Abstract
Purpose :
Exfoliation Syndrome (XFS) is a prevalent age-related disorder defined by the accumulation of extracellular fibrillar aggregates in the anterior segment of the eye. These aggregates are a major risk factor for the development of Exfoliation Glaucoma (XFG), a secondary open-angle glaucoma affecting about 70 million people worldwide. A genome-wide association study (GWAS) uncovered a linkage between XFG and Cytochrome P450 gene CYP39A1 variants. The objective of this study is to investigate the structural changes of CYP39A1 protein variants that may contribute to altered enzymatic activity.
Methods :
cDNAs containing five nonsynonymous single nucleotide polymorphisms of CYP39A1 (Arg103His, Leu174Pro, Phe175Leu, Asn324Lys, and Phe385Val) were compared to WT cDNA. cDNAs were overexpressed in Human Embryonic Kidney (HEK) cells. The structural changes of CYP39A1 variants were studied using SDS-PAGE western blotting from cell lysates and membrane fractions. Microscopy was used to detect differences in cell localization in immortalized hTERT fibroblasts.
Results :
Each CYP39A1 point mutation has varying enzymatic activity. A cell lysing protocol was optimized to enhance extraction of the target protein into the soluble fraction for the purpose of analyzing differences in post-translational modifications. The CYP39A1 variants were normalized to WT enzymatic activity. CYP39A1 Leu174Pro and Phe385Val have a greater than 95% reduction in enzymatic activity compared to WT. These variants displayed an increase in molecular weight (MW) and also have an aggregated phenotype in hTERT-immortalized fibroblasts compared to WT.
Conclusions :
CYP39A1 variants Leu174Pro and Phe385Val have increased MW and altered cellular distribution suggesting that structural changes or post-translational modification may contribute to the loss of WT enzymatic activity.
This abstract was presented at the 2024 ARVO Annual Meeting, held in Seattle, WA, May 5-9, 2024.