Hypophosphorylation of aqueous humor sCD44 and primary open-angle glaucoma.
OBJECTIVE
The ectodomain of CD44, the principal receptor for hyaluronic acid (HA), is shed as a 32-kDa fragment-soluble CD44 (sCD44)-which is cytotoxic to trabecular meshwork (TM) cells and retinal ganglion cells (RGCs) in culture. The purpose of this study was to characterize sCD44 further by determining the phosphorylation of aqueous humor sCD44 in normal and primary open-angle glaucoma (POAG).


METHODS
Aqueous humor samples of patients were subjected to CD44 enzyme-linked immunosorbent assay (ELISA) and two-dimensional (2-D) polyacrylamide gel electrophoresis, followed by Western blot analysis with anti-CD44, anti-serine/threonine, and anti-tyrosine phosphospecific antibodies, to determine sCD44 concentration, isoelectric point (pI), and phosphorylation, respectively. The bioactivity of hypophosphorylated sCD44 was tested in cell culture and HA affinity columns.


RESULTS
Two-dimensional Western blot analysis revealed that the representative pI of the 32-kDa sCD44 was 6.96 +/- 0.07 in POAG versus 6.38 +/- 0.08 in normal (P < 0.0004). Enzymatic dephosphorylation of sCD44 resulted in a basic shift in the pI. The normal aqueous humor sCD44 was positive for serine-threonine phosphorylation; however, POAG sCD44 was hypophosphorylated. Hypophosphorylated sCD44 was more toxic to TM and RGC cells than standard sCD44, and hypophosphorylated sCD44 had decreased affinity to HA, particularly with increased pressure.


CONCLUSIONS
POAG aqueous is characterized by posttranslational change in the pI of sCD44 and hypophosphorylation, which clearly distinguished POAG from normal aqueous humor. The high toxicity and low HA-binding affinity of hypophosphorylated sCD44 may represent specific pathophysiologic features of the POAG disease process.
Knepper
Paul A
PA
Laboratory for Oculo-Cerebrospinal Investigation, Division of Neurosurgery, Children's Memorial Medical Center, Northwestern University Medical School, Chicago, IL, USA. pknepper@northwestern.edu
Miller
Adam M
AM
Choi
John
J
Wertz
Robert D
RD
Nolan
Michael J
MJ
Goossens
William
W
Whitmer
Susan
S
Yue
Beatrice Y J T
BY
Ritch
Robert
R
Liebmann
Jeffrey M
JM
Allingham
R Rand
RR
Samples
John R
JR
D001082:Aqueous Humor / Q000378:metabolism*; D015153:Blotting, Western; D018929:Cell Culture Techniques; D015180:Electrophoresis, Gel, Two-Dimensional; D004797:Enzyme-Linked Immunosorbent Assay; D005902:Glaucoma, Open-Angle / Q000378:metabolism*; D006801:Humans; D018960:Hyaluronan Receptors / Q000378:metabolism* / Q000494:pharmacology; D006820:Hyaluronic Acid / Q000378:metabolism; D047468:Immunoprecipitation; D007526:Isoelectric Point; D010766:Phosphorylation; D012165:Retinal Ganglion Cells / Q000187:drug effects; D014129:Trabecular Meshwork / Q000187:drug effects
D018960:Hyaluronan Receptors; D006820:Hyaluronic Acid