title: γ375W fibrinogen-synthesizing CHO cells indicate the accumulation of variant fibrinogen within endoplasmic reticulum.
abstract: BACKGROUND
  Hepatic endoplasmic reticulum (ER) storage disease (HERSD) associated with hypofibrinogenemia has been reported in patients with four types of heterozygous γ-chain variant fibrinogen in the C terminal region. Of interest, substitution of γR375W induced hypofibrinogenemia and HERSD, whereas γR375G induced dysfibrinogenemia.
  
  
  OBJECTIVE
  To analyze the synthesis of variant fibrinogen and morphological characteristics, we established variant fibrinogen-producing cells and compared them with wild-type fibrinogen-synthesizing cells.
  
  
  METHODS
  The fibrinogen γ-chain expression vectors coding γ375W and γ375G were altered by oligonucleotide-directed mutagenesis and transfected into Chinese hamster ovary (CHO) cells. Synthesis of fibrinogen (media and cell lysates) was measured by ELISA for each cloned cell line and morphological characteristics were observed by immunofluorescence and transmission electron microscopy.
  
  
  RESULTS
  The medium/cell lysate fibrinogen ratio of γ375W-CHO cells was markedly lower than that of the normal cells and γ375G-CHO cells. Immunostaining with anti-fibrinogen antibody showed only γ375W-CHO cells, but revealed two types of cells containing cytoplasmic inclusion bodies, scattered large-granular bodies and fibrous forms. Observation by confocal microscopy indicated that both inclusion bodies were colocalized with fibrinogen and ER-membrane protein; furthermore, transmission electron microscopic observation demonstrated dilatation of the ER by large-granular inclusion bodies and fibrous forms filled with regularly structured fibular materials within the dilated ER.
  
  
  CONCLUSIONS
  These results demonstrated that assembled and non-secreted γ375W fibrinogen was accumulated in the dilated ER and aggregated variant fibrinogen was seen as regularly structured fibular materials, which was similar to the fingerprint-like pattern observed at inclusion bodies in patients' hepatocytes affected with HERSD.
authors.affiliation: Department of Clinical Laboratory Investigation, Shinshu University, Matsumoto, Japan.
authors.forename: Tamaki
authors.initials: T
authors.lastname: Kobayashi
authors.affiliation: Department of Laboratory Medicine, Shinshu University Hospital, Matsumoto, Japan; Department of Laboratory Medicine, Graduate School of Medicine, Shinshu University, Matsumoto, Japan.
authors.forename: Shinpei
authors.initials: S
authors.lastname: Arai
authors.affiliation: Department of Laboratory Medicine, Shinshu University Hospital, Matsumoto, Japan.
authors.forename: Naoko
authors.initials: N
authors.lastname: Ogiwara
authors.affiliation: Department of Laboratory Medicine, Shinshu University Hospital, Matsumoto, Japan; Department of Health and Medical Sciences, Graduate School of Medicine, Shinshu University, Matsumoto, Japan.
authors.forename: Yuka
authors.initials: Y
authors.lastname: Takezawa
authors.affiliation: Department of Clinical Laboratory Investigation, Shinshu University, Matsumoto, Japan.
authors.forename: Mai
authors.initials: M
authors.lastname: Nanya
authors.affiliation: Department of Clinical Laboratory Investigation, Shinshu University, Matsumoto, Japan; Department of Health and Medical Sciences, Graduate School of Medicine, Shinshu University, Matsumoto, Japan.
authors.forename: Fumiko
authors.initials: F
authors.lastname: Terasawa
authors.affiliation: Department of Clinical Laboratory Investigation, Shinshu University, Matsumoto, Japan; Department of Health and Medical Sciences, Graduate School of Medicine, Shinshu University, Matsumoto, Japan. Electronic address: nobuoku@shinshu-u.ac.jp.
authors.forename: Nobuo
authors.initials: N
authors.lastname: Okumura
mesh_terms: D000347:Afibrinogenemia / Q000097:blood / Q000235:genetics* / Q000378:metabolism; D000818:Animals; D016466:CHO Cells; D006224:Cricetinae; D003412:Cricetulus; D004721:Endoplasmic Reticulum / Q000235:genetics / Q000378:metabolism*; D005340:Fibrinogen / Q000235:genetics* / Q000378:metabolism*; D006801:Humans; D046529:Microscopy, Electron, Transmission
chemical_list: D005340:Fibrinogen
keywords: Endoplasmic reticulum; Fibrinogen storage disease; Fibrinogen γ-chain mutation; Hepatic endoplasmic reticulum storage disease; Hypofibrinogenemia