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-red-stainable 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, NH2-terminal, 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.

Peritubular dentin formation: crystal organization and the macromolecular constituents in human teeth.

Peritubular dentin (PTD) is a relatively dense mineralized tissue that surrounds the tubules of coronal tooth dentin. It is composed mainly of crystals of carbonated apatite together with a small amount of collagen. Its mode of formation has been investigated by studying the relatively dense particles isolated from a powdered preparation. Electron microscopic examination of the PTD particles, including 3-dimensional image reconstruction and electron diffraction, shows that the organization of the crystals of PTD is very similar to that of the adjacent intertubular dentin (ITD). The latter contains relatively large amounts of collagen and the carbonated apatite crystals are closely associated with the collagen matrix. The proteins present in the PTD particles are soluble after decalcification and stain with Stains All. The principal protein has higher molecular weight and a quite different amino acid composition than the phosphophoryns of the intertubular dentin. The interface between the PTD and the ITD shows structural continuity. These data show how two distinct carbonated apatite-based mineralized tissues can be organized and formed contiguously within the same organ by utilizing different sets of matrix proteins.

Type I collagen-phosphophoryn interactions: specificity of the monomer-monomer binding.

It has been postulated that phosphophoryn (PP) molecules bind specifically to type I collagen fibrils as the key event in inducing matrix mineralization in dentin. The nature and specificity of the collagen molecule-PP interaction has been examined by rotary shadowing-electron microscopy of mixtures of native, monomeric lathyritic rat skin collagen and purified rat incisor PP. An antibody to the amino-telopeptide of the collagen alpha1(I)-chain was used to determine the N-terminal end of the collagen molecules. Solutions of collagen and PP in 0.01 M ammonium formate (+/- antibody) were mixed and spread in 70% glycerol-30% 0.01 M ammonium formate on freshly cleaved mica surfaces using the sandwich technique. After rotary shadowing with Pt and backcoating with a carbon film, the spreads were viewed in a JEOL 1200EX TEM. The PP appeared as 15-nm diameter globules, the collagen as semi-flexible 270 nm filaments. At neutral pH and low PP/collagen mixing ratios, a single interaction site was evident, centered at approximately 210 nm from the N-terminus. The binding interaction induced a local conformational change in the collagen, bending the molecule and reducing its effective length. The sequence within the collagen-PP-binding domain has a net-positive charge but contains both positively and negatively charged groups.

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

Endoplasmic reticulum protein Hsp47 binds specifically to the N-terminal globular domain of the amino-propeptide of the procollagen I alpha 1 (I)-chain.

Hsp47, an endoplasmic reticulum-resident heat shock protein in fibroblasts, has gelatin-binding properties. It had been hypothesized that it functions as a chaperone regulating procollagen chain folding and/or assembly, but the mechanism of the hsp47-procollagen I interaction was not clear. Hsp47 could bind to both denatured and native procollagen I. A series of competition studies were carried out in which various collagens and collagen domain peptides were incubated with 35[S]-methionine-labeled murine 3T6 cell lysates prior to mixing with gelatin-Sepharose 4B beads. The gelatin-bound proteins were collected and analyzed by gel electrophoresis and autoradiography. Collagenase digested procollagen I had the same effect as denatured intact procollagen, indicating that the propeptides were the major interaction sites. The addition of intact pro alpha 1(I)-N-propeptide at 25 micrograms/ml completely inhibited hsp47 binding to the gelatin-Sepharose. Even the pentapeptide VPTDE, residues 86-90 of the pro alpha 1(I)-N-propeptide, inhibits hsp47-gelatin binding. These data implicating the pro alpha 1(I)-N-propeptide domain were confirmed by examination of polysome-associated pro alpha chains. The nascent pro alpha 1(I)-chains with intact N-propeptide regions could be precipitated by monoclonal hsp47 antibody 11D10, but could not be precipitated by monoclonal anti-pro alpha 1 (I)-N-propeptide antibody SP1.D8 unless dissociated from the hsp47. GST-fusion protein constructs of residues 23-108 (NP1), 23-151 (NP2), and 23-178 (NP3) within the pro alpha 1 (I)- N-propeptide were coupled to Sepharose 4B and used as affinity beads for collection of hsp47 from 3T6 cell lysates. NP1 and NP2 both showed strong specific binding for lysate hsp47. Finally, the interaction was studied in membrane-free in vitro cotranslation systems in which the complete pro alpha 1(I)- and pro alpha 2(I)-chain RNAs were translated alone and in mixtures with each other and with hsp47 RNA. There was no interaction evident between pro alpha 2(I)-chains and hsp47, whereas there was strong interaction between pro alpha 1(I)-chains and nascent hsp47. SP1.D8 could not precipitate pro alpha 1(I)-chains from the translation mix if nascent hsp47 was present. These data all suggest that if hsp47 has a "chaperone" role during procollagen chain processing and folding it performs this specific role via its preferential interaction with the pro alpha 1 (I) chain, and the pro alpha 1(I) amino-propeptide region in particular.

Characterization of a novel dentin matrix acidic phosphoprotein. Implications for induction of biomineralization.

