Hyperglycemic microenvironment compromises the homeostasis of communication between the bone-brain axis by the epigenetic repression of the osteocalcin receptor, Gpr158 in the hippocampus.

Diabetes mellitus (DM) is a chronic metabolic disease, mainly characterized by increased blood glucose and insulin dysfunction. In response to the persistent systemic hyperglycemic state, numerous metabolic and physiological complications have already been well characterized. However, its relationship to bone fragility, cognitive deficits and increased risk of dementia still needs to be better understood. The impact of chronic hyperglycemia on bone physiology and architecture was assessed in a model of chronic hyperglycemia induced by a single intraperitoneal administration of streptozotocin (STZ; 55 mg/kg) in Wistar rats. In addition, the bone-to-brain communication was investigated by analyzing the gene expression and methylation status of genes that encode the main osteokines released by the bone [Fgf23 (fibroblast growth factor 23), Bglap (bone gamma-carboxyglutamate protein) and Lcn2 (lipocalin 2) and their receptors in both, the bone and the brain [Fgfr1 (fibroblast growth factor receptor 1), Gpr6A (G-protein coupled receptor family C group 6 member A), Gpr158 (G protein-coupled receptor 158) and Slc22a17 (Solute carrier family 22 member 17)]. It was observed that chronic hyperglycemia negatively impacted on bone biology and compromised the balance of the bone-brain endocrine axis. Ultrastructural disorganization was accompanied by global DNA hypomethylation and changes in gene expression of DNA-modifying enzymes that were accompanied by changes in the methylation status of the osteokine promoter region Bglap and Lcn2 (lipocalin 2) in the femur. Additionally, the chronic hyperglycemic state was accompanied by modulation of gene expression of the osteokines Fgf23 (fibroblast growth factor 23), Bglap (bone gamma-carboxyglutamate protein) and Lcn2 (lipocalin 2) in the different brain regions. However, transcriptional regulation mediated by DNA methylation was observed only for the osteokine receptors, Fgfr1(fibroblast growth factor receptor 1) in the striatum and Gpr158 (G protein-coupled receptor 158) in the hippocampus. This is a pioneer study demonstrating that the chronic hyperglycemic state compromises the crosstalk between bone tissue and the brain, mainly affecting the hippocampus, through transcriptional silencing of the Bglap receptor by hypermethylation of Gpr158 gene.

Long non-coding RNA Linc01133 promotes osteogenic differentiation of human periodontal ligament stem cells via microRNA-30c / bone gamma-carboxyglutamate protein axis.

Periodontitis is a chronic inflammation caused by the deposition of dental plaque on the tooth surface. Human periodontal ligament stem cells (hPDLSCs) have the potential of osteogenic differentiation. Long non-coding RNAs (lncRNAs) are collectively involved in periodontitis. This study was designed to explore the roles of Linc01133 in osteogenic differentiation of hPDLSCs. hPDLSCs obtained from the periodontal ligament (PDL) of patients with periodontitis were used to collect Linc01133, microRNA-30c (miR-30c), and bone gamma-carboxyglutamate protein (BGLAP) expression data, and their expression changes were traced during osteogenic differentiation of hPDLSCs. Quantitative reverse-transcription polymerase chain reaction as well as western blotting were used to analyze the levels of RNAs and proteins. Dual-luciferase reporter and RNA pull-down assays demonstrated the relationship between Linc01133, miR-30c, and BGLAP. Furthermore, alkaline phosphatase (ALP) staining and alizarin red staining were applied to evaluate the degree of osteogenic differentiation. Linc01133 was downregulated in the PDL of patients with periodontitis. Upregulated Linc01133 promoted osteogenic differentiation of hPDLSCs. Linc01133 could inhibit miR-30c expression by sponging miR-30c. miR-30c suppressed osteogenic differentiation. Additionally, miR-30c targeted BGLAP. Knockdown of BGLAP abrogated the effects of decreased miR-30c on osteogenic differentiation of hPDLSCs. Linc01133 acted as a ceRNA to regulate osteogenic differentiation of hPDLSCs via the miR-30c/BGLAP axis. Therefore, Linc01133 may participate in the progress of periodontitis.

