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Coagulation factor carboxylation in mammalian cells
matic activity of GGCX was first discovered in the 1970s, showing that radioactive 14CO2 was incorporated into PT in rats, and that the amount incorporated was dependent upon the administration of vitamin K.4 Two decades later, the GGCX gene was cloned5 and the enzyme was puri- fied6 by our laboratory, making it possible to study GGCX function at the molecular level.
Gamma-glutamyl carboxylase recognizes its protein substrate by binding tightly to the propeptide of the sub- strate, which tethers the substrate to the enzyme.7 The Glu residues within the Gla domain of the substrate pro- tein are progressively modified so that multiple carboxy- lation reactions occur during a single enzyme and sub- strate binding event.8 In addition, binding of the propep- tide to GGCX has been shown to significantly stimulate the activity of the enzyme toward non-covalently linked Glu-containing substrates.9,10 The propeptide of most VKD proteins is located at the N-terminus of the precursor pro- tein that is proteolytically removed after carboxylation to form the mature protein. Notably, a propeptide can also be found at the C-terminus of the precursor protein11 or even within the mature VKD protein.12 Removal of the propeptide from the precursor of coagulation factors abol- ishes their carboxylation,7,13 suggesting the pivotal role of the propeptide for carboxylation. Nevertheless, the propeptide of osteocalcin (or referred to as bone Gla pro- tein, BGP) appears to be unnecessary for its carboxylation.14 Moreover, high-affinity binding sites within the mature BGP were identified, which appeared to bind to GGCX through a different binding site to the propeptide binding site.15
Propeptides of coagulation factors are essential for the carboxylation of precursor proteins. These propeptide sequences are highly conserved, especially at residues -16, -10, -6, -4, and -1. It has been proposed that the N-terminal sequence of the propeptide is necessary for GGCX recog- nition, while the C-terminal sequence is required for propeptidase recognition.13 Despite the high sequence con- servation, in an in vitro study, the apparent affinities of the coagulation factors’ propeptide for GGCX varied over 100-fold.16 Nevertheless, these coagulation factors appear to be fully carboxylated in physiological conditions. It has been shown that replacing FX propeptide with a reduced affinity propeptide (PT’s propeptide) enhanced the car- boxylation of FX, which presumably increased substrate turnover.17 However, a similar strategy of replacing FIX propeptide failed to increase the carboxylation efficiency of FIX,18 although the reason for this discrepancy remains unclear.
It is worth noting that most of our knowledge of GGCX function and its interaction with natural protein substrates was obtained from in vitro studies carried out under artifi- cial conditions using the pentapeptide FLEEL as the sub- strate.19 Consequently, we do not know how GGCX car- boxylates natural VKD proteins in their native milieu.
Here, we studied the carboxylation of coagulation fac- tors in a cellular environment with different chimeric reporter-proteins using our recently established cell-based assay.20 We compared the contribution of the propeptide, the Gla domain, and the C-terminal functional domain of the coagulation factor to its carboxylation. In addition, we examined the effect of naturally occurring mutations in the propeptide on the coagulation factor’s carboxylation to gain an insight into the corresponding phenotype of warfarin hypersensitivity. Our results confirmed the piv-
otal role of the propeptide in coagulation factor carboxy- lation and interpreted the clinical phenotype of the hyper- sensitivity of warfarin during anticoagulation therapy.
Methods
Materials and cell lines
The mammalian expression vector pcDNA3.1Hygro(+), mouse anti-carboxylated BGP monoclonal antibody, Alexa Fluor-488 con- jugated donkey anti-mouse IgG, Alexa Fluor-568 conjugated don- key anti-sheep IgG, and Hoechst 33342 were from ThermoFisher Scientific (Waltham, MA, USA). The fluorescent protein-tagged marker proteins of the endoplasmic reticulum (ER) (mCherry- Sec61-N-18) and Golgi apparatus (pmScarlet_Giantin_C1) were gifts from Dr. Michael Davidson (Addgene plasmid # 55130) and Dr. Dorus Gadella (Addgene plasmid # 85048), respectively. The Ca2+-dependent monoclonal antibody to carboxylated Gla domain of PC (PCgla) was a gift from Dr. Paul Bajaj (University of California, Los Angeles, CA, USA).21 The horseradish peroxidase- conjugated goat anti-mouse IgG was from Jackson ImmunoResearch Laboratories Inc. (West Grove, PA, USA). The monoclonal antibody against Gla residues was from Sekisui Diagnostics LLC (Stamford, CT, USA). The HEK293 and COS-7 cell lines were from ATCC (Manassas, VA, USA).
DNA manipulations and plasmid constructions
The pcDNA3.1Hygro(+) vector, with the cDNA of FIXgla-PC (PC with its Gla domain exchanged with that of FIX) cloned onto the XbaI site, was used as the cloning and expression vector, as previously described.20 All other chimeric reporter-proteins, with the different propeptides and/or Gla domains used in this study, were obtained by overlap polymerase chain reaction (PCR). Replacement of FIX epidermal growth factor (EGF) domain and the following domains with cell organelle marker proteins was performed by PCR. The nucleotide sequences of all constructs were verified by DNA sequencing at Eton Bioscience Inc. (RTP, NC, USA).
Reporter-protein carboxylation in HEK293 cells
The efficiency of reporter-protein (FIXgla-PC) carboxylation was determined in HEK293 cells, as previously described.20 For the warfarin resistance study, HEK293 cells stably expressing the cor- responding mutant reporter-protein were cultured in complete medium containing 20 nM vitamin K with increasing concentra- tions of warfarin. The cell culture medium was collected 48 hours (h) later and used for the sandwich-based ELISA to determine the level of carboxylated reporter-proteins.20 For the BGP-PC reporter- protein (PC with its Gla domain replaced by BGP) detection, sheep anti-human PC IgG was used as the capture antibody, and mouse anti-carboxylated BGP antibody was used as the detection anti- body. Experimental data were analyzed using GraphPad Prism.
To purify carboxylated chimeric reporter-proteins for use as a standard for ELISA, different chimeric proteins were stably expressed in HEK293/VKOR cells (HEK293 cells over-expressing VKOR). Carboxylated reporter-proteins were purified from the collected medium using 2-step chromatography, as previously described.17 Protein concentrations were quantified using the BCA protein assay kit.
Immunofluorescence confocal imaging
The subcellular localization of reporter-proteins were examined by immunofluorescence confocal imaging, as previously described.22 To examine the effect of the propeptide on reporter- protein carboxylation, different propeptide attached reporter-pro-
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