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M J Glimcher

Publications and source records attributed to M J Glimcher.

At least 37 records · Page 2Linked to original sources
PubMed1997

Identification of the phosphorylated sites of metabolically 32P-labeled osteopontin from cultured chicken osteoblasts.

Osteopontin (OPN) is one of the major secretory phosphoproteins in both calcifying and non-calcifying tissues. Evidence has accumulated for the biological importance of the phosphoproteins and, in particular, the phosphate groups in bone formation, resorption, and calcification. The precise locations of the phosphate groups in the OPN molecule were determined by metabolically labeling OPN with 32P in cultured chicken osteoblasts, followed by purification to homogeneity. N-terminal sequencing showed a single sequence of WPVSKRQHAISA, consistent with that deduced from both cDNA, and previous amino acid sequencing of the protein isolated from chicken bone. Three 32P-labeled peptides were isolated by reverse-phase high performance liquid chromatography of thrombin-digested, 32P-labeled OPN. The N-terminal sequencing of each of these thrombin fragments gave single sequences as follows: WPVSKSRQHAIS, SHHTHRYHQDHVD, and ASKLRKAARKL, with approximate molecular masses of 5, 30, and 20 kDa. These data demonstrate that 32P was incorporated throughout the N- to C-terminal sequence of the protein. Thrombin specifically cleaved chicken OPN at two sites: between Arg-22 and Ser-23, which generated the 5-kDa N-terminal end fragment, and another between Lys-138 and Ala-139, which generated the 30- and 20-kDa fragments. To further define the exact locations of the phosphorylated amino acids and the surrounding amino acid sequences, OPN was digested with trypsin, which generated seven major 32P-labeled peptides whose amino acid sequences were determined. The phosphorylated peptide regions of osteopontin were identified as amino acids 8-18 (QHAIS*AS*S*EEK), 39-54 (LASQQTHYS*S*EENAD), 150-171 (LIEDDAT*AEVGDSQLAGLWLPK), 179-191 (ELAQHQSVENDSR), 194-205 (FDS*PEVGGDSK), 214-219 (ES*LASR), and 239-248 (HSIENNEVTR). The phosphorylated amino acid sites are followed by an asterisk (*). Of the seven identified phosphorylated peptide regions, three were localized on the N-terminal end of the osteopontin molecule (with five phosphorylated serines) and contained the sequence motifs that were phosphorylated by casein kinase II type(s), whereas the remaining four peptides are concentrated toward the C-terminal half of the molecule (with five phosphorylated residues) and contained recognition motifs for other kinases as well as casein kinase II.

Amino Acid Sequence↗
PubMed1996

Phosphorylation of purified bovine bone sialoprotein and osteopontin by protein kinases.

The large number of covalently bound phosphates on the extracellular phosphoproteins osteopontin (OPN) and bone sialoprotein (BSP) have been implicated in biological functions such as mineral deposition and osteoclast binding. In the present study the state of phosphorylation of BSP and OPN was evaluated by in vitro 32P labeling using a series of protein kinases and quantification. Both the purified bovine BSP and OPN were radiolabeled by [32P]ATP and factor-independent protein kinase. Quantification of 32P radioactivity incorporated on dephosphorylated BSP and OPN provided 6.6 and 8.9 mol of phosphate incorporated/mol, respectively. Native OPN incorporated 1.07 and BSP 2.46 mol of phosphate/mol by factor-independent protein kinase. These data led to calculations that OPN and BSP, respectively, contain 7.83 and 4.14 mol of phosphate/mol in their natural state. Thrombin digests of 32P-labeled BSP showed radioactivity to be associated with fragment of approximately molecular mass values 30 kDa (N-terminal half), with no observable radioactivity associated with the 40-kDa fragment (C-terminal half). Similar experiments with 32P-labeled OPN provided two radiolabeled thrombin fragments, with molecular mass 30 kDa (N-terminal half) and 20 kDa (C-terminal half), both were radioactive. The major phosphorylation was associated with the N-terminal half containing 7.0 mol of phosphate, and 1.9 mol of phosphate were associated with the C-terminal half. Additional experiments of in vitro phosphorylation of OPN and BSP by several other known protein kinases were carried out. cAMP-dependent protein kinase showed no phosphorylation of OPN or BSP, while protein kinase C and cGMP-dependent protein kinase led to minor phosphorylation, each of the latter introduced about 1 mol of phosphate/mol of OPN and BSP molecule.

Amino Acid Sequence↗
PubMed1996

Receptor-ligand interaction between CD44 and osteopontin (Eta-1).

