Gla Residues Research Articles

Synthesis and Evaluation of [18F]SiFA-Conjugated Ligands for Fibroblast Activation Protein Imaging.

In recent years, fibroblast activation protein (FAP) has emerged as an important target for the diagnosis and therapy of various tumors due to its high expression on the cell surface of cancer-associated fibroblasts, which are the major components of the tumor stroma. In this study, we synthesized and evaluated 18F-labeled FAP inhibitors (FAPIs) for FAP imaging. Two silicon fluoride acceptor (SiFA)-conjugated FAPIs were synthesized: one containing a γ-carboxy-l-glutamic acid (Gla) residue (1) and another containing two Gla residues (2). Both ligands exhibited high binding affinities for FAP. 18F/19F exchange reactions on both ligands were performed in the presence of 2% water. This resulted in the formation of radioligands [18F]1 and [18F]2 in high radiochemical yields. Radioligand [18F]2, with a more favorable partition coefficient, was selected for the U87MG cell binding study, and the results showed FAP-specific binding of the radioligand to the cells. An ex vivo biodistribution study in U87MG tumor-bearing mice 60 min after injection demonstrated a 5.8-fold higher tumor accumulation of [18F]2 than that of [18F]1. Furthermore, PET and ex vivo biodistribution studies of [18F]2 in U87MG tumor-bearing mice showed high and persistent tumor uptake over time, which was significantly blocked by the preinjection of FAPI-04. Our results indicate that [18F]SiFA-(Gla)2-conjugated FAPI ([18F]2) has the potential for FAP imaging.

Anti-cancer Effect of Unique Cartilage Matrix-associated Protein in Breast Cancer Cells Depends on γ-Carboxylation.

Unique cartilage matrix-associated protein (UCMA), a recently discovered vitamin K-dependent protein (VKDP) with a large number of γ-carboxyglutamic acid (Gla) residues, is associated with ectopic calcifications. Although the function of VKDPs is related to their γ-carboxylation status, the carboxylation status of UCMA in breast cancer is still unknown. Here, we investigated the inhibitory effect of UCMA with differing γ-carboxylation status on breast cancer cell lines, such as MDA-MB-231, 4T1, and E0771 cells. Undercarboxylated UCMA (ucUCMA) was generated by mutating the γ-glutamyl carboxylase (GGCX) recognition sites. The ucUCMA and carboxylated UCMA (cUCMA) proteins were collected from culture media of HEK293-FT cells that had been transfected with mutated GGCX and wild-type UCMA expression plasmids, respectively. Boyden Transwell and colony formation assays were performed to evaluate cancer cell migration, invasion, and proliferation. Culture medium containing cUCMA protein inhibited the migration, invasion, and colony formation of MDA-MB-231 and 4T1 cells to a greater degree than medium containing ucUCMA protein. Significant reductions in the migration, invasion, and colony formation were also observed in cUCMA-treated E0771 cells compared to those in ucUCMA-treated cells. The inhibitory role of UCMA in breast cancer is closely related to its γ-carboxylation status. The results of this study may be a basis for the development of UCMA-based anti-cancer drugs.

Vitamin K Status Influences Brain Sulfatide Metabolism in Young Mice and Rats

The established role of vitamin K in nutrition is as a cofactor in the post-translational conversion of specific glutamyl to γ-carboxyglutamyl (Gla) residues in a limited number of proteins. Administration of the vitamin K antagonist warfarin has previously been shown to decrease brain sulfatide concentrations and decrease brain galactocerebroside sulfotransferase (GST) activity in young mice. A dietary deficiency of vitamin K has now been shown to decrease (P < 0.01) brain sulfatide concentrations of 30-d-old mice significantly (by 21%). Male 21-d-old rats fed an excess of vitamin K for 7 or 14 d had 26 and 31% (P < 0.05) greater GST activity and 15 and 18% (P < 0.05) greater brain sulfatide concentrations, respectively, than controls fed a vitamin K-deficient diet. Male 21-d-old rats fed a diet containing 500 mg of phylloquinone/kg diet had an intermediate response and were vitamin K sufficient by normal criteria. The vitamin K response was observed when either phylloquinone or menaquinone-4 was fed as a source of the vitamin. These data suggest that in addition to its recognized role in Gla synthesis, vitamin K status is important in the maintenance of normal complex lipid sulfatide metabolism in young rats and mice.

Identification of non-neutralizing anti-factor X autoantibodies in three Japanese cases of autoimmune acquired factor X deficiency.

