Effects of EDTA on the hydration mechanism of mineral trioxide aggregate.

Ethylenediaminetetraacetic acid (EDTA) is commonly used during the preparation of obstructed root canals that face a high risk of root perforation. Such perforations may be repaired with mineral trioxide aggregate (MTA). Due to EDTA's ability to chelate calcium ions, we hypothesized that EDTA may disrupt the hydration of MTA. Using scanning electron microscopy and energy-dispersive x-ray spectroscopy, we found that MTA specimens stored in an EDTA solution had no crystalline structure and a Ca/Si molar ratio considerably lower than those obtained for specimens stored in distilled water and normal saline. Poor cell adhesion in EDTA-treated MTA was also noted. X-ray diffraction indicated that the peak corresponding to portlandite, which is normally present in hydrated MTA, was not shown in the EDTA group. The microhardness of EDTA-treated specimens was also significantly reduced (p < 0.0001). These findings suggest that EDTA interferes with the hydration of MTA, resulting in decreased hardness and poor biocompatibility.

The presence of multiple rat DSP-PP transcripts.

Phosphoproteins or phosphophoryns (PPs) are the most abundant (>50%) non-collagenous proteins (NCPs) in dentin. PPs bind to calcium and hydroxyapatite and are believed to play a crucial role in dentin mineralization. Dentin sialoprotein (DSP), a highly glycosylated protein, comprised 5-8% of NCPs in dentin. The coding sequences for these two major NCPs are known to be contiguously located (i.e. DSP-PP) at the cDNA and genomic DNA levels in both rat and mouse. Previous studies have demonstrated the presence of multiple DSP-PP transcripts in the total RNA of adult rat incisors. To further understand the nature of these multiple transcripts, we performed reverse transcription-PCR and obtained a PP cDNA variant which encoded a 171 amino acid peptide (PP(171)) that shares many of the same characteristics as that of the published rat PP(240) sequence [Ritchie, H.H. and Wang, L.-H., J. Biol. Chem. 271 (1996) 21695-21698]. Due to its reduced size, as compared to PP(240), this cDNA encodes a phosphorylated protein with a reduced negative charge that may differentially affect mineralization processes. We provide evidence that there are multiple DSP-PP transcripts with various sizes of PP sequences in rat.

A novel rat 523 amino acid phosphophoryn: nucleotide sequence and genomic organization.

Phosphophoryns (PP), the major noncollagenous proteins (NCPs) in dentin, are believed to play a crucial role in mineral nucleation and hydroxyapatite growth during dentin mineralization. Previously we identified two mature rat PP transcripts, one coding for a 240 amino acid protein (designated as PP(240)) (H.H. Ritchie, L.-H. Wang, J. Biol. Chem. 271 (1996) 21695-21698), and another coding for a 171 amino acid protein (PP(171)) (H. Ritchie, L. Wang, Biochim. Biophys. Acta 1493 (2000) 27-32). We now have identified a third novel dentin sialoprotein (DSP)-PP cDNA transcript that encodes a 523 amino acid protein (PP(523)) with typical PP characteristics including DSS and DS motifs suitable as potential casein kinase I and II phosphorylation sites. Based on amino acid composition, the PP(523) protein product is identical to native rat HP2. We also show that the PP(523) sequence is identical to the corresponding genomic DNA sequence. Taken together, the existence of multiple DSP-PP transcripts, each significantly different from the other in net negative charge, suggests that dentin mineralization processes may be under fine-tune control by these PP protein isoforms.

Effects of dexamethasone, vitamin A and vitamin D3 on DSP-PP mRNA expression in rat tooth organ culture.

