Primary structure of monkey osteocalcin

Hauschka, Peter V.; Carr, Steven A.; Biemann, K.

doi:10.1021/bi00533a006

Cited by 45 publications

(18 citation statements)

References 22 publications

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“…Rat BGP differs most noticeably from other BGPs at its COOH terminus. The COOH-terminal hexapeptide Ile-TyrGly-Thr-Thr-Val of the rat protein contrasts with the pentapeptide Phe-Tyr-Gly-Pro-Val of chicken, calf, monkey, and human BGPs (34,43). This difference can be invoked to explain the unique antigenic properties of rat BGP.…”

Section: Resultsmentioning

confidence: 95%

The propeptide of rat bone gamma-carboxyglutamic acid protein shares homology with other vitamin K-dependent protein precursors.

Pan¹,

Price²

1985

Proc. Natl. Acad. Sci. U.S.A.

191

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The molecular cloning of bone r-carboxyglutamic acid (Gla) protein (BGP; osteocalcin) was accomplished by constructing a phage 1gtll cDNA library from the rat osteosarcoma cell line ROS 17/2 and screening this library with antibodies raised against BGP from rat bone. By sequencing several cloned cDNAs, we have established a 489-base-pair sequence that predicts a mature BGP of 50 amino acid residues with an NH2-terminal extension of 49 residues. The leader peptide consists of a hydrophobic signal peptide followed by a basic propeptide of 26 or 27 residues that is cleaved after an Arg-Arg dipeptide prior to secretion from the cell. Mature rat BGP is extremely homologous to BGPs from other species except for its COOH-terminal sequence. A stretch of 9 residues proximal to the NH2 terminus of secreted BGP is strikingly similar to the corresponding regions in known propeptides of the y-carboxyglutamic acid-containing blood coagulation factors. We suggest that this common structural feature may be involved in the posttranslational targeting of these polypeptides for vitamin K-dependent r-carboxylation.Bone y-carboxyglutamic acid (Gla) protein (BGP) is a small, highly conserved protein associated with the mineralized matrix of bone (1). Its interaction with synthetic hydroxyapatite in vitro is absolutely dependent on three residues of -carboxyglutamic acid (2), the vitamin K-dependent amino acid formed posttranslationally from glutamic acid. Mineralization defects develop in vitamin K-deficient rats that produce a non-y-carboxylated BGP incapable of binding to bone (3, 4). BGP production is developmentally regulated (5) and is stimulated by the calcitropic steroid hormone 1,25-dihydroxyvitamin D3 (6, 7). We undertook the molecular cloning of BGP in order to generate probes for further investigation of its synthesis and regulation. The present report describes the construction of an osteosarcoma cDNA expression library and determination of the complete nucleotide sequence ofrat BGP cDNA. Ofparticular interest is the information obtained about the structure of the precursor polypeptide that may provide a clue to the mechanism by which the vitamin K-dependent y-carboxylating system recognizes specific proteins for modification. MATERIALS AND METHODSRNA Isolation. RNA was isolated from both osteosarcoma cells and rat calvariae by the guanidinium thiocyanate/CsCl method (8). The rat osteosarcoma cell line ROS 17/2 (9) was cultured as described (10). Confluent cultures at a density of 1-2 x 105 cells per cm2 were treated with daily changes of serum-free medium containing 1 ng of 1,25-dihydroxyvitamin D3 (Hoffmann-La Roche) per ml for a total of 54 hr. Approximately 6 hr after the last media change, cells were lysed directly on the plate with the guanidinium thiocyanate solution. Calvariae of weanling Sprague-Dawley rats (Simonsen Laboratories, Gilroy, CA) were dissected free of adhering membranes, frozen in liquid nitrogen, and pulverized with a chilled mortar and pestle. The frozen powder was transferred immediately ...

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Section: Resultsmentioning

confidence: 95%

The propeptide of rat bone gamma-carboxyglutamic acid protein shares homology with other vitamin K-dependent protein precursors.

Pan¹,

Price²

1985

Proc. Natl. Acad. Sci. U.S.A.

191

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“…Osteocalcin was extracted and purified from bone powder as described in ref. 7. Tryptic digestion, HPLC purification of peptides, amino acid analysis, and Edman sequencing followed ref.…”

Section: Methodsmentioning

confidence: 99%

“…Tryptic digestion, HPLC purification of peptides, amino acid analysis, and Edman sequencing followed ref. 7, except that 50-to 500-pmol peptide samples were analyzed on an ABI-477A pulsed-liquid phase protein sequencer (Applied Biosystems).…”

Section: Methodsmentioning

confidence: 99%

“…In all vertebrate species so far investigated, the osteocalcin protein sequence is highly conserved within the central portion of the molecule, containing the three ␥-carboxyglutamic acid (Gla) residues and Ca 2ϩ binding sites ( Fig. 1) (6)(7)(8), whereas the N terminus displays considerable variation and is the most genetically informative region of the molecule.…”

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confidence: 99%

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Osteocalcin protein sequences of Neanderthals and modern primates

Nielsen-Marsh

Richards

Hauschka

et al. 2005

Proc. Natl. Acad. Sci. U.S.A.

