title: Impaired secretion of carboxyl-terminal truncated factor VII due to an F7 nonsense mutation associated with FVII deficiency.
abstract: BACKGROUND
  Factor VII (FVII) is a vitamin K-dependent glycoprotein secreted into the blood circulation from hepatic cells. We investigated the molecular basis of the congenital FVII deficiency found in a Japanese patient.
  
  
  METHODS
  We analyzed the F7 gene of the patient, who was diagnosed with a FVII deficiency at pregnancy. We expressed a carboxyl-terminal truncated FVII (Arg462X FVII) corresponding to the identified mutation in CHO-K1 cells. To study roles of the carboxyl-terminus in the secretion of FVII, we also expressed a series of recombinant FVIIs deleted of limited numbers of carboxyl-terminal amino acids (462Arg-466Pro).
  
  
  RESULTS
  We identified a nonsense mutation (c.1384C>T: p.Arg462X) in F7, leading to a lack of five amino acids in the carboxyl-terminus. In expression experiments, Arg462X FVII was undetectable not only by Western blotting, but also by ELISA. A Western blot analysis of the truncated FVIIs revealed that all mutants were expressed in the cells the same as the wild type, but were secreted into the culture medium in lesser amounts than the wild type depending on the length of the deletion, which was confirmed by ELISA. Arg462X FVII did not colocalize with the Golgi on immunofluorescence staining, suggesting that it might be retained in the ER and degraded in the cell.
  
  
  CONCLUSIONS
  The carboxyl-terminal amino acids of FVII play an important role in its secretion, and the p.Arg462X mutation was likely to have caused the FVII deficiency in this patient.
authors.affiliation: Department of Pathophysiological Laboratory Sciences, Nagoya University Graduate School of Medicine, 1-1-20 Daiko-Minami, Higashi-Ku, Nagoya 461-8673, Japan.
authors.forename: Ryoko
authors.initials: R
authors.lastname: Tanaka
authors.forename: Daisuke
authors.initials: D
authors.lastname: Nakashima
authors.forename: Atsuo
authors.initials: A
authors.lastname: Suzuki
authors.forename: Yuhri
authors.initials: Y
authors.lastname: Miyawaki
authors.forename: Yuta
authors.initials: Y
authors.lastname: Fujimori
authors.forename: Takayuki
authors.initials: T
authors.lastname: Yamada
authors.forename: Akira
authors.initials: A
authors.lastname: Takagi
authors.forename: Takashi
authors.initials: T
authors.lastname: Murate
authors.forename: Koji
authors.initials: K
authors.lastname: Yamamoto
authors.forename: Akira
authors.initials: A
authors.lastname: Katsumi
authors.forename: Tadashi
authors.initials: T
authors.lastname: Matsushita
authors.forename: Tomoki
authors.initials: T
authors.lastname: Naoe
authors.forename: Tetsuhito
authors.initials: T
authors.lastname: Kojima
mesh_terms: D000328:Adult; D000818:Animals; D016466:CHO Cells; D018929:Cell Culture Techniques; D018389:Codon, Nonsense; D006224:Cricetinae; D003412:Cricetulus; D003470:Culture Media / Q000737:chemistry; D016895:Culture Media, Serum-Free; D005167:Factor VII / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism*; D005168:Factor VII Deficiency / Q000097:blood / Q000235:genetics*; D005260:Female; D019084:Fluorescent Antibody Technique, Indirect; D006801:Humans; D012150:Polymorphism, Restriction Fragment Length; D011247:Pregnancy; D011994:Recombinant Proteins / Q000737:chemistry / Q000378:metabolism; D017422:Sequence Analysis, DNA
chemical_list: D018389:Codon, Nonsense; D003470:Culture Media; D016895:Culture Media, Serum-Free; D011994:Recombinant Proteins; D005167:Factor VII