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Ursula Schlotzer-Schrehardt, Takako Sasaki, Matthias Zenkel, Friedrich Kruse, Anita Krysta; Functional Analysis of LOXL1 Missense Variants in the Pathophysiology of Pseudoexfoliation Syndrome/Glaucoma. Invest. Ophthalmol. Vis. Sci. 2013;54(15):6248.
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© ARVO (1962-2015); The Authors (2016-present)
To evaluate a potential functional role of the two non-synonymous coding polymorphisms rs1048661 (R141L) and rs3825942 (G153D) of LOXL1 (lysyl oxidase-like 1) in the pathophysiology of pseudoexfoliation (PEX) syndrome/glaucoma, a complex disorder of the elastic fiber system.
Haplotypes (G-G, T-G, G-A, T-A) of the LOXL1 variants rs1048661 and rs3825942 were generated by site-directed mutagenesis of full-length human LOXL1 cDNA. HEK-EBNA cells and primary Tenon’s capsule fibroblasts (hTCF) obtained from normal and PEX patients were stably transfected with the LOXL1 constructs. Cell lysates, supernatants, and purified LOXL1 proteins were comparatively analyzed by Western blotting, proteolytic processing, activity assays, and protein binding assays. Their effect on extracellular matrix synthesis and assembly in vitro was analyzed by real-time PCR and immunocytochemistry with and without stimulation by TGF-β1.
Stable clones produced a high steady-state level of LOXL1, which was 50-fold increased compared to non-transfected cells. Quantitative analysis of LOXL1 mRNA and protein expression/secretion, which was significantly increased by TGF-β1, did not show any significant differences between the four haplotypes. Proteolytic cleavage by endoproteases BMP-1 and furin as well as amine oxidase activities were also not different between LOXL1 variants. In the hTCF expression system, TGF-β1 stimulated co-expression of LOXL1 and elastic proteins (tropoelastin, fibrillin-1, fibulin-4 and -5) up to 30-fold and promoted incorporation of elastin into a microfibrillar scaffold 14-18 days post-confluence. The risk G-G variant displayed earlier and significantly higher expression levels of tropoelastin and fibulin-5 compared to the non-risk variant G-A. In contrast to LOXL1 (G-A), LOXL1 (G-G) also showed distinct colocalization with fibulin-5 on elastic microfibrils. Binding assays using purified LOXL1 proteins confirmed significantly increased binding of the risk variant G-G to fibulin-4 and -5, by tendency also to tropoelastin, but not to fibrillin-1, compared to the non-risk variant G-A.
Subtle functional differences regarding induction of elastogenesis and elastic matrix binding properties of LOXL1 risk variants may, in cooperation with TGF-β1, contribute to the formation and aggregation of elastotic PEX material and thus to the development of PEX syndrome/glaucoma.
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