title: Mutated mouse and human myocilins have similar properties and do not block general secretory pathway.
abstract: OBJECTIVE
  The present study compared properties of wild-type and mutated mouse and human myocilin (Myoc) proteins as a prerequisite for development of a mouse model of glaucoma.
  
  
  METHODS
  cDNA encoding full-length mouse Myoc was cloned into the p3XFLAG-CMV-14 vector. Tyr423His and Ile463Ser mutations were introduced into the mouse Myoc protein by in vitro mutagenesis. Intracellular localization and secretion of wild-type and mutated mouse Myoc proteins were studied in immunostaining and Western blotting experiments, respectively, after transfection into COS-7 cells.
  
  
  RESULTS
  Similar to human MYOC, wild-type and mutated mouse Myoc demonstrated vesicular staining in transfected cells. However, while wild-type human and mouse Myoc were preferentially located in both the endoplasmic reticulum and Golgi, mutated human and mouse Myoc were located mainly in the endoplasmic reticulum and were excluded from Golgi. Similar to mutations in human MYOC, mutations in mouse Myoc dramatically reduced its secretion from transfected cells. Secretion of mutated Myoc was partially restored by culturing cells at 30 degrees C instead of 37 degrees C. The presence of mutated human MYOC prevented secretion of wild-type mouse Myoc but did not dramatically affect secretion of alkaline phosphatase, thrombospondin, Timp3 or olfactomedin-1.
  
  
  CONCLUSIONS
  Properties of the mouse Myoc protein are similar to those of the human MYOC. The presence of mutated mouse or human Myoc does not block a general secretory pathway. Expression of mutated Myoc in the eye in mice may mimic human glaucoma and lead to development of a genetic mouse model of glaucoma.
authors.affiliation: Section of Molecular Mechanisms of Glaucoma, Laboratory of Molecular and Developmental Biology, National Eye Institute, National Institutes of Health, Bethesda, Maryland, USA.
authors.forename: Irina
authors.initials: I
authors.lastname: Malyukova
authors.forename: Hee-Sheung
authors.initials: HS
authors.lastname: Lee
authors.forename: Robert N
authors.initials: RN
authors.lastname: Fariss
authors.forename: Stanislav I
authors.initials: SI
authors.lastname: Tomarev
mesh_terms: D000595:Amino Acid Sequence; D000818:Animals; D015153:Blotting, Western; D019556:COS Cells / Q000378:metabolism; D002522:Chlorocebus aethiops; D003598:Cytoskeletal Proteins / Q000502:physiology*; D004721:Endoplasmic Reticulum / Q000378:metabolism; D016326:Extracellular Matrix Proteins / Q000235:genetics; D005136:Eye Proteins / Q000502:physiology*; D005822:Genetic Vectors; D006023:Glycoproteins / Q000235:genetics / Q000502:physiology*; D006056:Golgi Apparatus / Q000378:metabolism; D006801:Humans; D051379:Mice; D008969:Molecular Sequence Data; D016297:Mutagenesis, Site-Directed; D009154:Mutation / Q000502:physiology; D010957:Plasmids; D014162:Transfection; D000099179:Myocilin
chemical_list: D003598:Cytoskeletal Proteins; D016326:Extracellular Matrix Proteins; D005136:Eye Proteins; D006023:Glycoproteins; D000099179:Myocilin; C081451:olfactomedin