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ABSTRACT: One of the major limitations of studying cancer-bone metastasis has been the lack of an appropriate ex-vivo model which can be used under defined conditions that simulates closely the in vivo live bone microenvironment in response to cancer-bone interactions. We have developed and utilized a three-dimensional (3D) cancer-bone metastasis model using free-floating live mouse calvarial bone organs in the presence of cancer cells in a roller tube system. In such co-cultures under hypoxia and a specifically defined bone remodeling stage, viz., resorption system, cancer cells showed a remarkable affinity and specificity for the "endosteal side" of the bone where they colonize and proliferate. This was concurrent with differentiation of resident stem/progenitor cells to osteoclasts and bone resorption. In contrast, under bone formation conditions this model revealed different pathophysiology where the breast cancer cells continued to induce osteoclastic bone resorption whereas prostate cancer cells led to osteoblastic bone formation. The current 3D model was used to demonstrate its application to studies involving chemical and biochemical perturbations in the absence and presence of cancer cells and cellular responses. We describe proof-of-principle with examples of the broad versatility and multi-faceted application of this model that adds another dimension to the ongoing studies in the cancer-bone metastasis arena.
Biomaterials 11/2011; 33(4):1065-78. · 7.40 Impact Factor
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ABSTRACT: Hepatocyte nuclear factor-4α (HNF-4α), a liver-enriched transcription factor, is essential for liver development and function. HNF-4α regulates a large number of liver-specific genes, many of which are modulated by injury. While HNF-4α function is regulated by phosphorylation, only a limited number of phosphorylation sites in HNF-4α have been identified, and the roles of HNF-4α phosphorylation after injury are unexplored. To address these issues, we have carried out an extensive quantitative mass spectrometry (MS)-based analysis of HNF-4α serine and threonine phosphorylation in response to cytokine stimulation. Studies were performed in HNF-4α-enriched HepG2 cells treated with cytokines for 3 h or left untreated, followed by chemical derivatization of the phosphoserine and phosphothreonine residues using stable isotopic variants of dithiothreitol (DTT) and MS analysis. This has allowed the identification and relative quantification of 12 serine/threonine phosphorylation sites in HNF-4α. Eight of these phosphorylation sites and their sensitivity to cytokine stimulation have not been previously reported. We found that cytokine treatment leads to an increase of HNF-4α phosphorylation in several phosphopeptides. The phosphorylation of HNF-4α mediated by protein kinase A (PKA) significantly reduces HNF-4α binding activity, which mimics the repressive effect of cytokines on HNF-4α binding, and the inhibition of PKA activity by PKA inhibitor can partially recover the reduced HNF-4α binding activity induced by cytokines. These results suggest that the mechanism that alters HNF-4α binding after cytokine stimulation involves modulation of specific HNF-4α phosphorylation dependent, in part, on a PKA signaling pathway.
Biochemistry 06/2011; 50(23):5292-300. · 3.42 Impact Factor
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ABSTRACT: To date, only a handful of phosphoproteins with important biological functions have been identified and characterized in oral fluids, and these include some of the abundant protein constituents of saliva. Whole saliva (WS) samples were trypsin digested, followed by chemical derivatization using dithiothreitol (DTT) of the phospho-serine/threonine-containing peptides. The DTT-phosphopeptides were enriched by covalent disulfide-thiol interchange chromatography and analysis by nanoflow liquid chromatography and electrospray ionization tandem mass spectrometry (LC-ESI-MS/MS). The specificity of DTT chemical derivatization was evaluated separately under different base-catalyzed conditions with NaOH and Ba(OH)(2), blocking cysteine residues by iodoacetamide and enzymatic O-deglycosylation prior to DTT reaction. Further analysis of WS samples that were subjected to either of these conditions provided supporting evidence for phosphoprotein identifications. The combined chemical strategies and mass spectrometric analyses identified 65 phosphoproteins in WS; of these, 28 were based on two or more peptide identification criteria with high confidence and 37 were based on a single phosphopeptide identification. Most of the identified proteins (∼80%) were previously unknown phosphoprotein components. This study represents the first large-scale documentation of phosphoproteins of WS. The origins and identity of WS phosphoproteome suggest significant implications for both basic science and the development of novel biomarkers/diagnostic tools for systemic and oral disease states.
