Proteomics implicates peptidyl arginine deiminase 2 and optic nerve citrullination in glaucoma pathogenesis.
OBJECTIVE
Proteomic analyses of normal and glaucomatous human optic nerve were pursued for insights into the molecular pathology of primary open-angle glaucoma (POAG). Peptidyl arginine deiminase 2 (PAD2), an enzyme that converts protein arginine to citrulline, was found only in POAG optic nerve and was probed further for a mechanistic role in glaucoma.


METHODS
Protein identification used liquid chromatography-tandem mass spectrometry. Northern, Western, and immunohistochemical analyses measured PAD2 expression and/or protein citrullination and arginyl methylation in human and mouse optic nerve and in astrocyte cultures before and after pressure treatment. Proteins were identified after anticitrulline immunoprecipitation. In vitro translation of PAD2 was monitored in polyA RNA depleted optic nerve extracts. PAD2 shRNA transfections were evaluated in pressure-treated astrocytes.


RESULTS
Western and immunohistochemical analyses confirmed elevated PAD2 and citrullination in POAG optic nerve and decreased arginyl methylation. PAD2 was also detected in optic nerve from older, glaucomatous DBA/2J mice, but not in younger DBA/2J or control C57BL6J mice. Myelin basic protein was identified as a major citrullinated protein in POAG optic nerve. Pressure-treated astrocytes exhibited elevated PAD2 and citrullination without apparent change in PAD2 mRNA. Addition of exogenous polyA RNA to depleted optic nerve extracts yielded increased PAD2 expression in POAG but not in control extracts. Transfection with shRNA restored PAD2 and citrullination to control levels in pressure-treated astrocytes.


CONCLUSIONS
Current results support translational modulation of PAD2 expression and a possible role for the enzyme in POAG optic nerve damage through citrullination and structural disruption of myelination.
Bhattacharya
Sanjoy K
SK
Cole Eye Institute, Cleveland Clinic Foundation, Cleveland, Ohio, USA. sbhattacharya@med.miami.edu
Crabb
John S
JS
Bonilha
Vera L
VL
Gu
Xiaorong
X
Takahara
Hidenari
H
Crabb
John W
JW
D000368:Aged; D000369:Aged, 80 and over; D000818:Animals; D001120:Arginine / Q000378:metabolism; D001253:Astrocytes / Q000378:metabolism; D015153:Blotting, Western; D002956:Citrulline / Q000378:metabolism*; D004195:Disease Models, Animal; D015536:Down-Regulation; D005260:Female; D005902:Glaucoma, Open-Angle / Q000201:enzymology*; D006801:Humans; D006867:Hydrolases / Q000235:genetics / Q000378:metabolism*; D007150:Immunohistochemistry; D008297:Male; D008745:Methylation; D051379:Mice; D008810:Mice, Inbred C57BL; D008811:Mice, Inbred DBA; D008875:Middle Aged; D009901:Optic Nerve Diseases / Q000201:enzymology*; D040901:Proteomics; D012333:RNA, Messenger / Q000378:metabolism; D051381:Rats; D017207:Rats, Sprague-Dawley
D012333:RNA, Messenger; D002956:Citrulline; D001120:Arginine; D006867:Hydrolases