Arthur Veis

Phosphorylated proteins and control over apatite nucleation, crystal growth, and inhibition

Living organisms are capable of inducing the crystallization and deposition of a wide variety of minerals1 but the vertebrates mainly utilize the calcium phosphates in constructing their mineral phases in both normal circumstances in bone, dentin and tooth enamel and in pathological ectopic mineral deposits. The predominant form of the mineral in all situations is as carbonated apatite. However, the extent of mineralization in a particular tissue or organ is quite variable and crystallite size, crystal shape, and the packing and organization of the mineral crystals may also be variable. It is clear that the same physical chemical principles must apply to all, but it is equally clear that the organism must tightly regulate the local environment where the mineral is formed. This is an intrinsically complex problem because the mineral crystals of bone and dentin form in the extracellular matrix, external to the cells which are the ultimate regulators of the process. Years of study of this complex set of problems has led to the general consensus that the cell-controlled processes of mineralization begin with the manufacture of an organic structure within which, or a compartment surface upon which, the mineral crystals may be initiated. The cells secrete and organize macromolecular structures which determine the ultimate character and orientations of the crystals subsequently initiated and grown, but in general, these structures do not themselves have the capacity to initiate mineralization. The incipient crystal nucleation event depends upon the interaction of the structural macromolecules with another set of secreted interactive macromolecules which locate specifically on the structural framework. These interactive molecules are doubly interactive, binding specifically to the framework on the one hand, and attracting the requisite mineral ions on the other to initiate the mineral ion clustering to a critical size that nucleates crystal growth. The biological fluids are supersaturated in mineral ion content with respect to the solubility of their biogenic mineral crystal phase, but unrestricted and uncontrolled crystal growth are not permissible in the vertebrate animal so there is a third line of macromolecular components interactive with the growing crystal surfaces that can limit growth rates, and do so selectively, controlling the crystal shape as well as size. A final component of the regulation is the availability of the crystal microions, pumped in controlled fashion from the cell into the extracellular matrix. The situation is shown schematically in Figure 1, wherein the solid, heavy arrows trace the construction of the structural matrix (a), the addition of the interactive macromolecules that activate the structural matrix (b) to bind the mineral ions (c), leading to growth of the oriented crystals (d), and finally, the addition of further macromolecules (e) to shape and delimit the crystal size to attain the mature mineralized matrix form. Note that in this paragraph, and in Figure 1 the nature of the macromolecules and mineral phase has not been specified. This is intentional to emphasize the point that in the majority of mineralizing tissues in animal systems the same overall scheme of events is likely to apply, although the structural scaffolds and mineral types can vary greatly. Figure 1 A proposed generalized scheme for matrix-regulated mineralization reactions. Modified and reprinted with permission from Reference 280 [Veis, Reviews in Mineralogy & Geochemistry, V. 54]. Copyright 2003 Mineralogical Society of America. The calcium and phosphate ion product (Ca × P) in the extracellular fluids (ECF) of the vertebrates are greater than the solubility products of most crystalline forms of calcium phosphate, with hydroxylapatite (HA) being the major least soluble form of calcium phosphate. Hence, HA would precipitate spontaneously if it were not for the inhibition of crystal formation by the reduction of the activity of these ions, and their sequestration, by various macromolecular components. Thus, the ECF proteins in Ca-rich milk and blood, for example, play important roles as inhibitors of apatite precipitation. The initial form of the Ca – P solid phase deposits in vitro have been shown to be disordered and exhibit a diffuse X-ray diffraction pattern, indicating that initially only a very short range ordering of calcium and phosphate ions relative to each other takes place, producing a structure called “amorphous calcium phosphate” (ACP) 2,3,4. Depending upon the conditions of temperature, pH, and ion concentrations, the solid phase may grow through a variety of forms from the ACP to (in order of decreasing solubility) brushite [CaHPO4·2H2O], whitlockite [Ca3(PO4)2 ·3H20], octacalcium phosphate [Ca8H2(PO4)6·5H2O], and hydroxyapatite [Ca10(PO4)6(OH)2]. In bone, dentin, cementum and enamel, in vivo, where the nucleation process and the final crystal composition are determined by the local environment created by the combination of the collagen or amelogenin structural matrix and particular NCP components, it is evident that the transition from ion aggregate to crystalline form is tightly regulated. A question of great importance for those interested in development of biomimetic models of bone is how the NCP might template the desired crystal forms. The principal focus of this review, however, will be on the influence of the phosphoproteins of the matrix on the formation and growth of crystals in two systems: the small, plate-like crystals and crystal aggregates of carbonated apatite found in bone, dentin and cementum; and the large, high axial ratio rod-like crystal aggregates found in dental enamel. As pointed out many years ago5, the dentin and enamel are formed in apposition to each other and provide direct contrasts between the nature of the compartments created by the cells, the structural matrices produced, and the extracellular matrix macromolecules that control the nucleation, growth and structure of their carbonated apatite mineral phases. Here we consider these two systems separately.