Vitamin A, 1,25-dihydroxyvitamin D3 and dexamethasone are well-characterized hydrophobic molecules whose biological actions are mediated via different members of the nuclear hormone receptor family. We report here their actions on tooth formation at the molecular level. We have tested the effects of these compounds on osteopontin (OPN), dentin sialoprotein (DSP-PP), and collagen type I expression in pre-mineralization and mineralization stage rat tooth organ cultures which mirror in vivo developmental patterns. These proteins are all believed to participate in the mineralization of dentin. 1,25-Dihydroxyvitamin D3 up-regulated OPN, but had no effect on DSP-PP mRNA expression. Vitamin A up-regulated DSP-PP expression as did dexamethasone. Dexamethasone also up-regulated collagen type I expression. Our results suggest that 1,25-dihydroxyvitamin D3 does not modulate dentin mineralization by directly affecting DSP-PP expression. Vitamin A likely contributes to dentin mineralization by up-regulating DSP-PP expression. Finally, the up-regulation of DSP-PP expression in tooth germ cultures treated with dexamethasone suggests that its application to patient's dental pulp might promote increased extracellular matrix synthesis and mineralization in the pulp and may explain the narrowing of the dental pulp cavity in patients undergoing long-term dexamethasone administration.

Phosphate and calcium uptake by rat odontoblast-like MRPC-1 cells concomitant with mineralization.

It has been suggested that odontoblasts are instrumental in translocating Ca2+ and inorganic phosphate (Pi) ions during the mineralization of dentin. The aim of this study was to characterize cellular Pi and Ca2+ uptake in the novel rat odontoblast-like cell line mineralizing rat pulpal cell line (MRPC) 1 during mineralization to see if changes in the ion transport activity would occur as the cultures develop and begin forming a mineralized matrix. MRPC-1 cells were cultured in chemically defined medium containing ascorbate and Pi, and cultures were specifically analyzed for cellular P, and Ca2+ uptake activities and expression of type II high-capacity Na+-Pi cotransporters. The odontoblast-like phenotype of the cell line was ascertained by monitoring the expression of collagen type I and dentin phosphopoprotein (DPP). Mineralized nodule formation started at day 9 after confluency and then rapidly increased. Ca2+ uptake by the cells showed a maximum during the end of the proliferative phase (days 5-7). Pi uptake declined to a basal level during proliferation and then was up-regulated simultaneously with the onset of mineralization to a level fourfold of the basal uptake, suggesting an initiating and regulatory role for cellular Pi uptake in mineral formation. This up-regulation coincided with a conspicuously increased glycosylation of NaPi-2a, indicating an activation of this Na+-Pi cotransporter. The study showed that MRPC-1 cells express an odontoblast-like phenotype already at the onset of culture, but that to mineralize the collagenous extracellular matrix (ECM) that formed, a further differentiation involving their ion transporters is necessary.

Partial cDNA sequencing of mouse dentine sialoprotein and detection of its specific expression by odontoblasts.

Dentine sialoprotein (DSP), a 53-kDa acidic glycoprotein, is expressed by odontoblasts and secreted into the dentine extracellular matrix. Although little is known about its biological function, it might play a part in dentinogenesis. Because DSP has only been shown to occur in rat dentine, it is important to demonstrate its existence in another species. Here, the presence of DSP gene in the mouse genome, and the cloning of a mouse DSP cDNA coding for about one-fifth of the molecule with a nucleotide sequence similar to that for rat cDNA, are reported. Using in-situ hybridization, DSP mRNA was uniquely detected in mouse odontoblasts.

Dentin-pulp complex responses to carious lesions.

To understand the molecular events underlying the dentin-pulp complex responses to carious progression, we systematically analyzed tissue morphology and dentin matrix protein distribution in non-carious teeth and in teeth with enamel and dentin caries. Dentin matrix proteins analyzed included collagen type I, phosphophoryn (PP) and dentin sialoprotein (DSP), all of which play decisive roles in the dentin mineralization process. Human non-carious and carious third molar teeth were freshly collected, demineralized, and processed for hematoxylin and eosin staining. The ABC-peroxidase method was used for immunohistochemical staining of collagen type I, PP and DSP proteins using specific antibodies. In situ hybridization was also performed. In contrast to elongated odontoblasts in non-carious teeth, odontoblasts subjacent to dentin caries were cuboidal and fewer in number. The predentin zone was also dramatically reduced in teeth with dentin caries. The staining intensity for collagen type I, PP and DSP in the dentin-pulp complex increased progressively from non-carious teeth, to teeth with enamel and dentin caries. In situ hybridization studies showed DSP-PP mRNA expression in odontoblasts and dental pulp that was consistent with our immunohistochemical results. These results suggest that carious lesions stimulate the dentin-pulp complex to actively synthesize collagen type I, PP and DSP proteins. This response to carious lesions is likely to provide a basis for reparative and/or reactionary dentin formation.

