-
The Biochemical Journal Jul 1999Vitamin K-dependent proteins contain a propeptide that is required for recognition by the enzyme gamma-glutamylcarboxylase. Substrates used in vitro for carboxylation...
Vitamin K-dependent proteins contain a propeptide that is required for recognition by the enzyme gamma-glutamylcarboxylase. Substrates used in vitro for carboxylation studies lacking a prosequence are characterized by Km values in the millimolar range, whereas the Km for peptides containing a prosequence is three or four orders of magnitude smaller. Here we report that descarboxy-osteocalcin is an exception in this respect. With descarboxy-osteocalcin in purified propeptide-free recombinant carboxylase, the Km was 1.8 microM. Furthermore, osteocalcin was an inhibitor of descarboxy-osteocalcin carboxylation with a Ki of 76 microM. In contrast with the other vitamin K-dependent proteins, free propeptides do not inhibit descarboxy-osteocalcin carboxylation. Moreover, propeptide-containing substrates were inhibited neither by osteocalcin nor by its propeptide. From our studies we conclude that descarboxy-osteocalcin must have an internal recognition sequence that binds to gamma-glutamylcarboxylase at a site different from the propeptide-recognition site.
Topics: Amino Acid Sequence; Animals; Binding Sites; Carbon-Carbon Ligases; Cell Line; Molecular Sequence Data; Osteocalcin; Peptides; Protein Binding; Substrate Specificity; Vitamin K
PubMed: 10393081
DOI: No ID Found -
Clinical Endocrinology Feb 2013Recent evidence indicates that the osteoblast-derived protein osteocalcin is able to influence adiposity and glucose homeostasis in mice. Little is known about this...
BACKGROUND
Recent evidence indicates that the osteoblast-derived protein osteocalcin is able to influence adiposity and glucose homeostasis in mice. Little is known about this relationship in humans.
OBJECTIVE
To investigate the association of plasma osteocalcin levels with the metabolic syndrome in a community-dwelling cohort of older persons in the Netherlands.
DESIGN AND PARTICIPANTS
Data were used from the Longitudinal Aging Study Amsterdam (LASA), an ongoing multidisciplinary cohort study in a representative sample of the older Dutch population (≥65 years old). A total of 1284 subjects (629 men and 655 women) between the age of 65 and 88 years participated in this study.
MEASUREMENTS
Metabolic syndrome (U.S. National Cholesterol Education Program definition) and its individual components were assessed as well as plasma osteocalcin levels.
RESULTS
Among the participants, the prevalence of the metabolic syndrome was 37·1%. The median osteocalcin level was 2·0 nmol/l. Plasma osteocalcin was inversely associated with the metabolic syndrome. The odds ratio (OR) was 3·68 with 95% confidence interval (CI) 2·53-5·34 for the lowest osteocalcin quartile compared to the highest quartile. The association between osteocalcin and the metabolic syndrome was mainly determined by high triglycerides, low HDL, waist circumference and hypertension.
CONCLUSION
Low plasma osteocalcin levels are strongly associated with the metabolic syndrome in an older community-dwelling population.
Topics: Aged; Aged, 80 and over; Female; Humans; Male; Metabolic Syndrome; Netherlands; Osteocalcin; Risk Factors
PubMed: 22435398
DOI: 10.1111/j.1365-2265.2012.04391.x -
Rapid Communications in Mass... Oct 2016Osteocalcin is a small, abundant bone protein that is difficult to detect using high-throughput tandem mass spectrometry (MS/MS) proteomic approaches from bone protein...
RATIONALE
Osteocalcin is a small, abundant bone protein that is difficult to detect using high-throughput tandem mass spectrometry (MS/MS) proteomic approaches from bone protein extracts, and is predominantly detected by non-MS immunological methods. Here, we analyze bovine osteocalcin and its post-translational modifications to determine why a protein of this size goes undetected.
METHODS
Osteocalcin was purified from cow bone using well-established methods. Intact osteocalcin or trypsin-digested osteocalcin were separated using an Agilent 1200 series high-performance liquid chromatography (HPLC) system and analyzed using a ThermoScientific LTQ-Orbitrap XL after fragmentation with higher-energy collision dissociation. Data were analyzed using Mascot or Prosight Lite.
