Validation of the bone Gla protein (osteocalcin) assay.
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Biomarkers↗
Biomedical subjects
K G Mann
Publications and source records attributed to K G Mann.
Bone Gla protein (osteocalcin) assay standardization report.
The major conclusion of this study is that the different laboratory assays for serum BGP give a reasonably consistent picture of bone metabolism in the metabolic bone diseases examined only if the results are expressed as a percentage of serum BGP levels in normal individuals. This requires that all laboratories establish a mean control serum BGP value in an appropriate population of normal individuals. Since BGP levels determined by different laboratories on the same serum sample can vary by more than fourfold, the absolute serum BGP levels determined in one laboratory cannot be compared directly with the serum BGP levels determined in another. Although we cannot comment on the efficacy of different laboratory assays for serum BGP as measures of bone metabolism in disease states that were not examined, such as osteoporosis, it is clear that the large differences between laboratory assays make it imperative that all interlaboratory comparisons be based on values expressed as a percentage of serum BGP in an appropriate population of normal individuals.
Animals↗
Comparison of monoclonal and polyclonal antibody-based immunoassays for osteocalcin: a study of sources of variation in assay results.
We have compared two assays for osteocalcin (BGP, bone gla protein), one based on a monoclonal antibody, the other based on a polyclonal serum, in an effort to better understand the factors that contribute to the wide variation reported for osteocalcin normal ranges. The two assays compared well using serum samples, with a correlation coefficient of 0.9071 (n = 48). However, the monoclonal antibody assay returns values increased over the polyclonal assay by approximately 1.5-fold. Gel permeation studies indicate that these larger values are due primarily to increased reactivity in the monoclonal assay with high- and low-molecular-weight forms of osteocalcin in plasma; both assays give similar reactions to intact osteocalcin. Analysis of samples from individuals with increased bone resorption due to parathyroid hormone administration reveals that the decrease seen in osteocalcin values after hormone infusion occurs primarily in the fraction that corresponds to intact osteocalcin. During the course of these studies, we re-evaluated the extinction coefficient for osteocalcin, arriving at the value E = 1.33 mg/ml-1, cm-1. We also observed a significant negative interference in both assays (and a commercial assay) caused by hemolysis. This interference is due to proteolysis of osteocalcin by enzymatic activity released from the lysed red cells.
Animals↗
Heterogeneity of human bone.
Matched samples of bone from the lumbar spine and tibia were obtained at autopsy from three adult males who had no known evidence of metabolic bone disease at the time of their demise. The soluble noncollagenous bone proteins were quantitatively extracted from these samples and assayed for the relative content of two bone-associated proteins, osteocalcin and osteonectin. When compared to trabecular bone, cortical bone had higher levels of osteocalcin and much lower levels of osteonectin. When concentration is expressed per gram of dried bone, the osteocalcin excess in cortical bone ranged from 30- to 32-fold, and the osteonectin excess in trabecular bone ranged from 21- to 47-fold. These differences were significant (P less than 0.01) using analysis of variance. We conclude that the human skeleton is not homogeneous with regard to these biochemical markers and that cortical and trabecular bone are biochemically quite distinct. This implies that these two types of bone may be subject to distinct regulatory mechanisms and that global assessments of skeletal function and bone quality based upon soluble markers should be applied with caution. The data also imply that a differential assessment of skeletal performance may be possible using biochemical serum markers.
Aged↗
Detection of procollagen biosynthesis using peptide-specific antibodies.
Peptides corresponding to selected sequences of the alpha 1 chain of the COOH propeptide of type I and type III human procollagen were synthesized and used as antigens to develop polyclonal and monoclonal antibodies. The antibodies were shown to be epitope specific using a peptide-based solid phase enzyme-linked immunoadsorbent assay. The antibodies were specific for the appropriate procollagens and the COOH propeptides isolated from serum-free culture supernatants of human skin fibroblasts. The rabbit antisera directed to the type I synthetic peptide bound the intact procollagen molecule and both the procollagen alpha 1(I) and alpha 2(I) chains after the reduction of the disulfide bonds. In addition, the antisera bound intact type I COOH propeptide, generated by bacterial collagenase treatment of procollagen, and the individual chains of the propeptide after reduction. In contrast, a monoclonal antibody to the type I peptide was able to bind only to the reduced form of the COOH propeptide. Both rabbit polyclonal and murine monoclonal antibodies directed to the type III synthetic peptide bound the intact and the individual chains of type III procollagen as well as the intact and reduced forms of the type III COOH propeptide. The antibodies have been used to detect procollagen synthesis in two human osteosarcoma cell lines and the differential expression of procollagen in the culture medium of rat lung fibroblasts grown in the presence or absence of glucocorticoids.
