RESUMO
Although acellular cementum is essential for tooth attachment, factors directing its development and regeneration remain poorly understood. Inorganic pyrophosphate (PPi), a mineralization inhibitor, is a key regulator of cementum formation: tissue-nonspecific alkaline phosphatase (Alpl/TNAP) null mice (increased PPi) feature deficient cementum, while progressive ankylosis protein (Ank/ANK) null mice (decreased PPi) feature increased cementum. Bone sialoprotein (Bsp/BSP) and osteopontin (Spp1/OPN) are multifunctional extracellular matrix components of cementum proposed to have direct and indirect effects on cell activities and mineralization. Studies on dentoalveolar development of Bsp knockout (Bsp-/-) mice revealed severely reduced acellular cementum, however underlying mechanisms remain unclear. The similarity in defective cementum phenotypes between Bsp-/- mice and Alpl-/- mice (the latter featuring elevated PPi and OPN), prompted us to examine whether BSP is operating by modulating PPi-associated genes. Genetic ablation of Bsp caused a 2-fold increase in circulating PPi, altered mRNA expression of Alpl, Spp1, and Ank, and increased OPN protein in the periodontia. Generation of a Bsp knock-out (KO) cementoblast cell line revealed significantly decreased mineralization capacity, 50% increased PPi in culture media, and increased Spp1 and Ank mRNA expression. While addition of 2µg/ml recombinant BSP altered Spp1, Ank, and Enpp1 expression in cementoblasts, changes resulting from this dose were not dependent on the integrin-binding RGD motif or MAPK/ERK signaling pathway. Decreasing PPi by genetic ablation of Ank on the Bsp-/- mouse background reestablished cementum formation, allowing >3-fold increased acellular cementum volume compared to wild-type (WT). However, deleting Ank did not fully compensate for the absence of BSP. Bsp-/-; Ank-/- double-deficient mice exhibited mean 20-27% reduced cementum thickness and volume compared to Ank-/- mice. From these data, we conclude that the perturbations in PPi metabolism are not solely driving the cementum pathology in Bsp-/- mice, and that PPi is more potent than BSP as a cementum regulator, as shown by the ability to override loss of BSP by lowering PPi. We propose that BSP and PPi work in concert to direct mineralization in cementum and likely other mineralized tissues.
Assuntos
Calcificação Fisiológica , Cementogênese/efeitos dos fármacos , Difosfatos/farmacologia , Sialoproteína de Ligação à Integrina/metabolismo , Animais , Calcificação Fisiológica/efeitos dos fármacos , Cemento Dentário/efeitos dos fármacos , Cemento Dentário/metabolismo , Deleção de Genes , Regulação da Expressão Gênica/efeitos dos fármacos , Sialoproteína de Ligação à Integrina/deficiência , Camundongos Knockout , Periodonto/metabolismo , Fenótipo , Proteínas de Transporte de Fosfato/metabolismo , Fosforilação/efeitos dos fármacosRESUMO
Bone sialoprotein (BSP) is an acidic phosphoprotein with collagen-binding, cell attachment, and hydroxyapatite-nucleating properties. BSP expression in mineralized tissues is upregulated at onset of mineralization. Bsp-null (Bsp(-/-)) mice exhibit reductions in bone mineral density, bone turnover, osteoclast activation, and impaired bone healing. Furthermore, Bsp(-/-) mice have marked periodontal tissue breakdown, with a lack of acellular cementum leading to periodontal ligament detachment, extensive alveolar bone and tooth root resorption, and incisor malocclusion. We hypothesized that altered mechanical stress from mastication contributes to periodontal destruction observed in Bsp(-/-) mice. This hypothesis was tested by comparing Bsp(-/-) and wild-type mice fed with standard hard pellet diet or soft powder diet. Dentoalveolar tissues were analyzed using histology and micro-computed tomography. By 8 wk of age, Bsp(-/-) mice exhibited molar and incisor malocclusion regardless of diet. Bsp(-/-) mice with hard pellet diet exhibited high incidence (30%) of severe incisor malocclusion, 10% lower body weight, 3% reduced femur length, and 30% elevated serum alkaline phosphatase activity compared to wild type. Soft powder diet reduced severe incisor malocclusion incidence to 3% in Bsp(-/-) mice, supporting the hypothesis that occlusal loading contributed to the malocclusion phenotype. Furthermore, Bsp(-/-) mice in the soft powder diet group featured normal body weight, long bone length, and serum alkaline phosphatase activity, suggesting that tooth dysfunction and malnutrition contribute to growth and skeletal defects reported in Bsp(-/-) mice. Bsp(-/-) incisors also erupt at a slower rate, which likely leads to the observed thickened dentin and enhanced mineralization of dentin and enamel toward the apical end. We propose that the decrease in eruption rate is due to a lack of acellular cementum and associated defective periodontal attachment. These data demonstrate the importance of BSP in maintaining proper periodontal function and alveolar bone remodeling and point to dental dysfunction as causative factor of skeletal defects observed in Bsp(-/-) mice.
