title: Manipulating the quality control pathway in transfected cells: low temperature allows rescue of secretion-defective fibrinogen mutants.
abstract: BACKGROUND
  Congenital afibrinogenemia is characterized by the absence of fibrinogen, a hexamer composed of two copies of three polypeptides, Aalpha. Bbeta and gamma. The disease is caused by mutations in one of the three fibrinogen-encoding genes, FGA, FGB and FGG. Among these, several mutations have been reported to specifically impair fibrinogen secretion. We previously showed that secretion-defective fibrinogen mutants are retained in a pre-Golgi compartment and demonstrated the importance of the homologous betaC and gammaC domains in secretion. Here our aim was to restore the secretion of these mutants and study the properties of the rescued mutant molecules.
  
  
  METHODS
  COS-7 cells were transfected and incubated with chemical chaperones or at low temperature. Clotting assays and plasmin digestion studies were performed to characterize secreted fibrinogen molecules.
  
  
  RESULTS
  The secretion defect of two missense mutants but not that of late-truncating mutants could be partially corrected by incubating cells at 27 degrees C. By contrast, exposure of cells to chemical chaperones i.e. 4-phenylbutyrate, dimethyl sulfoxide or trimethylamine N-oxide had no effect. The mutants rescued at 27 degrees C were incorporated into fibrin clots and formed factor XIII-mediated gamma-gamma dimers in contrast to the dysfibrinogenemia Vlissingen/Frankfurt IV mutant, a negative control for these assays. However, plasmin digestion analyses revealed aberrant patterns for the mutants compared to normal fibrinogen.
  
  
  CONCLUSIONS
  Low temperature can restore the secretion of a subset of mutant fibrinogen molecules demonstrating that therapeutic manipulation of the quality control pathway is feasible for afibrinogenemia even though functional assays suggested a non-native conformation for the mutant molecules analyzed.
authors.affiliation: Department of Genetic Medicine and Development, University Medical Center, Geneva, Switzerland.
authors.forename: Dung
authors.initials: D
authors.lastname: Vu
authors.forename: Corinne
authors.initials: C
authors.lastname: Di Sanza
authors.forename: Marguerite
authors.initials: M
authors.lastname: Neerman-Arbez
mesh_terms: D000347:Afibrinogenemia / Q000235:genetics / Q000378:metabolism; D000818:Animals; D019556:COS Cells; D002522:Chlorocebus aethiops; D003080:Cold Temperature; D005176:Factor XIII / Q000378:metabolism; D005340:Fibrinogen / Q000378:metabolism*; D005341:Fibrinolysin / Q000378:metabolism; D006056:Golgi Apparatus / Q000235:genetics / Q000378:metabolism*; D006801:Humans; D018832:Molecular Chaperones / Q000494:pharmacology; D020125:Mutation, Missense; D011994:Recombinant Proteins / Q000235:genetics / Q000378:metabolism; D014162:Transfection / Q000379:methods
chemical_list: D018832:Molecular Chaperones; D011994:Recombinant Proteins; D005340:Fibrinogen; D005176:Factor XIII; D005341:Fibrinolysin