Difference in electrophoretic mobility and plasmic digestion profile between four recombinant fibrinogens, gamma 308K, gamma 308I, gamma 308A, and wild type (gamma 308N).
We have produced recombinant gamma-chain variant fibrinogens, gamma308K, gamma308I, and gamma308A simultaneously with wild-type fibrinogen, gamma-308N, by genetic protein engineering using Chinese hamster ovary cells. Although all three variant fibrinogens are a result of a single amino acid substitution, the aberrant gamma-chains of gamma308K and gamma308I fibrinogens migrated faster than gamma308N. Furthermore, plasmic digestion profiles were examined in the presence of 5 mM ethylene glycol-bis(beta-aminoethyl ether)-N,N,N',N'-tetraacetic acid (EGTA) or 1 mM CaCl2. In the presence of EGTA, the three variant fibrinogens were digested into D1 and D2 fragments slightly faster than wild type. In addition, the D2 fragment derived from gamma308K was further digested into D3 by plasmin much faster than that from gamma308N. These data suggest that cleavage of gamma356Lys-gamma357Ala bond by plasmin in gamma308K, gamma308I, and gamma308A is slightly accelerated and the gamma302Lys-gamma303Phe bond is cleaved by plasmin rapidly in only the gamma308K variant. Furthermore, the substitution of Lys for gamma308Asn results in the generation of a new plasmin cleavage site between gamma308Lys and gamma309Gly in the presence of EGTA. In conclusion, a substitution at residue gamma308Asn may cause a conformational change in the gammachain of fragment D affecting polymerization and plasmin cleavage.
Department of Medical Technology, School of Allied Medical Sciences, Shinshu University, Matsumoto, Japan. nobuoku@gipac.shinshu-u.ac.jp
N
N
Okumura
F
F
Terasawa
K
K
Fujita
M
M
Tozuka
H
H
Ota
T
T
Katsuyama
D000818:Animals; D016466:CHO Cells; D006224:Cricetinae; D004591:Electrophoresis, Polyacrylamide Gel / Q000379:methods; D005340:Fibrinogen / Q000737:chemistry* / Q000378:metabolism; D005341:Fibrinolysin / Q000378:metabolism; D005343:Fibrinolytic Agents / Q000378:metabolism; D020033:Protein Isoforms / Q000737:chemistry / Q000378:metabolism; D011994:Recombinant Proteins / Q000378:metabolism
D005343:Fibrinolytic Agents; D020033:Protein Isoforms; D011994:Recombinant Proteins; C027539:fibrinopeptides gamma; D005340:Fibrinogen; D005341:Fibrinolysin