Assuntos
Sialoproteína de Ligação à Integrina/fisiologia , Periodonto/patologia , Animais , Sialoproteína de Ligação à Integrina/genética , Camundongos , Camundongos KnockoutRESUMO
Bone sialoprotein (BSP) is a multifunctional extracellular matrix protein found in mineralized tissues, including bone, cartilage, tooth root cementum (both acellular and cellular types), and dentin. In order to define the role BSP plays in the process of biomineralization of these tissues, we analyzed cementogenesis, dentinogenesis, and osteogenesis (intramembranous and endochondral) in craniofacial bone in Bsp null mice and wild-type (WT) controls over a developmental period (1-60 days post natal; dpn) by histology, immunohistochemistry, undecalcified histochemistry, microcomputed tomography (microCT), scanning electron microscopy (SEM), transmission electron microscopy (TEM), and quantitative PCR (qPCR). Regions of intramembranous ossification in the alveolus, mandible, and calvaria presented delayed mineralization and osteoid accumulation, assessed by von Kossa and Goldner's trichrome stains at 1 and 14 dpn. Moreover, Bsp(-/-) mice featured increased cranial suture size at the early time point, 1 dpn. Immunostaining and PCR demonstrated that osteoblast markers, osterix, alkaline phosphatase, and osteopontin were unchanged in Bsp null mandibles compared to WT. Bsp(-/-) mouse molars featured a lack of functional acellular cementum formation by histology, SEM, and TEM, and subsequent loss of Sharpey's collagen fiber insertion into the tooth root structure. Bsp(-/-) mouse alveolar and mandibular bone featured equivalent or fewer osteoclasts at early ages (1 and 14 dpn), however, increased RANKL immunostaining and mRNA, and significantly increased number of osteoclast-like cells (2-5 fold) were found at later ages (26 and 60 dpn), corresponding to periodontal breakdown and severe alveolar bone resorption observed following molar teeth entering occlusion. Dentin formation was unperturbed in Bsp(-/-) mouse molars, with no delay in mineralization, no alteration in dentin dimensions, and no differences in odontoblast markers analyzed. No defects were identified in endochondral ossification in the cranial base, and craniofacial morphology was unaffected in Bsp(-/-) mice. These analyses confirm a critical role for BSP in processes of cementogenesis and intramembranous ossification of craniofacial bone, whereas endochondral ossification in the cranial base was minimally affected and dentinogenesis was normal in Bsp(-/-) molar teeth. Dissimilar effects of loss of BSP on mineralization of dental and craniofacial tissues suggest local differences in the role of BSP and/or yet to be defined interactions with site-specific factors.
Assuntos
Cementogênese , Dentinogênese , Ossos Faciais/patologia , Osteogênese , Osteopontina/genética , Crânio/patologia , Animais , Reabsorção Óssea , Cartilagem/metabolismo , Cemento Dentário/metabolismo , Dentina/metabolismo , Matriz Extracelular/metabolismo , Ossos Faciais/diagnóstico por imagem , Imageamento Tridimensional , Sialoproteína de Ligação à Integrina/metabolismo , Camundongos , Camundongos Knockout , Microscopia Eletrônica de Transmissão , Dente Molar/metabolismo , Odontogênese , Osteoclastos/metabolismo , Osteopontina/metabolismo , Reação em Cadeia da Polimerase , Ligante RANK/metabolismo , Crânio/diagnóstico por imagem , Dente/fisiologia , Raiz Dentária/metabolismo , Microtomografia por Raio-XRESUMO
Although osteopontin (OPN) is recognized generally as a secreted protein, an intracellular form of osteopontin (iOPN), associated with the CD44 complex, has been identified in migrating fibroblastic cells. Because both OPN and CD44 are expressed at high levels in osteoclasts, we have used double immunofluorescence analysis and confocal microscopy to determine whether colocalization of these proteins has functional significance in the formation and activity of osteoclasts. Analysis of rat bone marrow-derived osteoclasts revealed strong surface staining for CD44 and beta1- and beta3-integrins, whereas little or no staining for OPN or bone sialoprotein (BSP) was observed in nonpermeabilized cells. In permeabilized perfusion osteoclasts and multinucleated osteoclasts, staining for OPN and CD44 was prominent in cell processes, including filopodia and pseudopodia. Confocal microscopy revealed a high degree of colocalization of OPN with CD44 in motile osteoclasts. In cells treated with cycloheximide (CHX), perinuclear staining for OPN and BSP was lost, but iOPN staining was retained within cell processes. In osteoclasts generated from the OPN-null and CD44-null mice, cell spreading and protrusion of pseudopodia were reduced and cell fusion was impaired. Moreover, osteoclast motility and resorptive activity were significantly compromised. Although the area resorbed by OPN-null osteoclasts could be rescued partially by exogenous OPN, the resorption depth was not affected. These studies have identified an intracellular form of OPN, colocalizing with CD44 in cell processes, that appears to function in the formation and activity of osteoclasts.
