title: Perfusion with the olfactomedin domain of myocilin does not affect outflow facility.
abstract: OBJECTIVE
  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.
authors.affiliation: Department of Anatomy, Molecular Anatomy, and Embryology, University of Erlangen-Nürnberg, Erlangen, Germany.
authors.forename: Andreas
authors.initials: A
authors.lastname: Goldwich
authors.forename: C Ross
authors.initials: CR
authors.lastname: Ethier
authors.forename: Darren W-H
authors.initials: DW
authors.lastname: Chan
authors.forename: Ernst R
authors.initials: ER
authors.lastname: Tamm
mesh_terms: D000818:Animals; D001082:Aqueous Humor / Q000378:metabolism*; D015153:Blotting, Western; D018929:Cell Culture Techniques; D003598:Cytoskeletal Proteins; D015180:Electrophoresis, Gel, Two-Dimensional; D004847:Epithelial Cells / Q000821:virology; D019309:Epstein-Barr Virus Nuclear Antigens / Q000235:genetics; D016326:Extracellular Matrix Proteins / Q000008:administration & dosage / Q000302:isolation & purification / Q000502:physiology*; D005136:Eye Proteins / Q000008:administration & dosage / Q000302:isolation & purification / Q000502:physiology*; D006023:Glycoproteins / Q000008:administration & dosage / Q000302:isolation & purification / Q000502:physiology*; D004854:Herpesvirus 4, Human / Q000502:physiology; D006801:Humans; D009924:Organ Culture Techniques; D010477:Perfusion; D010957:Plasmids; D017434:Protein Structure, Tertiary; D011817:Rabbits; D011994:Recombinant Proteins / Q000008:administration & dosage / Q000302:isolation & purification; D013552:Swine; D014129:Trabecular Meshwork / Q000187:drug effects / Q000378:metabolism* / Q000473:pathology; D014162:Transfection; D000099179:Myocilin
chemical_list: D003598:Cytoskeletal Proteins; D019309:Epstein-Barr Virus Nuclear Antigens; D016326:Extracellular Matrix Proteins; D005136:Eye Proteins; D006023:Glycoproteins; D011994:Recombinant Proteins; D000099179:Myocilin; C081451:olfactomedin