The CD44 family of surface receptors regulates adhesion, movement, and activation of normal and neoplastic cells. The cytokine osteopontin (Eta-1), which regulates similar cellular functions, was found to be a protein ligand of CD44. Osteopontin induces cellular chemotaxis but not homotypic aggregation, whereas the inverse is true for the interaction between CD44 and a carbohydrate ligand, hyaluronate. The different responses of cells after CD44 ligation by either osteopontin or hyaluronate may account for the independent effects of CD44 on cell migration and growth. This mechanism may also be exploited by tumor cells to promote metastasis formation.

Amino Acid Sequence↗
PubMed1996

Chondrodysplasia and neurological abnormalities in ATF-2-deficient mice.

Activating transcription factor-2 (ATF-2) is a basic region leucine zipper protein whose DNA target sequence is the widely distributed cAMP response element (CRE). We report here that mice carrying a germline mutation in ATF-2 demonstrated unique actions of ATF-2 not duplicated by other ATF/CREB family members. Mutant mice had decreased postnatal viability and growth, with a defect in endochondral ossification at epiphyseal plates similar to human hypochondroplasia. The animals had ataxic gait, hyperactivity and decreased hearing. In the brain, there were reduced numbers of cerebellar Purkinje cells, atrophic vestibular sense organs and enlarged ventricles. Unlike CREB alpha/delta-deficient mice whose main defect is in long-term potentiation, the widespread abnormalities in ATF-2 mutant mice demonstrate its absolute requirement for skeletal and central nervous system development, and for maximal induction of select genes with CRE sites, such as E-selectin.

Abnormalities, Multiple↗
PubMed1996

Protein kinases of cultured osteoblasts: selectivity for the extracellular matrix proteins of bone and their catalytic competence for osteopontin.

The enzyme activities of the major kinases found within the cytosolic and microsomal fractions of embryonic avian calvaria osteoblasts were assayed for their specificity for various noncollagenous extracellular matrix (ECM) proteins of bone. At least 6 proteins with M(r)'s of 66, 58, 50, 36, 30, and 22 kD out of more than 30 of the noncollagenous proteins of the bone ECM were phosphorylated by the kinase(s) found in both osteoblast cellular fractions. The purification and N-terminal sequence analysis of three of the above proteins, M(r)'s 66 and 58 kD (+50 kD), identified them as chicken bone sialoprotein (BSP) and osteopontin (OPN), respectively. Heparin, a specific inhibitor of factor-independent protein kinase (FIPK) activity, blocked the phosphorylation of all six ECM proteins by the microsomal kinase(s) but only inhibited the phosphorylation of the 66, 50, and 36 kD by the cytosolic enzyme(s). Casein kinase II (a known FIPK) showed a similar phosphorylation pattern of the same bone ECM proteins as the FIPK(s) found in osteoblast cell extracts, while purified cyclic adenosine monophosphate (cAMP)-dependent protein kinase did not phosphorylate any of the ECM proteins. Use of dephosphorylated casein showed that in comparison with casein kinase II, casein was a poor substrate for the FIPK found in the osteoblast cellular extracts. Further studies, using FIPK(s) of osteoblasts and purified chicken OPN or bacterially produced recombinant murine OPN as a substrate, showed that both species of OPN were excellent substrates for the FIPK(s) found in osteoblasts. The phosphorylation of the purified chicken and recombinant mouse OPNs were evaluated by quantitative analysis using commercially available protein kinases. cAMP-dependent kinase showed no phosphorylation of either protein, and cyclic guanodine monophosphate (cGMP)-dependent kinase and protein kinase C incorporated 1.2 and 0.5 mol phosphate/mol OPN, respectively. However, both chicken and mouse OPNs were significantly phosphorylated by casein kinase II (9.3 and 9.0 mol of phosphate/mol of OPN, respectively). These results demonstrate that the noncollagenous proteins of the bone ECM, and in particular OPN, are predominantly phosphorylated by FIPK(s), and this class of kinase is the major enzyme found within the microsomal fraction of osteoblasts.

Amino Acid Sequence↗
PubMed1996

X-ray diffraction, electron microscopy, and Fourier transform infrared spectroscopy of apatite crystals isolated from chicken and bovine calcified cartilage.