Assuntos
Fusão Celular , Movimento Celular/fisiologia , Receptores de Hialuronatos/metabolismo , Osteoclastos/metabolismo , Sialoglicoproteínas/metabolismo , Animais , Imunofluorescência , Receptores de Hialuronatos/fisiologia , Camundongos , Camundongos Endogâmicos C57BL , Microscopia Confocal , Osteoclastos/citologia , Osteopontina , Ratos , Ratos Wistar , Sialoglicoproteínas/fisiologiaRESUMO
During endochondral ossification and bone remodeling, osteoprogenitors (OP) attach to the matrix and differentiate into osteoblasts. To identify matrix proteins binding specifically these precursors, fetal rat calvaria (RC) cells were plated for 5-20 min in serum-free medium, on wells coated with various proteins and saturated with bovine serum albumin (BSA) to block nonspecific binding sites. Adherent cells were either counted or grown to assess bone colony (nodule) formation. As each nodule originates from the clonal division of one OP, the ratio (nodules/100 cells attached) measures the proportion of OP among adherent cells. Of numerous purified matrix proteins tested, laminin-1 and tenascin inhibited cell attachment, whereas fibronectin, bone sialoprotein, and type I collagen increased cell attachment and others had no effect. Only laminin-1 and, to a lesser extent, tenascin, enriched the cell population in OP. Laminin-1 acted time- and dose-dependently. In experiments in which cell attachment to laminin-coated but unsaturated wells was ensured by plating for 24 h in 10% fetal calf serum, laminin-1 had no effect on cell attachment nor on OP differentiation. In contrast, repeated plating of RC cells on laminin-1-coated/saturated wells depleted the population in OP, confirming that OP selection was a cell-attachment effect. When RC cell populations isolated by successive collagenase extractions were compared, the highest rate of OP enrichment on laminin-1 was obtained with the earliest populations, which were the most responsive to dexamethasone, a marker of early OP stages. In conclusion, laminin-1 recruits in vitro, through a cell-attachment effect, OP present in early RC cell populations, of which laminins are abundant extracellular matrix components.
Assuntos
Laminina/fisiologia , Osteoblastos/fisiologia , Células-Tronco/fisiologia , Animais , Bovinos , Adesão Celular/fisiologia , Células Cultivadas , Proteínas da Matriz Extracelular/análise , Proteínas da Matriz Extracelular/fisiologia , RatosRESUMO
Osteopontin (OPN), a phosphorylated bone matrix glycoprotein, is an Arg-Gly-Asp (RGD)-containing protein that interacts with integrins and promotes in vitro attachment of a number of cell types, including osteoclasts. Gene knockout experiments support the idea that OPN is important in osteoclastic activity. We hypothesize that posttranslational modifications (PTMs) of OPN can influence its physiological function. Previous studies have suggested that phosphorylation of OPN and bone sialoprotein (BSP) is necessary for promoting osteoclast adhesion. However, no reports have explored the importance of phosphoserines and other PTMs in OPN-promoted bone resorption. To study this question, we determined the activities of different forms of OPN and BSP in three in vitro assays: attachment of osteoclasts; formation of actin rings; and bone resorption. For each assay, cells were incubated for 4-24 h, in the presence or absence of RGDS or RGES peptides, to test the involvement of integrin binding. In addition to OPN, activities of milk OPN (fully phosphorylated) and recombinant OPN (rOPN, no phosphate) were compared. We purified two forms of OPN (OPN-2 and OPN-5), which differ in the level of phosphorylation, and compared their activities. For comparison, the activities of BSP and recombinant BSP (rBSP) were determined. All forms of OPN, including rOPN, significantly increased attachment of tartrate-resistant acid phosphatase (TRAP)-positive osteoclasts. BSP and rBSP also promoted cell attachment. After 4 h of incubation, the proportion of cells with actin rings was increased with OPN, milk OPN, and BSP. In the presence of RGDS peptide, osteoclast retraction and the disruption of actin rings were observed, whereas no effect was seen with RGES. In the resorption assay, the number of pits and the total resorbed area per slice were increased in the presence of OPN, milk OPN, and BSP. As in other assays, the OPN enhancement of resorption was inhibited by RGDS, but not RGES, peptides. Significantly, rOPN and rBSP did not promote bone resorption. OPN-5 promoted resorption to a greater extent than OPN-2, and milk OPN significantly stimulated resorption to a greater extent than OPN. Our data suggest that: (1) the RGD sequence of OPN is essential in OPN-mediated cell attachment, actin ring formation, and bone resorption; and (2) some form of PTM, possibly phosphorylation, is necessary for in vitro osteoclastic bone resorption, but not for cell attachment and actin ring formation.