Autoimmune factor X (FX or F10) deficiency (AiF10D) is an extremely rare acquired haemorrhagic disorder characterized by a severe reduction in FX activity due to autoantibodies against FX. Anti-FX autoantibodies were investigated in four patients with suspected AiF10D, and their properties were analysed. Anti-FX auto antibodies in plasma were detected by ELISA with three of four cases. One case of anti-FX autoantibody negativity was later diagnosed as AL-amyloidosis. IgG1 and IgG3 coexisted in all anti-FX autoantibodies of the three patients with AiF10D (cases X1, X2, and X3). Western blot analysis showed that the antibodies were bound to the FX light chain for cases X2 and X3, but the binding was weak for case X1. When the fusion proteins of a secretory luciferase with full-length FX or its γ-carboxylated glutamic acid (Gla) domain were added to the plasma of the three patients, both fusion proteins were immunoprecipitated as antigen-antibody complexes. Contrarily, the latter fusion protein produced in the presence of warfarin demonstrated a decrease in the collection rate, suggesting that their autoantibodies recognized the light chain and regions containing Gla residues. Since all three patients were essentially negative for FX inhibitors, it was concluded that the anti-FX autoantibodies for these cases were predominantly non-neutralizing. The concentration of the FX antigen also significantly reduced in these patients, suggesting that anti-FX autoantibodies promote the clearance of FX. Immunological anti-FX autoantibody detection is highly recommended to ensure that AiF10D cases are not overlooked, and to start necessary immunosuppressive therapies.

HK is the apple of FXI's eye

HK is the apple of FXI's eye

Vitamin K-Dependent Protein Activation: Normal Gamma-Glutamyl Carboxylation and Disruption in Disease.

Vitamin K-dependent (VKD) proteins undergo an unusual post-translational modification, which is the conversion of specific Glu residues to carboxylated Glu (Gla). Gla generation is required for the activation of VKD proteins, and occurs in the endoplasmic reticulum during their secretion to either the cell surface or from the cell. The gamma-glutamyl carboxylase produces Gla using reduced vitamin K, which becomes oxygenated to vitamin K epoxide. Reduced vitamin K is then regenerated by a vitamin K oxidoreductase (VKORC1), and this interconversion of oxygenated and reduced vitamin K is referred to as the vitamin K cycle. Many of the VKD proteins support hemostasis, which is suppressed during therapy with warfarin that inhibits VKORC1 activity. VKD proteins also impact a broad range of physiologies beyond hemostasis, which includes regulation of calcification, apoptosis, complement, growth control, signal transduction and angiogenesis. The review covers the roles of VKD proteins, how they become activated, and how disruption of carboxylation can lead to disease. VKD proteins contain clusters of Gla residues that form a calcium-binding module important for activity, and carboxylase processivity allows the generation of multiple Glas. The review discusses how impaired carboxylase processivity results in the pseudoxanthoma elasticum-like disease.

Progressionof Prothrombin Induced by Vitamin K Absence-II in Hepatocellular Carcinoma.

Hepatocellular carcinoma (HCC) is the fifth most common cancer and the third leading cause of cancer-related death worldwide. Due to the lack of efficient tools for early detection, asymptomatic HCC patients are diagnosed at an advanced stage, leading to a poor prognosis. To improve survival, serum biomarker prothrombin induced by vitamin K absence-II (PIVKA-II) was under investigation. PIVKA-II is an abnormal protein produced in HCC. The coagulation function was insufficient due to the lack of Gla residues. Elevated PIVKA-II was associated with bad tumor behavior in terms of proliferation, metastasis, and invasion. Three major signaling pathways were proposed to clarify the mechanism. With the advantages including affordability, minimal invasiveness, convenience, and efficiency, PIVKA-II could improve HCC management consisting of four aspects. First, PIVKA-II was an effective and dynamic tool for improving HCC surveillance in high-risk population. Changes in the serum levels of PIVKA-II provided valuable molecular alteration information before imaging discovery. Second, PIVKA-II offered a complementary approach for HCC early detection. Compared to traditional diagnostic approaches, the combination of PIVKA-II and other biomarkers had better performance. Third, PIVKA-II was an indicator for the assessment of response to treatment in HCC. Preoperative assessment was for selecting personalized therapy, and postoperative measurement was for assessing treatment efficacy. Fourth, PIVKA-II was considered as a prognostic predictor for HCC. Patients with elevated PIVKA-II were more likely to develop microvascular invasion, metastasis, and recurrence.