Specific Amelogenin Gene Splice Products Have Signaling Effects on Cells in Culture and in Implants in Vivo

Low molecular mass amelogenin-related polypeptides extracted from mineralized dentin have the ability to affect the differentiation pathway of embryonic muscle fibroblasts in culture and lead to the formation of mineralized matrix in in vivo implants. The objective of the present study was to determine whether the bioactive peptides could have been amelogenin protein degradation products or specific amelogenin gene splice products. Thus, the splice products were prepared, and their activities were determined in vitro and in vivo. A rat incisor tooth odontoblast pulp cDNA library was screened using probes based on the peptide amino acid sequencing data. Two specific cDNAs comprised from amelogenin gene exons 2,3,4,5,6d,7 and 2,3,5,6d,7 were identified. The corresponding recombinant proteins, designated r[A+4] (8.1 kDa) and r[A−4] (6.9 kDa), were produced. Both peptides enhanced in vitrosulfate incorporation into proteoglycan, the induction of type II collagen, and Sox9 or Cbfa1 mRNA expression. In vivoimplant assays demonstrated implant mineralization accompanied by vascularization and the presence of the bone matrix proteins, BSP and BAG-75. We postulate that during tooth development these specific amelogenin gene splice products, [A+4] and [A−4], may have a role in preodontoblast maturation. The [A+4] and [A−4] may thus be tissue-specific epithelial mesenchymal signaling molecules.

The Carboxyl-terminal Domain of Phosphophoryn Contains Unique Extended Triplet Amino Acid Repeat Sequences Forming Ordered Carboxyl-Phosphate Interaction Ridges That May Be Essential in the Biomineralization Process

Phosphophoryns (PPs), a family of Asp and Ser(P)-rich dentin proteins, are considered to be archetypal regulators of several aspects of extracellular matrix (ECM) biomineralization. We have cloned a rat incisor PP gene, Dmp2, from our odontoblast cDNA library and localized it to mouse chromosome 5q21 within 2 centimorgans of Dmp1, another tooth-specific ECM protein. The carboxyl-terminal region of Dmp2 protein (60 residue % Ser, 31 residue % Asp) is divided into two domains, one with unique repetitive blocks of [DSS]n,3≤14, the other with [SD]m = 2,3. Conformational analysis shows the phosphorylated form of the [DS*S*]n repeats to have a unique structure with well defined ridges of phosphates and carboxyls available for counter ion binding. The [S*D]m domains have different phosphate and carboxylate interaction edges and thus different calcium ion and apatite surface binding properties. These two domains and the colocalization of Dmp1 and Dmp2 genes at a position equivalent to the dentinogenesis imperfecta type II location on human 4q21 all suggest that the PPs are indeed involved in some aspect of ECM mineralization.

Inflammatory and immunological aspects of dental pulp repair

The repair of dental pulp by direct capping with calcium hydroxide or by implantation of bioactive extracellular matrix (ECM) molecules implies a cascade of four steps: a moderate inflammation, the commitment of adult reserve stem cells, their proliferation and terminal differentiation. The link between the initial inflammation and cell commitment is not yet well established but appears as a potential key factor in the reparative process. Either the release of cytokines due to inflammatory events activates resident stem (progenitor) cells, or inflammatory cells or pulp fibroblasts undergo a phenotypic conversion into osteoblast/odontoblast-like progenitors implicated in reparative dentin formation. Activation of antigen-presenting dendritic cells by mild inflammatory processes may also promote osteoblast/odontoblast-like differentiation and expression of ECM molecules implicated in mineralization. Recognition of bacteria by specific odontoblast and fibroblast membrane receptors triggers an inflammatory and immune response within the pulp tissue that would also modulate the repair process.

