sCD44 phosphorylation in normal and Primary Open–Angle Glaucoma aqueous humor

Abstract

Abstract: : Purpose: Phosphorylation defects or alterations have been discovered in several neurodegenerative diseases. Recently, we observed that a 32–kDa ectodomain fragment of CD44, soluble CD44 (sCD44), is characteristic of primary open–angle glaucoma (POAG), is cytotoxic to retinal ganglion cells, and is hypo–phosphorylated in POAG aqueous humor. This study investigated the putative phosphorylation sites of sCD44, which may influence its functional properties. Methods: The amino acid sequence of the ectodomain fragment of sCD44 contains consensus phosphorylation motifs for PKA, PKC, CK2, and proline–directed kinases, such as MAPK. We purified sCD44 by immunoprecipitation of normal and POAG aqueous humor with an anti–CD44 monoclonal antibody recognizing the ectodomain of sCD44. The isolated sCD44 was subjected to SDS–PAGE under reducing conditions; transferred to nitrocellulose membranes; immuno–probed with a battery of phospho–specific antibodies against the following sites: serine/threonine, serine, threonine, PKA motif, PKB motif, PKC motif, MAPK/CDK motif, serine/threonine–phenylalanine, and tyrosine; incubated with the appropriate secondary HRP conjugated antibody; and finally visualized by X–ray film using enhanced chemiluminescence. Results: Western blot analysis showed that sCD44 from normal aqueous was strongly immunopositive for serine/threonine phosphorylation with immunopositive staining of 32–kDa and ∼55–kDa bands. In marked contrast, sCD44 from POAG aqueous was slightly immunopositive for serine/threonine phosphorylation, indicative that POAG sCD44 is hypo–phosphorylated in comparison to normal sCD44. Similarly, normal aqueous was immunopositive for PKC and POAG aqueous was weakly immunopositive for PKC. Both normal and POAG aqueous were immunopositive for MAPK/CDK antibody and immunonegative for tyrosine phosphorylation. Conclusions: We have identified phosphorylation sites in the ectodomain fragment of sCD44 using phospho–specific antibodies. These results will help identify the control points that lead to sCD44 hypo–phosphorylation and the increased toxicity of hypo–phosphorylated sCD44 in the POAG disease process.

Keywords: protein modifications–post translational • outflow: trabecular meshwork • signal transduction