A possible mechanism for increased IOP as a risk factor in Primary Open–Angle Glaucoma

Abstract

Abstract: : Purpose: Increased IOP is a known risk factor for primary open–angle glaucoma (POAG).The aqueous humor of patients with normal pressure glaucoma and POAG contains increased concentration of sCD44, the ectodomain fragment of CD44. CD44 is a receptor of hyaluronic acid(HA) whereas sCD44 is a neurotoxin to retinal ganglion cells in vitro. In the present study, we determined the binding affinity of standard sCD44 and hypo– phosphorylated sCD44 to HA. Methods: sCD44 was isolated from human sera by urea solubilization and immunoprecipitation and subjected to in vitro hypo–phosphorylation by incubation with 30 units of alkaline phosphatase for 1 hour at 37C and repeated. Highly purified HA was immobilized on EAH–Sepharose, packed in mini–columns, and graded amounts of standard and hypo–phosphorylated sCD44 were loaded on the column and eluted at operating pressures of 0 to 40 mmHg. The column was rinsed and bound sCD44 was eluted with glycine. Fractions were concentrated and analyzed by ELISA. Scatchard plots were constructed using Graphpad Prism 4.0.Results: The dissociation constant of hypo–phosphorylated sCD44 was 0.0868 nM to HA which was five–fold greater than the dissociation constant of standard sCD44. As the operating pressure of the column was increased to 40 mmHg, the dissociation constant increased so that less sCD44 bound to the HA. Additionally, though the maximum binding was comparable (Vmax), standard sCD44 bound 44 umol/mol HA and the hypo–phosphorylated sCD44 bound 60 umol/mol HA. Conclusions: An intriguing hypothesis could be drawn from these results. HA in the vitreous acts as a reservoir, an extracellular "dump" site, for sCD44. As IOP increases, the vitreous releases the neurotoxic sCD44, particularly hypo–phosphorylated sCD44, which could explain the risk factor of increased IOP in POAG.

Keywords: protein structure/function • apoptosis/cell death • proteoglycans/glycosaminoglycans