Molecular recognition in the assembly of collagens: Terminal noncollagenous domains are key recognition modules in the formation of triple-helical protomers

The α-chains of the collagen superfamily are encoded with information that specifies self-assembly into fibrils, microfibrils, and networks that have diverse functions in the extracellular matrix. A key self-organizing step, common to all collagen types, is trimerization that selects, binds, and registers cognate α-chains for assembly of triple helical protomers that subsequently oligomerize into specific suprastructures. In this article, we review recent findings on the mechanism of chain selection and infer that terminal noncollagenous domains function as recognition modules in trimerization and are therefore key determinants of specificity in the assembly of suprastructures. This mechanism is also illustrated with computer-generated animations.

Phosphophoryns-major noncollagenous proteins of rat incisor dentin.

SummaryFreshly excised rat incisors were immediately cleaned and demineralized in 0.5M ethylene diaminetetracetic acid at pH 7.5. The extracts were freed of calcium, diffusible phosphate and low molecular weight polypeptide components by dialysis in membranes with cut-off of 3500 molecular weight. The extract was resolved into at least 7 protein components by chromatography on DEAE-cellulose at pH 8.2. The composition of each protein component was determined. Two proteins, rich in serine, phosphorous and aspartic acid were unlike any proteins attributed to enamel, and hence were considered to be components of incisor dentin. These were the principal noncollagenous components of the teeth. Further purification was carried out under dissociative conditions on Sepharose CL-6B gel filtration columns in 3.0M guanidine hydrochloride. The two phsophoproteins have mol wts, by this method, of 71,000 and 65,000, respectively, and differ in content of apolar amino acids, although both contain >70 residue % of seryl (or phosphoseryl) and aspartyl residues. The name “phosphophoryns” is proposed to describe these dentinal proteins. The insoluble collagenous matrix remaining after the original demineralizing extraction was degraded with cyanogen bromide. Several non-collagenous protein components were released as well as the typical collagen derived peptides. Two collagen phosphoprotein complex peptides were also isolated, demonstrating as in bovine dentin, the probable direct covalent interaction of a dentin phosphoprotein with the collagen of the mineralized matrix.

Microanalysis and characterization of acidic glycosaminoglycans in human tissues.

Abstract The acid glycosaminoglycans (AG) were isolated from small quantities of human tissue (1.0 gm wet weight or less) and were separated, identified, and measured by zone electrophoresis on cellulose acetate. The major AG components in human skin, sclera, and cornea were identified and measured. The amount of Alcian Blue (dye used to stain the AG) bound to each AG polymer on the cellulose acetate strip was dependent on the number of moles of disaccharide repeating units (DRU) times the number of dissociated carboxylic and sulfate groups per DRU. The degree of dissociation of these groups depended on their corresponding pKs and the pH of the Alcian Blue solution. The glucosamine/galactosamine fraction agreed closely with the hyaluronic acid/sulfated AG (dermatan sulfate + chondroitin 4 6 - sulfate ) fraction obtained by electrophoresis. The mobility of each AG polymer was affected more by the number of negative charges per DRU than by the molecular weight. The identity and number of charged groups per DRU could be deduced from the electrophoretic mobilities and the dye binding data. The combined use of chemical analyses and zone electrophoresis permitted the major AG polymers to be identified and measured in tissues without the use of ion-exchange columns.