Analytical Biochemistry 12/2010; 407(1):19-33. · 3.00 Impact Factor
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ABSTRACT: Past studies of bone extracellular matrix phosphoproteins such as osteopontin and bone sialoprotein have yielded important biological information regarding their role in calcification and the regulation of cellular activity. Most of these studies have been limited to proteins extracted from mammalian and avian vertebrates and nonvertebrates. The present work describes the isolation and purification of two major highly glycosylated and phosphorylated extracellular matrix proteins of 70 and 22 kDa from herring fish bones. The 70-kDa phosphoprotein has some characteristics of osteopontin with respect to amino acid composition and susceptibility to thrombin cleavage. Unlike osteopontin, however, it was found to contain high levels of sialic acid similar to bone sialoprotein. The 22-kDa protein has very different properties such as very high content of phosphoserine (∼270 Ser(P) residues/1000 amino acid residues), Ala, and Asx residues. The N-terminal amino acid sequence analysis of both the 70-kDa (NPIMA(M)ETTS(M)DSKVNPLL) and the 22-kDa (NQDMAMEASSDPEAA) fish phosphoproteins indicate that these unique amino acid sequences are unlike any published in protein databases. An enzyme-linked immunosorbent assay revealed that the 70-kDa phosphoprotein was present principally in bone and in calcified scales, whereas the 22-kDa phosphoprotein was detected only in bone. Immunohistological analysis revealed diffusely positive immunostaining for both the 70- and 22-kDa phosphoproteins throughout the matrix of the bone. Overall, this work adds additional support to the concept that the mechanism of biological calcification has common evolutionary and fundamental bases throughout vertebrate species.
Journal of Biological Chemistry 11/2010; 285(46):36170-8. · 4.77 Impact Factor
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ABSTRACT: Individual aspects of the mode of action of histatin 5, a human salivary antifungal protein, have been partially elucidated, but the mechanism likely involves a complex set of events that have not been characterized. Previous evidence points toward histatin-induced alterations in mitochondrial function. The purpose of the present study was to verify and quantify changes in the mitochondrial proteome of Candida albicans treated with histatin 5. Cell killing was determined by plating and differential protein expression levels in the mitochondrial samples were determined by quantitative proteomics approaches employing mTRAQ and ICAT labeling and Western blotting. Relative quantitation ratios were established for 144 different proteins. Up-regulated mitochondrial proteins were predominantly involved in genome maintenance and gene expression, whereas proteins that constitute the respiratory enzyme complexes were mostly down-regulated. The differential expression of ATP synthase gamma chain and elongation factor 1-alpha were confirmed by Western blotting by comparison to levels of cytochrome c which were unchanged upon histatin treatment. The mTRAQ and ICAT proteomics results suggest that key steps in the histatin 5 antifungal mechanism involve a bioenergetic collapse of C. albicans, caused essentially by a decrease in mitochondrial ATP synthesis.
Journal of Proteome Research 11/2010; 10(2):646-55. · 5.11 Impact Factor
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ABSTRACT: Human salivary statherin inhibits both primary and secondary calcium phosphate precipitation and, upon binding to hydroxyapatite, associates with a variety of oral bacteria. These functions, crucial in the maintenance of tooth enamel integrity, are located in defined regions within the statherin molecule. Proteases associated with saliva, however, cleave statherin effectively, and it is of importance to determine how statherin functional domains are affected by these events. Statherin was isolated from human parotid secretion by zinc precipitation and purified by reversed-phase high performance liquid chromatography (RP-HPLC). To characterize the proteolytic process provoked by oral proteases, statherin was incubated with whole saliva and fragmentation was monitored by RP-HPLC. The early formed peptides were structurally characterized by reversed phase liquid chromatography electrospray-ionization tandem mass spectrometry. Statherin was degraded 3.6× faster in whole saliva than in whole saliva supernatant. The main and primary cleavage sites were located in the N-terminal half of statherin, specifically after Arg(9), Arg(10), and Arg(13); after Phe(14) and Tyr(18); and after Gly(12), Gly(15), Gly(17) and Gly(19) while the C-terminal half of statherin remained intact. Whole saliva protease activities separated the charged N-terminus from the hydrophobic C-terminus, negatively impacting on full length statherin functions comprising enamel lubrication and inhibition of primary calcium phosphate precipitation. Cryptic epitopes for bacterial binding residing in the C-terminal domain were likewise affected. The full characterization of the statherin peptides generated facilitates the elucidation of their novel functional roles in the oral and gastro-intestinal environment.