Measuring serum prothrombin in patients treated with warfarin or direct oral anticoagulants

BackgroundWarfarin therapy influences generation of γ-carboxyglutamyl (Gla) residues in prothrombin, causing reduced coagulation activity. It will leave such inactive prothrombin in serum after clot formation, resulting in serum prothrombin constituting total inactive prothrombin in these patients. MethodsAn ELISA was developed to measure biologically inactive prothrombin in serum, and applied to serum from warfarin therapy causing a decrease in Gla residues or direct oral anticoagulant (DOAC) therapy as its contrast. ResultsThe concentrations of serum prothrombin in both the warfarin and DOAC groups were higher than those in the healthy group (p < 0.01 and p < 0.001, respectively). When serum in the previous three groups was treated with barium carbonate to exclude prothrombin, which lost several Gla residues, the prothrombin concentration in the DOAC group decreased to the same level as that in the healthy group, indicating that prothrombin was obtained at a high level only in the warfarin group (p < 0.01). ConclusionsWarfarin and DOAC led to increase in serum prothrombin concentration. The reason is that DOAC decreases prothrombin recruitment during fibrin clot formation, while warfarin leads to the accumulation of inactive prothrombin, which have a decreased number of Gla residues.

Evolutionary adaptation of the γ‐carboxyglutamic acid domain in blood coagulation factor X in the Serpentes suborder

The serine protease factor Xa plays an important role in blood coagulation as it, in complex with its cofactor Va, proteolytically activates prothrombin to thrombin, the latter being a key enzyme in coagulation. To perform this vital function, factor Xa undergoes numerous posttranslational modifications including vitamin K-dependent γ-carboxylation of glutamic acid (Glu) residues into so-called Gla residues. A correctly γ-carboxylated GLA domain is essential for interaction with the negatively charged membrane surface of activated platelets or endothelial cells at the site of injury where clot formation is required. Moreover, assembly of factor Xa with the cofactor Va into the prothrombinase enzyme complex occurs on a negatively charged membrane surface only. Interestingly, the venom of some Australian Elapid snakes comprises powerful prothrombin-activating enzyme complexes consisting of factor Xa- and Va-like proteins that are specifically expressed in the venom gland. We have previously demonstrated that this venom enzyme complex is capable of functioning independently of a negatively charged phospholipid membrane. We aim to uncover the putative differential role of the GLA domain between human and snake venom (Pseudonaja textilis, pt) factor Xa. To do so, we exchanged the human factor Xa (hFX) GLA domain for that of the snake venom ortholog (ptFX), thereby generating hFX-wt, hFX-ptGLA, ptFX-wt, ptFX-hGLA. The FX variants were recombinantly expressed and purified employing chromatography techniques. FX activation by either the human extrinsic or intrinsic coagulation tenase complex was analyzed using a peptidyl substrate specific for activated FX. Introduction of the ptGLA domain into hFX modestly improved the catalytic rate of the extrinsic reaction, while for the intrinsic reaction the substrate binding affinity was enhanced. Conversely, using similar conditions no FXa formation could be detected following incubation of ptFX or ptFX-hGLA with the human intrinsic or extrinsic tenase complex. Assessment of the FX-specific plasma clotting activity in which thrombin-dependent fibrin clot formation is monitored following either an extrinsic or intrinsic coagulation trigger revealed that while the extrinsic clotting activity of hFX was drastically reduced when hGLA was exchanged for ptGLA (3% residual activity), its intrinsic activity was unperturbed. Use of human versus rabbit reagents for the extrinsic pathway may explain the different observations. Conversely, exchange of ptGLA for hGLA resulted in an overall substantial increase in the FX-specific clotting activity of ptFX-hGLA. Finally, exchanging the ptGLA domain for the hGLA domain did not abolish the requirement for a negatively charged membrane surface in Va-dependent prothrombin activation. These data indicate that the snake venom GLA domain interacts with the intrinsic tenase complex and contributes to substrate conversion and subsequent thrombin formation, which may point to evolutionary adaptation of this snake venom GLA domain.

Influence of Warfarin Therapy on Prothrombin Production and Its Posttranslational Modifications.