RESULTS
Our results support previous findings that the cow osteocalcin has up to three carboxylations using both intact osteocalcin and digested forms. Using Mascot, we were able to detect osteocalcin peptides, but no fragments that localized the carboxylations. Full annotation using Prosight Lite of the intact (three carboxylations), N-terminal peptide (one carboxylation), and middle peptide (two carboxylations) showed complete fragmentation was present, but complete neutral loss was observed.
CONCLUSIONS
Osteocalcin carboxylation, and its associated neutral losses, makes high-throughput detection of this protein challenging; however, alternative fragmentation or limited purification can overcome these challenges. Copyright © 2016 John Wiley & Sons, Ltd.
Topics: Animals; Cattle; Mass Spectrometry; Osteocalcin; Peptides; Protein Processing, Post-Translational; Proteomics
PubMed: 27470908
DOI: 10.1002/rcm.7692 -
American Journal of Physiology. Cell... Nov 2019Exosome secretion is an important paracrine way of endothelial progenitor cells (EPCs) to modulate resident endothelial cells. The osteocalcin (OCN)-expressing EPCs have...
Exosome secretion is an important paracrine way of endothelial progenitor cells (EPCs) to modulate resident endothelial cells. The osteocalcin (OCN)-expressing EPCs have been found to be increased in cardiovascular disease patients and are considered to be involved in the process of coronary atherosclerosis. Since OCN has been proven to prevent endothelial dysfunction, this study aimed to evaluate the effect of exosomes derived from OCN-overexpressed EPCs on endothelial cells. Exosomes derived from EPCs (Exos) and OCN-overexpressed EPCs (OCN-Exos) were isolated and incubated with rat aorta endothelial cells (RAOECs) with or without the inhibition of OCN receptor G protein-coupled receptor family C group 6 member A (GPRC6A). The effects of exosomes on the proliferation activity of endothelial cells were evaluated by CCK-8 assay, and the migration of endothelial cells was detected by wound healing assay. A tube formation assay was used to test the influence of exosomes on the angiogenesis performance of endothelial cells. Here, we presented that OCN was packed into Exos and was able to be transferred to the RAOECs via exosome incorporation, which was increased in OCN-Exos groups. Compared with Exos, OCN-Exos had better efficiency in promoting RAOEC proliferation and migration and tube formation. The promoting effects were impeded after the inhibition of GPRC6A expression in RAOECs. These data suggest that exosomes from OCN-overexpressed EPCs have a beneficial regulating effect on endothelial cells, which involved enhanced OCN-GPRC6A signaling.
Topics: Animals; Cell Movement; Cell Proliferation; Endothelial Progenitor Cells; Exosomes; Gene Expression; Neovascularization, Physiologic; Osteocalcin; Rats
PubMed: 31411920
DOI: 10.1152/ajpcell.00534.2018 -
Journal of Diabetes Research 2022Osteocalcin (OCN) has been proved to be closely related with the development of type 2 diabetes mellitus (T2DM). We aimed to study if OCN could improve the disorder of...
BACKGROUND
Osteocalcin (OCN) has been proved to be closely related with the development of type 2 diabetes mellitus (T2DM). We aimed to study if OCN could improve the disorder of islet cell caused by lipotoxicity.
METHODS
Alizarin red staining was used to investigate the mineralization. Western blotting and ELISA methods were used to measure protein expression. Immunofluorescence staining was used to investigate the protein nuclear transfer.
RESULTS
High glucose and high fat inhibited the differentiation of osteoblast precursors. Overexpression of insulin receptor (InsR) significantly promoted the Runx2 and OCN expression. The increase of insulin, Gprc6a, and Glut2 by osteoblast culture medium overexpressing insulin receptor was reversed by osteocalcin neutralizing antibody. Undercarboxylated osteocalcin (ucOC) suppressed the lipotoxic islet -cell damage caused by palmitic acid. The FOXO1 from intranuclear to extranuclear was also significantly increased after ucOC treatment compared with the group PA. Knockdown of Gprc6a or suppression of PI3K/AKT signal pathway could reverse the upregulation of GPRC6A/PI3K/AKT/FoxO1/Pdx1 caused by ucOC.
CONCLUSION
OCN could activate the FOXO1 signaling pathway to regulate GLUT2 expression and improve the insulin secretion disorder caused by lipotoxicity.