Journal of Proteome Research 10/2010; 9(10):5413-21. · 5.11 Impact Factor
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ABSTRACT: Proteases present in oral fluid effectively modulate the structure and function of some salivary proteins and have been implicated in tissue destruction in oral disease. To identify the proteases operating in the oral environment, proteins in pooled whole saliva supernatant were separated by anion-exchange chromatography and individual fractions were analyzed for proteolytic activity by zymography using salivary histatins as the enzyme substrates. Protein bands displaying proteolytic activity were particularly prominent in the 50-75 kDa region. Individual bands were excised, in-gel trypsinized and subjected to LC/ESI-MS/MS. The data obtained were searched against human, oral microbial and protease databases. A total of 13 proteases were identified all of which were of mammalian origin. Proteases detected in multiple fractions with cleavage specificities toward arginine and lysine residues, were lactotransferrin, kallikrein-1, and human airway trypsin-like protease. Unexpectedly, ten protease inhibitors were co-identified suggesting they were associated with the proteases in the same fractions. The inhibitors found most frequently were alpha-2-macroglobulin-like protein 1, alpha-1-antitrypsin, and leukocyte elastase inhibitor. Regulation of oral fluid proteolysis is highly important given that an inbalance in such activities has been correlated to a variety of pathological conditions including oral cancer.
PROTEOMICS - CLINICAL APPLICATIONS 07/2009; 3(7):810-820. · 1.81 Impact Factor
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ABSTRACT: We have determined transmembrane protein tyrosine phosphorylation (outside-in signaling) in cultured osteoclasts and macrophages in response to added native purified bone sialoprotein (nBSP) and its dephosphorylated form (dBSP). There were selective/differential and potent inhibitory effects by dBSP and minimal effect by nBSP on intracellular tyrosine phosphorylation in macrophages and osteoclasts. Further studies on the downstream gene expression effects led to identification of a large number of differentially expressed genes in response to nBSP relative to dBSP in both macrophages and osteoclasts. These studies were extended to a bone resorption model using live mouse neonatal calvarial bone organ cultures stimulated by parathyroid hormone (PTH) to undergo bone resorption. Inclusion of nBSP in such cultures showed no effect on type I collagen telopeptide fragment release, hence overall bone resorption, whereas addition of dBSP abolished the PTH-induced bone resorption. The inhibition of bone resorption by dBSP was shown to be unique since in complementary experiments use of integrin receptor binding ligand, GRGDS peptide, offered only partial reduction on overall bone resorption. Quantitative RANKL analysis indicated that mechanistically the PTH-induced bone resorption was inhibited by dBSP via down-regulation of the osteoblastic RANKL production. This conclusion was supported by the RANKL analysis in cultured MC3T3-E1 osteoblast cells. Overall, these studies provided direct evidence for the involvement of covalently bound phosphates on BSP in receptor mediated "outside-in" signaling via transmembrane tyrosine phosphorylation with concurrent effects on downstream gene expressions. The use of a live bone organ culture system augmented these results with further evidence that links the observed in vivo variable state of phosphorylation with bone remodeling.
Biochemistry 07/2009; 48(29):6876-86. · 3.42 Impact Factor
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ABSTRACT: The present study was undertaken to investigate the rate and mode of degradation of individual histatin proteins in whole saliva to establish the impact on its functional domains. Pure synthetic histatins 1, 3, and 5 were incubated with whole saliva supernatant as the enzyme source, and peptides in the resultant digests were separated by reverse-phase-HPLC and structurally characterized by electrospray ionization-tandem mass spectrometry. The overall V(max)/K(m) ratios, a measure of proteolytic efficiency, were on the order of histatin-5 > histatin-3 > histatin-1. Mathematical models predict that histatins 1, 3, and 5 levels in whole saliva stabilize at 5.1, 1.9, and 1.2 microM, representing 59, 27, and 11% of glandular histatins 1, 3, and 5 levels, respectively. Monitoring of the appearance and disappearance of histatin fragments yielded the identification of the first targeted enzymatic cleavage sites as K(13) and K(17) in histatin 1, R(22), Y(24), and R(25) in histatin 3, and Y(10), K(11), R(12), K(13), H(15), E(16), K(17), and H(18) in histatin 5. The data indicate that metal-binding, antifungal, and wound-healing domains are largely unaffected by the primary cleavage events in whole saliva, suggesting a sustained functional activity of these proteins in the proteolytic environment of the oral cavity.
