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It is widely accepted that cellular processes are controlled by protein phosphorylation and has become increasingly clear that protein degradation, localization and conformation as well as protein-protein interaction are the examples of subsequent cellular events modulated by protein phosphorylation. Enamel matrix proteins belong to members of the secretory calcium binding phosphoprotein (SCPP) family clustered on chromosome 4q21, and most of the SCPP phosphoproteins have at least one S-X-E motifs (S; serine, X; any amino acid, E; glutamic acid). It has been reported that mutations in C4orf26 gene, located on chromosome 4q21, are associated with autosomal recessive type of Amelogenesis Imperfecta (AI), a hereditary condition that affects enamel formation/mineralization. The enamel phenotype observed in patients with C4orf26 mutations is hypomineralized and partially hypoplastic, indicating that C4orf26 protein may function at both secretory and maturation stages of amelogenesis. The previous in vitro study showed that the synthetic phosphorylated peptide based on C4orf26 protein sequence accelerates hydroxyapatite nucleation. Here we show the molecular cloning of Gm1045, mouse homologue of C4orf26, which has 2 splicing isoforms. Immunohistochemical analysis demonstrated that the immunolocalization of Gm1045 is mainly observed in enamel matrix in vivo. Our report is the first to show that FAM20C, the Golgi casein kinase, phosphorylates C4orf26 and Gm1045 in cell cultures. The extracellular localization of C4orf26/Gm1045 was regulated by FAM20C kinase activity. Thus, our data point out the biological importance of enamel matrix-kinase control of SCPP phosphoproteins and may have a broad impact on the regulation of amelogenesis and AI.

The 24 kD form of secreted phosphoprotein (SPP-24), a cytokine-binding bone matrix protein with various truncated C-terminal products, is primarily synthesized by the liver. SPP-24 shares homology with fetuin-A, a potent vascular and soft tissue calcification inhibitor and SPP-24 is one component of calciprotein particles (CPPs), a circulating fetuin–mineral complex. The limited molecular evidence to date suggests that SPP-24 may also function as an inhibitor of bone formation and ectopic vascular calcification, potentially through bone morphogenic protein 2 (BMP-2) and Wnt-signaling mediated actions. The C-terminal products of SPP-24 bind to BMP-2 and attenuate BMP-2-induced bone formation. The aim of this study was to assess circulating SPP-24 in relation to kidney function and in concert with markers of mineral metabolism in humans. SPP-24 was measured in the serum of total of 192 subjects using ELISA-based measurements. Subjects were participants of one of two cohorts: (1) mGFR Cohort (n = 80) was participants of a study of measured GFR (mGFR) using inulin urinary clearance, recruited mostly from a chronic kidney disease clinic with low-range kidney function (eGFR 38.7 ± 25.0 mL/min/1.73 m2) and (2) CaMOS Cohort (n = 112) was a subset of randomly selected, community-dwelling participants of year 10 of the Canadian Multicentre Osteoporosis Study with eGFR in the normal range of 75.0 ± 15.9 mL/min/1.73 m2. In the combined cohort, the mean SPP-24 was 167.7 ± 101.1 ng/mL (range 33.4–633.6 ng/mL). The mean age was 66.5 ± 11.3, 57.1% female and mean eGFR (CKD-EPI) was 59.9 ± 27.0 mL/min/1.73 m2 (range 8–122 mL/min/1.73 m2). There was a strong inverse correlation between SPP-24 and eGFR (R = − 0.58, p

