Manipulating the quality control pathway in transfected cells: low temperature allows rescue of secretion-defective fibrinogen mutants

• Published in: Haematologica (Roma) 2008-02, Vol.93 (2), p.224-231
• Main Authors: Vu, Dung, Di Sanza, Corinne, Neerman-Arbez, Marguerite
• Format: Article
• Language: English
• Subjects: Afibrinogenemia - genetics; Afibrinogenemia - metabolism; Animals; Biological and medical sciences; Cercopithecus aethiops; Cold Temperature; COS Cells; Factor XIII - metabolism; Fibrinogen - secretion; Fibrinolysin - metabolism; Golgi Apparatus - genetics; Golgi Apparatus - secretion; Hematologic and hematopoietic diseases; Humans; Medical sciences; Molecular Chaperones - pharmacology; Mutation, Missense; Platelet diseases and coagulopathies; Recombinant Proteins - genetics; Recombinant Proteins - secretion; Transfection - methods
• ISSN: 0390-6078; 1592-8721
• DOI: 10.3324/haematol.11868

1 Department of Genetic Medicine and Development, University Medical Center, Geneva; 2 Division of Angiology and Hemostasis, University Hospital, Geneva, Switzerland Correspondence: Marguerite Neerman-Arbez, 1 rue Michel Servet, CH-1211 Geneva, Switzerland. E-mail: marguerite.arbez{at}medecine.unige.ch Background: Congenital afibrinogenemia is characterized by the absence of fibrinogen, a hexamer composed of two copies of three polypeptides, A . Bβ and . 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 βC and C domains in secretion. Here our aim was to restore the secretion of these mutants and study the properties of the rescued mutant molecules. Design and 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°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°C were incorporated into fibrin clots and formed factor XIII-mediated - 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. Key words: fibrinogen, afibrinogenemia, secretion, chemical chaperones, protein quality control, fibrinogen-related domain.