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Suresh Regmi, Michael L. Samsom, Gregory D. Jay, Benjamin D Sullivan, Tannin A Schmidt; Degradation of Proteoglycan 4 / Lubricin by Cathepsin S: Potential Mechanism for Diminished Ocular Surface Lubrication in Sjögren’s Syndrome. Invest. Ophthalmol. Vis. Sci. 2015;56(7 ):323.
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Sjögren’s syndrome (SS) is an autoimmune disease affecting the lacrimal and salivary glands with hallmark clinical symptoms of dry eye and dry mouth. Recently, markedly increased levels of cathepsin S (CTSS) activity has been observed in the tears of SS patients. Proteoglycan 4 (PRG4), or lubricin, is an effective ocular surface boundary lubricant that is naturally present on the ocular surface. PRG4 has previously been shown to be susceptible to proteolytic digestion by matrix metalloproteases 1 and 7, serine proteases elastase and plasmin, and the cysteine protease cathepsin B. However, the potential effect of CTSS on PRG4 remains unknown. As such, the objective of this study was to determine the ability of CTSS to enzymatically degrade PRG4.
CTSS was obtained from R&D Systems, and full-length recombinant human PRG4 (rhPRG4) was provided by Lubris. To assess the potential time course and dose-dependency of rhPRG4 digestion by CTSS, rhPRG4 was incubated at 37°C with or without CTSS in an enzymatic digestion buffer of (50 mM sodium acetate + 250 mM sodium chloride + 5 mM DTT at pH 4.5) for 2, 8, and 20 h at a CTSS:rhPRG4 mass ratios of 0.01, and 0.03 for 20 hours. Digestion products were separated on 3-8% SDS-PAGE and visualised by incubation with fluorescent Sypro Ruby Protein Stain.
CTSS enzymatically digested rhPRG4 in a time and dose dependent manner. CTSS digestion of rhPRG4 at a ratio 0.01 resulted in a time dependent decrease in the full-length ~460 KDa MW rhPRG4 band, and an appearance of lower MW (<40kDa) fragments. After 20 hours, no full-length rhPRG4 was observed. Furthermore, with an increased relative enzyme ratio of 0.03, no protein bands were observed after 20 h, indicating complete digestion of the rhPRG4 to fragments. Control samples showed no degradation of the rhPRG4.
These results demonstrate that rhPRG4 is susceptible to proteolytic digestion by CTSS. Given the elevated levels of CTSS in SS and the role intact PRG4 plays in ocular surface health and lubrication, degradation of PRG4 by CTSS could be a potential mechanism for diminished ocular surface lubrication in SS. Collectively these results suggest that supplementation of PRG4 may be warranted for SS patients.
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