Identification of Functionally Important Residues in the Protease Domain of Factor Ix That Are Critical for Binding Factor Xia, Tfpi, and Antibodies
Resource
BLOOD v.100 n.11 pp.1006 PART 1
Journal
BLOOD
Journal Volume
v.100
Journal Issue
1
Pages
-
Date Issued
2002
Date
2002
Author(s)
LIN, SHU-RUNG
HSU, YA-CHU
LIN, SHU-WHA
Abstract
This work describes the consequences of alanine scanning mutagenesis on 19 positions (within residues 185-301) of the catalytic domain of factor IX (FIX). R248 and K293 are critical for the secretion of FIX. Mutations at K265 and at E277 yielded novel FIX with 2-3 times better clotting activities compared to that of wild type. Both factor XIa ( FXIa) and the factor VIIa-tissue factor (TF) complex effectively catalyzed the activation of all the tested mutants except for three. Mutations at residues E235, E242, and E245 impaired factor IX's activation by FXIa but not by VIIa-TF, suggesting the mechanisms for cleavage of FIX by the two enzyme systems are different based on this and our previous finding (Chang YJ et al., 2002, J. Biol. Chem, p. 25393-9). All of the 19 mutations had little effect on the binding of FIX to factor VIIIa and antithrombin III ( ATIII). However, FIX with Ala at K265 exhibited 10-fold increase in binding TFPI, which suggests that this residue may render function of the 99-loop involved in the substrate specificity in the serine protease family. Besides being an important part of the substrate and inhibitor binding, this region (residues 185-301) is also an epitope for monoclonal antibody A-5. We found that an electrostatic surface composed of discontinuous amino acids of K201, D203, K228, R 252, and D276 is the epitope for A-5 (Smith KJ., and Ono K., 1984, Thrombosis Res. p. 211-4, and Hamaguchi et al., 1994, Blood, p. 1837-42). These residues constitute a highly charged surface opposite to the factor VIIIa binding surface . Since their Ala-mutant counterparts were either almost as active as, or slightly less active than wild-type FIX in clotting function, this side of FIX, although may not be very important for FIX in binding factor VIIIa, is a strong antigenic determinant. Taken together, we conclude that the surface-exposed residues within positions 185-301 of FIX are highly charged and antigenic. A-5 might exert its inhibitory effect by affecting the global conformation of this region. Moreover, this region although may not be important for the interaction of FIX with factor VIIIa, VIIa -TF and ATIII, is critical for interacting with FXIa and TFPI.
Subjects
Factor IX
Alanine scanning mutagenesis
Protease domain