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Editorials
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1996
Insights into vitamin K-dependent carboxylation: home field advantage
Francis Ayombil1 and Rodney M. Camire1,2
1Division of Hematology and the Raymond G. Perelman Center for Cellular and Molecular Therapeutics, The Children’s Hospital of Philadelphia and 2Department of Pediatrics, Perelman School of Medicine, University of Pennsylvania, Philadelphia, PA, USA
E-mail: RODNEY M. CAMIRE - rcamire@pennmedicine.upenn.edu doi:10.3324/haematol.2020.253690
Vitamin K-dependent (VKD) proteins play critical roles in blood coagulation, bone metabolism, and other physiologic processes. These proteins under- go a specific post-translational modification called gamma (γ)-carboxylation which is critical to their biologic function.1 The reaction, which occurs in the endoplasmic reticulum (ER) and requires reduced vitamin K, carbon dioxide and oxygen as co-factors, is catalyzed by γ-glu- tamyl carboxylase (GGCX). GGCX converts several glu- tamic acid residues (Glu) on its protein substrate [e.g. pro- thrombin, FVII, FIX, FX, PC, PS, PZ, and bone Gla protein (BGP)] to γ-carboxy-glutamic acid, otherwise known as Gla.2 How does this enzyme pick its protein substrate and modify specific glutamic acid residues? In work spanning over 30 years, researchers identified a critical sequence called the propeptide region that is N-terminal to the mature protein (Figure 1). GGCX binds the propeptide and directs carboxylation of 9-13 Glu residues on the so- called Gla domain in a processive fashion.2 The signal sequence and propeptide region are removed by pepti- dases prior to secretion of the mature VKD protein (Figure 1). For the VKD coagulation factors, the enhanced net negative charge following carboxylation in the Gla domain allows for high affinity divalent metal ion bind- ing.3 This changes the structural conformation of the Gla domain which facilitates binding to anionic phospho- lipids and localizes these proteins to the site of vascular injury.3,4 Defects of VKD protein carboxylation cause bleeding disorders, and inhibition of this pathway is the basis of warfarin anticoagulation.2
Acquiring mechanistic information about GGCX and deciphering how the propeptide influences carboxylation has been challenging. Since GGCX is an integral membrane ER protein (Figure 1), extracting it in a functional state is dif- ficult and requires artificial conditions to study it. Early work used crude microsomal extracts or detergent-solubi- lized liver microsomes following warfarin treatment or vitamin K-deficient animals which contained the enzyme and small amounts of endogenous protein substrate (e.g. prothrombin).1 Advancements to this system incorporated artificial peptide substrates for GGCX such as FLEEL (residues 5-9 of rat prothrombin).5 In the late 1980s, it was
recognized that the propeptide sequence is critical for VKD protein carboxylation.6 This insight led to the development of GGCX substrates that contained a propeptide sequence and portions of the Gla domain which are superior when compared to FLEEL alone.7,8 These and other substrates have been used to demonstrate the importance of propep- tide affinity in substrate recognition using either crude preparations or purified forms of GGCX and increased our understanding about the enzyme.9 Further insights into the importance of the propeptide came from studies using mutant peptides and identification of naturally occurring mutations in the propeptide region of FIX.10,11 However, this knowledge about the function of GGCX was obtained out- side of its natural environment under artificial conditions. To better understand VKD carboxylation in its native milieu, Tie and Stafford developed a cell-based reporter assay to study γ-carboxylation and the entire VKD cycle.12 In this system, a chimeric reporter-protein, FIXgla-PC is used, in which the PC backbone was replaced at the N-ter- minus with the FIX Gla domain.12,13 This allowed for an ELISA-based quantification of carboxylated reporter pro- tein using a capture antibody that recognizes only a fully carboxylated FIX Gla domain and an antibody against PC. The advantage of the system is that it allows for functional assessment of the VKD cycle enzymes, including GGCX, in an environment that requires the enzymes to interact with their physiologic substrates, a departure from systems pre- viously employed.
In this issue of Haematologica, Hao et al. use this cell- based assay to study the role of the propeptide in directing carboxylation of VKD proteins.14 Previous studies indicate that the propeptide region of VKD coagulation factors show considerable variation in their affinities for GGCX with FX, FIX and PC showing high (Kd ~1 nM), intermedi- ate (Kd ~5 nM), and low affinity (Kd ~20 nM), respectively (Figure 1).15 It is thought that these disparate affinities con- tribute to the heterogeneity in carboxylation in mammalian expression systems. Furthermore, it is thought that there is likely an optimal propeptide affinity that best directs car- boxylation. To better understand how GGCX interacts with its protein substrates via propeptide binding in its nat- ural environment, the authors created a series of chimeric
haematologica | 2020; 105(8)