Decreased serum undercarboxylated osteocalcin is associated with cognitive impairment in male patients with type 2 diabetes.

BACKGROUND: Basic and clinical researches have suggested that type 2 diabetes (T2DM) is associated with cognitive impairment, and diabetes mellitus increases the risk of cognitive impairment and dementia. Recently, some reports found that undercarboxylated osteocalcin (ucOC) could affect brain functions, and decreased in patients with T2DM. We aimed to investigate the association of serum ucOC with cognitive impairment in T2DM patients. METHODS: A total of 196 male T2DM patients without medications known to affect bone metabolism or history of bone fracture, aged ≥18years were recruited and divided into impaired cognition group and normal cognition group. We use the scores of Minimum Mental State Examination (MMSE) to evaluate the subjects' cognitive function. Detailed cognitive performance was also evaluated by the Repeatable Battery for the Assessment of Neuropsychological Status (RBANS). Serum ucOC was measured by Enzyme-Linked Immunosorbent Assay (ELISA) kit. RESULTS: Compared to male T2DM patients with normal cognition, the mean osteocalcin concentrations were significantly lower in male T2DM patients with impaired cognition (P<0.05). RBANS total and all indexes scores were also lower in patients with impaired cognition (all P<0.05). After adjusted effects of confounding factors, serum ucOC was positively correlated with a variety indexes of RBANS except visuospatial/constructional. CONCLUSIONS: The serum ucOC is positively correlated with RBANS scores in male T2DM patients. It suggests that serum ucOC may be involved in the development and progression of cognitive dysfunction in T2DM patients.

Lipid specificity of the membrane binding domain of coagulation factor X.

Essentials Membrane-binding GLA domains of coagulation factors are essential for proper clot formation. Factor X (FX) is specific to phosphatidylserine (PS) lipids through unknown atomic-level interactions. Molecular dynamics simulations were used to develop the first membrane-bound model of FX-GLA. PS binding modes of FX-GLA were described, and potential PS-specific binding sites identified. SUMMARY: Background Factor X (FX) binds to cell membranes in a highly phospholipid-dependent manner and, in complex with tissue factor and factor VIIa (FVIIa), initiates the clotting cascade. Experimental information concerning the membrane-bound structure of FX with atomic resolution has remained elusive because of the fluid nature of cellular membranes. FX is known to bind preferentially to phosphatidylserine (PS). Objectives To develop the first membrane-bound model of the FX-GLA domain to PS at atomic level, and to identify PS-specific binding sites of the FX-GLA domain. Methods Molecular dynamics (MD) simulations were performed to develop an atomic-level model for the FX-GLA domain bound to PS bilayers. We utilized a membrane representation with enhanced lipid mobility, termed the highly mobile membrane mimetic (HMMM), permitting spontaneous membrane binding and insertion by FX-GLA in multiple 100-ns simulations. In 14 independent simulations, FX-GLA bound spontaneously to the membrane. The resulting membrane-bound models were converted from HMMM to conventional membrane and simulated for an additional 100 ns. Results The final membrane-bound FX-GLA model allowed for detailed characterization of the orientation, insertion depth and lipid interactions of the domain, providing insight into the molecular basis of its PS specificity. All binding simulations converged to the same configuration despite differing initial orientations. Conclusions Analysis of interactions between residues in FX-GLA and lipid-charged groups allowed for potential PS-specific binding sites to be identified. This new structural and dynamic information provides an additional step towards a full understanding of the role of atomic-level lipid-protein interactions in regulating the critical and complex clotting cascade.

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.

Oral anticoagulant drugs and the risk of osteoporosis: new anticoagulants better than old?

Several drugs have been associated with an increased risk of osteoporosis when used chronically. Coumarins (warfarin, acenocoumarol, phenprocoumon, and fluindione) are oral anticoagulants widely used for the prevention and treatment of arterial and venous thromboembolic diseases. These drugs are vitamin K antagonists that interfere with γ-carboxyglutamate formation, and consequently inhibit the carboxylation of glutamate residues of proteins that are synthesized in the bone. These effects on bone turnover and dietary restrictions in patients on anticoagulation are possible mechanisms inducing osteoporosis in coumarin users. However, conflicting evidence is available concerning the risk of osteoporosis and bone fractures in patients on treatment with these drugs. This risk is likely to be clinically relevant in long-term (more than 1 year) coumarin users. Novel direct oral anticoagulants, recently introduced in clinical practice, exert reduced interference on bone metabolism; however, limited in vitro and animal data are currently available, and their long-term effects will only become apparent in time.