Protein induced by vitamin K absence-II (PIVKA-II) is produced by the liver during hepatoma and upon warfarin administration. Those patients have disturbed protein synthesis and glycosylation in the liver. This decreases the number of γ-carboxyglutamyl (Gla) residues on prothrombin, converting prothrombin into PIVKA-II. The mechanism of this conversion, however, is not clearly understood. Prothrombin was isolated from healthy and warfarin-treated individuals whose liver function of protein production was quantitatively normal. Glycan structures in the purified prothrombin containing PIVKA-II were qualitatively analyzed by high performance liquid chromatography after labeling the glycan with fluorophore 2-aminobenzamide. The concentration of PIVKA-II was significantly higher in the warfarin-treated individuals than in the healthy individuals (P< 0.001). Although protein production in the liver was normal in both groups, the concentration of prothrombin was lower in the warfarin-treated individuals than in the healthy individuals (P < 0.001). The main glycan was A2 in the healthy and warfarin-treated individuals (86.6 ± 4.4% and 85.6 ± 3.4%, respectively). Eight types of glycan were characterized in both groups, although generation of PIVKA-II in the warfarin-treated individuals did not lead to variation in glycosylation of prothrombin. Warfarin therapy leads to lower amounts of prothrombin and Gla residues within prothrombin without exerting qualitative and quantitative change in glycan profile and protein synthetic function in the liver.

Warfarin use and risk of knee replacement

Purpose: Vitamin K is an essential co-factor in the post-translational gamma-carboxylation of glutamic acid to form gamma-carboxy-glutamic acid (Gla) residues. This confers functionality to vitamin K-dependent Gla proteins including those in bone and cartilage such as matrix Gla protein (MGP) which plays an important role in regulating mineralization. Inadequate vitamin K can lead to under-carboxylation of Gla proteins and therefore dysfunction of those proteins. Vitamin K deficiency and MGP polymorphisms have been associated with osteoarthritis (OA), and a randomized trial of vitamin K supplementation demonstrated trends towards less OA progression.

Use of an innovative system and nanotechnology-based strategy for therapeutic applications of Gla-rich protein (GRP)

Introduction: Gla-rich protein (GRP) is a vitamin K-dependent protein (VKDP) acting as a calcification inhibitor and anti-inflammatory agent in cardiovascular and articular systems, and THP1 monocyte/macrophage cells [1,2]. Calcification and inflammation processes are known to be involved in the etiology of several calcification-related chronic inflammatory diseases such as atherosclerosis, CKD and osteoarthritis, in a complex bi-directional interplay that drives disease progression. Here, we developed an innovative system to produce human γ-carboxylated GRP (cGRP), and a nanotechnology strategy based on GRP loading into extracellular vesicles (EVs) as a gold standard delivery system for GRP in therapeutic applications. Materials and methods: Human GRP protein was co-expressed with γ-carboxylase enzyme (GGCX), vitamin K oxidoreductase (GGCX) and furin, in the insect cell baculovirus system in the presence of vitamin K. GRP released in the cell culture media was characterized by mass spectrometry based techniques and Western blot analysis. EVs released by the insect cells overexpressing GRP were isolated by ultracentrifugation, and characterized for GRP content through TEM-immunogold staining, Western blot, ELISA, qPCR. Functional assays using isolated EVs containing GRP were performed in primary vascular smooth muscle cells (VSMCs) and THP1 monocyte/macrophage cells, for anti-mineralizing and anti-inflammatory screening. Results: GRP released in the cell culture media when co-expressed with GGCX, VKOR and furin in the presence of vitamin K, is processed at the pro-peptide and contain Gla residues. EVs released by the insect cells in this system were shown to be loaded with GRP protein and mRNA, and capable of reducing ECM calcium deposition of calcifying VSMCs and the production of TNFα in THP1 monocyte/macrophage cells stimulated with LPS. Discussion and conclusions: While the successful production of human cGRP constitutes a major achievement, this innovative methodology will open new opportunities for the production of other biological active VKDPs. Furthermore, EVs loaded with GRP were shown to have anti-mineralizing and anti-inflammatory properties, with promising therapeutic potentialities for calcification-related chronic inflammatory diseases.

Discerning the Role of the Hydroxyproline Residue in the Structure of Conantokin Rl-B and Its Role in GluN2B Subunit-Selective Antagonistic Activity toward N-Methyl-d-Aspartate Receptors.