A novel rat dentin mRNA coding only for dentin sialoprotein.

Dentin sialoprotein (DSP) is a major glycoprotein present in the mineralized dentin matrix that is expressed mainly by young and mature odontoblasts. Mutations in the DSP coding regions are linked to Dentinogenesis imperfecta I and II. indicating the importance of DSP in tooth formation. Previous studies have identified multiple mRNA transcripts in dentin that code for both DSP and phosphophoryns (PPs). Using reverse transcriptase-polymerase chain reaction (RT-PCR) to characterize these mRNA transcripts, we have identified a cDNA that codes for DSP, but not PP. This cDNA codes for a protein with 324 amino acids, 303 amino acids being identical to the published rat DSP sequence. However, the subsequent 21 amino acids are unique to this cDNA. Based on the coding sequence, the core protein is predicted to have a pI=4.24, a net charge of -34, and to contain four potential N-glycosylation sites and six potential sites for phosphorylation by casein kinase. That the corresponding mRNA was present in day 5 molar tooth germs was confirmed using RNA protection assays. These data, therefore, identify a novel transcript in rat tooth germs that codes only for DSP (designated as DSPII).

Sequence determination of an extremely acidic rat dentin phosphoprotein.

The mineralization process associated with the conversion of predentin to dentin is believed to be initiated and controlled by a set of acidic regulatory noncollagenous proteins (NCPs) which include phosphophoryn, the major NCP in dentin. Phosphophoryn binds tightly to collagen and is believed to initiate the formation of apatite crystals which play a central role in the mineralization process. During the process of analyzing the 3' end of an odontoblast-specific cDNA which codes for dentin sialoprotein (Ritchie, H. H., Hou, H., Veis, A., and Butler, W. T. (1994) J. Biol. Chem. 269, 3698-3702), we discovered a 801-base pair open reading frame. This downstream open reading frame encodes a putative leader sequence and a very acidic mature protein sequence having a deduced amino acid composition containing high percentages of both Ser (43%) and Asp (31%) residues which closely coincides with the amino acid composition of phosphophoryns from human, bovine, rat, and rabbit (i. e. Asp (30-40%) and Ser (38-50%)). This newly identified cDNA therefore encodes a protein with characteristics similar to phosphophoryn. Here we present the cDNA sequence, the deduced amino acid sequence, and the prospective Ser residue-specific casein kinase I and II phosphorylation sites for this putative phosphophoryn.

Junctional proteins and Ca2+ transport in the rat odontoblast-like cell line MRPC-1.

A transcellular bulk flow of Ca2+ ions through the odontoblast layer is of central importance during dentinogenesis. For this, specialized mechanisms may exist, which by a concerted action, gate Ca2+ into the proximal end of the cells and extrude the ions towards the mineralization front. To elucidate these mechanisms, an in vitro model would be useful. Mature odontoblasts are, however, post-mitotic cells and cannot be propagated in cell culture. The aim of the present study was, therefore, to characterize the odontoblast-like rat cell line MRPC-1(1) with regard to transcellular Ca2+ transport, barrier function, and intercellular junctions when cultured on membranes in Transwell chambers. The MRPC-1 cells grew as epithelial-like cells in a continuous bilayer separated by a thin collagenous matrix and with intercellular junctional complexes. They exhibited properties of a low-resistance epithelium, maintained a Ca(2+)-dependent diffusion barrier, and exhibited a functional diversity between the two cell layers. MRPC-1 cells expressed ZO-1, occludin, E-, and N-cadherins in addition to alpha-, beta-, gamma- and p120cat catenins, thereby demonstrating some traits in common with, but also differences from, epithelial cells and major differences from fibroblasts. The transcellular Ca2+ flux was inhibitable by nifedipine unidirectionally, giving evidence for an active intracellular Ca2+ transport through voltage-gated channels of the L-type. Similarities with native odontoblasts indicate that MRPC-1 cells may be useful for in vitro studies of transcellular Ca2+ transport mechanisms of importance for the calcification process.

Extracellular matrix proteins of dentine.