Evidence for the Proteolytic Processing of Dentin Matrix Protein 1 IDENTIFICATION AND CHARACTERIZATION OF PROCESSED FRAGMENTS AND CLEAVAGE SITES

Full-length cDNA coding for dentin matrix protein 1 (DMP1) has been cloned and sequenced, but the corresponding complete protein has not been isolated. In searching for naturally occurring DMP1, we recently discovered that the extracellular matrix of bone contains fragments originating from DMP1. Shortened forms of DMP1, termed 37K and 57K fragments, were treated with alkaline phosphatase and then digested with trypsin. The resultant peptides were purified by a two-dimensional method: size exclusion followed by reversed-phase high performance liquid chromatography. Purified peptides were sequenced by Edman degradation and mass spectrometry, and the sequences compared with the DMP1 sequence predicted from cDNA. Extensive sequencing of tryptic peptides revealed that the 37K fragments originated from the NH2-terminal region, and the 57K fragments were from the COOH-terminal part of DMP1. Phosphate analysis indicated that the 37K fragments contained 12 phosphates, and the 57K fragments had 41. From 37K fragments, two peptides lacked a COOH-terminal lysine or arginine; instead they ended at Phe173 and Ser180 and were thus COOH termini of 37K fragments. Two peptides were from the NH2 termini of 57K fragments, starting at Asp218 and Asp222. These findings indicated that DMP1 is proteolytically cleaved at four bonds, Phe173–Asp174, Ser180–Asp181, Ser217–Asp218, and Gln221–Asp222, forming eight fragments. The uniformity of cleavages at the NH2-terminal peptide bonds of aspartyl residues suggests that a single proteinase is involved. Based on its reported specificity, we hypothesize that these scissions are catalyzed by PHEX protein. We envision that the proteolytic processing of DMP1 plays a crucial role during osteogenesis and dentinogenesis.

Dentin Matrix Protein 1 Initiates Hydroxyapatite Formation In Vitro

Bone and dentin formation are interesting examples of matrix-mediated mineralization. However, factors and mechanisms regulating this process are poorly understood. Dentin matrix protein 1 (DMP1) is an acidic extracellular matrix protein found in dentin and bone, and based on its amino acid composition it could be postulated to play an important role in mineralization. Our present study examines the ability of recombinant DMP1 to initiate apatite formation in vitro. A 45 Ca-binding assay demonstrated that recombinant DMP1 (rDMP1) possesses calcium-binding ability under physiological conditions. The in vitro nucleation experiments when conducted with rDMP1-coated glass plates demonstrated hydroxyapatite nucleation, while amorphous mineral was deposited on blank or BSA-coated surface. This mineral deposition was found to be 10-fold higher on rDMP1-coated glass surface when compared with the control glass plates. These findings suggest that DMP1 could be considered as a nucleator for apatite deposition in vitro.

Identification of the Chondrogenic-inducing Activity from Bovine Dentin (bCIA) as a Low-molecular-mass Amelogenin Polypeptide

Dentin extracellular matrix has been shown to contain components capable of inducing chondrogenesis and osteogenesis at ectopic sites when implanted in vivo, and chondrogenesis in cultures of embryonic muscle-derived fibroblasts (EMF) in vitro. The polypeptide responsible, called the chondrogenic-inducing agent (CIA), has been isolated from a 4.0-M guanidinium hydrochloride extract of demineralized bovine dentin matrix. Following Sephacryl S-100 chromatography, CIA activity was identified in fractions by assay for uptake of [35S]-SO4 into proteoglycan by the EMF after 24 hrs in culture. The active fraction induced the EMF to produce type II collagen mRNA and decrease production of type I collagen mRNA after 5 days in culture. The EMF + CIA, cultured for 4 to 7 wks, formed toluidine-blue- and alizarin-redstainable nodules, indicative of chondrogenic induction. In vivo implants in rat muscle with collagen carrier produced ectopic bone after 7 wks. The CIA was brought to near-homogeneity by reverse-phase high-performance liquid chromatography, tested at each step by EMF [ 35S]-SO4-incorporation assays. The CIA components had masses in the ranges of 6000 to 10,000 Da by both mass spectroscopy and gel electrophoresis. The CIA amino acid composition, NH2terminal, and internal amino acid sequences were determined. These data showed unequivocally that the CIA peptides were derived from bovine amelogenin. The peptides contain the amino-terminal portion of the bovine amelogenin. The presence of these chondrogenic/osteogenic amelogenin-polypeptides in dentin matrix leads us to hypothesize that they may be involved in epithelial-mesenchymal signaling during tooth development interactions-the first time a function has been indicated for these molecules.