Residue gamma153Cys is essential for the formation of the complexes Aalphagamma and Bbetagamma, assembly intermediates for the AalphaBbetagamma complex and intact fibrinogen.
BACKGROUND
Fibrinogen Matsumoto IV was found in a hypofibrinogenemia caused by a heterozygous missense mutation, i.e., the substitution of the fibrinogen gamma-chain residue Cys153 by Arg.


METHODS
To examine the precise basis for the fibrinogen deficiency, mixtures of any two vectors, the fibrinogen Aalpha-, Bbeta-, gamma- (153Cys) or gammam-(153Ala) chain were transfected into Chinese hamster ovary cells (CHO-Aalpha/gamma, -Aalpha/gammam, -Bbeta/gamma, -Bbeta/gammam). Expression and constitution of each of two chains and their complexes in the individual CHO cell lines were identified by sodium dodecyl sulfate (SDS)-polyacrylamide gel electrophoresis (PAGE) and immunoblot analysis using polypeptide specific antibodies and two-dimensional electrophoresis (2-DE).


RESULTS
In the CHO-Aalpha/gamma and -Bbeta/gamma, the Aalpha/gamma- or Bbeta/gamma-complex was formed, whereas in the CHO-Aalpha/gammam and -Bbeta/gammam, no Aalpha/gammam- or Bbeta/gammam-complex was observed. These results demonstrate that gamma153Ala cannot assemble with the Aalpha- and Bbeta-chains, leading to impaired fibrinogen assembly and secretion.


CONCLUSIONS
gamma153Cys is an essential residue for the fibrinogen assembly which is dependent on Aalpha/gamma- and Bbeta/gamma-complex formation.
Terasawa
Fumiko
F
Department of Biomedical Laboratory Sciences, School of Health Sciences, Shinshu University, 3-1-1 Asahi, Matsumoto 390-8621, Japan. fterasa@gipac.shinshu-u.ac.jp
Fujita
Kiyotaka
K
Okumura
Nobuo
N
D000818:Animals; D015153:Blotting, Western; D016466:CHO Cells; D006224:Cricetinae; D003545:Cysteine / Q000737:chemistry*; D015180:Electrophoresis, Gel, Two-Dimensional; D005340:Fibrinogen / Q000737:chemistry / Q000378:metabolism*
D005340:Fibrinogen; D003545:Cysteine