Amino Acid Sequence↗
Mechanisms of vitamin K antagonism.
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Amino Acid Sequence↗
Expression of human bone-related proteins in the hematopoietic microenvironment.
Given the intimate relationship between bone and bone marrow, we hypothesized that the human bone marrow may function as a source (or reservoir) of bone-forming progenitor cells. We observed a population of cells within the bone marrow which produce bone-specific or bone-related proteins. The production of these proteins was developmentally regulated in human long-term bone marrow cell cultures; the bone protein-producing cells (BPPC) are observed under serum-free, short-term culture conditions, respond to bone-related and not hematopoietic growth factors, and are derived from a population of low-density, nonadherent, My10-negative (or low My10 density), marrow cells (My10 is an antigen found on most hematopoietic progenitor cells). Cultivation of marrow-derived BPPC in secondary, serum-containing cultures results in their differentiation into osteoblastlike cells. At this stage of development, BPPC produce an extracellular matrix which incorporates both bone-related proteins and radiolabeled calcium. Human bone marrow BPPC thus represent a newly described cell phenotype important to both bone and hematopoietic cell biology.
Antigens, Neoplasm↗
Discrimination of normal and abnormal prothrombin and protein C in plasma using a calcium ion-inhibited monoclonal antibody to a common epitope on several vitamin K-dependent proteins.
Vitamin K deficiency or administration of vitamin K antagonists results in the biosynthesis of abnormal des-gamma-carboxy forms of the vitamin K-dependent proteins. Monoclonal antibody H-11 binds several vitamin K-dependent proteins at a determinant that includes the first two residues of gamma-carboxyglutamic acid. Antibody H-11 binds fully carboxylated prothrombin and protein C in the presence of EDTA but binding is inhibited by the divalent metal ions, calcium, magnesium, and manganese. By contrast, des-gamma-carboxy prothrombin and protein C bind antibody H-11 the same in the presence of EDTA or calcium ion. Antibody H-11 thus appears to bind a conserved antigenic site containing gamma-carboxyglutamic acid that in the presence of divalent metal ion undergoes a conformational transition. This ability of antibody H-11 to bind des-gamma-carboxy prothrombin and protein C in the presence of calcium ion allowed the development of an immunoassay for these proteins in plasma. Prothrombin and protein C from stably anticoagulated individuals receiving warfarin were characterized by their ability to bind antibody H-11 in the presence of calcium ion. Binding of prothrombin and protein C to antibody H-11 in the presence of calcium correlated temporally with warfarin administration. The inability of calcium ion to inhibit binding of antibody H-11 to abnormal prothrombin and protein C in plasma suggests that the circulating forms of both proteins following warfarin administration cannot undergo the metal ion-dependent conformational transition that includes sequence residues 1 through 12.
Antibodies, Monoclonal↗
Evidence for a common metal ion-dependent transition in the 4-carboxyglutamic acid domains of several vitamin K-dependent proteins.
The murine monoclonal antibody H-11 binds a conserved epitope found at the amino terminal of the vitamin K-dependent blood proteins prothrombin, factors VII and X, and protein C. The sequence of polypeptide recognized by antibody H-11 contains 2 residues of gamma-carboxyglutamic acid, and binding of the antibody is inhibited by divalent metal ions. By using a solid-phase immunoassay with 125I-labeled antibody and immobilized vitamin K-dependent protein, binding of the antibody to the vitamin K-dependent proteins was inhibited by increasing concentrations of calcium, manganese, and magnesium ion. The transition midpoints for antibody binding were in the millimolar concentration range and were different for each metal ion. In general, the transition midpoints were lowest for manganese ion, intermediate for calcium ion, and highest for magnesium ion. Antibody H-11 bound specifically to a synthetic peptide corresponding to residues 1-12 of human prothrombin that was synthesized as the gamma-carboxyglutamic acid-containing derivative. Binding of the antibody to the peptide was not inhibited by calcium ion. These data suggest that inhibition of antibody H-11 binding by divalent metal ions is not due simply to neutralization of negative charge by Ca2+. This transition which is conserved in vitamin K-dependent proteins containing the H-11 antigenic site is likely due to a structural transition of the amino-terminal polypeptide possibly from a random (accessible) to ordered (inaccessible) structure.