Assuntos
Reabsorção Óssea/metabolismo , Osteoclastos/citologia , Sialoglicoproteínas/metabolismo , Actinas/metabolismo , Animais , Reabsorção Óssea/induzido quimicamente , Adesão Celular/efeitos dos fármacos , Adesão Celular/fisiologia , Técnicas In Vitro , Sialoproteína de Ligação à Integrina , Oligopeptídeos , Osteoclastos/efeitos dos fármacos , Osteoclastos/fisiologia , Osteopontina , Fosforilação , Processamento de Proteína Pós-Traducional , Ratos , Proteínas Recombinantes/farmacologia , Sialoglicoproteínas/química , Sialoglicoproteínas/farmacologiaRESUMO
Mammalian bone sialoprotein (BSP) is a mineralized tissue-specific protein containing an RGD (arginine-glycine-aspartic acid) cell-attachment sequence and two distinct glutamic acid (glu)-rich regions, with each containing one contiguous glu sequence. These regions have been proposed to contribute to the attachment of bone cells to the extracellular matrix and to the nucleation of hydroxyapatite (HA), respectively. To further delineate the domains responsible for these activities, porcine BSP cDNA was used to construct expression vectors coding for two partial-length recombinant BSP peptides: P2S (residues 42-87), containing the first glutamic acid-rich domain; and P1L (residues 69-300), containing the second glutamic acid-rich region and the RGD sequence. These peptides were expressed in Escherichia coli as his-tag fusion proteins and purified by nickel affinity columns and FPLC chromatography. Digestion with trypsin released the his-tag fusion peptide, which generated P2S-TY (residues 42-87) and P1L-TY (residues 132-239). Using a steady-state agarose gel system, P2S-TY promoted HA nucleation, whereas P2S, P1L, and P1L-TY did not. This implies that the minimum requirement for nucleation of HA resides within the amino acid sequence of the first glutamic acid-rich domain, whereas the second glutamic acid-rich domain may require posttranslational modifications for activity. P1L, but not P2S, promoted RGD-mediated attachment of human gingival fibroblasts in a manner similar to that of native BSP. Deletion of the RGD domain or conversion of it to RGE (arginine-glycine-glutamic acid) abolished the cell-attachment activity of P1L. This suggests that, at least for human gingival fibroblasts, the major cell-attachment activity in the recombinant BSP peptides studied (residues 42-87 and 69-300) requires the RGD sequence located at the C-terminal domain.
Assuntos
Osso e Ossos/química , Durapatita/química , Sialoglicoproteínas/química , Sialoglicoproteínas/genética , Sequência de Aminoácidos , Animais , Osso e Ossos/metabolismo , Calcificação Fisiológica/fisiologia , Células Cultivadas , Cristalização , Fibroblastos/química , Fibroblastos/citologia , Expressão Gênica/fisiologia , Gengiva/citologia , Humanos , Sialoproteína de Ligação à Integrina , Dados de Sequência Molecular , Mutagênese Sítio-Dirigida/fisiologia , Oligopeptídeos/genética , Oligopeptídeos/metabolismo , Estrutura Terciária de Proteína , Proteínas Recombinantes/química , Proteínas Recombinantes/genética , Proteínas Recombinantes/isolamento & purificação , Sialoglicoproteínas/metabolismo , SuínosRESUMO
Osteopontin (OPN) is one of the major non-collagenous proteins in root cementum and other mineralized tissues. Although most of this mineral-seeking protein is thought to be produced by local tissue cells, some of it might enter the mineralizing matrix from the blood. To test this hypothesis, we followed the distribution of a single dose of purified porcine or rat 125I-labeled OPN injected i.v. in rats, in mineralizing and non-mineralizing tissues and in subcutaneously implanted collagenous implants. The animals were killed 30 or 48 hrs after injection. Tissues (calvaria, tibia, lower and upper jaws) were harvested and processed for radioautography and biochemical analysis. Tissues as well as calcifying collagenous implants proved to have taken up radiolabel. In EDTA extracts of long bones, the majority of the radiolabel was demonstrated to be associated with intact OPN. The iodinated protein was also found in the acellular extrinsic fiber cementum (acellular cementum) layer investing the continuously growing incisors, in laminae limitantes, cement lines, and in forming bone near the mineralization front. Further, the label was present in the circumpulpal dentin of the incisors, and some of it appeared to have been incorporated into developing enamel. It is concluded that OPN in acellular cementum and other mineralizing tissues may-at least partially-originate from sources outside the direct environment following its transportation via serum.
