Abstract
Purpose: :
Recent evidence suggests that destabilized protein aggregates are toxic in a variety of neurodegenerative diseases. Our laboratory has identified soluble CD44 (sCD44), the shed extracellular domain of membrane CD44, as a toxic protein which may be a causative factor in primary open-angle glaucoma (POAG). This study examined whether the aggregated form of sCD44 is more toxic to trabecular meshwork (TM) cells than a de-aggregated preparation.
Methods: :
sCD44 was isolated from human serum by sequential anion exchange, hyaluronic acid affinity chromatography and immunoprecipitation. A de-aggregated sCD44 preparation was obtained by dialysis through a 50 kDa cut-off membrane which was chosen to ensure that the released proteins were monomeric. Aliquots of each preparation were followed by a thioflavin T binding fluorescence assay to measure the extent of aggregation. Blue native polyacrylamide gel electrophoresis (BNPAGE) and Western blot analysis using A11 (a beta-sheet specific antibody), serum amyloid A and CD44 antibodies were also performed to examine the extent of aggregation of each preparation. Human TM cells incubated with 1, 5 and 10 ng/ml aggregated sCD44, de-aggregated sCD44, heat-inactivated sCD44 (negative control) and 5 and 50 ug/ml amyloid beta [1-42] (positive control) were tested for cell survival after 24 hours.
Results: :
sCD44 formed extremely large aggregates and displayed a beta amyloid-like oligomer conformation by BNPAGE analysis using A11, a beta-sheet specific antibody. De-aggregation of sCD44 occurred in three days as determined by a thioflavin T fluorescence assay and BNPAGE. The sCD44 amyloid-like aggregates were toxic and decreased cell viability (P<0.005), whereas the freshly de-aggregated sCD44 preparation was non-toxic in a bioassay of cell toxicity using primary TM cells. After storage for 24 days, the de-aggregated sCD44 preparation was re-tested; sCD44 had re-aggregated by BNPAGE and regained toxicity toward TM cells.
Conclusions: :
Our results suggest that the extent of sCD44 aggregation predicates its cytotoxicity. Preventing aggregation and/or de-aggregating the toxic proteins may prove to be an effective treatment in neurodegenerative diseases such as POAG.
Keywords: trabecular meshwork • apoptosis/cell death • cell survival