis, such as tropoelastin, fibulin-4, and fibrillin-1, in vitro as well as co-localization with elastic microfibrils and fibers deposited by transfected hTCF in culture. LOXL1 protein aggregation was not directly affected by the rare alleles, but by protective haplotypes formed by rare and common variants, which showed a significantly lower propensity for in vitro aggregation and intracellular aggregate formation. No differences were observed in proteolytic processing of LOXL1 variants.

Conclusions : These findings suggest that the protective effect conferred by rare LOXL1 mutations could be mediated by promoting proper enzyme function, reduced LOXL1 aggregation and enhanced binding to elastic fiber components and elastogenesis.

This abstract was presented at the 2024 ARVO Annual Meeting, held in Seattle, WA, May 5-9, 2024.