Impact of γ-carboxylations in the Gla domain of rIX-FP on its functional properties
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Philipps-Universität Marburg
Abstract
Hemophilia B is a bleeding disorder caused by the deficiency of vitamin K-dependent coagulation factor IX (FIX). The standard therapy comprises intravenous administration with plasma-derived or recombinant FIX. In particular, the recombinant fusion protein linking coagulation factor IX with albumin (rIX FP) has emerged as an effective therapeutic option due to its extended half-life. One critical quality attribute is the γ carboxylation of up to 12 glutamates in the N terminal Gla domain. In the presence of divalent metal ions, these γ carboxylated glutamic acids (Gla) lead to a structural rearrangement that is essential for the functionality of FIX. However, in contrast to plasma-derived FIX that is known to be fully γ carboxylated, recombinant FIX products also contain partially γ carboxylated species and their effects on rIX FP are still not fully understood.
In this work, a purification process was established that allowed for the first time the isolation of partially γ carboxylated rIX-FP for a comprehensive in-depth characterization. After analysis of general quality attributes such as post-translational modifications and purity, their functional properties were assessed. rIX-FP species with 12 to 10 γ carboxylations showed similar activation rates upon both intrinsic and extrinsic activation. In contrast, rIX FP with 9 or fewer γ carboxylations showed clearly reduced FIXa release. This emphasizes the importance of a structurally intact Gla domain for the interaction with FXIa in case of the intrinsic activation and the TF/FVIIa complex in case of the extrinsic activation. In addition, the activity profiles of the isolated Gla species were analyzed using three orthogonal FIX activity assays. Across all assays, the 11Gla species showed the highest activity, followed by the 12Gla and 10Gla species. The 9Gla species demonstrated only residual activity, and species with fewer than 9 γ carboxylations exhibited almost no activity. This research project not only revealed a substantial loss of activity for rIX-FP with fewer than 10 γ carboxylations, but also provides explanations for this finding. These undercarboxylated rIX FP species showed a remarkably impaired ability to bind phospholipid membranes. In addition, further investigations demonstrated that the lacking γ carboxylations in rIX FP with fewer than 10 γ carboxylations caused an altered calcium-mediated protein folding.
It is assumed that these structural changes led to an impaired calcium-dependent membrane binding motif of the Gla domain. The resulting defective membrane binding is suggested to affect pivotal protein-protein interactions, such as the formation of the tenase complex. This leads to a massive loss of procoagulant functionality.
The second aspect of this work was to investigate sequence variants with additional point mutations in the Gla domain. The aim was to gain a deeper understanding of the role of these residues and to generate rIX FP mimicking partially γ carboxylated rIX FP.
FIX comprising the K5R mutation emerged as a promising candidate for hemophilia B treatment. This mutation is known to enhance the accumulation of FIX in the extravascular space, leading to modified pharmacokinetic properties. In this work, it was further found that the K5R mutation slightly enhances the procoagulant activity and the affinity to phospholipid membranes. Moreover, the number of γ carboxylations was found to have similar effects in rIX FP with and without the K5R mutation. Like in non-mutated rIX FP, a slight increase in activity was observed in the 11Gla species and a substantial loss of activity in the 9Gla species.
Furthermore, variants encompassing an increasing number of glutamate-to-aspartate substitutions (E_D) in addition to the K5R mutation were produced to obtain site-specific information on partially γ carboxylated species. The rIX FP/K5R E_D sequence variants exhibited distinct discrepancies to the corresponding rIX FP/K5R Gla variants. These were attributed to additional effects of the introduced aspartates. Nevertheless, in agreement with previous findings, these variants highlighted the essential role of the γ carboxylated glutamates at position 30 and 33 in terms of membrane binding and FIX activity.
In conclusion, these results underscore the pivotal role of γ carboxylations for the functional and physicochemical integrity of rIX FP and provided experimental data to elucidate the observed loss in activity in species with less than 10 γ carboxylations. In addition, significant functional differences were found between rIX FP species with 12, 11 and 10 γ carboxylations.
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