Apatite crystals of the calcified zone of the subarticular cartilaginous growth plates of the long bones of young growing chickens and calves were isolated by low temperature reaction with hydrazine and plasma ashing and examined by electron microscopy, electron diffraction and microprobe analysis, and computer-generated deconvolution of the spectra obtained by Fourier transform infrared spectroscopy. The crystal habit was that of wide, very thin, relatively long rectangular plates, which tended to form small clusters of crystals, possibly because reaction with hydrazine alone did not remove all of the organic matrix constituents. Further reaction with low power plasma ashing released more of the isolated crystals although to a lesser extent than was possible with bone. Stereograms of the small clusters showed that many of the crystals in the small isolated aggregates of crystals were bent and/or curved. Together with the resultant overlap of individual adjacent crystals, they also produced images of sharp, very dense lines, reminiscent of the electron-dense needle or rod-like appearances frequently observed by transmission electron microscopy of thin sections of calcified cartilage and thought to represent the habit of the apatite crystals. No true rod or needle-like crystals were observed in the isolated crystals. Although the overall general apatitic structure of the apatite crystals was similar to that of the apatitic crystals of bone, the individual crystals were significantly larger than those of bone from the same specimen, and there were small but significant differences in the concentrations of acid phosphate and carbonate groups and in their short range order.

Animals↗
PubMed1996

Protein kinases of cultured chicken osteoblasts that phosphorylate extracellular bone proteins.

Cytosolic and microsomal protein kinase preparations from cultured chicken osteoblasts were found to phosphorylate up to six major proteins with Mrs 66, 58, 50, 36, 32, and 22 kDa in chicken bone extract. Use of heparin led to the conclusion that these proteins were predominantly phosphorylated by factor-independent protein kinase (FIPK) present both in microsomal and cytosolic preparations. It was confirmed that microsomal preparation contained predominantly FIPK, whereas cytosolic preparation contained additional kinases, that can phosphorylate the bone proteins. Use of purified chicken bone osteopontin (OPN) (58 kDa) and recombinant OPN led to the same conclusions. The identify of the protein kinases was clearly established by using a series of synthetic peptide substrates. Quantitative analysis utilizing pure protein kinases and purified chicken bone OPN, recombinant mouse OPN, and bovine bone OPN and BSP led to introduction of approximately 9 moles of phosphate/mole of OPN and 6.6 moles phosphate/mole bovine bone sialoprotein (BSP) by casein kinase II. cGMP-dependent protein kinase and protein kinase C both introduced 0.5-1.2 moles phosphate/mole of OPN and BSP, whereas cAMP-dependent protein kinase led to no significant phosphorylation of OPN or BSP. Consistent with the above results, sites of phosphorylation identified for OPN (metabolically labeled) and BSP (labeled by casein kinase II) revealed that predominant phosphorylated sites have recognition sequences for FIPK.

Amino Acid Sequence↗
PubMed1996

Characterization of the apatite crystals of bone and their maturation in osteoblast cell culture: comparison with native bone crystals.

Calcium phosphate crystals deposited in the organic matrix synthesized by chick bone osteoblasts in culture were studied by x-ray and electron diffraction, Fourier transform infrared spectroscopy and chemical composition. The amounts of mineral phase deposited with time and the extent of calcification (% of mineral phase in the tissue) were also determined as a function of time, as were the nature of the changes in the short range order of the crystals. The amount of mineral deposited and the extent of calcification increased with time; the tissue not only contained more crystals of apatite, but the extent of calcification also increased with time as it does in vivo. After 30 days of culture the extent of calcification in the cell culture matrix was similar to that in late chick embryonic and early postnatal chick tibiae. The nature of the CO3 and HPD4 environments were similar to those found in vivo although the concentrations of these ions and the changes in their concentrations with time appeared to develop more slowly in cell culture than they do in vivo. However, the general overall pathway of maturation was similar in cell culture to that observed in vivo.

Animals↗
PubMed1995

Gene expression and phosphorylation of mouse osteopontin.

Osteopontin is expressed in many different cell types and has been proposed to play several functions. Distinct forms of the protein have been detected. Various tissues and cell lines from mouse, however, exhibit two classes of transcripts with different 5'-untranslated ends but with an identical coding region (exons II through VII). These transcripts do not arise from the alternative splicing of coding exons. These results suggest that posttranslational modifications of osteopontin, such as phosphorylation, are a major mechanism to generate different forms of the protein. Mouse osteopontin was expressed in E. coli and used as a model to study its phosphorylation.

Animals↗
PubMed1995

Characterization of the major non-collagenous proteins of chicken bone: identification of a novel 60 kDa non-collagenous phosphoprotein.