Conantokins (con) are short γ-carboxyglutamate (Gla)-containing polypeptides expressed by marine snails that function as antagonists of N-methyl-d-aspartate receptor (NMDAR) ion channels. The Gla residues govern structural conformations and antagonistic activities of the conantokins. In addition to Gla, some conantokins, e.g., conRl-B, also contain a hydroxyproline (HyP or O) residue, which in this case is centrally located in the peptide at position 10. Because conRl-B specifically inhibits ion channels of GluN2B subunit-containing heterotetrameric NMDARs, we evaluated the unusual role of HyP10 in this effect. To accomplish this goal, we examined synthetic variants of conRl-B in which HyP10 was either deleted (conRl-B[ΔO10]) or replaced with alanine (conRl-B[O10A]) or proline (conRl-B[O10P]). The solution structures of these variants were determined by nuclear magnetic resonance spectroscopy. Deletion of HyP10, or replacement of HyP10 with Ala10, attenuated the distortion in the central region of the apo-conRl-B helix and allowed Mg2+-complexed end-to-end α-helix formation. The inhibitory properties of these variants were assessed by measuring NMDA/Gly-stimulated intracellular Ca2+ influx in mice neurons. ConRl-B[O10P] retained its NMDAR ion channel inhibitory activity in wild-type (WT) neurons but lost its GluN2B specificity, whereas conRl-B[ΔO10] showed overall diminished inhibitory function. ConRl-B[O10A] showed attenuated inhibitory function but retained its GluN2B specificity. Thus, HyP10 plays a critical role in maintaining the structural integrity of conRl-B, which can be correlated with its GluN2B subunit-selective inhibition. Weakened inhibition by conRl-B was also observed in neurons lacking either the GluN2C or GluN2D subunit, compared to WT neurons. This suggests that GluN2C and GluN2D are also required for inhibition by conRl-B.

Novel Sites of Gamma Carboxylation in Human Factor VII

IntroductionGamma- (γ-) carboxylation of glutamate residues is a vitamin K-dependent post-translational modification critical to the function of several plasma proteins. Found in the N-terminal domains of specific proteins, most of which are involved in hemostasis, these γ-carboxyglutamate residues (Gla) help mediate binding of divalent cations and are essential to protein function. Coagulation factor VII(a) bears 10 known Gla residues as characterized by N-terminal sequencing, yet Thim et al.( Biochemistry 27:7785 1988) used amino acid analysis to quantify 11.0 mol of Gla/mol of protein for plasma-derived factor VII(a). We used mass spectrometry to map and validate Gla residues of coagulation factor VII(a) to potentially identify Gla residues outside of the Gla domain.MethodsFour sources of factor VII(a) (2 plasma-derived and 2 recombinant) were extracted, digested, and analyzed by tandem mass spectrometry (LC-MS/MS). Each sample was digested with trypsin and chymotrypsin to provide orthogonal coverage. Peptides derived from factor VII(a) proteolysis were analyzed on a nanoLC coupled to a quadrupole-Orbitrap-quadrupole linear ion trap mass spectrometer (Orbitrap Fusion Lumos Thermo Scientific). Multiple fragmentation methods were used to map and validate the sites including collisional based dissociation (CAD), higher energy collisional activated dissociation (HCD), electron transfer dissociation (ETD), and electron-transfer/higher-energy electron transfer activation (EThcD). Tandem MS spectra were collected at resolution 30K at 200 m/z, and data were processed using MaxQuant, COMPASS, and Proteome Discoverer. All identified sites were validated through manual annotation of spectra. We estimate that any site with >1% occupancy will be identified as Gla with this method.To validate select sites of novel gamma carboxylation, synthetic peptides were made for 4 different sites that were consistently identified in factor VII(a) from all 4 sources. Synthetic peptides were analyzed using MS methods described above, generating "true positives" to match with peptides identified from the factor VII(a) sources. Spectra from the synthetic peptides and factor VII from each source were compared using manual spectral annotation.ResultsIn addition to identifying known Gla residues at positions 6, 7, 19, 20, 29, and 35 of factor VII(a), we detected and validated 9 novel Gla residues outside of the N-terminal Gla domain. Novel sites include residues 94, 116, 132, 219, 215, 229, 265, 196 and 385. Four of these residues (210, 220, 296 and 385) were identified as Gla in all 4 sources of factor VII(a) and were validated with synthetic peptides using a combination of fragmentation methods, providing high confidence in their characterization. Published crystallographic data suggest that residues 210 and 220 of factor VIIa-tissue factor are closely approximated to a Ca2+ ion complexed to the C-terminal protease domain; this is not the case for residues 296 and 385. We continue to refine the technique to map the Gla residues (novel and known), and to quantify the fraction of factor VII(a) molecules from each source that contain the modification at each site, in order to better incorporate our data with established studies showing >90% occupancy at each of the 10 Gla domain sites. These data suggest there is room to expand our understanding of how carboxylation contributes to specific protein function, in order to provide more comprehensive understanding of this post-translational modification, and refine our understanding of hemostatic mechanisms. DisclosuresNo relevant conflicts of interest to declare.