1-Carboxyglutamic Acid↗
The activation of bovine protein C by factor Xa.
A complex composed of factor Xa and phospholipid vesicles assembled in the presence of calcium ions catalyzes a discrete cleavage of the heavy chain of bovine protein C that is indistinguishable from that produced by thrombin as judged by sodium dodecyl sulfate-polyacrylamide gel electrophoresis. This cleavage generates an active site capable of hydrolyzing small substrates and inactivating factor Va function in the prothrombinase complex. Activation of protein C by factor Xa requires both calcium ions and phospholipid vesicles and proceeds at a rate an order of magnitude greater than that observed for alpha-thrombin in solution. gamma-Carboxyglutamic acid-domainless protein C is not activated by factor Xa, consistent with the requirement for phospholipid and distinguishing this reaction from protein C activation by thrombin. Thrombomodulin serves as a cofactor for the factor Xa-catalyzed reaction, forming a 1:1 complex with factor Xa (apparent Kd = 5.7 X 10(-10) M) and stimulating the saturated rate of protein C activation by factor Xa (kcat = 149 min-1) to levels comparable with the thrombin-thrombomodulin complex. Protein C activation by factor Xa is not inhibited by the specific thrombin inhibitor dansyl-N-(3-ethyl-1,5-pentanediyl)amide but is inhibited by antithrombin III, tripeptide-chloromethyl ketones, and the monoclonal antibody alpha-BFX-2b that is highly specific for factor Xa. These data indicate that thrombomodulin is promiscuous in its role as a cofactor and suggest the existence of an alternative pathway for protein C activation in vivo.
Animals↗
Structure of human osteonectin based upon analysis of cDNA and genomic sequences.
Overlapping human bone osteonectin cDNAs were obtained by screening two independent human SaOS-2 lambda gt11 libraries using antibovine osteonectin monoclonal antibodies. One clone contains a 0.54-kb insert and the other a 1.9-kb insert. Insertion fragments from lambda clones were liberated by restriction digestion and subcloned into pUC19 for sequencing. Digestion of the 1.9-kb insert with EcoRI released 0.4- and 1.5-kb fragments. Sequencing analysis revealed that the 0.54- and 0.4-kb fragments are identical except for 150 nucleotides missing at the 5' region of the 0.4-kb fragment. The composite nucleotide sequence of human osteonectin has a total length of 2091 nucleotides and is comprised of 50 nucleotides of 5'-noncoding sequence, a coding segment for 303 amino acids, a termination codon, and 1114 nucleotides of 3'-noncoding sequence. The primary transcript codes for 286 amino acids of mature protein and a 17-residue amino-terminal hydrophobic signal peptide. Outstanding properties inferred from the primary structure are putative Ca2+ binding domains located in the glutamic acid rich NH2 terminus (residues 1-52) and two "EF"-hand domains in the C-terminal half of the protein (residues 165-176 and 257-286). The mature protein also contains a cysteine-rich, highly hydrophilic region homologous to the ovomucoid serine protease inhibitors (residues 76-132). Overlapping human genomic clones in lambda EMBL3 for osteonectin have been isolated and characterized. Intron/exon junction sequencing of the human osteonectin gene shows the presence of 10 exons and 9 introns. The mature protein is encoded by nine exons separated by eight introns.(ABSTRACT TRUNCATED AT 250 WORDS)
Amino Acid Sequence↗
Zymogen/enzyme discrimination using peptide chloromethyl ketones.