Chicken bone powder, sequentially extracted with 4M guanidine HCl (NG), 0.3M HCl (H), 0.5 NaCl at neutral pH (NS), and 4M guanidine HCl pH 7.4 (G), preferentially solubilized osteocalcin (OC), osteopontin (OPN), alpha 2 HS-glycoprotein, bone sialoprotein (BSP), and osteonectin (ON). These studies provide the first amino acid sequences for a non-mammalian form of ON and both N-terminal and internal amino acid sequences obtained for ON demonstrated that the avian form of this protein is more than 80% conserved for all the peptides sequenced in this study. A novel approximately 60 kDa protein was identified with a unique N-terminal amino acid sequence and several internal amino acid sequences. Amino acid composition of this protein indicates that it is similar to other acidic glycosylated phosphoprotein of bone, and it is phosphorylated principally on serine residues, although a small amount of phosphorylated threonine residues were also detected. Solubility characteristics of this protein differed from those of ON, OPN and Decorin, and, it was not recognized by polyclonal antibodies to ON, BSP, OPN or by a monoclonal antibody to Decorin. This protein was not present in chicken serum and no comparable sequences were found in the GenBank or EMBL protein sequence database. These studies provide the first sequence for a non-mammalian form of ON and identify a novel acidic bone protein.

Amino Acid Sequence↗
PubMed1995

Structural and chemical characteristics and maturation of the calcium-phosphate crystals formed during the calcification of the organic matrix synthesized by chicken osteoblasts in cell culture.

The calcium-phosphate (CA-P) crystals formed in the extracellular organic matrix synthesized by chicken osteoblasts in cell culture were examined after 30, 40, and 60 days of culture by a number of physical and chemical techniques including chemical analyses, X-ray diffraction, transmission electron microscopy of isolated crystals, and resolution-enhanced Fourier transform infrared spectroscopy. The data reveal that the solid inorganic calcium-phosphate phase consists of a very poorly crystalline apatite, having a low carbonate content and containing acid phosphate groups. The chemical and structural characteristics are generally similar to the apatite crystals found in young newly synthesized bone but there were small but significant differences found. The major significant differences noted were the rate at which maturational changes occurred in the crystals formed in cell culture compared with those noted in vivo and in synthetic carbonate apatite crystals equilibrated with the same cell culture medium, and the persistence of labile groups, especially HPO4(-2) ions during a relatively long period of incubation. Despite extensive chemical efforts to degrade the organic constituents and to disperse the individual crystals isolated from the organic matrix constituents, a large proportion of the crystals were found to be organized in both loosely and densely packed relatively large roughly spherical aggregates. A few of the aggregates were organized in the form of fibrils with the crystals oriented with their c-axes roughly parallel to the long axes of the crystal aggregate. With briefer periods of chemical treatment, larger aggregates of crystals were occasionally observed in which there was a distinct axial periodicity of approximately 70 nm. In such collagen-crystal fragments, the crystals were well-oriented with their c-axis roughly parallel to the long axes of the aggregate similar to the organization and relationships between crystals and collagen fibrils in native bone. Isolated crystals were in the shape of thin plates. At the end of 30 days of culture, many of the crystals were clearly larger than those observed in native chick bone, except for those in the very youngest (7- to 8-day-old) embryos. At the end of 40 and 60 days of culture, the crystal habit remained as thin plates but the crystals were predominantly smaller, similar to those found in older embryo and postnatal chicken bone. The marked tendency of the crystals to form relatively large aggregates that resist dispersion by techniques that readily disperse the crystals of bone, and the presence of a significant number of larger crystals has also been observed in studies of calcified cartilage. Resolution enhanced FTIR spectroscopy revealed the presence of a high concentration of labile phosphate groups, especially after 30 days of culture and just after the plateau of mineralization is reached.

Animals↗
PubMed1995

Isolation of calcium-phosphate crystals of bone by non-aqueous methods at low temperature.

We have developed low temperature nonaqueous solution methods as well as low power plasma ashing for the degradation of the organic matrix of bone power which have permitted us to obtain bone crystals essentially free of organic matrix constituents without any significant change in their composition, overall structure, or internal short-range order. We have also been able to disperse the crystals, which has made it possible to examine the isolated crystals by X-ray diffraction and resolution-enhanced Fourier transform infrared (FTIR) spectroscopy and isolated single crystals by high resolution transmission electron microscopy (TEM) and electron diffraction. TEM of isolated single crystals of fish, chicken, mouse and bovine bone have clearly demonstrated that the crystals are very thin plates. No rod or needle-like crystals were observed in any of the bone samples in the four species studied including the earliest crystals deposited. Although there were variations in the size distribution of the crystals in the different species studied, in general the average crystal dimensions were very similar.

Animals↗