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.
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.
Authors: Mohamed-Karim Ezzat; Kyle G Howell; Cindy K Bahler; Thomas G Beito; Nils Loewen; Eric M Poeschla; Michael P Fautsch Journal: Exp Eye Res Date: 2008-07-12 Impact factor: 3.467
Authors: Bum-Chan Park; Xiang Shen; Michael P Fautsch; Martin Tibudan; Douglas H Johnson; Beatrice Y J T Yue Journal: Mol Vis Date: 2006-07-31 Impact factor: 2.367
Authors: Walter Paper; Markus Kroeber; Sebastian Heersink; Dietrich A Stephan; Rudolf Fuchshofer; Paul Russell; Ernst R Tamm Journal: Exp Eye Res Date: 2008-06-17 Impact factor: 3.467
Authors: Carmen Knaupp; Cassandra Flügel-Koch; Andreas Goldwich; Andreas Ohlmann; Ernst R Tamm Journal: Graefes Arch Clin Exp Ophthalmol Date: 2004-01-29 Impact factor: 3.117