Acidic phosphorylated proteins have been shown to be prominent constituents of the extracellular matrix of bone and dentin. The acidic phosphoproteins of bone contain more glutamic acid than aspartic acid and a lower serine content than either. On the other hand, the major dentin acidic phosphoproteins, phosphophoryns, have been defined as aspartic acid- and serine-rich proteins, with a lesser content of glutamic acid. Both sets of phosphoproteins have been implicated as key participants in regulating mineralization, but it has been difficult to unify their mechanisms of action. We have now identified, by cDNA cloning, a new serine-rich acidic protein of the dentin matrix, AG1, with a composition intermediate between the bone acidic proteins and dentin phosphophoryns. AG1 has numerous acidic consensus sites for phosphorylation by both casein kinases I and II. Immunochemical and organ culture biosynthetic studies show that AG1 is present in phosphorylated form at low levels in the dentin matrix. If fully phosphorylated, AG1 would bear a net charge of -175/molecule of 473 residues. AG1 contains single RGD integrin binding and N-glycosylation sequences. The overall picture that emerges is that of a matrix-associated acidic phosphoprotein, with a potentially high calcium ion binding capacity, present at levels compatible with a regulatory role in dentin mineralization.

Dentin phosphophoryn binding to collagen fibrils.

The interaction of rat incisor phosphophoryn with native turkey tendon collagen fibers has been examined by electron microscopy. The binding of phosphophoryn to the tendon fibril surfaces is quite selective. The phosphophoryn is seen as positively or negatively stained globular particles predominantly at the "e" band in the collagen gap region in transmission electron micrographs of the phosphophoryn-reacted fibrils. The selectivity of binding to the fibrils was obtained in the presence of calcium ions, which bind avidly to phosphophoryn. The specific association of phosphophoryn at the "e" band suggests a possible regulation of mineral deposition within the gap regions of the collagen fibrils.

Domain structure and sequence distribution in dentin phosphophoryn.

Phosphophoryn (PP) is a protein unique to the mineralized matrix of dentin. It also has a unique composition, with aspartic acid and phosphoserine comprising greater than 85% of all amino acid residues. Because of this unique composition and high content of phosphoserine, it has been difficult to apply direct peptide sequencing procedures effectively. However, to understand its function, and to prepare suitable probes for screening cDNA libraries, some sequence distribution information is required. To this end, using bovine (b) and rat incisor (ri) PPs, partial mild acid hydrolysis has been used to cleave at the aspartic acid residues and generate free amino acids and small peptides. The nature of the released amino acids and peptides has been determined. Peptides have also been generated by limited digestion with trypsin. Some of the peptides have been purified by h.p.l.c. techniques and sequenced. About 90% of the bPP and riPP were resistant to trypsin, and the large resistant fragment was sharply depleted of the non-aspartic acid and non-phosphoserine [(P)Ser] residues. All peptides isolated were acidic, but the remaining residues (other than aspartic acid and serine) appeared to be collected in regions flanking the trypsin-resistant core. These data show directly the presence of regions [Asp]n, [(P)Ser]m and [Asp-(P)Ser-Asp]k as prominent sequence features. A domain structure model is proposed.

The isolation and partial characterization of a rat incisor dentin matrix polypeptide with in vitro chondrogenic activity.

In vivo implants of demineralized dentin matrix into muscle induce the formation of bone within the muscle. As with bone matrix implants, the bone induction appears to follow a chondrogenic pathway. Outgrowth cells from explants of neonatal rat muscle respond to bone matrix, in vitro, by expressing a heightened synthesis of sulfated proteoglycans and type II collagen, phenotypic of cartilage. The in vitro cell culture system has been used as an assay to monitor the isolation of the factor responsible for expression of this phenotypic transformation. Soluble proteins extracted from rat incisor dentin matrix during demineralization with EDTA, and not precipitable with 1.0 M CaCl2, were active in the in vitro system. The active extract was fractionated by Sephacryl S-100 chromatography in 6 M guanidine HCl, isoelectric focusing in Immobilines, and by reverse phase high performance liquid chromatography. All fractions were assayed for activity at every stage. The final active fraction from the reverse phase chromatography on a Zorbax Poly-F column was purified to homogeneity, and yielded a single spot on two-dimensional gel electrophoresis. The component, RP-4, had pI 5.4-5.5, and an apparent Mr 6,000-10,000, based on globular protein standards. Maximal activity with respect to both sulfate incorporation into proteoglycan and production of type II collagen was in the 1.0-10 ng/ml range. The RP-4 had a unique amino-terminal amino sequence and was rich in Gly, Pro, Glx, and Ala residues. It was different from transforming growth factor-beta and the bone morphogenetic protein family of proteins in these essential features.

Concentration-dependent effects of dentin phosphophoryn in the regulation of in vitro hydroxyapatite formation and growth.

