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Daniel Berner, Ursula Hoja, Matthias Zenkel, Francesca Pasutto, Mei Chin Lee, Tin Aung, Chiea Chuen Khor, André Reis, Friedrich E Kruse, Ursula Schlotzer-Schrehardt; Functional implication of the pseudoexfoliation-associated rare variant p.Y407F at LOXL1. Invest. Ophthalmol. Vis. Sci. 2019;60(9):1612.
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A recently performed deep sequencing study of the entire lysyl oxidase like 1 (LOXL1) locus in pseudoexfoliation (PEX) and control cases revealed a rare non-synonymous variant, p.Y407F, which conferred a 25-fold protection against PEX on a genome-wide level (Aung et al., 2017). Since this variant is localized in the catalytic domain of LOXL1, which catalyzes the crosslinking of extracellular matrix molecules such as tropoelastin, we analyzed the effect of this variant on LOXL1 enzyme function.
HEK293-EBNA cells were stably transfected with a pCEP-Pu/LOXL1-HA vector containing full length HA-tagged LOXL1 with one of six different haplotypes combining the two PEX-associated common nonsynonymous variants (p.R141L; rs1048661G>T and p.G153D; rs3825942G>A) and the rare variant (p.Y407F; rs201011613 A>T): GGA, GGT, GAA, GAT, TGA, TGT. LOXL1 proteins were affinity purified from concentrated supernatants using Anti-HA Agarose beads. LOXL1 oxidase activity was measured with an Amplex UltraRed fluorescence based assay using tropoelastin as substrate and ß-aminoproprionitril (BAPN) as control. To quantitatively monitor LOXL1 protein aggregation in solution, we used the fluorescence-based PROTEOSTAT Protein aggregation assay. Substrate binding of LOXL1 protein was measured by ELISA based solid phase binding assays using LOXL1 proteins as solid phase and human recombinant tropoelastin, fibulin-4 and fibulin-5 as soluble ligands.
Amine oxidase activity assay showed that the rare variant p.Y407F had no effect on enzymatic activity, but that the common haplotype variants GAA and TGA had significantly lower activity than GGA (p<0.05). The PROTEOSTAT assay revealed significant aggregation of LOXL1 protein compared to lysozyme monomer as control (p<0.001), but could not detect any differences between haplotype variants. Solid-phase binding assays revealed significantly increased binding of LOXL1 variants harboring the rare protective allele T to fibulin-4, but not to tropoelastin and fibulin-5, compared to proteins containing the A allele (p<0.05).
These findings indicate that the rare non-synonymous variant, p.Y407F, impacts on binding properties of LOXL1 and may exert a protective effect by increased binding to fibulin-4. These subtle differences may contribute to the formation and crosslinking of PEX material in the pathophysiology of PEX syndrome.
This abstract was presented at the 2019 ARVO Annual Meeting, held in Vancouver, Canada, April 28 - May 2, 2019.
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