Ectopic calcification is a major cause of bioprosthetic heart valve failure. New therapeutic opportunities are offered by the growing understanding that ectopic calcification is an actively regulated process involving several key gene products. One of these products, osteopontin (OPN), is a glycosylated phosphoprotein previously shown to inhibit apatite crystal formation, induce carbonic anhydrase II, and promote mineral resorption. In this study, OPN-deficient mice (OPN-/-) were utilized as an in vivo model to stimulate the ectopic calcification of glutaraldehyde-fixed bovine pericardium (GFBP) tissue and to examine OPN delivery and structure-function relationships with respect to its anti-calcific activity. Significant calcification of GFBP tissue was obtained within 7 days of subcutaneous implantation in OPN-/- mice. Direct rescue of the calcification phenotype was achieved by the administration of exogenous recombinant rat, histidine-fused OPN (rat His-OPN) to the implant site via soluble injection (up to 72% mitigation achieved) or adsorption onto the implant materials (up to 91% mitigation achieved). Effects were specific, since neither fibronectin nor polyhistidine alone could mitigate calcification of GFBP. The maximum anti-calcific effect was achieved only when rat His-OPN was adequately phosphorylated and contained a functional arginine-glycine-aspartate (RGD) cell adhesive domain. Furthermore, CAII levels in host cells surrounding GFBP were greatest when phosphorylated, RGD-containing rat His-OPN was adsorbed. These data suggest that both physical inhibition, mediated by phosphorylation sites in OPN, as well as the induction of CAII and mineral regression, mediated by the RGD domain, contribute to the unique ability of OPN to mitigate ectopic calcification of bioprosthetic valve tissue.

The present study further characterizes an extract from immature, human tooth apicies from which an intact dentin phosphoprotein has been identified. Third molar apicies from developing roots were decalcified in 10% EDTA until Ca2+ was undetectable in the decalcifying solution. The crude extract was run on 7.5% SDS-PAGE and stained with "Stains-All." Four distinct bands were found and the molecular weights were 140, 60, 50, and 34 k. When run on a SDS-PAGE under nonreducing conditions the 60, 50, and 34 k bands were absent. These results suggest that the lower molecular weight bands may be subunits of the larger protein. The extract was then further purified by adding CaCl2 and MgCl2 to precipitate the phosphoprotein. The precipitate was subjected to a DEAE-Sepharose CL6B column and eluted by 0-0.7 M NaCl gradient solution. The amino acid composition of the purified phosphoprotein was determined and the extract was found to be rich in serine and aspartic acid residues. The N-terminal peptide Asp-Asp-Pro was identified. The sequence of the three amino acids is identical to rat incisor phosphoprotein.

Localization of a ∼66 kD glycosylated phosphoprotein during morphogenesis of the embryonic chick tibia has been accomplished using immunohistochemistry. Although initial expression of the tibial osteoblast phenotype is detected as early as stage 28.5, with the deposition of osteoid matrix beginning at stage 30, little or no immunoreactivity against the ∼66 kD glycosylated phosphoprotein is observed in pre-osteoblasts, osteoblasts, osteocytes, or in the uncalcified osteoid matrix during the early events of tibia development. Immunoreactivity was first observed at stage 32 when mineralization of the osteoid matrix is initiated. At this and all later stages, the phosphoprotein is located almost exclusively in the extracellular matrix at the mineralization front with essentially no detectable staining in the adjacent unmineralized osteoid matrix. Similarly, no cellular staining is observed when even the lightly mineralized extracellular matrix is strongly immunoreactive. Only scant immunostaining is present over the heavily mineralized regions, although demineralization of these areas with EDTA exposes a low intensity, punctate staining pattern. Additionally, cryosections of developing calvaria stained with this antiserum only display reactivity in regions of bone matrix undergoing mineralization. These localization studies support the hypothesis that this phosphoprotein is intimately associated with the process of bone matrix mineralization in the developing chick long bone.

Histidinoalanine is a naturally occurring cross-linking amino acid found in mammalian bone, dentin, cartilage and aorta, and also in the extracellular fluid of bivalve clams. The exact components from which this amino acid derives have not been characterized in the case of bone. In this study, the origin of histidinoalanine in bone was investigated in order to clarify the possible role of the matrix component containing this amino acid in bone mineralization. An extract of bovine bone powder with 0.5 M EDTA/1M NaCl was desalted and separated by calcium-induced precipitation. The supernatant fraction was chromatographed successively on Sepharose CL-6B, hydroxyapatite, DEAE cellulose, and reverse-phase high-performance liquid chromatography columns. The fraction with the highest affinity for hydroxyapatite was found to contain the highest concentration of histidinoalanine. A phosphoprotein with a molecular weight of about 24 K that contained 1.2 mol of histidinoalanine per molecule was isolated. The amino acid composition of this fraction showed a high content of acidic amino acids and serine, at least 42% of which was phosphorylated. These results suggested a possible formation mechanism of histidinoalanine that includes the beta-elimination of phosphoserine, producing a dehydroalanine residue and an addition of histidine on it. It was concluded that this low molecular weight phosphoprotein is one of the major sources of histidinoalanine in bovine bone.

