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H. Kwon, H. Lee, S. I. Tomarev; Effects of Chemical Chaperones on Secretion and Cleavage of Wild Type and Mutated Human and Mouse Myocilin. Invest. Ophthalmol. Vis. Sci. 2007;48(13):5905.
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© ARVO (1962-2015); The Authors (2016-present)
To study the properties of wild type (wt) and mutated (mt) MYOCILIN (MYOC) and develop approaches to treat MYOC-associated glaucoma.
COS7 and HEK293 cells were transiently transfected with cDNAs encoding wt and/or mt MYOC. Secretion and stability of wt and mt MYOC was studied by western blot analysis and ELISA assay. A Rheoswitch inducible expression system was used to produce several HEK293 cell lines stably transfected with wt MYOC.
Human and mouse wt MYOC were secreted in both HEK293 and COS7 cells, but specific proteolytic cleavage of MYOC was observed only in HEK293 cells. The human MYOC cleavage products had molecular weights of 35 kDa and 22 kDa. Specific degradation products of mouse Myoc with molecular weighs of 35 kDa and 20 kDa were observed in eye angle tissues and sclera but not in heart of old (18-24 months) mice. Young animals did not produce intensive cleavage of Myoc. The presence of the Ile477Asn human MYOC mutant inhibited secretion and cleavage of wt MYOC, while the presence of the Ile463Ser mouse Myoc mutant did not completely block secretion and cleavage of wt mouse Myoc. Several known chemical chaperones, 3-Amino-1 propanesulfonic acid (APS), 4-phenylbutyric acid (PBA), trimethylamine N-oxide dihydrate, glycerol and dimethyl sulfoxide, and heat-shock proteins (Hsp40 and Hsp60) were tested for their ability to increase secretion of mt MYOC. A combination of 2.5 mM APS and 2.5 mM PBA produced the highest increase in secretion of mt human and mouse MYOC, while other reagents were less effective or did not improve secretion of mt MYOC.
Human and mouse MYOC have similar but not identical properties. We speculate that 35 kDa and 22 kDa fragments of MYOC may have properties different from full-length MYOC molecules. Chemical chaperones might be used to enhance secretion of mt MYOC.
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