The endothelial protein C receptor enhances hemostasis of FVIIa administration in hemophilic mice in vivo.

Recombinant activated human factor VII (rhFVIIa) is an established hemostatic agent in hemophilia, but its mechanism of action remains unclear. Although tissue factor (TF) is its natural receptor, rhFVIIa also interacts with the endothelial protein C receptor (EPCR) through its γ-carboxyglutamic acid (Gla) domain, with unknown hemostatic consequences in vivo. Here, we study whether EPCR facilitates rhFVIIa hemostasis in hemophilia using a mouse model system. Mouse activated FVII (mFVIIa) is functionally homologous to rhFVIIa, but binds poorly to mouse EPCR (mEPCR). We modified mFVIIa to gain mEPCR binding using 3 amino acid changes in its Gla domain (L4F/L8M/W9R). The resulting molecule mFVIIa-FMR specifically bound mEPCR in vitro and in vivo and was identical to mFVIIa with respect to TF affinity and procoagulant functions. In macrovascular injury models, hemophilic mice administered mFVIIa-FMR exhibited superior hemostatic activity compared with mFVIIa. This was abolished by blocking mEPCR and was absent in ex vivo whole blood coagulation assays, implicating a specific mFVIIa-FMR and endothelial mEPCR interaction. Because mFVIIa-FMR models the TF-dependent and EPCR binding properties of rhFVIIa, our data unmask a novel contribution of EPCR on the action of rhFVIIa administration in hemophilia, prompting the rational design of improved and safer rhFVIIa therapeutics.

Carboxylation-dependent conformational changes of human osteocalcin.

Osteocalcin (OCN) is a small noncollagenous protein mainly produced by osteoblasts and is highly represented in bones of most vertebrates. Human OCN contains up to three gamma-carboxyglutamic acid (Gla-OCN) residues at positions 17, 21 and 24 which are thought to increase calcium binding strength, improving mechanical properties of the bone matrix. Recent studies revealed that OCN exerts also important endocrine functions, affecting energy metabolism and male fertility. The latter effect seems to be mediated by the uncarboxylated form of OCN (Glu-OCN). We employed human and mouse OCN as models of fully carboxylated and uncarboxylated OCN forms to investigate, by the use of circular dichroism and molecular dynamics simulations, the respective conformational properties and Ca2+ affinity. Ca2+ binding was found to trigger a similar conformational transition in both Glu-OCN and Gla-OCN, from a disordered structure to a more compact/stable form. Notably, gamma-carboxylation increases the affinity of OCN for Ca2+ by > 30 fold suggesting that, in physiological conditions, Gla-OCN is essentially Ca2+-bound, whereas Glu-OCN circulates mainly in the Ca2+-free form.

Role of gamma carboxylated Glu47 in connexin 26 hemichannel regulation by extracellular Ca²âº: insight from a local quantum chemistry study.

Connexin hemichannels are regulated by several gating mechanisms, some of which depend critically on the extracellular Ca(2+) concentration ([Ca(2+)]e). It is well established that hemichannel activity is inhibited at normal (∼1 mM) [Ca(2+)]e, whereas lowering [Ca(2+)]e to micromolar levels fosters hemichannel opening. Atomic force microscopy imaging shows significant and reversible changes of pore diameter at the extracellular mouth of Cx26 hemichannels exposed to different [Ca(2+)]e, however, the underlying molecular mechanisms are not fully elucidated. Analysis of the crystal structure of connexin 26 (Cx26) gap junction channels, corroborated by molecular dynamics (MD) simulations, suggests that several negatively charged amino acids create a favorable environment for low-affinity Ca(2+) binding within the extracellular vestibule of the Cx26 hemichannel. In particular a highly conserved glutammic acid, found in position 47 in most connexins, is thought to undergo post translational gamma carboxylation (γGlu47), and is thus likely to play an important role in Ca(2+) coordination. γGlu47 may also form salt bridges with two conserved arginines (Arg75 and Arg184 in Cx26), which are considered important in stabilizing the structure of the extracellular region. Using a combination of quantum chemistry methods, we analyzed the interaction between γGlu47, Arg75 and Arg184 in a Cx26 hemichannel model both in the absence and in the presence of Ca(2+). We show that Ca(2+) imparts significant local structural changes and speculate that these modifications may alter the structure of the extracellular loops in Cx26, and may thus account for the mechanism of hemichannel closure in the presence of mM [Ca(2+)]e.