Osteopontin is an acidic phosphoprotein containing casein kinase II (CKII) phosphorylatable sites and an acidic amino acid cluster. The metabolically 32P-labelings of both serines and threonines in vitro in osteopontin immunoprecipitated from rat osteoblast-like ROS 17/2.8 cells may suggest that casein kinase II catalyzes this modification. The enzyme occurs in microsomal fractions of rat osteoblast-like ROS 17/2.8 cells. Subcellular fractions containing endoplasmic reticulum and Golgi apparatus were isolated by differential centrifugation and were identified according to their ultrastructures and the presence of marker enzymes such as glucose-6-phosphatase and thiamine pyrophosphatase, respectively. both fractions phosphorylated the partially dephosphorylated osteopontin and the specific substrate peptide RRREEETEEE. Endoplasmic reticulum-catalyzed peptide phosphorylation was 2.7 times lower than that of Golgi although both endoplasmic reticulum- and Golgi-catalyzed peptide reactions were 50% inhibited by 20 and 100 ng/ml heparin, respectively. Western blot analysis revealed that both fractions contained osteopontin and microsomal CKII. Furthermore, microsomal CKII was immunogold-labeled in endoplasmic reticulum and Golgi apparatus. Heparin inhibition and utilization of [gamma-32P]GTP as a phosphate donor by both fractions confirmed their capacity to phosphorylate osteopontin. The results suggest that microsomal CKII modifies the acidic matrix proteins during transportation. These matrix phosphoproteins may participate in the mineralization process of hard tissues.

Osteopontin, bone sialoprotein, and bone acidic glycoprotein-75 are three acidic phosphoproteins that are isolated from the mineralized phase of bone matrix, are synthesized by osteoblastic cells, and are generally restricted in their distribution to calcified tissues. Although each is a distinct gene product, these proteins share aspartic/glutamic acid contents of 30–36% and each contains multiple phosphoryl and sialyl groups. These properties, plus a strict relationship of acidic macromolecules with cell-controlled mineralization throughout nature, suggest functions in calcium binding and nucleation of calcium hydroxyapatite crystal formation. However, direct proof for such roles is still largely indirect in nature. The purpose of this review is to present two speculative hypotheses regarding acidic phosphoprotein function. The goal was to use new sequence information along with database comparisons to develop a structural rationalization of how these proteins may function in calcium handling by bone. For example, our analysis has identified a conserved polyacidic stretch in all three phosphoproteins which we propose mediates metal binding. Also, conserved motifs were identified that are analogous with those for casein kinase II phosphorylation sites and whose number correlates well with that of phosphoryl groups/protein. A two-state conformational model of calcium binding by bone matrix acidic phosphoproteins is described which incorporates these findings.

Ethylenediaminetetraacetate and hydrochloric acid (EDTA) (HCl) extracts of cementum were fractionated by molecular sieving, ion exchange chromotography, and reverse phase high precision liquid chromatography (HPLC). Nine fractions were isolated, all of which contained serine phosphate, threonine phosphate, and high concentrations of aspartic acid (asp) and glutamic acid (glu). Five of the fractions obtained by repeated HPLC consisted of a single band by SDS-PAGE; the others contained at least one other minor component. All of the protein bands stained with both Rhodamine B and alcian blue, the latter consistent with analytical determinations that demonstrated that the phosphoprotein component contained a significant amount of carbohydrate, including neuraminic acid.