Recombinant FIX/X-Bp from the Japanese Habu Snake Is a Universal Ligand for Purification of Highly Carboxylated Factor IX Variants and Factor X

Background: The purification of vitamin K-dependent clotting factors typically involves multiple chromatographic steps, including an ion exchange-based pseudo-affinity step to enrich for species with sufficiently high gamma-carboxyglutamic acid (Gla) content to achieve maximal specific activity. Variants of these factors have been engineered to improve their pharmacokinetic properties by appending or inserting a variety of elements, including the Fc domain of IgG, unstructured hydrophilic peptides of defined amino acid composition (XTEN), albumin, and polyethylene glycol (PEG). In most cases, however, such modification alters both the hydrodynamic and electrostatic properties of the resulting molecule relative to those of the predicate molecule, thereby complicating their purification, particularly with regard to Gla enrichment by pseudo-affinity chromatography. Factor IX (FIX)- and factor X (FX)-binding protein (FIX/X-bp) isolated from the venom of the Japanese Habu snake (T. flavoviridis) has been shown to bind with high affinity and specificity to both FIX and FX, and structural studies have demonstrated that FIX/X-bp binds to the highly carboxylated calcium-bound forms of the Gla domains of these proteins. We therefore reasoned that FIX/X-bp could serve as a novel affinity ligand for rapid and simple purification of variants of FIX and FX with high specific activity.Aims: To generate and purify recombinant FIX/X-bp (rFIX/X-bp) and assess its utility for the purification of FIX, FIX-XTEN, FIX-albumin, and FX with high Gla content.Methods: A two-chained rFIX/X-bp molecule in which a polyhistidine tag was appended to one chain was generated by stable co-transfection of Chinese hamster ovary (CHO) cells. Culture medium was concentrated by tangential-flow filtration (TFF), and rFIX/X-bp was purified by one of two methods: 1) immobilized metal ion affinity chromatography (IMAC), followed by anion-exchange chromatography, or 2) affinity chromatography on immobilized FIX in calcium-containing buffer and subsequent elution in EDTA-containing buffer. The potent anticoagulant activity of rFIX/X-bp was verified by prothrombin time (PT) and activated partial thromboplastin time (APTT) assays, and its ability to bind to human FIX, FX, factor VII (FVII), protein S, and prothrombin was evaluated by biolayer interferometry. The affinity of rFIX/X-bp for FIX and FX was determined by surface plasmon resonance (SPR). An affinity column was then generated by chemical conjugation of rFIX/X-bp to NHS-activated Sepharose. Recombinant FIX, FIX-albumin, and FIX-XTEN were first affinity purified on IXSelect resin from the culture medium of transiently transfected HEK293 cells, and the resulting protein preparations, which were heterogeneous with regard to Gla content, were then applied to the rFIX/X-bp affinity column in calcium- or magnesium-containing buffer and eluted with EDTA-containing buffer. Activity was assessed by APTT assay, and Gla content was determined by mass spectrometric peptide mapping. Recombinant FX was purified from the culture medium of transiently transfected HEK293 cells by sequential barium citrate adsorption, anion exchange chromatography, and affinity chromatography on a rFIX/X-bp column.Results: In the presence of calcium or magnesium ions, rFIX/X-bp binds to FIX and FX with high affinity (KD≈ 10 pM), to a lesser extent to protein S and prothrombin, but not to FVII. FIX and FIX-albumin that had been affinity purified on a rFIX/X-bp column had specific activities that were consistent with published data and greater than 11 Gla residues per molecule. The Gla content of FX that had been affinity purified on a rFIX/X-bp column was 10 Gla residues per molecule (out of 11 possible).Conclusions: rFIX/X-bp is a universal ligand for the purification of highly carboxylated FX and FIX variants, including FIX-albumin and FIX-XTEN. DisclosuresMercury:Biogen: Employment. Liu:Biogen: Employment. Ismail:Biogen: Employment. Zhang:Biogen: Employment. Lu:Biogen: Employment. Cameron:Biogen: Employment. Goodman:Biogen: Employment. Culyba:Biogen: Employment. Ravindran Nair:Biogen: Employment. Holthaus:Biogen: Employment. Kulman:Biogen: Employment. Peters:Biogen: Employment.