Factor VII and protein C are phosphatidic acid-binding proteins.

Seven proteins in the human blood clotting cascade bind, via their GLA (γ-carboxyglutamate-rich) domains, to membranes containing exposed phosphatidylserine (PS), although with membrane binding affinities that vary by 3 orders of magnitude. Here we employed nanodiscs of defined phospholipid composition to quantify the phospholipid binding specificities of these seven clotting proteins. All bound preferentially to nanobilayers in which PS headgroups contained l-serine versus d-serine. Surprisingly, however, nanobilayers containing phosphatidic acid (PA) bound substantially more of two of these proteins, factor VIIa and activated protein C, than did equivalent bilayers containing PS. Consistent with this finding, liposomes containing PA supported higher proteolytic activity by factor VIIa and activated protein C toward their natural substrates (factors X and Va, respectively) than did PS-containing liposomes. Moreover, treating activated human platelets with phospholipase D enhanced the rates of factor X activation by factor VIIa in the presence of soluble tissue factor. We hypothesize that factor VII and protein C bind preferentially to the monoester phosphate of PA because of its accessibility and higher negative charge compared with the diester phosphates of most other phospholipids. We further found that phosphatidylinositol 4-phosphate, which contains a monoester phosphate attached to its myo-inositol headgroup, also supported enhanced enzymatic activity of factor VIIa and activated protein C. We conclude that factor VII and protein C bind preferentially to monoester phosphates, which may have implications for the function of these proteases in vivo.

Staphylococcal superantigen-like protein 10 (SSL10) inhibits blood coagulation by binding to prothrombin and factor Xa via their γ-carboxyglutamic acid (Gla) domain.

The staphylococcal superantigen-like protein (SSL) family is composed of 14 exoproteins sharing structural similarity with superantigens but no superantigenic activity. Target proteins of four SSLs have been identified to be involved in host immune responses. However, the counterparts of other SSLs have been functionally uncharacterized. In this study, we have identified porcine plasma prothrombin as SSL10-binding protein by affinity purification using SSL10-conjugated Sepharose. The resin recovered the prodomain of prothrombin (fragment 1 + 2) as well as factor Xa in pull-down analysis. The equilibrium dissociation constant between SSL10 and prothrombin was 1.36 × 10(-7) M in surface plasmon resonance analysis. On the other hand, the resin failed to recover γ-carboxyglutamic acid (Gla) domain-less coagulation factors and prothrombin from warfarin-treated mice, suggesting that the Gla domain of the coagulation factors is essential for the interaction. SSL10 prolonged plasma clotting induced by the addition of Ca(2+) and factor Xa. SSL10 did not affect the protease activity of thrombin but inhibited the generation of thrombin activity in recalcified plasma. S. aureus produces coagulase that non-enzymatically activates prothrombin. SSL10 attenuated clotting induced by coagulase, but the inhibitory effect was weaker than that on physiological clotting, and SSL10 did not inhibit protease activity of staphylothrombin, the complex of prothrombin with coagulase. These results indicate that SSL10 inhibits blood coagulation by interfering with activation of coagulation cascade via binding to the Gla domain of coagulation factor but not by directly inhibiting thrombin activity. This is the first finding that the bacterial protein inhibits blood coagulation via targeting the Gla domain of coagulation factors.

Arachidonic acid: a bridge between traumatic brain injury and fracture healing.

