title: Characterization of rabbit myocilin: Implications for human myocilin glycosylation and signal peptide usage.
abstract: BACKGROUND
  Mutations in the gene encoding human myocilin (MYOC) have been shown to cause juvenile- and adult-onset glaucoma. In addition, myocilin has been associated with glucocorticoid-induced ocular hypertension and steroid-induced glaucoma. To better understand the role myocilin plays in steroid-induced glaucoma and open-angle glaucoma, we examined rabbit myocilin for use in the rabbit animal model of steroid-induced glaucoma.
  
  
  RESULTS
  We have cloned the rabbit ortholog of human MYOC. Rabbit MYOC consists of three exons and an open reading frame encoding a 490 amino acid, 54,882-Da protein, which is 14 amino acids shorter at the N-terminus than human myocilin but 84% identical overall. Rabbit myocilin migrates as a single electrophoretic band, vs. double-banded human myocilin, by SDS-PAGE/immunoblot analysis. We determined that the differential migration exhibited is due to an N-glycosylation site that is present in human (Asn57), monkey and mouse myocilin but absent in rabbit (Ser43), rat and bovine myocilin. Rabbit myocilin is secreted in vitro in trabecular meshwork cell culture and in vivo in aqueous humor. Secretion of human myocilin is shown to be dependent on the signal peptide and independent of the extra 14 amino acids not found in rabbit myocilin. Many of the amino acids in myocilin that are mutated in glaucoma patients are conserved across species.
  
  
  CONCLUSIONS
  We have cloned the rabbit MYOC cDNA and determined that rabbit myocilin is secreted but not N-linked glycosylated. Knowledge of the rabbit MYOC cDNA sequence will facilitate future studies in the rabbit animal model examining the role of myocilin in steroid-induced glaucoma and the gain-of-function hypothesis in open-angle glaucoma.
authors.affiliation: Alcon Research, Ltd, 6201 South Freeway, Fort Worth, TX 76134, USA. allan.shepard@alconlabs.com
authors.forename: Allan R
authors.initials: AR
authors.lastname: Shepard
authors.forename: Nasreen
authors.initials: N
authors.lastname: Jacobson
authors.forename: Ruifang
authors.initials: R
authors.lastname: Sui
authors.forename: H Thomas
authors.initials: HT
authors.lastname: Steely
authors.forename: Andrew J
authors.initials: AJ
authors.lastname: Lotery
authors.forename: Edwin M
authors.initials: EM
authors.lastname: Stone
authors.forename: Abbot F
authors.initials: AF
authors.lastname: Clark
mesh_terms: D000595:Amino Acid Sequence; D000818:Animals; D001082:Aqueous Humor / Q000378:metabolism; D001483:Base Sequence; D002417:Cattle; D002478:Cells, Cultured; D003598:Cytoskeletal Proteins; D018076:DNA, Complementary / Q000737:chemistry / Q000235:genetics; D004591:Electrophoresis, Polyacrylamide Gel; D005136:Eye Proteins / Q000235:genetics* / Q000378:metabolism; D006023:Glycoproteins / Q000235:genetics* / Q000378:metabolism; D006031:Glycosylation; D000882:Haplorhini; D006801:Humans; D015151:Immunoblotting; D051379:Mice; D008969:Molecular Sequence Data; D009154:Mutation; D021382:Protein Sorting Signals / Q000235:genetics*; D011817:Rabbits; D051381:Rats; D016415:Sequence Alignment; D017422:Sequence Analysis, DNA; D017386:Sequence Homology, Amino Acid; D014129:Trabecular Meshwork / Q000166:cytology / Q000378:metabolism; D000099179:Myocilin
chemical_list: D003598:Cytoskeletal Proteins; D018076:DNA, Complementary; D005136:Eye Proteins; D006023:Glycoproteins; D021382:Protein Sorting Signals; D000099179:Myocilin