Assuntos
Circulação Sanguínea/fisiologia , Cemento Dentário/metabolismo , Fosfoproteínas/metabolismo , Sialoglicoproteínas/metabolismo , Calcificação de Dente/fisiologia , Animais , Bovinos , Colágeno/efeitos dos fármacos , Colágeno/metabolismo , Cemento Dentário/efeitos dos fármacos , Cemento Dentário/ultraestrutura , Imuno-Histoquímica , Radioisótopos do Iodo , Masculino , Microscopia Eletrônica , Osteopontina , Fosfoproteínas/administração & dosagem , Fosfoproteínas/isolamento & purificação , Ratos , Ratos Wistar , Sialoglicoproteínas/administração & dosagem , Sialoglicoproteínas/isolamento & purificação , Suínos , Fatores de Tempo , Calcificação de Dente/efeitos dos fármacosRESUMO
To study this relationship, specific antibodies were used to determine the distribution of these proteins in mature rat dental tissues. Staining for SPARC with affinity-purified polyclonal antibodies was prominent throughout molar and incisor ligaments, endosteal tissue, dental pulp and muscle. More moderate staining was observed in other soft tissues including the lamina propria of gingiva, whereas the staining of demineralized bone was weak and in dentine was barely detectable. A monoclonal antibody (MBP 322), raised against a denatured form of a small collagenous bone protein, reacted strongly with osteoblastic cells but more moderately with alveolar bone. A strong reaction, indicative of unfolded collagen, was also evident throughout the dental pulp and molar ligament, whereas in the incisor ligament staining was largely restricted to the tooth-related half. Moderate staining with this antibody was also observed in other soft tissues and in dentine. The monoclonal antibody also stained the nuclei of certain cells; notably, whereas most of the fibroblasts in the tooth-related half of the incisor ligament were stained strongly, only occasional nuclei of fibroblasts in the molar ligament and in the bone-related half of the incisor ligament showed immunoreactivity. The differential staining of nuclei provides evidence for phenotypic differences between fibroblast populations within these tissues. The prominence of SPARC in the ligament tissues is consistent with their embryonic characteristics, whereas unfolded collagen recognized by the MBP 322 antibody may indicate sites of rapid collagen remodelling.
Assuntos
Colágeno/farmacocinética , Osteonectina/farmacocinética , Periodonto/metabolismo , Dente/metabolismo , Animais , Núcleo Celular/ultraestrutura , Polpa Dentária/citologia , Polpa Dentária/metabolismo , Dentina/citologia , Dentina/metabolismo , Músculos Faciais/citologia , Músculos Faciais/metabolismo , Imuno-Histoquímica , Mandíbula/citologia , Mandíbula/metabolismo , Ligamento Periodontal/citologia , Ligamento Periodontal/metabolismo , Periodonto/citologia , Ratos , Ratos Endogâmicos , Dente/citologiaRESUMO
Bone sialoprotein (BSP) is an extracellular matrix protein found in mineralized tissues of the skeleton and dentition. BSP is multifunctional, affecting cell attachment and signaling through an RGD integrin-binding region, and acting as a positive regulator for mineral precipitation by nucleating hydroxyapatite crystals. BSP is present in cementum, the hard tissue covering the tooth root that anchors periodontal ligament (PDL) attachment. To test our hypothesis that BSP plays an important role in cementogenesis, we analyzed tooth development in a Bsp null ((-/-)) mouse model. Developmental analysis by histology, histochemistry, and SEM revealed a significant reduction in acellular cementum formation on Bsp (-/-) mouse molar and incisor roots, and the cementum deposited appeared hypomineralized. Structural defects in cementum-PDL interfaces in Bsp (-/-) mice caused PDL detachment, likely contributing to the high incidence of incisor malocclusion. Loss of BSP caused progressively disorganized PDL and significantly increased epithelial down-growth with aging. Bsp (-/-) mice displayed extensive root and alveolar bone resorption, mediated by increased RANKL and the presence of osteoclasts. Results collected here suggest that BSP plays a non-redundant role in acellular cementum formation, likely involved in initiating mineralization on the root surface. Through its importance to cementum integrity, BSP is essential for periodontal function.