Glutamylglycinylarginyl chloromethyl ketone, tyrosylglycinylarginyl chloromethyl ketone, and phenylalanylprolylarginyl chloromethyl ketone have been labeled at their amino termini using fluorescein, rhodamine-X, lissamine-rhodamine, pyrene, and the 1,5-, 2,5-, and 2,6-dimethylaminonaphthalene-1-sulfonyl moieties. These peptidyl chloromethyl ketones have also been modified by incorporation of biotin and epsilon-amino caproyl biotin. The ability of these various chloromethyl ketones to be incorporated into a collection of zymogen-enzyme pairs has been evaluated using a variety of coagulation and fibrinolytic proteins. All labeled chloromethyl ketones were efficiently incorporated into the proteases tested, with the exception of urokinase which was refractory to inhibition by phenylalanylprolylarginyl chloromethyl ketone derivatives. No modification of any zymogen species was observed even under conditions designed to detect minimal reactivity. When enzymes were modified using chloromethyl ketones labeled with epsilon-amino caproyl biotin, the modified proteins readily reacted with avidin under a variety of different conditions. The observed reactivity with avidin was used in enzyme "blotting" following electrophoretic resolution of polypeptide chains and to remove active enzyme present in enzyme-zymogen mixtures. These reagents have been used to evaluate the potential for active site expression by the single-chain human factor VII molecule. Studies conducted with tissue factor, phospholipids, and calcium using factor X as substrate demonstrate that no activity can be obtained without initial activation of either factor X to factor Xa or factor VII to factor VIIa by an external source. We thus conclude that factor VII is a true zymogen, inert in the blood clotting process prior to its cleavage to factor VIIa.
Amino Acid Chloromethyl Ketones↗
The interaction of bone Gla protein (osteocalcin) with phospholipid vesicles.
The binding interaction of bone Gla protein (BGP), or osteocalcin, to phospholipid vesicles in the presence of calcium has been investigated. Two separate indirect methodologies involving displacement of pyrene-modified Factor Va bound to phospholipid vesicles, and competition with several coagulation proteins in a prothrombin activation assay were performed. Titration of BGP into a cuvette containing phospholipid vesicles (75:25, L-alpha-phosphatidylcholine/L-alpha-phosphatidylserine (PCPS] saturated with pyrene-modified Factor Va resulted in a systematic decrease in steady-state anisotropy, suggesting competition for membrane binding sites with pyrene-modified Factor Va. BGP was also found to inhibit thrombin generation in the prothrombin activation assay. Approximately 50% inhibition was observed at 3 microM BGP under phospholipid-limiting (0.5 microM PCPS) concentrations. No inhibition was observed under phospholipid excess (30 microM PCPS) concentrations. Direct measurement of phospholipid binding was measured using equilibrium gel filtration. Elution profiles using fixed lipid (3.4 mumol of PCPS) and varying BGP concentrations (1-17 microM) in the presence of 3 mM CaCl2 showed a BGP-phospholipid association. Quantitation of determined isotherm yielded a dissociation constant of 6 +/- 1 microM with a stoichiometry of 102 +/- 9 BGP molecules/vesicle at saturation (35 PCPS lipids/BGP) in the presence of 3 mM CaCl2. These results support the hypothesis that protein gamma-carboxylation events are coincident with membrane binding potential.
Binding, Competitive↗
Human D-Phe-Pro-Arg-CH2-alpha-thrombin crystallization and diffraction data.
Human alpha-thrombin, inhibited with the high-affinity irreversible inhibitor D-Phe-Pro-Arg-chloromethylketone, has been crystallized from polyethylene glycol 8000 solutions buffered with 0.1 M-sodium phosphate. The crystals are: orthorhombic, a = 67.9(1) A, b = 87.9(1) A, c = 61.0(1) A, space group P2(1)2(1)2(1) with four molecules per unit cell. This gives a protein fraction of 58% consistent with the excellent X-ray diffraction quality of the crystals. A mercury heavy-atom derivative is being prepared from a thioester analogue of D-Phe-Pro-Arg-CH2-alpha-thrombin in anticipation of a complete crystallographic structure determination.
Amino Acid Chloromethyl Ketones↗
Substitution of valine for glycine-558 in the congenital dysthrombin thrombin Quick II alters primary substrate specificity.
Thrombin Quick II is one of two dysfunctional forms of thrombin derived from the previously described congenital dysprothrombin prothrombin Quick. Thrombin Quick II does not clot fibrinogen, hydrolyze p-nitroanilide substrates of thrombin, or bind N2-[5-(dimethylamino)naphthalene-1-sulfonyl]arginine N,N-(3-ethyl-1,5-pentanediyl)amide, a high-affinity competitive inhibitor of thrombin. To determine the structural alteration in thrombin Quick II, the reduced, carboxymethylated protein was hydrolyzed by a lysyl endopeptidase. A peptide not present in a parallel thrombin hydrolysate was identified by reverse-phase chromatography. The peptide was purified by rechromatography and subjected to Edman degradation which showed that Gly-558 of human prothrombin had been replaced by Val. This corresponds to a point mutation of the Gly codon GGC to GUC. This Gly residue, which is highly conserved in the chymotrypsin family of serine proteases, forms part of the substrate binding pocket for bulky aromatic and basic side chains in chymotrypsin and trypsin, respectively. However, in porcine elastase 1, the corresponding residue is threonine. Consistent with the identified structural alteration, thrombin Quick II incorporates [3H]diisopropyl fluorophosphate stoichiometrically and hydrolyzes the elastase substrate succinyl-Ala-Ala-Pro-Leu-p-nitroanilide with a relative kcat/KM of 0.14 when compared to thrombin. This results from a 3-fold increase in KM and a 2.5-fold decrease in kcat for thrombin Quick II when compared to thrombin acting on the same substrate. These results and those of other investigators studying mutant trypsins support the conclusion that the catalytic activity of serine proteases is very sensitive to structural alterations in the primary substrate binding pocket.