Topics: Cell Line; Diabetes Mellitus, Type 2; Humans; Insulin; Insulin-Secreting Cells; Osteocalcin; Real-Time Polymerase Chain Reaction
PubMed: 35313683
DOI: 10.1155/2022/3025538 -
Endocrinology Dec 1996The Hyp mouse manifests rickets and renal wasting of phosphorus. We previously reported elevated circulating osteocalcin in Hyp mice, and a paradoxical decrease in...
The Hyp mouse manifests rickets and renal wasting of phosphorus. We previously reported elevated circulating osteocalcin in Hyp mice, and a paradoxical decrease in response to 1,25-dihydroxyvitamin D3 [1,25-(OH)2D3]. To investigate these abnormalities further, we characterized in detail the response of circulating osteocalcin to 1,25-(OH)2D3 and compared skeletal osteocalcin in normal and Hyp mice. The affinity of osteocalcin for hydroxyapatite and the protein's clearance were compared in Hyp and normal animals. Finally, the response of osteocalcin messenger RNA (mRNA) to 1,25-(OH)2D3 was examined in normal mice, Hyp mice, and normal mice subjected to dietary phosphorus deprivation. Multiple (n = 3-6) daily doses of 1,25-(OH)2D3 are required to increase serum osteocalcin levels in normal C57 BL/6 mice; the effect is apparent by 6 h and persists for at least 24 h after injection. Single doses of up to 50 ng have no significant effect. In contrast, an approximately 50% decrement in circulating osteocalcin occurs after a single dose of 1,25-(OH)2D3 in Hyp mice. Osteocalcin clearance in Hyp mice appears to be normal. Bone osteocalcin per U calcium or phosphorus is normal in Hyp mice, suggesting that its affinity for hydroxyapatite is normal. Osteocalcin mRNA from Hyp mice is expressed in greater abundance than that from normal animals, reflecting the differences in circulating levels of the protein. Similarly, osteocalcin mRNA from Hyp mice decreases in response to 1,25-(OH)2D3, whereas an increase in osteocalcin message is seen in normal animals. These studies indicate that normal mice are relatively resistant to 1,25-(OH)2D3 stimulation of osteocalcin production. Furthermore, the differences between Hyp and normal mice in circulating osteocalcin reflect differences in the regulation of gene expression.
Topics: Animals; Binding, Competitive; Bone and Bones; Calcitriol; Diet; Durapatite; Genetic Linkage; Hypophosphatemia; Male; Mice; Mice, Inbred C57BL; Minerals; Osteocalcin; Phosphorus; Reference Values; Rickets; X Chromosome
PubMed: 8940337
DOI: 10.1210/endo.137.12.8940337 -
Journal of Structural Biology Aug 2019Non-collagenous proteins such as osteocalcin function as regulators of the mineralization process in bone. Osteocalcin undergoes post-translational modification adding...
Non-collagenous proteins such as osteocalcin function as regulators of the mineralization process in bone. Osteocalcin undergoes post-translational modification adding an extra carboxylate group on three of its glutamate residues to enhance interaction with bone mineral. In this work, we examine regulation of biomimetic apatite formation by osteocalcin that was not modified after translation. We analyze the structural features in the protein and mineral-protein interfaces to elicit the unmodified protein's fold inside the mineral and to unveil the species that interact with the mineral surface. The results presented here give clues on the protein's active role in controlling the mineral phases that are formed on hydroxyapatite crystals and its ability to influence the extent of order in these crystals.
Topics: Apatites; Biomimetics; Calcification, Physiologic; Durapatite; Minerals; Osteocalcin; Protein Folding; Proteins; Surface Properties
PubMed: 31015050
DOI: 10.1016/j.jsb.2019.04.014 -
Endocrinology Jan 1998Rickets and osteomalacia are characteristic features of the Hyp mouse model of human X-linked hypophosphatemia. Hyp mice demonstrate elevated circulating osteocalcin...