The FASEB Journal 04/2009; 23(8):2691-701. · 5.71 Impact Factor
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ABSTRACT: The nonsterile environment of the oral cavity facilitates substantial proteolytic processing, not only of resident salivary proteins but also of dietary proteins. To gain insight into whole saliva enzymatic processes, the in vivo generated peptides in this oral fluid were subjected to nano-flow liquid chromatography electrospray ionization tandem mass spectrometry. The 182 peptides identified were predominantly derived from acidic and basic proline-rich proteins, statherin, and histatins. The proteolytic cleavages in the basic proline-rich proteins occurred preferentially after a Gln residue with predominant specificity for the tripeptide Xaa-Pro-Gln, where Xaa in the P(3) position was mostly represented by Lys. Using the synthetic substrates Lys-Pro-Gln-pNA and Gly-Gly-Gln-pNA, the overall K(m) values were determined to be 97 +/- 7.7 and 611 +/- 28 microm, respectively, confirming glutamine endoprotease activity in whole saliva and the influence of the amino acids in positions P(2) and P(3) on protease recognition. The pH optimum of Lys-Pro-Gln-pNA hydrolysis was 7.0, and the activity was most effectively inhibited by antipain and 4-(2-aminoethyl) benzenesulfonyl fluoride, was metal ion-dependent, and not inhibited by cysteine protease inhibitors. A systematic evaluation of enzyme activities in various exocrine and nonexocrine contributors to whole saliva revealed that the glutamine endoprotease is derived from dental plaque and likely microbial in origin. The P(1) site being occupied by a Gln residue is a nonarchetype with respect to known proteases and indicates the presence of novel glutamine-specific endoprotease(s) in oral fluid.
Journal of Biological Chemistry 08/2008; 283(29):19957-66. · 4.77 Impact Factor
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ABSTRACT: Osteopontin is a noncollagenous, phosphorylated extracellular glycoprotein, expressed in mineralized and nonmineralized tissues, organs and body fluids. The protein contains an RGD tripeptide cell-binding motif, and is subjected to a variety of posttranslational modifications that play important roles in its multiple biological functions, such as bone remodeling and inhibition of pathological calcification. In this study, we have expressed bovine osteopontin in a prokaryotic system and identified the seven amino acid residues phosphorylated in vitro by CKII.
Journal of Cellular Biochemistry 03/2008; 103(3):852-6. · 2.87 Impact Factor
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ABSTRACT: Salivary diagnostics for oral as well as systemic diseases is dependent on the identification of biomolecules reflecting a characteristic change in presence, absence, composition, or structure of saliva components found under healthy conditions. Most of the biomarkers suitable for diagnostics comprise proteins and peptides. The usefulness of salivary proteins for diagnostics requires the recognition of typical features, which make saliva as a body fluid unique. Salivary secretions reflect a degree of redundancy displayed by extensive polymorphisms forming families for each of the major salivary proteins. The structural differences among these polymorphic isoforms range from distinct to subtle, which may in some cases not even affect the mass of different family members. To facilitate the use of modern state-of-the-art proteomics and the development of nanotechnology-based analytical approaches in the field of diagnostics, the salient features of the major salivary protein families are reviewed at the molecular level. Knowledge of the structure and function of salivary gland-derived proteins/peptides has a critical impact on the rapid and correct identification of biomarkers, whether they originate from exocrine or non-exocrine sources.
