Abstract
Purpose: :
Our proteomic studies identify glaucomatous alterations in protein expression. This study focused on the complement system that is an essential effector of innate and adaptive immune activities involved in cytolysis and immune/inflammatory responses.
Methods: :
Human retinal protein samples were obtained from ten donors with (n:5) or without (n:5) glaucoma. Tryptic peptides were analyzed by 2D-LC-MS/MS. The mass spectral data generated by Sequest Sorcerer were submitted to BIGCAT statistical platforms to establish a protein abundance factor for comparison of protein enrichment within and between samples. Bioinformatic analysis utilized ARIADNE Pathway Studio and Ingenuity Pathways Analysis. For further validation of the proteomic findings, quantitative western blot analysis was performed using specific antibodies. In addition, the extent and cellular localization of specific immunolabeling for different complement components were determined in human donor eyes with glaucoma (n:34) in comparison to age-matched control eyes without glaucoma (n:20).
Results: :
Proteomic analysis detected expression and differential regulation of several complement components in glaucomatous samples, which included C1q, C4BP, C4b, C3, C5aR, C8, and C9. Besides these proteins involved in the classical complement pathway and lytic pathway, proteins differentially regulated in human glaucoma also included those linked to the lectin pathway of complement activity, such as mannose-binding lectin, galectin, and C-type lectin. In addition, several complement regulatory proteins, including CD35 (CR1), CD55 (DAF), and clusterin, were detected in the retinal proteome, and glaucomatous samples exhibited a trend toward down-regulation of complement factor H (HF1), a major inhibitor of the alternative complement pathway with additional regulatory impacts on the classical and lectin pathways. Western blot analysis and immunohistochemistry using specific antibodies validated protein expression and cellular localization.
Conclusions: :
These findings support the role of complement activity in the neurodegenerative injury in human glaucoma. The complement cascade may initially participate in healing of the injured tissue by eliminating cell debris; however, an intrinsic deficiency in the regulation of complement activity may then facilitate tissue injury. Ongoing studies should further illuminate cellular mechanisms involved in the regulation and dysregulation of immune activity in glaucoma, thereby providing neuroprotective treatment possibilities by immunomodulation.
Keywords: proteomics • ganglion cells • immunomodulation/immunoregulation