Bone and dentine extracellular matrix proteins are similar, consisting primarily of type I collagen, acidic proteins and proteoglycans. Although collagen forms the lattice for deposition of calcium and phosphate for formation of carbonate apatite, the non-collagenous proteins are believed to control initiation and growth of the crystals. Despite this similarity, dentine contains three unique proteins apparently absent from bone and other tissue: dentine phosphophoryn (DPP), dentine matrix protein 1 (DMP1) and dentine sialoprotein (DSP). DPP and DMP1 are acidic phosphoproteins probably involved in the control of mineralization processes. DPP may localize in gap regions of collagen and initiate apatite crystal formation by binding large quantities of calcium in a conformation that promotes this process. Extensive studies have been conducted in our laboratory on the nature, biosynthesis, localization and gene structure of DSP. Immunolocalization studies showed that rat DSP, a 53 kDa sialic acid-rich glycoprotein, was synthesized by young and mature odontoblasts, and by dental pulp cells and pre-ameloblasts, but not by ameloblasts, osteoblasts, chondrocytes or other cell types. The cDNA sequence indicated that DSP was a 366-residue protein with several potential N-glycosylation sites, as well as phosphorylation sites, but that the amino acid sequence was dissimilar to that of other known proteins. Northern blot analysis detected several mRNA species near 4.6 and 1.5 kb, indicative of alternative splicing events. Evidence for two DSP genes was obtained, further complicating this picture. Recent in situ hybridization studies utilizing rat and mouse molars and incisors indicated that DSP mRNA was expressed by young odontoblasts and odontoblasts in animals of all ages. Transcripts were also observed in pre-ameloblasts. The expression of DSP mRNA ceased when these cells matured to become secretory ameloblasts. DSP transcripts were not detected in osteoblasts or other cell types. The transient expression in pre-ameloblasts suggests a role of epithelial-mesenchymal interactions in the formation of the tooth.

Six decades of dentinogenesis research. Historical and prospective views on phosphophoryn and dentin sialoprotein.

The molecular basis underlying the mineralization process associated with the conversion of predentin to dentin is poorly understood. What is clear is that a unique set of non-collagenous proteins (NCPs) participate in the nucleation process and in hydroxyapatite growth during dentin formation. Phosphophoryn (PP), the most abundant NCP in dentin, is secreted by odontoblasts and appears at the mineralization front. Dentin sialoprotein (DSP), another NCP, also appears at the mineralization front, but only accounts for 5-8% of the weight of dentin NCPs. Functionally, PP is believed to be directly involved in tile nucleation process by virtue of its ability to bind to collagen type I, and its high affinity for calcium ions. Based on the analysis of the putative rat PP amino acid sequence, this latter activity is believed due to the highly phosphorylated character of PP, which results from the dual actions of casein kinases I and II at selected domains within PP. The precise role of DSP is currently unknown. In situ studies demonstrate that DSP is substantially expressed in odontoblasts and transiently expressed in preameloblasts. However, no information is currently available to directly explain DSP's role in mineralization. Genetically, we and others have now identified a novel DSP-PP bicistronic mammalian transcriptional unit, suggesting that the functional roles of these two NCPs may also be tightly coupled with respect to dentinogenesis. Certainly, further exciting studies are now needed to explain how this DSP-PP transcriptional unit is finally expressed: whether DSP and PP associate with one another, or with collagen at the mineralization front: and how selective mutations in either gene may influence dentin mineralization.

The conservation and regulation of rat DSP-PP gene.

Two highly expressed noncollagenous proteins associated with dentin mineralization, dentin sialoprotein (DSP) and phosphophoryn (PP), are encoded by a single DSP-PP transcript. To better understand how DSP-PP transcripts are regulated, we have determined the DSP-PP transcription start site, sequenced its 5' flanking region, and analyzed the transcriptional activity of the gene promoter out to -1615 bp. Comparison of the rat cDNA sequence with the mouse, rat and human genes clearly indicates high sequence conservation within the DSP-PP 5' flanking region, implicating the possible presence of highly conserved gene regulatory cis elements. Among a number of conserved transcription sites identified in the 5' flanking region, we demonstrate that the conserved Y box sequence (ATTGG) can specifically bind nuclear extracts from mouse MDPC23 cells. This sequence (located within the -57 bp/-52 bp 5' flanking region) therefore likely represents one DSP-PP transcriptional regulatory sequence.