Annals of the New York Academy of Sciences 04/2007; 1098:22-50. · 3.15 Impact Factor
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ABSTRACT: Bone sialoprotein (BSP) is one of the major non-collagenous glycosylated phosphoproteins of the extracellular matrix in bone. In vitro studies suggest that BSP may play important roles in the initiation and/or growth of calcium-phosphate crystals. To investigate the potential role of BSP in more complex in vivo environments, we implanted purified bovine BSP with type-I collagen as a carrier into surgically created rat calvarial defects and thoracic subcutaneous pouches. The responses to the implants were assessed by histochemistry, immunohistochemistry, in situ hybridization, quantitative real-time PCR, and biochemical analyses. BSP-collagen, but not collagen alone, elicited mineral deposition in the matrix of proliferating cells near the dura at days 4-5 followed by osteoblast differentiation and synthesis of new bone in the mid-portion of the calvarial defects. In contrast, implantation of BSP-collagen into subcutaneous pouches did not induce calcification or osteogenesis over the same experimental period. We explored the underlying mechanisms for the site-specific responses to BSP-collagen implants and found that higher levels of calcium content and alkaline phosphatase activity at the cranial site at days 2-5 were associated with the BSP-mediated calcification. We also found that BSP stimulated osteoblast differentiation through up-regulation of cbfa1 and osterix, key transcription factors of osteoblast differentiation, which occurred in the calvarial defects but not in the subcutaneous tissue. These results demonstrate that BSP stimulates calcification and osteogenesis in a site-specific manner, and that local environment and the specificities of responding cells may play critical roles in the function of BSP in vivo.
Calcified Tissue International 10/2006; 79(3):179-89. · 2.38 Impact Factor
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ABSTRACT: Bone sialoprotein is an extracellular noncollagenous acidic protein that plays a role in bone mineralization and remodeling. Its expression is restricted to mineralized tissues and is subjected to variety of posttranslational modifications including phosphorylation and glycosylation. We have expressed the full-length and half domains of bovine bone sialoprotein in a prokaryotic system and identified the phosphorylation sites of casein kinase II. The N-terminal automated solid-phase sequencing defined four phosphorylated peptides: residues 28-38 (LEDS(P)EENGVFK), 51-86 (FYPELKRFAVQSSS(P)DS(P)S(P)EENGNGDS(P)S(P)EEEEEEEETS(P)), 151-165 (EDES(P)DEEEEEEEEEE), and 295-305 (GRGYDS(P)YDGQD). Nine phosphoserines were identified within the four peptides. Seven of them were in the N-terminus (S31, S64, S66, S67, S75, S76, and S86) and two were in the C-terminus (S154 and S300) of the protein.
Biochemical and Biophysical Research Communications 08/2005; 333(2):443-7. · 2.48 Impact Factor
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ABSTRACT: The objective of this study was to determine the relative efficacy of currently available alloplastic bone repair materials in the healing of rat calvarial defects histologically, histomorphometrically and biochemically. A representative material was selected from six major classes of bone repair materials and placed in 4 mm diameter calvarial defects of 6-week-old male Sprague-Dawley rats (five animals in the control and each of the six experimental groups). The outcomes were assessed after 2 months for alkaline phosphatase (ALP) activity and after 4 months of healing for histomorphometry. The tested alloplastic implant materials did not significantly increase ALP activity or the amount of new bone formation in the healing of rat calvarial defects relative to controls (P > 0.05). However, when the implant material itself was included in the analysis, significant differences were observed (P < 0.05). Additionally, the tested materials varied in their ability to bridge the bony defect. These data suggest that the rate of bone formation cannot be increased beyond control levels, rather the advantage of implant materials may be in their efficiency in filling the defect through incorporation of the material into the healing site and rapidly bridging the wound.
The European Journal of Orthodontics 10/2004; 26(5):475-82. · 0.89 Impact Factor
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ABSTRACT: Bone sialoprotein (BSP) is a multifunctional, highly phosphorylated, and glycosylated protein with key roles in biomineralization and tissue remodeling. This work identifies the complete topographical distribution and precise location of both the in vitro and in vivo phosphorylation sites of bovine BSP by a combination of state-of-the-art techniques and approaches. In vitro phosphorylation of native and deglycosylated BSPs by casein kinase II identified seven phosphorylation sites by solid-phase N-terminal peptide sequencing that were within peptides 12-22 (LEDS(P)EENGVFK), 42-62 (FAVQSSSDSS(P)EENGNGDS(P)S(P)EE), 80-91 (EDS(P)DENEDEES(P)E), and 135-145 (EDES(P)DEEEEEE). The in vivo phosphorylation regions and sites were identified by use of a novel thiol reagent, 1-S-mono[(14)C]carboxymethyldithiothreitol. This approach identified all of the phosphopeptides defined by in vitro phosphorylation, but two additional phosphopeptides were defined at residues, 250-264 (DNGYEIYES(P)ENGDPR), and 282-289 (GYDS(P)YDGQ). Furthermore, use of native BSP and matrix-assisted laser desorption ionization time-of-flight mass spectrometry identified several of the above peptides, including an additional phosphopeptide at residues 125-130 (AGAT(P)GK) that was not defined in either of the in vitro and in vivo studies described above. Overall, 7 in vitro and 11 in vivo phosphorylation sites were identified unequivocally, with natural variation in the quantitative extent of phosphorylation at each in vivo phosphorylation site.
