Expression, purification and characterization of factor IX derivatives using a novel vector system.
Recent studies have indicated that the loop harboring the S1 specificity site (residues 185-189 in chymotrypsin numbering) of coagulation proteases has several charged residues with important structural and functional roles for the catalytic activity of these proteases. This loop is allosterically linked to the Na(+)-binding site in both factor Xa and thrombin. There are three candidate residues (His-185, Glu-186, and Arg-188) on this loop of factor IXa (fIXa) whose side chains can influence the Na(+) binding and the catalytic function of the protease in the intrinsic Xase complex. In this study, we developed a novel expression/purification vector system, substituted all three residues of factor IX individually with Ala, and expressed the mutant zymogens in mammalian cells. Following activation, all three fIXa mutants exhibited normal activity towards a fIXa-specific chromogenic substrate in the presence of Ca(2+) with no obvious requirement for Na(+) in the reaction. Furthermore, all three mutants interacted with factor VIIIa with near normal affinity and catalyzed the activation of factor X in the intrinsic Xase complex with a normal catalytic efficiency. These results suggest that, unlike thrombin and factor Xa, the charged residues of this loop do not play a functional role in modulating the catalytic function of fIXa in the intrinsic Xase complex.
Yang
Likui
L
Edward A. Doisy Department of Biochemistry and Molecular Biology, St. Louis University School of Medicine, 1402 S. Grand Blvd., St. Louis, MO 63104, USA.
Gopalakrishna
Kota
K
Manithody
Chandrashekhara
C
Rezaie
Alireza R
AR
D000409:Alanine / Q000235:genetics / Q000378:metabolism; D002118:Calcium / Q000378:metabolism; D002384:Catalysis; D003546:Cysteine Endopeptidases / Q000378:metabolism; D004789:Enzyme Activation; D015949:Factor IXa / Q000096:biosynthesis / Q000235:genetics / Q000302:isolation & purification*; D015944:Factor VIIIa / Q000378:metabolism; D005170:Factor X / Q000378:metabolism; D005822:Genetic Vectors; D006801:Humans; D008958:Models, Molecular; D009154:Mutation; D009363:Neoplasm Proteins / Q000378:metabolism; D010957:Plasmids; D011485:Protein Binding; D011487:Protein Conformation; D011994:Recombinant Proteins / Q000096:biosynthesis / Q000235:genetics / Q000302:isolation & purification*; D012964:Sodium / Q000378:metabolism
D009363:Neoplasm Proteins; D011994:Recombinant Proteins; D015944:Factor VIIIa; D005170:Factor X; D012964:Sodium; D015949:Factor IXa; D003546:Cysteine Endopeptidases; C030910:cancer procoagulant; D000409:Alanine; D002118:Calcium