Protein engineering of the hydrophobic domain of human factor IX.
Vitamin K-dependent plasma proteins contain a highly conserved hydrophobic domain located between the gamma-carboxyglutamic acid (Gla) domain and the first epidermal growth factor (EGF)-like domain. Here we have used protein engineering of the hydrophobic domain in human factor IX to investigate its function in intact factor IX. Mutant proteins were generated by site-directed mutagenesis and in vitro expression in Madin-Darby canine kidney (MDCK) cells. All of our mutants, including one with a deletion of the entire hydrophobic domain, were activated by factor XIa, showing that this domain is not required for factor IX activation. The results with the mutant Phe41-->Val suggest that the hydrophobic domain interacts with the adjacent EGF-like domain. Our data for the Phe41-->Asp mutant is consistent with, but cannot prove, a role for this residue in the maintenance of a phospholipid-binding structure required for factor IX function.
Chemical Pathology Unit, Sir William Dunn School of Pathology, University of Oxford, UK.
P E
PE
Hughes
P A
PA
Handford
D E
DE
Austen
G G
GG
Brownlee
D000595:Amino Acid Sequence; D000818:Animals; D000911:Antibodies, Monoclonal; D001483:Base Sequence; D001665:Binding Sites / Q000235:genetics; D002460:Cell Line; D018076:DNA, Complementary / Q000235:genetics; D004285:Dogs; D005164:Factor IX / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism; D015949:Factor IXa / Q000378:metabolism; D005170:Factor X / Q000378:metabolism; D006801:Humans; D066298:In Vitro Techniques; D008969:Molecular Sequence Data; D016297:Mutagenesis, Site-Directed; D010743:Phospholipids / Q000378:metabolism; D017354:Point Mutation; D015202:Protein Engineering; D017384:Sequence Deletion
D000911:Antibodies, Monoclonal; D018076:DNA, Complementary; D010743:Phospholipids; D005164:Factor IX; D005170:Factor X; D015949:Factor IXa