Journal of Biological Chemistry 06/2004; 279(19):19808-15. · 4.77 Impact Factor
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Erdjan Salih
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ABSTRACT: A novel radioactive thiol reagent, 1-S-[3H]carboxymethyl-dithiothreitol (DTT-S-C[3H(2)]CO(2)H, [3H]CM-DTT), was designed and synthesized at the micromole level by reaction of dithiothreitol with tritiated iodoacetic acid (I-C[3H(2)].CO(2)H). The reaction progress was followed by reverse-phase high-performance liquid chromatography (RP-HPLC) and matrix-assisted laser desorption/ionization time-of-flight mass spectrometry. The usefulness of the synthesized reagent was evaluated in a series of experimental approaches. (i) The synthetic phosphopeptide, NSVS(P)EEGRGDSV, was derivatized by [3H]CM-DTT separated from excess reagent by RP-HPLC. The extent of derivatization was quantitated in terms of the mol of P-Ser/mol of peptide by 3H counting, and the location of the phosphoserine was defined by the N-terminal Edman degradation sequence analysis as being the fourth amino acid residue from the N terminus. (ii) A sample of trypsin-digested alpha-casein was derivatized with [3H]CM-DTT, peptides were separated by RP-HPLC, and aliquots of each fraction were counted for 3H label within the peptide map which rapidly pinpointed the original four phosphoserine-containing peptides. This demonstrated the utility of the synthesized radioactive thiol agent in rapid purification and identification of phosphopeptides from HPLC peptide mapping of proteolytic digests of phosphoproteins. (iii) The [3H]CM-DTT was also used to determine the extent of phosphorylation of phosphoproteins both qualitatively by sodium dodecyl sulfate-polyacrylamide gel electrophoresis and autoradiography and quantitatively by 3H counting. The synthesized radioactive thiol reagent [3H]CM-DTT proved to be very efficient and sensitive and should be adaptable to a wide range of routinely utilized laboratory approaches in many fields of the biological sciences.
Analytical Biochemistry 09/2003; 319(1):143-58. · 3.00 Impact Factor
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Erdjan Salih
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ABSTRACT: The present study for the first time evaluated both the in vitro and in vivo phosphorylation regions of bone sialoprotein (BSP) by utilizing multiple approaches and techniques. The in vitro phosphorylation sites were determined by 32P-labeling of native BSP using purified casein kinase II (CKII), followed by peptide mapping and solid-phase N-terminal sequence analyses. The in vivo phosphorylation sites were determined by (i) derivatization with 1-S-[14C]carboxymethyl-dithiothreitol ([14C] CM-DTT) of the proteolytic digests of BSP, followed by isolation and N-terminal peptide sequence analysis; and (ii) analyzing the proteolytic peptides of native BSP using matrix-assisted laser desorption/ionization-time of flight-mass spectrometry (MALDI-TOF-MS). Native BSP incorporated approximately 2.5 mol of phosphate/mol of BSP by CKII, which were distributed over four major peptide peaks and three shoulder peaks within the peptide map with varying degrees of phosphorylation. Further studies using the [14C] CM-DTT thiol reagent indicated that native and deglycosylated BSP incorporated 5.84 and 5.80 mol of 14C/mol of BSP, respectively. This confirmed that there were approximately 5.8 mol P-Ser/mol of BSP naturally (in vivo) occurring phosphorylation sites and that there was no overlap between the phosphorylation and glycosylation sites. The 5.8 mol P-Ser/mol BSP reflects the total number of mols of naturally occurring phosphorylation, phosphorylated in vivo by CKII (4.1 mol), protein kinase C (0.9 mol), and cGMP-dependent kinase (0.8 mol). Peptide N-terminal sequence analyses of both in vitro (32P) and in vivo (14C) phosphorylated peptides indicated that the phosphorylated residues were predominantly on the N-terminal half of the protein that included recognition sequences for CKII, e.g., LESDEENGVFK (residues 12-22).