Epitope characterization of an anti-PIVKA-II antibody and evaluation of a fully automated chemiluminescent immunoassay for PIVKA-II

ObjectivesProtein induced by vitamin K absence or antagonist-II (PIVKA-II) has been used as a tumor marker to aid in the diagnosis of hepatocellular carcinoma (HCC). We developed an anti-PIVKA-II monoclonal antibody, 3C10, and a fully automated quantitative immunoassay for PIVKA-II on the ARCHITECT® i-systems. The aim of this study was to characterize the epitope of 3C10 and to evaluate the reactivity to PIVKA-II of this assay. MethodsThe epitope characterization was examined by using prothrombin γ-carboxyglutamic acid residues (Gla) domain polypeptides which are amino acid residues 17–27 that include four Gla residues at positions 19, 20, 25 and 26. The correlation with Picolumi PIVKA-II MONO (Eidia, Tokyo, Japan) and tube type equivalency was evaluated by using the developed fully automated quantitative immunoassay. ResultsPeptides having glutamic acid residues (Glu) at Gla domains strongly reacted to 3C10 but lost reactivity when the Glu at positions 19 or 20 was changed to Gla. The results were equivalent with an existing in vitro diagnostics product for PIVKA-II using the MU-3 antibody. A correlation study with the Picolumi PIVKA-II MONO gave a correlation coefficient of 0.99 and a regression slope of 0.92. No difference between a plain serum tube and a rapid serum tube including thrombin (RST) was observed on ARCHITECT PIVKA-II. ConclusionsThe results demonstrate that this anti-PIVKA-II antibody detects equivalent epitopes with MU-3 and has equivalent reactivity to PIVKA-II as MU-3. Moreover, the ARCHITECT PIVKA-II assay has good correlation with the existing PIVKA-II product, and is applicable for use with RST.

Hydroxyproline-induced Helical Disruption in Conantokin Rl-B Affects Subunit-selective Antagonistic Activities toward Ion Channels of N-Methyl-d-aspartate Receptors

Conantokins are ~20-amino acid peptides present in predatory marine snail venoms that function as allosteric antagonists of ion channels of the N-methyl-d-aspartate receptor (NMDAR). These peptides possess a high percentage of post-/co-translationally modified amino acids, particularly γ-carboxyglutamate (Gla). Appropriately spaced Gla residues allow binding of functional divalent cations, which induces end-to-end α-helices in many conantokins. A smaller number of these peptides additionally contain 4-hydroxyproline (Hyp). Hyp should prevent adoption of the metal ion-induced full α-helix, with unknown functional consequences. To address this disparity, as well as the role of Hyp in conantokins, we have solved the high resolution three-dimensional solution structure of a Gla/Hyp-containing 18-residue conantokin, conRl-B, by high field NMR spectroscopy. We show that Hyp(10) disrupts only a small region of the α-helix of the Mn(2+)·peptide complex, which displays cation-induced α-helices on each terminus of the peptide. The function of conRl-B was examined by measuring its inhibition of NMDA/Gly-mediated current through NMDAR ion channels in mouse cortical neurons. The conRl-B displays high inhibitory selectivity for subclasses of NMDARs that contain the functionally important GluN2B subunit. Replacement of Hyp(10) with N(8)Q results in a Mg(2+)-complexed end-to-end α-helix, accompanied by attenuation of NMDAR inhibitory activity. However, replacement of Hyp(10) with Pro(10) allowed the resulting peptide to retain its inhibitory property but diminished its GluN2B specificity. Thus, these modified amino acids, in specific peptide backbones, play critical roles in their subunit-selective inhibition of NMDAR ion channels, a finding that can be employed to design NMDAR antagonists that function at ion channels of distinct NMDAR subclasses.

Gamma-Carboxylation and Fragmentation of Osteocalcin in Human Serum Defined by Mass Spectrometry*