Cloning and sequence determination of rat dentin sialoprotein, a novel dentin protein.

Dentin sialoprotein (DSP) is a 53-kDa protein isolated from rat dentin. It contains 29.6% carbohydrate (including 9% sialic acid) and has an overall composition similar to that of the bone sialoproteins osteopontin and bone sialoprotein (i.e. rich in Asp, Ser, Glu, and Gly). Using a monospecific anti-DSP polyclonal antibody to screen a rat incisor odontoblast cDNA library, a cDNA clone was isolated and sequenced. This approximately 750-base pair clone contained a DNA sequence corresponding to the NH2-terminal 9 amino acids of DSP. A second cDNA clone was isolated by using the first cDNA as a probe to rescreen the library. This second clone had the full-length DSP coding region. From the sequence, we deduced that the DSP cDNA coded for 366 amino acids, predominantly Asp, Ser, Glu, and Gly. The amino acid composition calculated for this sequence was very similar to that for purified DSP reported earlier; likewise the deduced molecular weight (53,045) was essentially identical to that determined by sedimentation equilibrium. Six potential N-linked glycosylation sites were present in the predicted DSP sequence. No Arg-Gly-Asp sequence was found, and the sequence for DSP was dissimilar to those of osteopontin and bone sialoprotein. Multiple transcripts near 4.6 and 1.5 kilobases were detected by Northern blot analysis in the incisor of 21-day-old rat and the tooth germ of the newborn rat. Consistent with previous immunohistochemical findings, no transcripts were detected in brain, salivary gland, heart, muscle, spleen, kidney, intestine, lung, liver, pancreas, tibia, calvaria, or osteoblast-like osteosarcoma (ROS 17/2.8) cells, indicating that DSP is specifically expressed by odontoblasts and related cells.

Molecular cloning of a human dentin sialophosphoprotein gene.

Dentin sialoprotein (DSP) and dentin phosphoprotein (DPP; phosphophoryn) are two principal dentin-specific non-collagenous proteins. DPP is extremely acidic and is rich in aspartic acid and serine. By virtue of this structure, DPP may bind large amounts of calcium and may facilitate initial mineralization of dentin matrix collagen as well as regulate the size and shape of the crystals. The function of DSP is not known. DSP and DPP are encoded by a single gene in both rat and mouse, and are uniquely expressed in odontoblasts and transiently in pre-ameloblasts. Because DSP and DPP are isolated from dentin as distinct proteins and appear to be present in different amounts, the nascent dentin sialophosphoprotein (DSPP) is likely cleaved to yield DSP and DPP. However, when, where and how the DSPP is cleaved into DSP and DPP is not clear. To further elucidate the structure and function of human DSP and DPP, we have cloned DPP and DSP cDNA by reverse transcriptase-polymerase chain reaction (RT-PCR) strategies, and then cloned and initiated characterization of a human dentin sialophosphoprotein gene. The genomic organization of human DSPP is very similar to that of mouse, containing five exons and four introns, suggesting it is a homologue of mouse dentin sialophosphoprotein (DSPP). Exons 1-4 encode for DSP, while exon 5 encodes for the C-terminus of DSP and the whole DPP. A 4.6-kb RNA transcript was detected on Northern blot analyses of total RNA extracted from immature (open root apices) human teeth using either a human DPP or DSP probe.

Molecular analysis of rat dentin sialoprotein.

