title: Heterozygous expression of myocilin glaucoma mutants increases secretion of the mutant forms and reduces extracellular processed myocilin.
abstract: OBJECTIVE
  Heterozygous mutations in the myocilin gene (MYOC) cause glaucoma by an unknown mechanism. MYOC encodes an extracellular protein of unidentified function that undergoes intracellular endoproteolytic processing in the secretory pathway. It has been described that co-expression of wild-type/mutant myocilin reduces the secretion of the wild-type protein and that single expression of glaucoma myocilin mutants reduces its proteolytic processing. However, the effect of wild-type myocilin on mutant myocilin secretion and how mutant myocilin affects the proteolytic processing of wild-type myocilin have not been investigated. We herein analyze these two issues.
  
  
  METHODS
  We modeled the heterozygous state for 4 missense (E323K, R346T, P370L, D380A) and 1 nonsense (Q368X) myocilin mutants by transiently co-expressing each mutant with the wild-type protein in HEK-293T cells. Recombinant mutant and wild-type myocilin in both culture media and cellular fractions were quantified by western immunoblot and densitometry.
  
  
  RESULTS
  A 24 h transient co-expression of each myocilin mutant with the wild-type protein elicited an augmented secretion of the mutant forms from 1.5 fold (D380A) to 5.4 fold (E323K). Under such conditions, extracellular mutant myocilin represented up to 20% of the total mutant protein. Other than this effect, secreted wild-type myocilin significantly decreased from 2.6 fold (E323K) to 36 fold (Q368X). When myocilin proteolytic processing was enhanced (96 hour co-expression) the extracellular amount of wild-type processed myocilin diminished from approximately 2.1 fold (E323K) to 6.3 fold (P370L). Nonreducing SDS-PAGE indicated that extracellular myocilin resulting from 24 h co-expression of wild-type myocilin and each of the 4 missense mutants forms hetero-oligomers and that glaucoma mutations do not increase the size of myocilin aggregates.
  
  
  CONCLUSIONS
  Increased extracellular levels of mutant myocilin expressed in heterozygosis may play a relevant role in glaucoma pathogenesis. This effect is likely the result of intracellular mutant/wild-type myocilin hetero-oligomerization.
authors.affiliation: Area de Genética, Facultad de Medicina/Centro Regional de Investigaciones Biomédicas (CRIB), Universidad de Castilla-La Mancha, Albacete, Spain.
authors.forename: José-Daniel
authors.initials: JD
authors.lastname: Aroca-Aguilar
authors.forename: Francisco
authors.initials: F
authors.lastname: Sánchez-Sánchez
authors.forename: Francisco
authors.initials: F
authors.lastname: Martínez-Redondo
authors.forename: Miguel
authors.initials: M
authors.lastname: Coca-Prados
authors.forename: Julio
authors.initials: J
authors.lastname: Escribano
mesh_terms: D002460:Cell Line; D003598:Cytoskeletal Proteins / Q000737:chemistry / Q000235:genetics* / Q000378:metabolism*; D018076:DNA, Complementary / Q000235:genetics; D005110:Extracellular Space / Q000378:metabolism*; D005136:Eye Proteins / Q000737:chemistry / Q000235:genetics* / Q000378:metabolism*; D005901:Glaucoma / Q000235:genetics*; D006023:Glycoproteins / Q000737:chemistry / Q000235:genetics* / Q000378:metabolism*; D006579:Heterozygote; D006801:Humans; D008954:Models, Biological; D050505:Mutant Proteins / Q000235:genetics / Q000378:metabolism*; D009154:Mutation / Q000235:genetics; D011499:Protein Processing, Post-Translational; D020836:Protein Structure, Quaternary; D000099179:Myocilin
chemical_list: D003598:Cytoskeletal Proteins; D018076:DNA, Complementary; D005136:Eye Proteins; D006023:Glycoproteins; D050505:Mutant Proteins; D000099179:Myocilin