Amino Acid Sequence↗
The reassociation of factor Va from its isolated subunits.
Factor Va is an essential cofactor for the activation of prothrombin catalyzed by factor Xa. The cofactor is a heterodimer composed of a light chain and a heavy chain that are associated noncovalently in the presence of divalent metal ions. The kinetics of the formation of factor Va from the isolated and separated subunits was examined by the time-dependent regain in cofactor activity using direct assays of prothrombin activation catalyzed by prothrombinase. The rate of reassociation at saturating concentrations of calcium ions was slow with a strong temperature dependence. The product of the association reaction was indistinguishable from native factor Va on the basis of activity. The second order rate constant for the process at 37 degrees C in the presence of 2 mM CaCl2 was 1.58 X 10(5) M-1.min-1. Manganese ion increased the rate of regain of activity without influencing the extent of the reaction. The previous identification of a single reactive sulfhydryl in each subunit of factor Va permitted the modification of the separated subunits with sulfhydryl-directed fluorophores. Subunit reassociation was directly measured by fluorescence energy transfer using light chain modified with 6-acryloyl-2-dimethylaminonaphthalene (fluorescence donor) and heavy chain modified with fluorescein 5-maleimide (fluorescence acceptor). Fluorescence measurements indicate that the heavy and light chains associate tightly (Kd = 5.9 x 10(-9) M) and reversibly with a stoichiometry of 1:1. The dissociation of the subunits from the cofactor is first order with a rate constant of 1.03 X 10(-3) min-1. These interpretations were confirmed by physical measurements of subunit reassociation by sedimentation velocity studies.
2-Naphthylamine↗
Factor V: a prototype pro-cofactor for vitamin K-dependent enzyme complexes in blood clotting.
The relative abundance of factor V, factor X and prothrombin has enabled detailed analyses of the prothrombinase complex. Determination of the primary structure for factor V has provided the basis for examination of structure-function relationships. The imminent in vitro expression of recombinant factor V will provide the opportunity for site-specific mutagenesis and a verification of these structure-function relationships. A comparison of the physical properties and primary structures for factors V and VIII has revealed extensive similarities in these two cofactor proteins. This observation indicates that a direct application of the technology developed for the analysis of prothrombinase will lead to an equal understanding of the factor Xase complex. Whether similar relationships exist for other blood coagulation enzyme complexes remains to be determined.
Factor V↗
Identification of a vitamin D-responsive protein on the surface of human osteosarcoma cells.
Monoclonal antibodies were elicited to membrane constituents of the osteoblastic human osteosarcoma cell line Saos-2. Two types of antibody reactivities were characterized: one group of antibodies identified fibroblastic and osteoblastic cultured cells, whereas the other group was specific for the parent cell line, Saos-2. Primary endothelial cells and hepatoma cells were not recognized by either group of antibodies. Through indirect immunofluorescent microscopy, the Saos-2-specific antigen was demonstrated to reside on the surface of these osteosarcoma cells. Metabolic radiolabeling of cultured Saos-2 cells and subsequent immunoprecipitation, electrophoretic separation, and autoradiography revealed this protein to have a Mr of 80,000. Similar experiments in the presence of hormones showed that the expression of this cell surface protein was influenced in an opposing fashion by the bone-regulating hormones parathyroid hormone and vitamin D. Vitamin D stimulated expression by 300%, whereas parathyroid hormone depressed expression by 50%. Thus, Saos-2 human osteoblastic cells demonstrate hormonal regulation through an apparently specific membrane protein.
Antibodies, Monoclonal↗