Characterization of the Felix domesticus (cat) glaucoma-associated protein myocilin.
The domestic cat (Felix domesticus) is a useful model for understanding the implications of long-term expression and function of normal and mutant myocilin. To better understand the role myocilin has in the cat eye, we isolated and characterized cat myocilin. Oligonucleotides designed against conserved nucleotide regions of myocilin mammalian orthologs were used to PCR amplify a partial cat myocilin cDNA clone. Rapid amplification of cDNA ends (5' and 3' RACE) was used to obtain full-length cat myocilin. The 2125 nucleotide cat myocilin cDNA contains a 490 amino acid open reading frame. Comparison of cat myocilin to human myocilin shows a 87% similarity, including conservation of the N-terminal leucine zipper, N-linked glycosylation site, C-terminal olfactomedin domain, and all five cysteine residues thought to be involved in disulfide bond formation. Expression in a transformed human trabecular cell line or in Crandall feline kidney cells showed cat myocilin was secreted from these cells, similar to human myocilin, suggesting cat myocilin contains a functional signal peptide sequence. In contrast, expression of cat myocilin containing a known human glaucoma-associated mutation (Y423H in cat; Y437H in human) was not secreted. Characterization of cat myocilin will enable long-term studies be performed in Felix domesticus to analyze changes to intraocular pressure and the aqueous outflow pathway following expression of myocilin and glaucoma causing mutations.
Fautsch
Michael P
MP
Department of Ophthalmology, Mayo Clinic College of Medicine, 200 First Street SW, Rochester, MN 55905, USA. fautsch.michael@mayo.edu
Vrabel
Anne M
AM
Johnson
Douglas H
DH
D000595:Amino Acid Sequence / Q000235:genetics; D000596:Amino Acids / Q000235:genetics; D000818:Animals; D001483:Base Sequence; D002415:Cats / Q000235:genetics*; D002460:Cell Line; D003001:Cloning, Molecular / Q000379:methods; D003545:Cysteine / Q000235:genetics; D003598:Cytoskeletal Proteins / Q000235:genetics*; D004270:DNA, Circular / Q000235:genetics; D016326:Extracellular Matrix Proteins / Q000235:genetics; D005136:Eye Proteins / Q000235:genetics*; D015870:Gene Expression / Q000235:genetics; D005901:Glaucoma / Q000235:genetics; D006023:Glycoproteins / Q000235:genetics*; D006801:Humans; D007668:Kidney / Q000166:cytology; D016350:Leucine Zippers / Q000235:genetics; D023421:Models, Animal; D008957:Models, Genetic; D050505:Mutant Proteins / Q000235:genetics; D009154:Mutation / Q000235:genetics; D010802:Phylogeny; D014129:Trabecular Meshwork / Q000166:cytology; D000099179:Myocilin
D000596:Amino Acids; D003545:Cysteine; D003598:Cytoskeletal Proteins; D004270:DNA, Circular; D016326:Extracellular Matrix Proteins; D005136:Eye Proteins; D006023:Glycoproteins; D050505:Mutant Proteins; D000099179:Myocilin; C081451:olfactomedin