Perfusion with the olfactomedin domain of myocilin does not affect outflow facility.
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.
Goldwich
Andreas
A
Department of Anatomy, Molecular Anatomy, and Embryology, University of Erlangen-Nürnberg, Erlangen, Germany.
Ethier
C Ross
CR
Chan
Darren W-H
DW
Tamm
Ernst R
ER
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
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