Assuntos
Cementogênese/fisiologia , Cemento Dentário/patologia , Sialoproteína de Ligação à Integrina/fisiologia , Fosfatase Alcalina/análise , Perda do Osso Alveolar/patologia , Animais , Dentina/ultraestrutura , Epitélio/patologia , Incisivo/ultraestrutura , Sialoproteína de Ligação à Integrina/genética , Queratinas/análise , Camundongos , Camundongos Transgênicos , Microscopia Eletrônica de Varredura , Dente Molar/ultraestrutura , Odontogênese/genética , Odontogênese/fisiologia , Osteoclastos/patologia , Osteopontina/análise , Perda da Inserção Periodontal/patologia , Ligamento Periodontal/patologia , Ligante RANK/análise , Reabsorção da Raiz/patologia , Calcificação de Dente/genética , Calcificação de Dente/fisiologia , Colo do Dente/ultraestrutura , Microtomografia por Raio-XRESUMO
Histatins are salivary proteins that exhibit a high affinity for hydroxyapatite and contribute to the acquired enamel pellicle. Previous studies have observed that, despite the high proteolytic activity in saliva, significant numbers of histatin molecules in acquired enamel pellicle are intact. Our working hypothesis was that histatins are less susceptible to proteinases present in saliva when adsorbed on the hydroxyapatite. To test this premise, we incubated histatin 1 with hydroxyapatite and human whole saliva. Proteolytic products of this incubation were then characterized by PAGE, HPLC, and mass spectrometry. This study shows for the first time that binding to hydroxyapatite confers intact histatin 1 with resistance to proteolytic degradation.
Assuntos
Esmalte Dentário/metabolismo , Película Dentária/metabolismo , Durapatita/metabolismo , Histatinas/metabolismo , Adsorção , Adulto , Análise de Variância , Cromatografia Líquida de Alta Pressão/métodos , Eletroforese em Gel de Poliacrilamida/métodos , Feminino , Hemólise , Humanos , Masculino , Espectrometria de Massas , Fragmentos de Peptídeos/análise , Ligação Proteica , Adulto JovemRESUMO
A neutral proteinase, distinct from collagenase, was isolated by gel-filtration from medium that had been conditioned by the culture of explanted porcine gingiva. This enzyme was shown to cleave the alpha 1 (I) chain carboxyterminal telopeptide in native collagen proximal to (i.e. nearer to the helix than) the lysyl residue at position 17 which is known to be important in the formation of covalent intermolecular cross-links. We refer to this activity as 'telopeptidase'. The enzyme had an apparent Mr of 70,000, as determined by gel-filtration. It was inhibited by ethylene diamine tetraacetic acid but not by N-ethyl-maleimide nor by phenylmethylsulphonyl fluoride. It is therefore probably a metalloproteinase. The pH optimum for this activity was 7.0-7.5. Incubation of the enzyme with fibrillar (acid-insoluble) calf-skin collagen resulted in solubilization of collagen in which shortening of the carboxy-terminal telopeptides could be demonstrated. It is suggested that the telopeptidase, acting within a region of the Type I collagen molecule which is known to be essential for the stability of collagen fibrils, could potentially play an important role in collagen degradation in vivo.
Assuntos
Endopeptidases , Endopeptidases/isolamento & purificação , Gengiva/enzimologia , Metaloendopeptidases , Animais , Colágeno/metabolismo , Endopeptidases/metabolismo , Gengiva/citologia , Cinética , Neprilisina , Técnicas de Cultura de Órgãos , Fragmentos de Peptídeos/análise , SuínosRESUMO
Bone sialoprotein (BSP) is a bone-specific glycoprotein containing phosphoserine and sulphotyrosine residues and regions of contiguous glutamic acid residues. Recent studies in this laboratory have shown that BSP is capable of nucleating the bone mineral hydroxyapatite in a steady-state agarose gel system. We show here that chemical modification of carboxylate groups abolishes the nucleation activity of BSP, but enzymic dephosphorylation has no effect. Formation of hydroxyapatite is also induced by poly(L-glutamic acid) and poly(D-glutamic acid), but not by poly(L-aspartic acid) or poly(L-lysine). Calreticulin, a muscle protein with short sequences of contiguous glutamic acid residues, also lacks nucleation activity. These findings suggest that the nucleation of hydroxyapatite by BSP involves one or both of the glutamic acid-rich sequences. Based on these findings and others, we propose that polycarboxylate sequences represent a general site for growth-modulating interactions between proteins and biological crystals.
