gamma-Carboxyglutamic acids 36 and 40 do not contribute to human factor IX function

Area of Science:

• Biochemistry
• Hematology
• Molecular Biology

Background:

• Vitamin K-dependent proteins, including coagulation factor IX, undergo gamma-carboxylation of glutamic acid residues.
• Factor IX possesses unique gamma-carboxyglutamic acid (Gla) residues at positions 36 and 40, in addition to the conserved Gla residues in its Gla domain.
• The functional significance of these unique Gla residues in factor IX remains to be fully elucidated.

Purpose of the Study:

• To investigate the functional importance of gamma-carboxyglutamic acid (Gla) residues at positions 36 and 40 in human factor IX.
• To determine if these unique Gla residues are necessary for factor IX's calcium-dependent conformational changes, phospholipid binding, endothelial cell binding, and coagulant activity.
• To assess the role of Gla 36 and Gla 40 in the epitope recognized by specific anti-factor IX antibodies.

Main Methods:

• Generation and purification of recombinant human factor IX species with specific uncarboxylated residues (factor IX/gamma 40E and factor IX/gamma 36,40E).
• Biochemical characterization using ion-exchange chromatography, peptide mapping, mass spectrometry, and N-terminal sequencing.
• Assessment of calcium-dependent conformational changes, phospholipid binding, endothelial cell binding, and coagulant activity using various assays and anti-factor IX antibodies.

Main Results:

• Partially carboxylated recombinant factor IX species (factor IX/gamma 40E and factor IX/gamma 36,40E) were successfully purified and identified.
• All tested factor IX species, including those lacking Gla 36 and 40, exhibited normal calcium-induced conformational transitions and equivalent binding to phospholipid vesicles.
• Coagulant activity and participation in factor X activation within the tenase complex were similar across all factor IX variants.
• Antibody binding studies revealed that Gla 36 and Gla 40 are part of the epitope for anti-factor IX:Mg(II)-specific antibodies.

Conclusions:

• The gamma-carboxylation of glutamic acid residues 36 and 40 in human factor IX is not required for its essential functions, including phospholipid binding and coagulant activity.
• These unique Gla residues do not influence the calcium-dependent conformational changes necessary for factor IX's procoagulant functions.
• Gla 36 and Gla 40 are important for antibody recognition, specifically for antibodies targeting the Mg(II)-dependent epitope of factor IX.