An immunological species comparison, using a monospecific rabbit polyclonal antibody directed against rat incisor alpha-phosphophoryn, has been undertaken to assess the similarity in epitope regions among various dentin phosphophoryns (PP) that were prepared from human, monkey, bovine, ovine, and echinoderm teeth. Dentin extracellular matrix proteins were extracted with a standard method using 0.5 M EDTA in the presence of enzyme inhibitors. Final phosphophoryn purification was performed on DEAE ion exchange HPLC. Cross-reactivity of the polyclonal antibody was examined by enzyme-linked immunosorbant assay (ELISA) and dot-blot. The results of this investigation demonstrate a cross-reactivity of the rat-alpha-phosphophoryn antibody (anti-RIPP) with at least one phosphophoryn component in each dentin studied, indicating the existence of similar antigenic determinants among these proteins. It would seem that these epitope regions have been strongly conserved since the epitope region is also present in the phosphoprotein of echinoderm teeth. No cross-reactivity was found with phosvitin (a phosphoserine-rich phosphoprotein), rat serum albumin, bovine serum albumin, or collagen type IV. However, a new and distinct second cross-reactive phosphophoryn, not calcium ion-precipitable, was found in the EDTA insoluble fraction from the teeth. These results indicate that dentin phosphophoryns are specific phenotypic markers for odontoblast expression. Because of the species cross-reactivity, the polyclonal anti-RIPP antibody may be a useful probe in studying the distribution of phosphophoryns in other species, such as human teeth.

When compared to that from sham-operated controls, sarcoplasmic reticulum isolated from skeletal muscle of uremic rabbits had a lower rate of calcium uptake and storing capacity. In vivo administration of 1,25-dihydroxycholecalciferol [1,25(OH)2D3] restored the values in uremic animals toward normal. To obtain information about the mechanisms responsible for these differences, phosphorylation of the calcium transport ATPase was studied. The steady-state levels of phosphoprotein in uremic membranes were lower and returned to normal when the secosteroid was administered. Electrophoresis of the membranes phosphorylated with 32P-inosine triphosphate (32P-ITP) showed that the differences were related to a 100,000 dalton protein. The rate of phosphoprotein formation, determined with 32P-ITP and at 0 degrees C, was considerably lower in uremic than in control animals. Pretreatment with 1,25(OH)2D3 prevented this change. The hypothesis is advanced that the vitamin D metabolite affects the steady-state concentration and rate constant of formation of active sites in the Ca-ATPase. These results may partly explain the altered Ca transport function of the sarcoplasmic reticulum in experimental uremia.

A monoclonal antibody was raised against phosphophoryn, a unique noncollagenous phosphoprotein in dentin. Mouse myeloma NS-I cells were fused with spleen cells obtained from BALB/c mice immunized with phosphophoryn from fetal calf tooth germs. Mice inoculated with the hybridoma produced ascites fluid containing the antibody and this reacted only with a band of phosphophoryn transblotted from polyacrylamide gel. Immunohistochemical studies with the antibody showed that phosphophoryn was present in odontoblasts, odontoblastic processes and dentin, but not in the matrix of predentin, and that the phosphophoryn content of the dentin layer was high at and around the predentin-dentin junction and gradually decreased toward the enamel layer. The area corresponding to mantle dentin was not stained with the antibody.

Trypsin digestion and successive calcium-induced precipitation of the insoluble bovine dentin matrix effectively separated the collagen and phosphoprotein fractions which were firmly associated together in this material. Amino acid analysis by four different systems revealed that the lysinoalanine and histidinoalanine, which had been previously reported to occur in the human dentin collagen, were concentrated in the phosphoprotein fraction but were not present in the collagen fraction. Furthermore, it was found that the free-type phosphoprotein which was isolated from EDTA extract of dentin powder also contained both "cross-linking" amino acids. The results indicated the both "cross-links" distributed within the dentin phosphoprotein and were not likely to contribute the unique stability of dentin collagen.

Monoclonal and polyclonal antibodies were raised against 150k, 60k, 32k, and 15k phosphoprotein components of 14-week chicken bone. Hybridomas were prepared by immunizing Balb/c mice with 150k, 60k, or 32k phosphoproteins followed by fusion of their spleen cells with X63-Ag8.653 myeloma cells. Polyclonal antibodies to the 60k, 32k, and 15k phosphoprotein components were produced in immunized rabbits. Immunological cross-reactivity between antigen and antibody was examined by enzyme-linked immunosorbent assay (ELISA) and dot-blotting. There was cross-reactivity between the 150k, 32k, and 15k phosphoprotein components and between the 150k, 90k, and 60k components. The antibodies raised against 60k component do not bind 32k and 15k antigens.