Assuntos
Osso e Ossos/metabolismo , Durapatita/metabolismo , Glutamatos/metabolismo , Fosfoproteínas/metabolismo , Sialoglicoproteínas/metabolismo , Sequência de Aminoácidos , Animais , Proteínas de Ligação ao Cálcio/farmacologia , Calreticulina , Ácidos Carboxílicos/metabolismo , Cristalização , Ácido Glutâmico , Sialoproteína de Ligação à Integrina , Dados de Sequência Molecular , Peptídeos/farmacologia , Fosforilação , Ribonucleoproteínas/farmacologia , SuínosRESUMO
A number of acidic proteins, such as those found in bone and dentin, are poorly resolved on acrylamide gels using Coomassie blue or silver nitrate staining. The cationic dye Stains-all allows visualization and identification of these proteins due to their differential staining: highly acidic proteins stain blue and intact proteoglycans stain purple, whereas less acidic proteins stain pink. However, the use of Stains-all is limited due to relatively poor staining sensitivity and lack of stability to light. A procedure which addresses these deficiencies has been developed utilizing established protocols for Stains-all staining followed by silver nitrate incubation and development. In this way, phosphoproteins such as osteopontin, bone sialoprotein, dentin phosphophoryn, and other acidic glycoproteins are visualized at higher sensitivity (greater than fivefold) and staining stability than normally achieved with just Stains-all. The protocol stains a greater variety of proteins than a combined alcian blue/silver staining procedure previously described. Utilizing the Stains-all/silver protocol, porcine bone osteopontin, a protein not visualized by standard silver staining, can be observed in amounts as little as 0.25 ng on polyacrylamide gels. Furthermore, densitometric scans demonstrate that the staining intensity is proportional to osteopontin amount and can be used for quantification over a range from 0.25 to 50 ng.
Assuntos
Carbocianinas , Corantes , Proteínas/análise , Coloração pela Prata/métodos , Resinas Acrílicas , Animais , Eletroforese em Gel de Poliacrilamida , Glicoproteínas/análise , Sialoproteína de Ligação à Integrina , Osteopontina , Fosfoproteínas/análise , Sensibilidade e Especificidade , Sialoglicoproteínas/análise , SuínosRESUMO
Medium conditioned by the culture of porcine gingival explants was shown to contain, in addition to collagenase, proteolytic activity capable of releasing small fragments, devoid of hydroxyproline but containing hydroxynorleucine, from reduced (tritiated) type I collagen in solution at neutral pH. Quantitative comparison of this effect with that of cathepsin D, at pH 4, revealed that the fragments were derived at least in part from the carboxy-terminal, extra-helical portion of the collagen alpha 1-chains. Incubation of concentrated conditioned medium with fibrillar acetic acid-insoluble collagen resulted in the solubilization of the TC 3/4 and TC 1/4 fragments characteristic of the action of collagenase. However, alpha 1-chain fragments isolated from the latter were found to lack the antigenic determinant normally present on the amino-terminal side of the (hydroxy-)lysine residue which is known to be involved in intermolecular cross-linking. It is therefore suggested that the proteolytic activity described above was involved in the solubilization process. Both the release of low molecular fragments from soluble collagen and the solubilization effect were abolished by ethylenediaminetetra-acetic acid.
Assuntos
Gengiva/enzimologia , Colagenase Microbiana/metabolismo , Pró-Colágeno/metabolismo , Sequência de Aminoácidos , Animais , Técnicas de Cultura , Solubilidade , SuínosRESUMO
Bone sialoprotein (BSP) and osteopontin, the major phosphorylated proteins of mammalian bone, have been proposed to function in the initiation of mineralization. To test this hypothesis, the effects of BSP and osteopontin on hydroxyapatite crystal formation were determined by using a steady-state agarose gel system. At low calcium phosphate concentrations, no accumulation of calcium and phosphate occurred in control gels or gels containing osteopontin. Gels containing BSP at 1-5 micrograms/ml, however, exhibited a visible precipitation band and significantly elevated Ca + PO4 contents. By powder x-ray diffraction, the precipitate formed in the presence of BSP was shown to be hydroxyapatite. These findings suggest that bone sialoprotein may be involved in the nucleation of hydroxyapatite at the mineralization front of bone.
Assuntos
Hidroxiapatitas/metabolismo , Sialoglicoproteínas/metabolismo , Animais , Osso e Ossos/química , Osso e Ossos/metabolismo , Fosfatos de Cálcio/metabolismo , Cromatografia em Gel , Cromatografia por Troca Iônica , Cromatografia Líquida , Durapatita , Hidroxiapatitas/química , Sialoproteína de Ligação à Integrina , Cinética , Osteopontina , Fosfoproteínas/metabolismo , Sefarose , Sialoglicoproteínas/isolamento & purificação , Suínos , Difração de Raios XRESUMO
Osteopontin is a phosphorylated sialoprotein containing a conserved sequence of contiguous aspartic acid residues. This protein is expressed at high levels in mineralized tissues and has previously been shown to inhibit the in vitro formation of hydroxyapatite (HA). In the present study, protein modification and model compound studies have been used to identify the structural features of osteopontin that are responsible for its crystal-modulating properties. Using metastable calcium phosphate solutions buffered by autotitration, osteopontin caused half-maximal inhibition of HA formation at a concentration (IC50) of 0.06 microgram/ml. The hen egg yolk phosphoprotein phosvitin was a much weaker inhibitor, while dextran sulphate had no effect. The synthetic polypeptide poly(aspartic acid) was almost as effective an inhibitor of HA formation as osteopontin (IC50 0.11 microgram/ml), whereas poly(glutamic acid) was more than a thousand times less potent (IC50 155 micrograms/ml). In a steady-state agarose gel system, much higher polypeptide concentrations were required for inhibition of HA formation, but a similar relative order of inhibitory effectiveness was observed. Treatment of osteopontin with alkaline phosphatase removed 84% of the covalently bound phosphate and reduced its HA-inhibiting activity by more than 40-fold. Treatment with glycine ethyl ester in the presence of carbodi-imide modified 86% of the carboxylate groups in osteopontin and reduced its inhibitory activity by 6-fold. These findings indicate that osteopontin is a potent inhibitor of HA formation. This activity requires phosphate and carboxylate groups, possibly including the conserved sequence of contiguous aspartic acid residues. Osteopontin may act as an inhibitor of phase separation in physiological fluids of high supersaturation.
