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Kristine M. Porter, Nallathambi Jeyabalan, Nikolai P. Skiba, David L. Epstein, Paloma B. Liton; Proteomic Analysis Of The Lysosomal Fraction In Trabecular Meshwork Cells Subjected To Chronic Oxidative Stress. Invest. Ophthalmol. Vis. Sci. 2012;53(14):3248.
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© ARVO (1962-2015); The Authors (2016-present)
Previous work in our laboratory reported impaired lysosomal function in trabecular meshwork (TM) cells subjected to chronic oxidative stress. Here we compare the proteomic composition of isolated lysosomes from TM cells grown under physiological and oxidative stress conditions.
Confluent cultures of porcine TM cells were grown for two weeks under physiological (5% O2) and chronic oxidative stress (40% O2) conditions. The lysosomal fraction was isolated by ultracentrifugation and cell fractioning in OptiPrep gradients. Lysosomal proteins were separated in SDS-PAGE gels, followed by in-gel trypsin digestion. The tryptic-digested peptides were identified by peptide mass finger printing analyses and tandem mass spectrometry. Protein expression levels were quantified by WB analysis using specific antibodies against LAMP1, Rab7, CTSB, CTSD, and CD63. Cathepsin activities were assayed using fluorogenic substrates (z-FR-AMC, z-RR-AMC, z-GPR-AMC, z-VVR-AMC, CTSD/E substrate).
Using an acceptance criteria for protein identification of at least two peptides with confidence interval percentage over 95%, we identified a total of 83 proteins. These included lysosomal matrix proteins and lysosomal enzymes, structural glycoproteins (LAMP1, LAMP2, LIMP2, CD63), proteins involved in translocation and lysosomal acidification (vacuolar H+ATPases), and membrane trafficking proteins. The most remarkable difference was the absence of CD63 and lysosomal acid phosphatase peptides in the lysosomal fraction from TM cells grown at 40% O2. Confirming our results obtained using whole lysates, the lysosomal fraction from oxidatively stressed cultures displayed increased LAMP1, CTSB and CTSD protein levels. However, no increased in cathepsin activities were observed. Furthermore, the proteolytic processing of CSTB from single-chain to double-chain was significantly blocked in the stressed cultures.
Here we have characterized for the first time the proteome composition of lysosomes from oxidatively stressed TM cells. Our results indicate that chronic exposure to oxidative stress induces changes in the lysosomal proteomic composition and defective lysosomal function. Since the lysosomal system is responsible for the turnover of cellular organelles and degradation of phagocytosed material, diminished lysosomal activity may lead to progressive failure of the cellular TM function with age and contribute to the pathogenesis of primary open angle glaucoma.
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