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Sanjoy K Bhattacharya, Teresia Carreon, Genea T Edwards; Degradation of Basement membrane Protein is Significantly Reduced in Glaucomatous Trabecular meshwork. Invest. Ophthalmol. Vis. Sci. 201657(12):.
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© 2017 Association for Research in Vision and Ophthalmology.
We tested the hypothesis that the basement membrane (BM) proteins undergo significantly reduced degradation in glaucomatous trabecular meshwork (TM).
To prepare basement membrane (BM) proteins, the TM was dissected from normal and glaucomatous cadaver eyes (n=10 each) and incubated in 1% sodium deoxycholate buffer containing 2% Triton X-100, 0.5% Tween-20, and 0.5% Genapol C-100 overnight. The detergent-insoluble BMs were separated by centrifugation (15000 xg) and transferred multiple times into new detergent. The BMs were then deglycosylated with heparitinase and chondroitinase ABC lyase (100 U/mL) for 3 h, then solubilized in 8 M urea/mercaptoethanol. The solubilized sample was separated on a 4−15% 1D SDS PAGE gel under reducing conditions. Bands were visualized with colloidal Coommassie blue gel stain according to the manufacturer′s protocol. The gel bands from normal and glaucomaotus TM derived BM preparations were trypsin digested and subjected to iTRAQ labeling following established procedures and were analyzed on a Q-exactive instrument. Western blot and immunohistochemistry (n=10 normal and glaucomatous eyes) was performed for selected proteins. The BM samples were also subjected to intact protein mass analyses using a MALDI-Orbitrap imaging mass spectrometer (IMS). The University of Miami Institutional Review Board deemed the anonymous use of cadaveric eye tissue to be exempt.
The selected low abundance BM proteins (LTBP2, Gasdermin, Wolframin, Alpha-tectorin) show elevated levels in glaucomatous TM. Some of these proteins are known to be of low expression in other systems. Western blot and intact IMS analyses show that increased levels of these proteins (such as Wolframin, Collagen type IV) are comprised of full length molecular weight proteins. Intact collagen IV levels were 0.2 ± 0.05 and 0.5 ± 0.07 μg/5 μg total protein extracted in normal and glaucomatous TM respectively (mean ± standard deviation, p≤0.005). Taken together with the IMS results, our results are consistent with the decreased degradation of BM proteins in glaucomatous TM compared to controls and support our hypothesis.
Basement membrane protein degradation is significantly reduced in the glaucomatous TM for selected proteins thus supporting our hypothesis.
This is an abstract that was submitted for the 2016 ARVO Annual Meeting, held in Seattle, Wash., May 1-5, 2016.
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