Impaired secretion of carboxyl-terminal truncated factor VII due to an F7 nonsense mutation associated with FVII deficiency.
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.
Tanaka
Ryoko
R
Department of Pathophysiological Laboratory Sciences, Nagoya University Graduate School of Medicine, 1-1-20 Daiko-Minami, Higashi-Ku, Nagoya 461-8673, Japan.
Nakashima
Daisuke
D
Suzuki
Atsuo
A
Miyawaki
Yuhri
Y
Fujimori
Yuta
Y
Yamada
Takayuki
T
Takagi
Akira
A
Murate
Takashi
T
Yamamoto
Koji
K
Katsumi
Akira
A
Matsushita
Tadashi
T
Naoe
Tomoki
T
Kojima
Tetsuhito
T
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
D018389:Codon, Nonsense; D003470:Culture Media; D016895:Culture Media, Serum-Free; D011994:Recombinant Proteins; D005167:Factor VII