Recombinant variant fibrinogens substituted at residues gamma326Cys and gamma339Cys demonstrated markedly impaired secretion of assembled fibrinogen.
BACKGROUND
To study the functions of residues gamma326Cys and gamma339Cys in the assembly and/or secretion of fibrinogen, recombinant fibrinogens were synthesized to replicate naturally occurring gamma326Tyr and gamma326Ser variants, along with gamma326Ala and gamma339Ala variants.


METHODS
A fibrinogen gamma-chain expression vector was altered and transfected into Chinese hamster ovary (CHO) cells. Cell lysates and culture media of the established cell lines were subjected to ELISA and immunoblotting analysis. In addition, pulse-chase analysis was performed.


RESULTS
The CHO cells synthesized mutant gamma-chains and assembled these into fibrinogen in the cells, although these variant fibrinogens were barely secreted into the culture media. Pulse-chase analysis indicated that the rates of both assembly and secretion of the variant fibrinogens were lower than that of normal fibrinogen.


CONCLUSIONS
The present study indicated that the 326-339 intrachain disulfide bond has a crucial role in maintaining the tertiary structure of the C-terminal domain of the gamma-module, which is necessary for fibrinogen assembly and specifically secretion. A combination of the present results and observations from naturally occurring heterozygous cases of gamma326Tyr and gamma326Ser suggest that heterozygous fibrinogen molecules containing variant gamma-chains might be secreted into plasma and show impaired fibrin polymerization, resulting in a phenotype of hypodysfibrinogenemia.
Haneishi
Ayumi
A
Department of Laboratory Medicine, Shinshu University School of Medicine, Matsumoto, Japan.
Terasawa
Fumiko
F
Fujihara
Noriko
N
Yamauchi
Kazuyoshi
K
Okumura
Nobuo
N
Katsuyama
Tsutomu
T
D019943:Amino Acid Substitution; D000818:Animals; D001665:Binding Sites; D016466:CHO Cells; D006224:Cricetinae; D003412:Cricetulus; D019281:Dimerization; D005340:Fibrinogen / Q000737:chemistry / Q000235:genetics* / Q000378:metabolism*; D014644:Genetic Variation; D016297:Mutagenesis, Site-Directed; D011485:Protein Binding; D011994:Recombinant Proteins / Q000737:chemistry / Q000378:metabolism*; D013329:Structure-Activity Relationship
D011994:Recombinant Proteins; D005340:Fibrinogen