Traumatic brain injury (TBI) is associated with enhanced osteogenesis. The aim of this study was to investigate the effect of serum from TBI rats on fracture healing. Results from this study showed that the serum from TBI rats enhanced the expression of bone gamma carboxyglutamate protein (BGLAP), and promoted in vitro proliferation of MC3T3-E1 cells, a mouse osteoblastic cell line. Furthermore, gas chromatography/mass spectrometry (GC/MS) coupled with multivariate statistical analysis was used to identify the changes in global serum metabolites after TBI. We found that arachidonic acid (AA) was significantly enhanced in serum metabolites in TBI subjects, while hydroxybutyric acid, leucine, malic acid, 5-oxyproline, isocitric acid, mannose, and uric acid were reduced. Finally, we examined the effects of AA on BGLAP expression and cell proliferation in MC3T3-E1 cells. We found that BGLAP expression and proliferation of osteoblasts were positively regulated in the presence of AA. These findings suggest that the increased AA in serum after TBI may play a key role in enhancing the speed of fracture healing.

Computational prediction and analysis of protein γ-carboxylation sites based on a random forest method.

The glutamate γ-carboxylation plays a pivotal part in a number of important human diseases. However, traditional protein γ-carboxylation site detection by experimental approaches are often laborious and time-consuming. In this study, we initiated an attempt for the computational prediction of protein γ-carboxylation sites. We developed a new method for predicting the γ-carboxylation sites based on a Random Forest method. As a result, 90.44% accuracy and 0.7739 MCC value were obtained for the training dataset, and 89.83% accuracy and 0.7448 MCC value for the testing dataset. Our method considered several features including sequence conservation, residual disorder, secondary structures, solvent accessibility, physicochemical/biochemical properties and amino acid occurrence frequencies. By means of the feature selection algorithm, an optimal set of 327 features were selected; these features were considered as the ones that contributed significantly to the prediction of protein γ-carboxylation sites. Analysis of the optimal feature set indicated several important factors in determining the γ-carboxylation and a possible consensus sequence of the γ-carboxylation recognition site (γ-CRS) was suggested. These may shed some light on the in-depth understanding of the mechanisms of γ-carboxylation, providing guidelines for experimental validation.

Gla-rich protein, a new player in tissue calcification?

A novel γ-carboxyglutamate (Gla)-containing protein, named Gla-rich protein (GRP) after its high content in Gla residues or upper zone of growth plate and cartilage matrix associated protein after its preferential expression by cartilage chondrocyte, was recently identified in sturgeon, mice, and humans through independent studies. GRP is the most densely γ-carboxylated protein identified to date and its structure has been remarkably conserved throughout vertebrate evolution but is apparently absent from bird genomes. Several transcript and genomic variants affecting key protein features or regulatory elements were described and 2 paralogs were identified in the teleost fish genome. In the skeleton, most relevant levels of GRP gene expression were observed in cartilaginous tissues and associated with chondrocytes, suggesting a role in chondrogenesis. But GRP expression was also detected in bone cells, indicative of a more widespread role for the protein throughout skeletal formation. Although the molecular function of GRP is yet unknown, the high content of Gla residues and its accumulation at sites of pathological calcification in different human pathologies affecting skin or the vascular system and in breast cancer tumors suggest that GRP may function as a modulator of calcium availability. Because of its association with fibrillar collagens, GRP could also be involved in the organization and/or stabilization of cartilage matrix. Although transgenic mice did not reveal obvious phenotypic alterations in skeletal development or structure, zebrafish morphants lack craniofacial cartilage and exhibit limited calcification, suggesting a role for GRP during skeletal development, but additional functional data are required to understand its function.

Ucmaa (Grp-2) is required for zebrafish skeletal development. Evidence for a functional role of its glutamate γ-carboxylation.

