Amino acid sequence and posttranslational modifications of human factor VIIa from plasma and transfected baby hamster kidney cells.
Blood coagulation factor VII is a vitamin K dependent glycoprotein which in its activated form, factor VIIa, participates in the coagulation process by activating factor X and/or factor IX in the presence of Ca2+ and tissue factor. Three types of potential posttranslational modifications exist in the human factor VIIa molecule, namely, 10 gamma-carboxylated, N-terminally located glutamic acid residues, 1 beta-hydroxylated aspartic acid residue, and 2 N-glycosylated asparagine residues. In the present study, the amino acid sequence and posttranslational modifications of recombinant factor VIIa as purified from the culture medium of a transfected baby hamster kidney cell line have been compared to human plasma factor VIIa. By use of HPLC, amino acid analysis, peptide mapping, and automated Edman degradations, the protein backbone of recombinant factor VIIa was found to be identical with human factor VIIa. Neither recombinant factor VIIa nor human plasma factor VIIa was found to contain beta-hydroxyaspartic acid. In human plasma factor VIIa, the 10 N-terminally located glutamic acid residues were found to be fully gamma-carboxylated whereas 9 full and 1 partial gamma-carboxylated residues were found in the corresponding positions of the recombinant factor VIIa molecule. Asparagine residues 145 and 322 were found to be fully N-glycosylated in human plasma factor VIIa. In the recombinant factor VIIa, asparagine residue 322 was fully glycosylated whereas asparagine residue 145 was only partially (approximately 66%) glycosylated. Besides minor differences in the sialic acid and fucose contents, the overall carbohydrate compositions were nearly identical in recombinant factor VIIa and human plasma factor VIIa.(ABSTRACT TRUNCATED AT 250 WORDS)
Thim
L
L
Novo Research Institute, Bagsvaerd, Denmark.
Bjoern
S
S
Christensen
M
M
Nicolaisen
E M
EM
Lund-Hansen
T
T
Pedersen
A H
AH
Hedner
U
U
D000595:Amino Acid Sequence; D000818:Animals; D002478:Cells, Cultured; D006224:Cricetinae; D005167:Factor VII / Q000235:genetics / Q000378:metabolism*; D015942:Factor VIIa; D006031:Glycosylation; D006801:Humans; D006900:Hydroxylation; D008969:Molecular Sequence Data; D011499:Protein Processing, Post-Translational; D014162:Transfection
D005167:Factor VII; D015942:Factor VIIa