The effect of dentin phosphophoryn on hydroxyapatite formation and growth was studied in an in vitro gelatin gel diffusion system. Phosphophoryn, in low concentrations (0.010-1 microgram/ml) promoted de novo hydroxyapatite formation; at a higher concentration (100 micrograms/ml) in the same system, the dentin matrix protein inhibited hydroxyapatite growth. Similar inhibition of hydroxyapatite growth was seen in solution. The intact phosphophoryn was not essential for either inhibition of seeded growth or promotion of mineralization, since the formic acid degraded protein was comparably effective. Transmission electron microscopy of the precipitates formed at 7 days showed no significant differences in crystallite size distribution in the presence and absence of phosphophoryn. However there was a dose-dependent decrease in the number of mineral clusters formed in the presence of increasing amounts of phosphophoryn, suggesting inhibition of secondary nucleation. These data provide support for the postulated 'multifunctional' role of the dentin phosphoprotein in the mineralization process.

Rat incisor dentine contains a factor which alters the phenotypic expression and stimulates chondrogenesis in fibroblast-like cells in vitro.

A low molecular weight protein fraction isolated under dissociative conditions during the demineralization of rat incisor dentine has the ability to modulate, in culture, the expression of fibroblast-like cells explanted from neonatal rat muscle. The protein fraction enhances the incorporation of 35S-sulphate into a proteoglycan larger in weight than that produced by the uninduced cells; furthermore it induces the production of type II collagen. These changes take place in the absence of cell proliferation as measured by 3H-thymidine incorporation. The altered fibroblast-like cells form nodules and secrete an abundant extracellular matrix which stains for proteoglycan after 7-9 days in culture. These data show that the dentine matrix does contain a factor which can initiate a mitogenesis-independent alteration in the expression of the muscle-explant outgrowth cells. Those changes are consistent with a shift to a chondrogenic mode.

Isolation of an acidic protein from cholesterol gallstones, which inhibits the precipitation of calcium carbonate in vitro.

In seeking to identify nucleating/antinucleating proteins involved in the pathogenesis of cholesterol gallstones, a major acidic protein was isolated from each of 13 samples of cholesterol gallstones. After the stones were extracted with methyl t-butyl ether to remove cholesterol, and methanol to remove bile salts and other lipids, they were demineralized with EDTA. The extracts were desalted with Sephadex-G25, and the proteins separated by PAGE. A protein was isolated, of molecular weight below 10 kD, which included firmly-bound diazo-positive yellow pigments and contained 24% acidic, but only 7% basic amino acid residues. The presence of N-acetyl glucosamine suggested that this was a glycoprotein. This protein at concentrations as low as 2 micrograms/ml, but neither human serum albumin nor its complex with bilirubin, inhibited calcium carbonate precipitation from a supersaturated solution in vitro. This protein could be precipitated from 0.15 M NaCl solution by the addition of 0.5 M calcium chloride. Considering that cholesterol gallstones contain calcium and pigment at their centers, and that small acidic proteins are important regulators in other biomineralization systems, this protein seems likely to play a role in the pathogenesis of cholesterol gallstones.

Characterization of the molecular weights of bovine molar, rat incisor, and other phosphophoryns.

The phosphophoryns show anomalous behavior in solution, and are easily degraded during extraction. They appear in varied forms in the teeth of different species and differ in the teeth of the same species in a developmental and age dependent fashion. This set of properties has made the characterization of the phosphophoryns by biochemical means a difficult and controversial subject. Bovine molar phosphophoryn, bPP, has been characterized in detail by a variety of physical methods, and then compared with the PP from other species by DEAE-HPLC, gel electrophoresis, and immunological cross reactivity. The possible existence of a proPP biosynthetic precursor has been investigated by rat incisor organ culture and examination of the 32P and 14C labeled products. These studies all show marked differences in the Mr values for the PP from teeth of different species, even when they are antigenically cross-reactive.

Matrix proteins of the teeth of the sea urchin Lytechinus variegatus.

The teeth of the sea urchin Lytechinus variegatus grow continuously. The mineral phase, a high magnesium calcite, grows into single crystals within numerous compartments bounded by an organic matrix deposited by the odontoblasts. Electron microscopic examination of glutaraldehyde-fixed Ethylene Diamine Tetra acetic acid (EDTA) demineralized teeth shows the compartment walls to be organized from multiple layers of cell membrane which might contain cytoplasmic protein inclusions. Proteins extracted during demineralization of unfixed teeth were examined by gel electrophoresis, high performance liquid chromatography, and amino acid analysis. The tooth proteins were acidic, they contained phosphoserine, and they were rich in aspartic acid. By contrast, the proteins of similarly extracted mineralized Aristotle's lantern skeletal elements were nonphosphorylated and were rich in glutamic acid. Vertebrate tooth and bone matrix proteins show similar differences. Surprisingly, an antibody to the principle rat incisor phosphoprotein showed a significant cross-reactivity with the urchin tooth protein, by dot-blot and enzyme-linked immunosorbent assay procedures. Thus, the urchin tooth proteins contain epitope regions similar to those which are phenotypic markers of vertebrate odontoblasts. Whether this is an expression of convergent or divergent evolutionary processes, it is likely that the matrix proteins play a similar role in matrix mineralization. The sea urchin tooth may thus be an excellent model for the study of odontoblast-mediated mineral-matrix relationships.