Myocilin Regulates Metalloprotease 2 Activity Through Interaction With TIMP3

Invest Ophthalmol Vis Sci. 2017 Oct 1;58(12):5308-5318. doi: 10.1167/iovs.16-20336.


Purpose: To elucidate functions of wild-type myocilin, a secreted glycoprotein associated with glaucoma.

Methods: Lysates of mouse eyes were used for immunoprecipitation with affinity-purified antibodies against mouse myocilin. Shotgun proteomic analysis was used for the identification of proteins interacting with myocilin. Colocalization of myocilin and tissue inhibitor of metalloproteinases 3 (TIMP3) in different eye structures was investigated by a multiplex fluorescent in situ hybridization and immunofluorescent labeling with subsequent confocal microscopy. Matrix metalloproteinase 2 (MMP2) activity assay was used to test effects of myocilin on TIMP3 inhibitory action.

Results: TIMP3 was identified by a shotgun proteomic analysis as a protein that was coimmunoprecipitated with myocilin from eye lysates of wild-type and transgenic mice expressing elevated levels of mouse myocilin but not from lysates of transgenic mice expressing mutated mouse myocilin. Interaction of myocilin and TIMP3 was confirmed by coimmunoprecipitation of myocilin and TIMP3 from HEK293 cells transiently transfected with cDNAs encoding these proteins. The olfactomedin domain of myocilin is essential for interaction with TIMP3. In the eye, the main sites of myocilin and TIMP3 colocalization are the trabecular meshwork, sclera, and choroid. Using purified proteins, it has been shown that myocilin markedly enhanced the inhibitory activity of TIMP3 toward MMP2.

Conclusions: Myocilin may serve as a modulator of TIMP3 activity via interactions with the myocilin olfactomedin domain. Our data imply that in the case of MYOCILIN null or some glaucoma-causing mutations, inhibitory activity of TIMP3 toward MMP2 might be reduced, mimicking deleterious mutations in the TIMP3 gene.

Publication types

  • Research Support, N.I.H., Extramural
  • Research Support, N.I.H., Intramural

MeSH terms

  • Animals
  • Blotting, Western
  • Ciliary Body / metabolism
  • Cytoskeletal Proteins / physiology*
  • Eye Proteins / physiology*
  • Female
  • Fluorescent Antibody Technique, Indirect
  • Gene Expression
  • Glycoproteins / physiology*
  • HEK293 Cells
  • Hep G2 Cells
  • Humans
  • Immunoprecipitation
  • In Situ Hybridization, Fluorescence
  • Male
  • Matrix Metalloproteinase 2 / metabolism*
  • Mice
  • Mice, Inbred C57BL
  • Mice, Transgenic
  • Microscopy, Confocal
  • Proteomics
  • RNA, Messenger / genetics
  • Tissue Inhibitor of Metalloproteinase-3 / genetics
  • Tissue Inhibitor of Metalloproteinase-3 / metabolism*
  • Trabecular Meshwork / metabolism
  • Transfection


  • Cytoskeletal Proteins
  • Eye Proteins
  • Glycoproteins
  • RNA, Messenger
  • Tissue Inhibitor of Metalloproteinase-3
  • trabecular meshwork-induced glucocorticoid response protein
  • Matrix Metalloproteinase 2
  • Mmp2 protein, mouse