γ‐Carboxyglutamic acids 36 and 40 do not contribute to human factor IX function

Abstract

The gamma-carboxyglutamic acid (Gla) domains of the vitamin K-dependent blood coagulation proteins contain 10 highly conserved Gla residues within the first 33 residues, but factor IX is unique in possessing 2 additional Gla residues at positions 36 and 40. To determine their importance, factor IX species lacking these Gla residues were isolated from heterologously expressed human factor IX. Using ion-exchange chromatography, peptide mapping, mass spectrometry, and N-terminal sequencing, we have purified and identified two partially carboxylated recombinant factor IX species; factor IX/gamma 40E is uncarboxylated at residue 40 and factor IX/gamma 36,40E is uncarboxylated at both residues 36 and 40. These species were compared with the fully gamma-carboxylated recombinant factor IX, unfractionated recombinant factor IX, and plasma-derived factor IX. As monitored by anti-factor IX:Ca (II)-specific antibodies and by the quenching of intrinsic fluorescence, all these factor IX species underwent the Ca(II)-induced conformational transition required for phospholipid membrane binding and bound equivalently to phospholipid vesicles composed of phosphatidylserine, phosphatidylcholine, and phosphatidylethanolamine. Endothelial cell binding was also similar in all species, with half-maximal inhibition of the binding of 125I-labeled plasma-derived factor IX at concentrations of 2-6 nM. Functionally, factor IX/gamma 36,40E and factor IX/gamma 40E were similar to fully gamma-carboxylated recombinant factor IX and plasma-derived factor IX in their coagulant activity and in their ability to participate in the activation of factor X in the tenase complex both with synthetic phospholipid vesicles and activated platelets. However, Gla 36 and Gla 40 represent part of the epitope targeted by anti-factor IX:Mg(II)-specific antibodies because these antibodies bound factor IX preferentially to factor IX/gamma 36,40E and factor IX/gamma 40E. These results demonstrate that the gamma-carboxylation of glutamic acid residues 36 and 40 in human factor IX is not required for any function of factor IX examined.

Prolonged activity of factor IX as a monomeric Fc fusion protein

“…21 Peptide mapping confirmed the ␥-carboxylation pattern to be similar to rFIX, with 10 of the 12 sites fully occupied, and the residues primarily at position 36 and 40 being under ␥-carboxylated, which has been shown to have no effect on activity. 14 In addition to ␥-carboxylation and propeptide processing, several other posttranslational modifications were assessed for rFIXFc and compared with rFIX (BeneFIX) and pdFIX (Mononine). In general, rFIXFc was found to be comparable with rFIX, with respect to Ser 158 phosphorylation and Tyr 155 sulfation (Table 1).…”

“…14 Gla content was confirmed by mass spectroscopy after digestion with LysC and subsequent peptide mapping.…”

Prolonged half-life and preserved enzymatic properties of factor IX selectively PEGylated on native N-glycans in the activation peptide

“…Twelve acceptor sites are present but occupancy of 10 are sufficient for FIX to attain full activity. 29 Gla profiling by anion exchange HPLC demonstrated a predominance of 11 (33%) and 12 (64%) Gla forms with an average content of 11.6 Gla per molecule, which is similar to the composition of rFIX but less than the 12 Gla residues present in plasma-derived FX (pdFX; Figure 1A). 28 Further analysis of posttranslational modifications identified as expected partial ␤-hydroxylation of Asp64 and O-glycans attached to Ser53 and Ser61 in the EGF1 module.…”

Crystal Structure of the Calcium-stabilized Human Factor IX Gla Domain Bound to a Conformation-specific Anti-factor IX Antibody

“…The ternary complex of Ca-6, phosphatidylserine, and Factor IX may be character- ized by tighter binding of calcium than this complex in the absence of phosphatidylserine. Ca-8, coordinated by Gla-36 and Gla-40, has no apparent functional role because elimination of these ␥-carboxyglutamic acid residues does not alter the biological assays tested (34).…”

“…Given crystallization conditions using high concentrations of ammonium sulfate, we suspect that this is a weak calcium binding site that is not occupied by calcium under the conditions employed. The carboxylation at these two positions were not required for Factor IX function in Factor X activation and coagulant activity (34).…”