Serum osteocalcin (Oc) concentration is a highly specific measure of bone turnover, but its circulating proteoform(s) have not been well defined. Based on immunological methods, the major forms are thought to be the intact polypeptide and a large N-terminal-mid molecule fragment for which there is no consensus on the precise sequence. Vitamin K-dependent gamma (γ)-carboxylated variants of Oc are also found in circulation but there have been no methods that can define how many of the three potential γ-carboxyglutamic acid (Gla) residues are γ-carboxylated or provide their relative abundances. Recent reports that uncarboxylated and partially γ-carboxylated Oc forms have hormonal function underscore the need for precise evaluation of Oc at all three potential γ-carboxylation sites. Herein, mass spectrometric immunoassay (MSIA) was used to provide qualitative and semiquantitative (relative percent abundance) information on Oc molecular variants as they exist in individual plasma and serum samples. Following verification that observable Oc proteoforms were accurately assigned and not simply ex vivo artifacts, MALDI-MSIA and ESI-MSIA were used to assess the relative abundance of Oc truncation and γ-carboxylation, respectively, in plasma from 130 patients enrolled in vitamin K supplementation trials. Human Oc was found to circulate in over a dozen truncated forms with each of these displaying anywhere from 0-3 Gla residues. The relative abundance of truncated forms was consistent and unaffected by vitamin K supplementation. In contrast, when compared with placebo, vitamin K supplementation dramatically increased the fractional abundance of Oc with three Gla residues, corresponding to a decrease in the fractional abundance of Oc with zero Gla residues. These findings unequivocally document that increased vitamin K intake reduces the uncarboxylated form of Oc. Several reports of a positive effect of vitamin K intake on insulin sensitivity in humans have shown that un- or undercarboxylation of Oc, unlike in mice, is not associated with insulin resistance. Analyses similar to those described here will be useful to understand the functional significance of Oc γ-carboxylation in human health and disease.

Quantifying vitamin K-dependent holoprotein compaction caused by differential γ-carboxylation using high-pressure size exclusion chromatography

This study uses high-pressure size exclusion chromatography (HPSEC) to quantify divalent metal ion (X2+)-induced compaction found in vitamin K-dependent (VKD) proteins. Multiple X2+ binding sites formed by the presence of up to 12 γ-carboxyglutamic acid (Gla) residues are present in plasma-derived FIX (pd-FIX) and recombinant FIX (r-FIX). Analytical ultracentrifugation (AUC) was used to calibrate the Stokes radius (R) measured by HPSEC. A compaction of pd-FIX caused by the filling of Ca2+ and Mg2+ binding sites resulted in a 5 to 6% decrease in radius of hydration as observed by HPSEC. The filling of Ca2+ sites resulted in greater compaction than for Mg2+ alone where this effect was additive or greater when both ions were present at physiological levels. Less X2+-induced compaction was observed in r-FIX with lower Gla content populations, which enabled the separation of biologically active r-FIX species from inactive ones by HPSEC. HPSEC was sensitive to R changes of approximately 0.01nm that enabled the detection of FIX compaction that was likely cooperative in nature between lower avidity X2+ sites of the Gla domain and higher avidity X2+ sites of the epidermal growth factor 1 (EGF1)-like domain.

Phylloquinone supplementation improves glycemic status independent of the effects of adiponectin levels in premonopause women with prediabetes: a double-blind randomized controlled clinical trial.

BackgroundVitamin K, as a cofactor in the gamma carboxylation of certain glutamic acid (Gla) residues, has been related to glucose metabolism and insulin sensitivity. Osteocalcin, also known as bone γ-carboxyglutamic acid, increases β-cell proliferation as well as insulin and adiponectin secretion, which improve glucose tolerance and insulin sensitivity. Thus, the purpose of the present study was to examine the possible role of adiponectin as a mediator of glucose homeostasis following phylloquinone supplementation in premonopause women with prediabetes.MethodsEighty two women were randomized to consume vitamin k1 supplement (n = 39) or placebo (n = 43) for four weeks. Participants in vitamin K1 treatment group received one pearl softgel capsule containing 1000 micrograms phylloquinone while the placebo group received one placebo capsules daily for four weeks. The Blood samples were collected at baseline and after a four-week intervention to quantify osteocalcin, adiponectin, leptin and relevant variables.ResultsPhylloquinone supplementation significantly increased serum adiponectin concentration (1.24 ± 1.90 compared with −0.27 ± 1.08 μg/ml), and did not alter total osteocalcin (0.50 ± 4.11 compared with 0.13 ± 1.85 ng/ml) and leptin (−0.29 ± 8.23 compared with −1.15 ± 5.25 ng/ml) compared with placebo. Adjustments for total osteocalcin and adiponectin using analysis of covariance (ANCOVA) did not affect the association of glycemic status with related variables.ConclusionsIn conclusion our study demonstrated that phylloquinone supplementation improved glycemic status in premonopausal prediabetic women independent of adiponectin.Trial registrationThis trial was registered in Iranian Registry of Clinical Trials with ID number of IRCT2013120915724N1.