Rickets and osteomalacia are characteristic features of the Hyp mouse model of human X-linked hypophosphatemia. Hyp mice demonstrate elevated circulating osteocalcin levels, as well as altered regulation of osteocalcin by 1,25(OH)2D3. Whether this osteocalcin abnormality is intrinsic to the osteoblast, or mediated by the in vivo milieu, has not been established. We therefore characterized osteocalcin production and its regulation by 1,25(OH)2D3 in primary cultures of murine osteoblasts and examined osteocalcin and its messenger RNA in response to 1,25(OH)2D3 in cultures of Hyp mouse-derived osteoblasts. Cell viability and osteocalcin production are optimal when murine cells are harvested within 36 h of age. Murine primary osteoblast cultures mineralize and produce osteocalcin in a maturation-dependent fashion (as demonstrated in other species), and continuous exposure to 1,25(OH)2D3, beginning at day 9 of culture, inhibits osteoblast differentiation and osteocalcin production and prevents mineralization of the culture. However, in contrast to other species, exposure to 1,25(OH)2D3, added later (days 17-25) in culture, does not stimulate osteocalcin but arrests osteocalcin production at current levels. Ambient media levels of osteocalcin were no different in cultures from Hyp mice and their normal litter mates, and the down-regulatory response to 1,25(OH)2D3 was comparable in cultures from normal and Hyp mice. Furthermore, expression of osteocalcin messenger RNA in murine cultures is reduced with exposure to 1,25(OH)2D3, and there is no difference between normal and Hyp cultures in this response. Thus, primary murine osteoblasts manifest a species-specific effect of 1,25(OH)2D3 on osteocalcin production. Furthermore, the increased serum osteocalcin production seen in intact Hyp mice, and the altered response to 1,25(OH)2D3 in Hyp mice, are not observed in osteoblast cultures derived from the mutant strain. These data indicate that abnormalities of osteocalcin described in intact Hyp mice require factors other than those present in cultured cells.
Topics: Animals; Calcitriol; Cells, Cultured; Female; Hypophosphatemia; Male; Mice; Mice, Inbred C57BL; Osteoblasts; Osteocalcin; RNA, Messenger; Rats; Species Specificity
PubMed: 9421395
DOI: 10.1210/endo.139.1.5677 -
Journal of Bone and Mineral Research :... Jan 1992Osteocalcin is initially synthesized as an 11 kD molecule consisting of a 23-residue translocation signal peptide that is cleaved during translation, a 26-residue...
Osteocalcin is initially synthesized as an 11 kD molecule consisting of a 23-residue translocation signal peptide that is cleaved during translation, a 26-residue propeptide that targets the protein for gamma-carboxylation, and the 49-residue mature protein. Although the majority of newly synthesized osteocalcin is deposited into bone matrix, a small amount can be detected in blood, and it is this characteristic that has led to its current clinical use as a specific index of osteoblastic activity. Nothing is known, however, about the fate of the propeptide. If osteocalcin and the propeptide are cosecreted, then the concentration of the propeptide could also be useful as a marker of osteoblastic function and, further, may be superior to osteocalcin because it would be unaffected by binding to bone. To test this hypothesis, we synthesized a peptide corresponding to 21 residues of the osteocalcin propeptide from humans and produced a polyclonal antibody to this peptide. Human sera were screened for the presence of the propeptide, and the human osteosarcoma cell line MG-63 was tested for secretion of the propeptide. We could not detect any osteocalcin propeptide in sera from normal adults or individuals with renal failure or primary hyperparathyroidism or those on long-term coumadin therapy. Likewise there was no propeptide present in media from cells grown in the presence of vitamin K, 1,25-(OH)2D3, warfarin, or warfarin plus 1,25-(OH)2D3. In contrast, the cell extract, characterized by high-performance liquid chromatography, contained mature osteocalcin, free propeptide, and the proosteocalcin precursor when cells were grown in the presence of 1,25-(OH)2D3 alone.(ABSTRACT TRUNCATED AT 250 WORDS)
Topics: Calcitriol; Chromatography, High Pressure Liquid; Humans; Osteoblasts; Osteocalcin; Protein Precursors; Radioimmunoassay; Tumor Cells, Cultured; Warfarin
PubMed: 1549960
DOI: 10.1002/jbmr.5650070111 -
Nihon Yakurigaku Zasshi. Folia... Apr 2015
Review
Topics: Female; Glucagon-Like Peptide 1; Humans; Insulin; Insulin Secretion; Male; Metabolic Syndrome; Osteocalcin
PubMed: 25864831
DOI: 10.1254/fpj.145.201