A novel gene mutation in the 60s loop of human coagulation factor VII - inhibition of interdomain crosstalk.
A novel mutation in the factor VII gene resulting in procoagulant activity of 7.5% and antigen levels of 23% is presented. Single-stranded conformational polymorphism and DNA sequencing analysis revealed heterozygous shifts, and mutations were detected in exons 5, 7 and 8. The mutant L204P in exon 7 was novel, while the common polymorphisms, H115H and R353Q, were located in exons 5 and 8, respectively. The molecular effect of the L204P mutation was characterized using recombinant mammalian expression in Chinese hamster ovary cells. Low levels (4 ng/ml) of secreted mutant protein were found in transiently transfected cells compared to wild-type factor VII (83 ng/ml). Metabolic labeling demonstrated that the rate of mutant protein synthesis was similar to that of wild-type FVII, and the mutant protein accumulated intracellularly with no signs of increased degradation during a four-hour chase. No interaction between secreted P204 protein and immobilized soluble tissue factor was detected using surface plasmon reso-nance. The activation rate of recombinant mutant FVII protein was strongly reduced compared to wild-type FVII. A 9-fold reduction in the rate of FX activation was detected whereas Km was nearly the same for wild-type and the mutant. This slow rate was caused by a correspondingly lowered rate of P204 activation. A synthetic peptide sequence comprising amino acids 177-206 blocked binding of FVIIa to the TF-chip, and the subsequent factor X activation with an IC(50) value of 0.5 micro M in a chromogenic factor Xa assay. Additionally, evaluation of the peptide by surface plasmon resonance analysis resulted in inhibition of complex formation with an apparent K(I) of 7 micro M.
Kavlie
Anita
A
Biotechnology Centre of Oslo, University of Oslo, Oslo, Norway.
Wiiger
Merete Thune
MT
Husbyn
Mette
M
Stormorken
Helge
H
Prydz
Hans
H
D000111:Acetylcysteine / Q000031:analogs & derivatives* / Q000737:chemistry; D000818:Animals; D000911:Antibodies, Monoclonal / Q000737:chemistry; D001921:Brain / Q000378:metabolism; D020126:Brefeldin A / Q000737:chemistry; D016466:CHO Cells; D002942:Circular Dichroism; D006224:Cricetinae; D015853:Cysteine Proteinase Inhibitors / Q000494:pharmacology; D004305:Dose-Response Relationship, Drug; D004591:Electrophoresis, Polyacrylamide Gel; D004792:Enzyme Precursors / Q000737:chemistry; D005091:Exons; D005167:Factor VII / Q000737:chemistry* / Q000235:genetics*; D005170:Factor X / Q000737:chemistry; D015951:Factor Xa / Q000737:chemistry; D006579:Heterozygote; D006801:Humans; D020128:Inhibitory Concentration 50; D007700:Kinetics; D008958:Models, Molecular; D009154:Mutation; D010455:Peptides / Q000737:chemistry; D016133:Polymerase Chain Reaction; D011110:Polymorphism, Genetic; D018807:Polymorphism, Single-Stranded Conformational; D011233:Precipitin Tests; D011485:Protein Binding; D017434:Protein Structure, Tertiary; D011500:Protein Synthesis Inhibitors / Q000494:pharmacology; D011994:Recombinant Proteins / Q000737:chemistry; D015183:Restriction Mapping; D017422:Sequence Analysis, DNA; D012697:Serine Endopeptidases / Q000737:chemistry; D020349:Surface Plasmon Resonance; D013997:Time Factors; D014162:Transfection
D000911:Antibodies, Monoclonal; D015853:Cysteine Proteinase Inhibitors; D004792:Enzyme Precursors; D010455:Peptides; D011500:Protein Synthesis Inhibitors; D011994:Recombinant Proteins; C067713:lactacystin; D020126:Brefeldin A; D005167:Factor VII; D005170:Factor X; D012697:Serine Endopeptidases; D015951:Factor Xa; D000111:Acetylcysteine