UCMA (alternatively named GRP) is a novel member of the family of γ-carboxyglutamate (Gla) containing proteins that is mainly expressed in cartilage. We have used the zebrafish as a model organism to study UCMA function. Due to the whole genome duplication two Ucma genes are present in zebrafish, ucmaa and ucmab, located on chromosomes 25 and 4, respectively. UCMA gene structure, alternative splicing and protein sequence are highly conserved between mammals and zebrafish and Ucmaa and Ucmab are expressed in zebrafish skeletal tissues. Ucmaa is first detected in the notochord at 18 hpf and expression continues during notochord development. In addition, it is widely present in the developing craniofacial cartilage. In contrast, the weakly expressed Ucmab can be first detected at specific sites in the craniofacial cartilage at 96 hpf, but not in notochord. Knockdown of ucmaa leads to severe growth retardation and perturbance of skeletal development. The cartilage of the morphants has a decreased aggrecan and collagen II content. Similar malformations were observed when glutamate γ-carboxylation was inhibited by warfarin treatment, indicating that glutamate γ-carboxylation is crucial for Ucma function and pointing to a role of UCMA in the pathogenesis of "warfarin embryopathies" and other human skeletal diseases.

Molecular modulation of osteocalcin and its relevance in diabetes (Review).

Osteocalcin (OC) is encoded by the bone γ-carboxyglutamate (Gla) protein (BGLAP) gene, and it is released by osteoblasts during osteogenesis. Its expression can be modulated by growth factors, hormones, cytokines and physical stimuli via signal transduction pathways, binding to the BGLAP gene promoter or interactions with nuclear transcription factors. It was recently demonstrated that uncarboxylated OC improves glucose tolerance and insulin sensitivity in mice by increasing the expression and secretion of insulin in ß-cells and of adiponectin in adipocytes. Humans with type 2 diabetes have significantly lower serum levels of OC than healthy individuals and indeed, serum OC levels have been inversely correlated with fasting plasma glucose, fasting insulin and the homeostasis model assessment of insulin resistance (HOMA-IR) index. Moreover, several drugs have been shown to influence OC expression and its serum concentration. This review summarizes the molecular mechanisms involved in the modulation of OC expression, and discusses the potential relevance of OC in the pathogenesis and treatment of diabetes.

Vitamin K, osteoporosis and degenerative diseases of ageing.

The function of vitamin K is to serve as a co-factor during the post-translational carboxylation of glutamate (Glu) residues into γ-carboxyglutamate (Gla) residues. The vital importance of the Gla-proteins essential for normal haemostasis is well recognized. During recent years, new Gla-containing proteins have been discovered and the vitamin K-dependent carboxylation is also essential for their function. It seems, however, that our dietary vitamin K intake is too low to support the carboxylation of at least some of these Gla-proteins. According to the triage theory, long-term vitamin K inadequacy is an independent, but modifiable risk factor for the development of degenerative diseases of ageing including osteoporosis and atherosclerosis.

Investigation and identification of protein γ-glutamyl carboxylation sites.

BACKGROUND: Carboxylation is a modification of glutamate (Glu) residues which occurs post-translation that is catalyzed by γ-glutamyl carboxylase in the lumen of the endoplasmic reticulum. Vitamin K is a critical co-factor in the post-translational conversion of Glu residues to γ-carboxyglutamate (Gla) residues. It has been shown that the process of carboxylation is involved in the blood clotting cascade, bone growth, and extraosseous calcification. However, studies in this field have been limited by the difficulty of experimentally studying substrate site specificity in γ-glutamyl carboxylation. In silico investigations have the potential for characterizing carboxylated sites before experiments are carried out. RESULTS: Because of the importance of γ-glutamyl carboxylation in biological mechanisms, this study investigates the substrate site specificity in carboxylation sites. It considers not only the composition of amino acids that surround carboxylation sites, but also the structural characteristics of these sites, including secondary structure and solvent-accessible surface area (ASA). The explored features are used to establish a predictive model for differentiating between carboxylation sites and non-carboxylation sites. A support vector machine (SVM) is employed to establish a predictive model with various features. A five-fold cross-validation evaluation reveals that the SVM model, trained with the combined features of positional weighted matrix (PWM), amino acid composition (AAC), and ASA, yields the highest accuracy (0.892). Furthermore, an independent testing set is constructed to evaluate whether the predictive model is over-fitted to the training set. CONCLUSIONS: Independent testing data that did not undergo the cross-validation process shows that the proposed model can differentiate between carboxylation sites and non-carboxylation sites. This investigation is the first to study carboxylation sites and to develop a system for identifying them. The proposed method is a practical means of preliminary analysis and greatly diminishes the total number of potential carboxylation sites requiring further experimental confirmation.