Characterization and Novel Activation of 72-kDa Metalloproteinase in Retinal Interphotoreceptor Matrix and Y-79 Cell Culture Medium

B. Eric Jones, Payman Moshyedi, Samuel Gallo, Joyce Tombran-Tink, Gloria Arand, Deborah A. Reid, Erik W. Thompson, Gerald J. Chader, Robert J. Waldbillig

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Abstract

Analysis of bovine interphotoreceptor matrix and conditioned medium from human Y-79 retinoblastoma cells by gelatin SDS-PAGE zymography reveals abundant activity of a 72-kDa Mr gelatinase. The 72-kDa gelatinase from either source is inhibited by EDTA but not aprotinin or NEM, indicating that it is a metalloproteinase (MMP). The 72-kDa MMP is converted to a 62-kDa species with APMA treatment after gelatin sepharose affinity purification, typical of previously described gelatinase MMP-2. The latent 72-kDa gelatinase from either bovine IPM or Y-79 media autoactivates without APMA in the presence of calcium and zinc after 72 hr at 37°C, producing a fully active mixture of proteinase species, 50 (48 in Y-79 medium), 38 and 35 kDa in size. The presence of inhibitory activity was examined in both whole bovine IPM and IPM fractions separated by SDS-PAGE. Whole IPM inhibited gelatinolytic activity of autoactivated Y-79-derived MMP in a dose-dependent manner. Inhibitory activities are observed in two protein fractions of 27-42 and 20-25 kDa. Western blots using antibodies to human tissue inhibitor of metalloproteinase 1 and 2 (TIMP-1 and -2) reveal the presence of two TIMP-1-like proteins at 21 and 29 kDa in inhibitory fractions of the bovine IPM. TIMP-2 was not detected in the inhibitory IPM fractions, consistent with the observed autoactivation of bovine IPM 72-kDa gelatinase. Potential roles for this IPM MMP-TIMP system include physiologic remodelling of the neural retina-RPE cell interface and digestion of shed rod outer segment as well as pathological processes such as retinal detachment, PE cell migration, neovascularization and tumor progression. Cultured Y-79 cells appear to be a good model for studying the production and regulation of this proteinase system.

Original languageEnglish (US)
Pages (from-to)257-269
Number of pages13
JournalExperimental Eye Research
Volume59
Issue number3
DOIs
StatePublished - Jan 1 1994

All Science Journal Classification (ASJC) codes

  • Ophthalmology
  • Sensory Systems
  • Cellular and Molecular Neuroscience

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    Jones, B. E., Moshyedi, P., Gallo, S., Tombran-Tink, J., Arand, G., Reid, D. A., Thompson, E. W., Chader, G. J., & Waldbillig, R. J. (1994). Characterization and Novel Activation of 72-kDa Metalloproteinase in Retinal Interphotoreceptor Matrix and Y-79 Cell Culture Medium. Experimental Eye Research, 59(3), 257-269. https://doi.org/10.1006/exer.1994.1107