Abstract
Purpose :
Clusterin is an N-glycosylated secretory molecular chaperone involved in the prevention of non-native protein aggregation. This study aims to understand the functional role of clusterin in trabeceular meshwork (TM) aqueous humor (AH) outflow pathway by examining its expression, glycosylation and secretion.
Methods :
Using immunoblotting and immunofluorescence analyses, we assessed - A) the expression and secretion of clusterin in - primary human trabecular meshwork (HTM) cells, normal (NTM) and glaucomatous (GTM) cell lines and human AH and cell culture media, B) the effects of stressors including TGF-β2, dexamethasone, elevated pressure (2X), oxidative stress induced by hydrogen peroxide and endoplasmic reticulum (ER) stress induced by thapsigargin on clusterin expression, glycosylation and secretion, C) the role of clusterin glycosylation in HTM by expressing wild type secretory clusterin or mutant clusterin containing mutations at glycosylation sites on – i) the phagocytic activity by challenging HTM cells with pHRodo-labeled E.coli, and ii) apoptosis using annexin-V-FITC and Akt signaling. All experiments had N≥6. Paired student's t test was used to calculate statistical significance and results were significant if p<0.05.
Results :
Immunoblotting revealed the presence of fully glycosylated and nascent unglycosylated clusterin in HTM cells and secretory clusterin in both human AH and cell culture media. Clusterin immunofluorescence showed punctate cytoplasmic staining. Significant findings are as follows: GTM compared to NTM had higher levels (~2.3 fold) of unglycosylated and decreased glycosylated intracellular and secreted clusterin (~1.6 fold). In response to stressors, clusterin expression, glycosylation and secretion was decreased significantly (>2 folds) with increase in hypo/unglycosylated intracellular clusterin. HTM cells transfected with mutant clusterin plasmids compared to controls showed-decreased phagocytic activity (~38% lower), increased annexin-V as detected by immunostaining and decreased phosphorylated Akt (~1.8 fold) indicating the induction of apoptosis.
Conclusions :
The data collectively indicate that clusterin glycosylation is an important post-translational modification, which regulates the phagocytic function and cellularity of the TM indicating the potential involvement of clusterin in AH outflow regulation and glaucoma pathogenesis.
This is an abstract that was submitted for the 2017 ARVO Annual Meeting, held in Baltimore, MD, May 7-11, 2017.