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We report here protein sequences of fossil hominids, from two Neanderthals dating to Ϸ75,000 years old from Shanidar Cave in Iraq. These sequences, the oldest reported fossil primate protein sequences, are of bone osteocalcin, which was extracted and sequenced by using MALDI-TOF͞TOF mass spectrometry. Through a combination of direct sequencing and peptide mass mapping, we determined that Neanderthals have an osteocalcin amino acid sequence that is identical to that of modern humans. We also report complete osteocalcin sequences for chimpanzee (Pan troglodytes) and gorilla (Gorilla gorilla gorilla) and a partial sequence for orangutan (Pongo pygmaeus), all of which are previously unreported. We found that the osteocalcin sequences of Neanderthals, modern human, chimpanzee, and orangutan are unusual among mammals in that the ninth amino acid is proline (Pro-9), whereas most species have hydroxyproline (Hyp-9). Posttranslational hydroxylation of Pro-9 in osteocalcin by prolyl-4-hydroxylase requires adequate concentrations of vitamin C (L-ascorbic acid), molecular O2, Fe 2؉ , and 2-oxoglutarate, and also depends on enzyme recognition of the target proline substrate consensus sequence Leu-Gly-Ala-Pro-9-Ala-Pro-Tyr occurring in most mammals. In five species with Pro-9 -Val-10, hydroxylation is blocked, whereas in gorilla there is a mixture of Pro-9 and Hyp-9. We suggest that the absence of hydroxylation of Pro-9 in Pan, Pongo, and Homo may reflect response to a selective pressure related to a decline in vitamin C in the diet during omnivorous dietary adaptation, either independently or through the common ancestor of these species.MALDI-TOF͞TOF ͉ dietary adaptation ͉ biomolecular preservation ͉ vitamin C ͉ evolution

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“…Osteocalcin is a 5200-5900 MW protein (depending on species) containing 3 Gla residues/ molecule [14]. Although its physiologic function is as yet unknown, osteocalcin has several unique properties which suggest its potential involvement in mineral deposition.…”

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confidence: 99%

Calcergy and calciphylaxis: Timed appearance of γ-carboxyglutamic acid and osteocalcin in mineral deposits

Lian

Boivin²,

Patterson-Allen

et al. 1983

Calcif Tissue Int

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gamma-Carboxyglutamate (Gla), a calcium binding amino acid whose synthesis depends on vitamin K, has been found in association with pathologic calcifications. It is of interest therefore to examine the role of Gla-containing proteins in the formation of nonskeletal mineralized tissues. Calcergy and calciphylaxis, experimentally induced models of pathologic calcification, offer the opportunity to study the formation of mineral deposits in the absence of an endochondral sequence of bone formation. Before induction of subcutaneous calcinosis by topical treatment with the direct calcergen, KMnO4, or by challenging dihydrotachysterol-sensitized animals with FeCl2, control specimens contain no gamma-carboxyglutamic acid. With the initial formation of cytoplasmic vesicles, calcium content of the tissues increases and Gla is detected. Gla levels are further elevated with the appearance of poorly crystalline apatite-like crystallites. Origin of protein bound Gla was established by positive identification of osteocalcin by radioimmunoassay. Gla and osteocalcin appear concomitant with the earliest mineral deposits observed by electron microscopy and micro X-ray analysis. The formation of organized extracellular pattern by X-ray diffraction allowed sufficient mineral accumulation for detection with calcium, Gla, and osteocalcin increasing allowed sufficient mineral accumulation for detection of an apatite-like pattern by X-ray diffraction with calcium, Gla, and osteocalcin increasing proportionately as mineral is deposited.

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Primary structure of monkey osteocalcin

Cited by 45 publications

References 22 publications

The propeptide of rat bone gamma-carboxyglutamic acid protein shares homology with other vitamin K-dependent protein precursors.

The propeptide of rat bone gamma-carboxyglutamic acid protein shares homology with other vitamin K-dependent protein precursors.

Osteocalcin protein sequences of Neanderthals and modern primates

Calcergy and calciphylaxis: Timed appearance of γ-carboxyglutamic acid and osteocalcin in mineral deposits

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