Assuntos
Calcificação Fisiológica , Durapatita/química , Fosfoproteínas/química , Sialoglicoproteínas/química , Sequência de Aminoácidos , Animais , Ácido Aspártico , Fosfatos de Cálcio/química , Cristalografia , Técnicas In Vitro , Dados de Sequência Molecular , Osteopontina , Peptídeos/química , Crânio/química , Relação Estrutura-Atividade , SuínosRESUMO
Carbonated apatite (dahllite) is formed within and between collagen fibrils in the mineralization of connective tissues. However, the mechanism of crystal nucleation at these sites has not been resolved. To identify non-collagenous proteins that may be involved in the nucleation process we have utilized a dissociative extraction procedure to isolate proteins associated non-covalently with the de-mineralized collagen matrix of dentine isolated from tooth roots of adult porcine incisors. Following extraction of dentine fragments with 4M GuHCl (G1-extract) and 0.5M EDTA (E-extract), de-mineralized collagen matrix-associated proteins were isolated with a second series of extractions with 4M GuHCl (G2-extract). Analysis of the G2-extracts on SDS-PAGE revealed two major 32 kDa and 24 kDa protein bands, comprising > 80% of the extracted non-collagenous proteins. The 32 kDa protein was purified by FPLC on hydroxyapatite and Mono Q resins, followed by HPLC reverse-phase chromatography. Small amounts of 26 kDa and 6 kDa proteins, which appear to represent proteolytically processed, disulphide-linked fragments of the 32 kDa protein, co-eluted with the major protein. The 32 kDa protein was identified as lysyl oxidase from amino acid sequence analysis of a 13 kDa CNBr peptide obtained from protein purified by preparative electrophoresis on SDS-PAGE. Fractionation of the 24 kDa protein on FPLC Mono Q resin generated < 5 closely eluting protein peaks. The proteins from these peaks were similar in size, staining properties, amino acid composition and CNBr digestion patterns. Each protein was immunoreactive with antibodies raised against a tyrosine-rich acidic matrix protein (TRAMP), reported previously to co-purify with lysyl oxidase. These studies, therefore, show that lysyl oxidase, which is important in collagen cross-link formation, and proteins with properties of TRAMP, a protein that can modulate collagen fibrillogenesis, are the major proteins in dissociative extracts of de-mineralized porcine dentine.
Assuntos
Proteoglicanas de Sulfatos de Condroitina/isolamento & purificação , Dentina/metabolismo , Proteínas da Matriz Extracelular/isolamento & purificação , Proteína-Lisina 6-Oxidase/isolamento & purificação , Alquilação , Sequência de Aminoácidos , Animais , Western Blotting , Técnica de Desmineralização Óssea , Osso e Ossos/química , Osso e Ossos/enzimologia , Osso e Ossos/metabolismo , Proteoglicanas de Sulfatos de Condroitina/metabolismo , Cromatografia Líquida de Alta Pressão , Cromatografia por Troca Iônica , Eletroforese em Gel de Poliacrilamida , Proteínas da Matriz Extracelular/metabolismo , Feto , Dados de Sequência Molecular , Oxirredução , Proteína-Lisina 6-Oxidase/metabolismo , SuínosRESUMO
An enzyme that was capable of releasing small fragments containing hydroxynorleucine from the c-terminal extra-helical region of the alpha 1 chain of reduced (tritiated) soluble type I collagen was found, along with collagenase, in medium that had been conditioned by the culture of human gingival fibroblasts (Gin-1). The enzyme was present in a latent form or forms and could be activated by treatment with either trypsin or p-hydroxymercuribenzoate. It was maximally active at neutral pH and inhibited by EDTA. It is suggested that this enzyme, acting within a region of the molecule which is of major importance in stabilizing fibrillar collagen through intermolecular cross-linking, could potentially play an important role in collagen turnover in vivo.