Abnormal secretion and function of recombinant human factor VII as the result of modification to a calcium binding site caused by a 15-base pair insertion in the F7 gene.
A case of a novel mutation in the F7 gene that results in factor VII coagulant activity (VII:c) of less than 1% and VII antigen (VII:Ag) levels of 10% is presented. DNA analysis revealed a homozygous 15-base pair (bp) in-frame insertion-type mutation at nucleotide 10554. This insertion consisted of a duplication of residues leucine (L)213 to aspartic acid (D)217 (leucine, serine, glutamic acid, histidine, and aspartic acid), probably arising by slipped mispairing between 2 copies of a direct repeat (GCGAGCACGAC) separated by 4 bp. Molecular graphic analyses showed that the insertion is located at the surface of the catalytic domain in an exposed loop stabilized by extensive salt-bridge and hydrogen bond formation at which the calcium binding site is located. The mutation probably interferes with protein folding during VII biosynthesis and/or diminishes functional activity through the loss of calcium binding. In vitro expression studies demonstrated that the levels of VII:Ag in lysates of cells transfected with wild type VII (VIIWT) were equivalent to those with mutant type VII (VIIMT), but the level of secreted VIIMT was 5% to 10% that of VIIWT. Pulse chase studies demonstrated that VIIMT did not accumulate intracellularly, and studies with inhibitors of protein degradation showed that recombinant VIIMT was partially degraded in the pre-Golgi compartment. Accordingly, only small amounts of VIIMT with undetectable procoagulant activity were secreted into conditioned media. These results demonstrate that a combination of secretion and functional defects is the mechanism whereby this insertion causes VII deficiency.
Peyvandi
F
F
Hematology Section, Department of Medicine, VA Boston Healthcare System, Harvard Medical School, Boston, MA, USA. flora.peyvandi@unimi.it
Carew
J A
JA
Perry
D J
DJ
Hunault
M
M
Khanduri
U
U
Perkins
S J
SJ
Mannucci
P M
PM
Bauer
K A
KA
D020137:Base Pair Mismatch; D001665:Binding Sites; D002118:Calcium / Q000378:metabolism; D002675:Child, Preschool; D003001:Cloning, Molecular; D003241:Consanguinity; D004252:DNA Mutational Analysis; D005167:Factor VII / Q000096:biosynthesis / Q000737:chemistry / Q000235:genetics* / Q000378:metabolism; D005168:Factor VII Deficiency / Q000235:genetics*; D005260:Female; D006801:Humans; D006860:Hydrogen Bonding; D008958:Models, Molecular; D016254:Mutagenesis, Insertional; D011487:Protein Conformation; D017434:Protein Structure, Tertiary; D011993:Recombinant Fusion Proteins / Q000737:chemistry / Q000378:metabolism / Q000502:physiology; D013329:Structure-Activity Relationship
D011993:Recombinant Fusion Proteins; D005167:Factor VII; D002118:Calcium