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Stanislav I Tomarev, Myung Kuk Joe; Myocilin is a modulator of TIMP3 activity. Invest. Ophthalmol. Vis. Sci. 2016;57(12):No Pagination Specified.
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© ARVO (1962-2015); The Authors (2016-present)
To gain better insight into the molecular mechanisms underlying myocilin action in different eye structures.
Lysates of adult mouse eyes with removed lenses were used for immunoprecipitation using polyclonal rabbit antibodies against mouse myocilin that were cross-linked to protein A/G beads. Immunoprecipitates were eluted from the beads and analyzed by shotgun proteomics analysis. Binding of myocilin to Timp3 was assessed by co-immunoprecipitation from eye lysates and from transiently transfected HepG2 cells. The distribution of myocilin and Timp3 was investigated by immunofluorescent labeling using a confocal laser microscope. The inhibitory action of Timp3 was tested by measuring the gelatinase activity of Mmp2 using a fluorescein conjugate-gelatin as a substrate.
Eye lysates from wild-type, myocilin null mutant and transgenic lines expressing a significantly higher level of myocilin or Y437H mutated myocilin in the eye drainage structures were immunoprecipitated with polyclonal antibodies against mouse myocilin. Several proteins were immunoprecipitated from wild-type eye lysates but not from myocilin null or myocilin mutant lysates as demonstrated by shotgun proteomics analysis of immunoprecipitates. One of these proteins was Timp3, a known inhibitor of metalloproteinases. The interaction between myocilin and Timp3 was confirmed by inverse co-immunoprecipitation with anti-Timp3 antibodies from the eye lysates of wild-type mice and by co-transfection experiments using transiently transfected HepG2 cells. These experiments also demonstrated that the C-terminal part of myocilin is sufficient for interaction with Timp3 and that some mutations in the C-terminal part of myocilin reduced its interaction with Timp3. In the eye tissues, myocilin and Timp3 were co-localized in the retinal pigmented epithelial cells, Bruch’s membrane, trabecular meshwork, sclera, and ciliary body epithelium as shown by immunostaining of mouse and monkey eye sections. Although purified myocilin did not suppress Mmp2 activity, the inhibitory effects of Timp3 on Mmp2 activity were enhanced by the addition of myocilin.
Myocilin is a modulator of Timp3 activity. Because mutations in the TIMP3 gene may lead to Sorsby fundus dystrophy and genome-wide association studies identified a locus located near TIMP3 as associated with age-related macula degeneration, myocilin may play a role in these eye pathologies.
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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