Connective Tissue Research 02/2003; 44 Suppl 1:223-9. · 1.20 Impact Factor
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ABSTRACT: Implants of allogenic demineralized bone matrix were placed in distinct in vivo environments, i.e. calvarial (bony) and subcutaneous (soft tissue) sites. Detailed analyses of the biochemical components were performed. Quantitative levels of osteopontin (OPN), bone sialoprotein (BSP) and calcium phosphate (Ca-P) deposition within each implant environment varied as a function of new bone formation, and were substantially different in samples from calvarial and subcutaneous sites. Quantification of the extent of phosphorylation of affinity-purified OPN and BSP from such implants indicated that: (i) the number of mols of phosphoserine (P-Ser)/mol of affinity-purified OPN or BSP varied as a function of implant time and bone formation within both implant sites, and (ii) the 'effective P-Ser concentration' provided by the total OPN and BSP within each implant site varied and increased as a function of time, being approx. 5-fold higher for BSP in calvarial compared with subcutaneous implants. Peak levels of mols of P-Ser/mol of BSP coincided with maximum rates of Ca-P deposition in calvarial implants. Levels of OPN phosphorylation from both calvarial and subcutaneous implants also indicated fluctuations as a function of bone formation. Hence the present study, for the first time, provides direct evidence of natural variation in the extent of phosphorylation of both OPN and BSP as a function of time of mineralized tissue formation. Further evaluation of the data provides the first evidence of a direct and linear relationship between the rate of Ca-P deposition and the ratio of P-Ser-BSP/P-Ser-OPN for calvarial implants. Data for subcutaneous implants failed to provide such correlation. Overall, the present work demonstrates that the natural biological progression of the process of biomineralization follows strict criteria consistent with the anatomical location. Biomineralization fails to proceed in the same way in a soft tissue environment.
Biochemical Journal 07/2002; 364(Pt 2):465-74. · 4.90 Impact Factor
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Erdjan Salih
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ABSTRACT: The general fields of biological sciences have seen phenomenal transformations in the past two decades at the level of data acquisition, understanding biological processes, and technological developments. Those advances have been made partly because of the advent of molecular biology techniques (which led to genomics) coupled to the advances made in mass spectrometry (MS) to provide the current capabilities and developments in proteomics. However, our current knowledge that approximately 30,000 human genes may code for up to 1 million or more proteins disengage the interface between the genome sequence database algorithms and MS to generate a major interest in independent de novo MS/MS sequence determination. Significant progress has been made in this area through procedures to covalently modify peptide N- and C-terminal amino-acids by sulfonation and guanidination to permit rapid de novo sequence determination by MS/MS analysis. A number of strategies that have been developed to perform qualitative and quantitative proteomics range from 2D-gel electrophoresis, affinity tag reagents, and stable-isotope labeling. Those procedures, combined with MS/MS peptide sequence analysis at the subpicomole level, permit the rapid and effective identification and quantification of a large number of proteins within a given biological sample. The identification of proteins per se, however, is not always sufficient to interpret biological function because many of the naturally occurring proteins are post-translationally modified. One such modification is protein phosphorylation, which regulates a large array of cellular biochemical pathways of the biological system. Traditionally, the study of phosphoprotein structure-function relationships involved classical protein chemistry approaches that required protein purification, peptide mapping, and the identification of the phosphorylated peptide regions and sites by N-terminal sequence analysis. Recent advances made in mass spectrometry have clearly revolutionized the studies of phosphoprotein biochemistry, and include the development of specific strategies to preferentially enrich phosphoproteins by covalent-modifications that incorporate affinity tags that use the physicochemical properties of phosphoaminoacids. The phosphoserine/phosphothreonine-containing proteins/peptides are derivatized under base-catalyzed conditions by thiol agents; mono- and di-thiol reagents both have been used in such studies. The thiol agent may have: (i) an affinity tag for protein enrichment; (ii) stable-isotopic variants for relative quantitation; or (iii) a combination of the moieties in (i) and (ii). These strategies and techniques, together with others, are reviewed, including their practical application to the study of phosphoprotein biochemistry and structure-function. The consensus of how classical protein chemistry and current MS technology overlap into special case of proteomics, namely "phosphoproteomics," will be discussed.
Mass Spectrometry Reviews 24(6):828-46. · 10.46 Impact Factor