Fractionation of the EDTA-soluble, noncollagenous proteins of the organic matrix of chicken bone by Sephadex G-100 molecular sieving has revealed that the majority of the organic phosphorus is present in two fractions, from one of which a homogeneous phosphoprotein has been isolated. The purified phosphoprotein has an apparent molecular weight of 12,000 and contains both O-phosphoserine and O-phosphothreonine. 31P-NMR spectroscopy demonstrates that all of the organic phosphorus exists in the form of phosphomonoesters which have an average pK2 of 6.8. The phosphoprotein is highly acidic due to its high content of dicarboxylic acids in addition to the presence of organic phosphorus. The characteristic amino acid composition of the phosphoprotein establishes its noncollagenous nature and highlights the differences among bone, dentin, and enamel phosphoproteins. The absence of gamma-carboxyglutamic acid distinguishes it from osteocalcin, the noncollagenous gamma-carboxyglutamic acid-containing peptide of bone matrix.

Normal human dentin was demineralized in 0.6 N HCl and then extracted in 1.0 M NaCl, 0.5 M Tris/HCl, pH 7.6 in the presence of neutral protease inhibitors. All of the soluble phosphorus-containing proteins were extracted directly in the 0.6 N HCl demineralizing solution; none were collected in the 1.0 M NaCl neutral pH extraction. The principal phosphoprotein was precipitated from solution by 1.0 M CaCl2 and subjected to further chromatographic purification. This fraction proved to be a typical phosphophoryn with Asp and Ser + PSer, in near equimolar amounts, accounting for approximately 75 residue percent of the protein. The second major organic phosphate-containing component was a peptide, Mr approximately 2,000. It was calcium precipitable and its amino acid composition showed a relationship to phosphophoryn. The residual collagenous matrix, which also contained organic phosphate, was digested with CNBr and the phosphate-containing moiety isolated. This had a composition indicative of a complex of collagen and phosphophoryn. Thus, in spite of the reports by Leaver and colleagues that human dentin contains neither soluble nor matrix-bound phosphophoryns, these data show that human dentin, like rat, hamster, rabbit, porcine, and bovine dentins, does contain a phosphophoryn as a major noncollagenous protein.

Amelogenins, enamelins, and soluble dentin proteins were sequentially separated under dissociative conditions from morphologically characterized molar tooth germs of 4-, 6-, and 7-day-old hamsters. Polyacrylamide gel electrophoretic, gel filtration chromatographic, and amino acid compositional data of neonatal hamster amelogenin extracts were in general agreement with those obtained from fetal bovine enamel under similar extraction conditions. As development progressed (e.g., 4 vs. 7 days of life), changes in amelogenin proteins were manifested by altered values of all biochemical parameters measured. A high molecular weight (approximately 160,000-200,000 daltons) Stains-All-positive protein band was observed for all hamster enamelin extracts on SDS gels. Amino acid compositional data from this "enamel crystal protein," partially purified by dissociative gel-filtration chromatography, are presented. The hamster dentin phosphoprotein was partially purified by ion-exchange chromatography in 7M urea. The molecular weight (75,000-80,000 daltons) and amino acid composition of this protein were similar to those of rat incisor dentin phosphoprotein, but different from those of the fetal bovine phosphoprotein.

Several phosphoprotein preparations (phosvitin, rat incisor and fetal calf molar dentin phosphoproteins) all inhibit apatite growth/replication from pre-existing crystal seeds in metastable solutions. Two stages of the crystal growth process were inhibited by these phosphoproteins. First an initial lag period was induced, probably associated with seed surface phenomena. This period was prolonged indefinitely when a combination of phosphoprotein precoated seeds was used together with soluble phosphoproteins in the crystal growth reaction. Second, the phosphoproteins prolonged that stage of the reaction where octacalcium phosphate is the predominant mineral phase present prior to its conversion to the final apatite product. Pre-treatment of the phosphoproteins with calcium diminished their inhibitory activity to seeded crystal growth as well as towards de novo apatite formation in synthetic extracellular fluids. The presence of collagen diminished the inhibitory activity of the phosphoproteins towards de novo precipitation but had no effect on phosphoprotein-modulated apatite crystal growth in the seeded systems. These results suggest a potential regulatory role for phosphoproteins in dentin mineralization.