Role of clusterin gene 3'-UTR polymorphisms and promoter hypomethylation in the pathogenesis of pseudoexfoliation syndrome and pseudoexfoliation glaucoma.

Pseudoexfoliation (PEX) is a multifactorial age-related disease characterized by the deposition of extracellular fibrillar aggregates in the anterior ocular tissues. This study aims to identify the genetic and epigenetic contribution of clusterin (CLU) in PEX pathology. CLU is a molecular chaperone upregulated in PEX and genetically associated with the disease. Sequencing of a 2.9 kb region encompassing the previously associated rs2279590 in 250 control and 313 PEX [(207 pseudoexfoliation syndrome (PEXS) and 106 pseudoexfoliation glaucoma (PEXG)] individuals identified three single nucleotide polymorphisms (SNPs), rs9331942, rs9331949 and rs9331950, in the 3'-UTR of CLU of which rs9331942 and rs9331949 were found to be significantly associated with PEXS and PEXG as risk factors. Following in silico analysis, in vitro luciferase reporter assays in human embryonic kidney cells revealed that risk alleles at rs9331942 and rs9331949 bind to miR-223 and miR-1283, respectively, suggesting differential regulation of clusterin in the presence of risk alleles at the SNPs. Further, through bisulfite sequencing, we also identified that CLU promoter is hypomethylated in DNA from blood and lens capsules of PEX patients compared to controls that correlated with decreased expression of DNA methyltransferase 1 (DNMT1). Promoter demethylation of CLU using DNMT inhibitor, 5'-aza-dC, in human lens epithelial cells increased CLU expression. Chromatin immunoprecipitation assays showed that the demethylated CLU promoter provides increased access to the transcription factor, Sp1, which might lead to enhanced expression of CLU. In conclusion, this study highlights the different molecular mechanisms of clusterin regulation in pseudoexfoliation pathology.

Purification, biochemical and biophysical characterization of a zinc dependent α-mannosidase isoform III from Custard Apple (Annona squamosa) seeds.

In the present study, out of three isoforms of α-mannosidase identified in the crude extract of defatted Custard apple seed powder, isoform III has been purified to homogeneity by two-step chromatography: hydrophobic interaction and gel filtration. The purified Custard apple α-mannosidase isoform III (CAM) hydrolyzed both chromogenic (p-nitrophenyl-α-D-mannopyranoside) and fluorescent (4-methylumbelliferyl α-D-mannopyranoside) substrates. Custard apple α-mannosidase migrated as a single band in native PAGE, showed about 220 kDa molecular mass in gel filtration and in SDS PAGE, dissociated into four bands (Mr ~ 75, 68, 56 and 50 kDa respectively). Temperature and pH optima were found to be 50 °C and 4.0-5.0 respectively and CAM was stable up to 60-70 °C. The enzymatic activity of CAM was inhibited by EDTA, Ag+, Hg2+, Ni2+ and swainsonine (IC50 value of 1.5 µM). CAM was observed to be a metallo enzyme requiring zinc for its activity. Kinetic parameters KM and Vmax were found to be 1.75 mM and 0.068 U/mL respectively. The CD spectral analysis at far UV region (190-300 nm) shows that purified CAM exists as helix (30.4%), ß turns (18%) and random coils (29.7%) in its secondary structure. Chemical modification studies with N-Bromosuccinimide revealed the presence of tryptophan in its active site.