Perfusion with the olfactomedin domain of myocilin does not affect outflow facility

Invest Ophthalmol Vis Sci. 2003 May;44(5):1953-61. doi: 10.1167/iovs.02-0863.


Purpose: Mutations in the MYOC gene coding for myocilin are associated with elevated intraocular pressure (IOP), and recombinant myocilin, produced in a prokaryotic expression system, has been reported to affect aqueous outflow facility. This study was conducted to test whether perfusion with a fragment of recombinant myocilin (containing the full-length olfactomedin domain), produced in a eukaryotic expression system, affects facility.

Methods: 293 EBNA cells were transfected by a vector containing the BM40 signal peptide, a human cDNA coding for myocilin, and a polyhistidine tag (HisTag) sequence. Recombinant protein was isolated by Ni-chelate chromatography, and characterized, and perfused into cultured anterior segments of human and porcine eyes.

Results: Recombinant myocilin was secreted as a approximately 55-kDa intact protein and two fragments arising from cleavage of the recombinant protein at amino acid 215. The C-terminal fragment, containing the entire olfactomedin domain, was successfully isolated. When perfused into human and porcine eyes, this C-terminal fragment did not appreciably affect outflow facility.

Conclusions: Although the olfactomedin domain appears to be important for the function of myocilin, perfusion with a recombinant myocilin fragment containing this domain does not change outflow facility. It is possible that both the olfactomedin and N-terminal domains (including the leucine zipper) must be present for myocilin to have full function. Alternatively, posttranslational modifications of myocilin may have a major impact on protein function.

Publication types

  • Research Support, Non-U.S. Gov't

MeSH terms

  • Animals
  • Aqueous Humor / metabolism*
  • Blotting, Western
  • Cell Culture Techniques
  • Cytoskeletal Proteins
  • Electrophoresis, Gel, Two-Dimensional
  • Epithelial Cells / virology
  • Epstein-Barr Virus Nuclear Antigens / genetics
  • Extracellular Matrix Proteins / administration & dosage
  • Extracellular Matrix Proteins / isolation & purification
  • Extracellular Matrix Proteins / physiology*
  • Eye Proteins / administration & dosage
  • Eye Proteins / isolation & purification
  • Eye Proteins / physiology*
  • Glycoproteins / administration & dosage
  • Glycoproteins / isolation & purification
  • Glycoproteins / physiology*
  • Herpesvirus 4, Human / physiology
  • Humans
  • Organ Culture Techniques
  • Perfusion
  • Plasmids
  • Protein Structure, Tertiary
  • Rabbits
  • Recombinant Proteins / administration & dosage
  • Recombinant Proteins / isolation & purification
  • Swine
  • Trabecular Meshwork / drug effects
  • Trabecular Meshwork / metabolism*
  • Trabecular Meshwork / pathology
  • Transfection


  • Cytoskeletal Proteins
  • Epstein-Barr Virus Nuclear Antigens
  • Extracellular Matrix Proteins
  • Eye Proteins
  • Glycoproteins
  • Recombinant Proteins
  • olfactomedin
  • trabecular meshwork-induced glucocorticoid response protein