Modification of the N-terminus of human factor IX by defective propeptide cleavage or acetylation results in a destabilized calcium-induced conformation: effects on phospholipid binding and activation by factor XIa

Abstract

The propeptide of human coagulation factor IX (FIX) directs the gamma-carboxylation of the first 12 glutamic acid residues of the mature protein into gamma-carboxyglutamic acid (Gla) residues. The propeptide is normally removed before secretion of FIX into the blood. However, mutation of Arg-4 in the propeptide abolishes propeptide cleavage and results in circulating profactor IX in the blood. We studied three such genetic variants, factor IX Boxtel (Arg-4-->Trp), factor IX Bendorf (Arg-4-->Leu) and factor IX Seattle C (Arg-4-->Gln). These variant profactor IX molecules bind normally to anti-FIX:Mg(II) antibodies, which indicates that the mutations do not seriously affect gamma-carboxylation. Metal ion titration of the binding of variant profactor IX to conformation-specific antibodies demonstrates that the calcium-induced conformation is destabilized in the variant molecules. Also the binding of FIX Boxtel to phospholipids and its activation by factor XIa requires a high (>5 mM) calcium concentration. The three-dimensional structure of the Gla domain of FIX in the presence of calcium indicates that the acylation of the amino-terminus, rather than the presence of the propeptide, was responsible for the destabilization of the calcium-induced conformation. In order to confirm this, the alpha-amino group of Tyr1 of FIX was acetylated. This chemically modified FIX showed a similar destabilization of the calcium-induced conformation to variant profactor IX. Our data imply that the amino-terminus of FIX plays an important role in stabilizing the calcium-induced conformation of the Gla domain of FIX. This conformation is important for the binding to phospholipids as well as for the activation by factor XIa. Our results indicate that mutations in FIX that interfere with propeptide cleavage affect the function of the protein mainly by destabilizing the calcium-induced conformation.

Vitamin K-dependent carboxylation of coagulation factors: insights from a cell-based functional study

“…Our result shows that, compared to the wild-type reporter-protein, the R-1S and R-4Q mutants appear to be properly carboxylated, but migrate slower (Figure 5). This result suggests that mutations at -1 and -4 do not affect reporter-protein carboxylation but prevent the cleavage of the propeptide, which agrees with previous observations 29,30,34 .…”

“…Together, these results suggest that mutations at -9 and -10 of the propeptide are more sensitive to warfarin inhibition. 1 3 Naturally occurring mutations at position -4 and -1 of the propeptide preclude post-translational cleavage of the propeptide, resulting in secretion of the procoagulation factor with its propeptide still attached 29,30,34,35 . To examine the effect of these mutations on carboxylation, we mutated R-1 (R-1S) or R-4 (R-4Q) of the propeptide in our reporter-protein and transiently expressed them in HEK293 cells.…”

The Insect Salivary Protein, Prolixin-S, Inhibits Factor IXa Generation and Xase Complex Formation in the Blood Coagulation Pathway

“…It is also suggested that the Gla domain plays an important role in the activation of factor IX by factor XIa as well as in the binding to phospholipids. In fact, Wojcik et al (29) have provided evidence that the conformational change of Gla domain is important for the factor IX activation by factor XIa.…”

The Factor IX γ-Carboxyglutamic Acid (Gla) Domain Is Involved in Interactions between Factor IX and Factor XIa

“…The noncatalytic portion of the molecule contains (from N terminus to C terminus) the Gla domain, a short aromatic stack region, two EGF-like domains, and the activation peptide (3). Some Gla domain mutations causing hemophilia B are associated with poor factor XIa mediated activation, as is the failure to remove the propeptide from the N terminus of the molecule (26,27). Similarly, single amino acid substitutions in the EGF-1 (factor IX New London) (51) and EGF-2 (52) domains associated with cross-reactive material-positive hemophilia B are activated poorly by factor XIa.…”

“…A Gla domain mutation at amino acid 4 associated with hemo-philia B interferes with factor XIa-mediated activation of FIX (26), as does failure to remove the FIX propeptide from the N terminus (27). Monoclonal antibodies directed against FIX-Gla block activation of FIX by factor XIa (28,29).…”