Dentin sialoprotein (DSP) is a noncollagenous protein originally isolated from rat dentin. Because it is made by odontoblasts that are actively synthesizing dentin. DSP may play an important role in dentinogenesis. We have isolated a full length DSP cDNA from a rat odontoblast/dental pulp cDNA library (Ritchie et al. [1994] J. Biol. Chem. 269:3698-3702) which codes for a 17 residue signal peptide and a 366 residue, 53 kDa mature protein. In situ hybridization revealed DSP mRNA expression by odontoblasts, but no other cells, in jaws from newborn rat. Northern analysis of various rat tissues demonstrated the presence of DSP transcripts in newborn tooth germs and 21 day old rat incisors. Moreover, multiple transcripts of 4.6 kb and 1.5 kb were found in these two tissues. To better understand the origin of these DSP mRNA multiple transcripts, we have isolated two rat genomic clones. Digestion of each clone with EcoRI followed by Southern analysis revealed that DSP cDNA hybridized to a 4 kb fragment in a lambda dash clone and to a 6 kb fragment in a cosmid clone. Since DSP cDNA hybridized to a 6 kb EcoRI fragment and a 4 kb EcoRI fragment obtained from a rat liver genomic cDNA digested with EcoRI, the multiple DSP mRNA transcripts are most likely derived from two related DSP genes which coexist in the rat genome.

Immunohistochemical characterization of rapid dentin formation induced by enamel matrix derivative.

The purpose of this study was to examine the pulpal expression of dentin-related proteins during enamel matrix derivative (EMD)-induced reparative dentin formation in a pulpotomy model in pig incisors. Pulpotomies were performed on 72 lower incisors in 24 adult miniature swine. The exposed pulp tissue was treated with EMD or covered with a calcium hydroxide paste (Dycal). At predefined time-points, ranging from 4 days to 12 weeks, experimental teeth were extracted and examined by use of light microscopy, and expression of dentin-related proteins in the pulps was investigated by immunohistochemistry, using antibodies against type I collagen, dentin sialoprotein (DSP), sheathlin, and EMD. In all EMD-treated teeth a substantial amount of reparative dentin formation was observed. The amount of reparative dentin in calcium hydroxide-treated teeth was significantly smaller than in EMD-treated teeth (P < 0.005) and was less effective in bridging the pulpal wounds. Immunohistochemistry demonstrated that enamel matrix proteins were present in detectable amounts at the application site for about 4 weeks. Moreover, the expression of proteins related to dentin formation in the wounded pulp tissue was about 2 weeks advanced in EMD-treated teeth. These findings demonstrate that enamel matrix molecules have the capacity to induce rapid pulpal wound healing in pulpotomized teeth, and suggest that the longevity and continued presence of enamel matrix macromolecules at the application site can be utilized to stimulate growth and repair of dentin over a period consistent with a favorable clinical outcome.

COUP-TF gene: a structure unique for the steroid/thyroid receptor superfamily.

Two different genomic genes for the COUP-transcription factor, COUP-TF I and COUP-TF II, have been isolated from a human cosmid genomic library using a [32P]-labeled cDNA probe. Data obtained from Southern blot analysis of these cosmid clones indicated that two closely related genes exist in the human genome and have a similar genomic organization. The genes are similar in the hormone and DNA binding domains but diverge from one another in the N-terminal region. Using DNA sequencing and polymerase chain reaction (PCR) techniques we have determined that the structure of COUP-TF I consists only of three exons and two introns. Surprisingly, both zinc fingers (i.e., F1 and F2) are located in the first exon. Therefore, COUP-TF I is unique among the members of the steroid/thyroid hormone receptor superfamily which have been described to date.

Development of an odontoblast in vitro model to study dentin mineralization.

The aim of the present work was to characterize the odontoblastic proliferation, differentiation, and matrix mineralization in culture of the recently established M2H4 rat cell line. Proliferation was assessed by cell counts, differentiation by RT-PCR analysis, and mineralization by alizarin red staining, atomic absorption spectrometry, and FTIR microspectroscopy. The results showed that M2H4 cell behavior closely mimics in vivo odontoblast differentiation, with, in particular, temporally regulated expression of DMP-1 and DSPP. Moreover, the mineral phase formed by M2H4 cells was similar to that in dentin from rat incisors. Finally, because in mice, transforming growth factor (TGF)-beta1 over-expression in vivo leads to an hypomineralization similar to that observed in dentinogenesis imperfecta type II, effects of TGF-beta1 on mineralization in M2H4 cell culture were studied. Treatment with TGF-beta1 dramatically reduced mineralization, whereas positive control treatment with bone morphogenetic protein-4 enhanced it, suggesting that M2H4 cell line is a promising tool to explore the mineralization mechanisms in physiopathologic conditions.

Human dentin phosphophoryn nucleotide and amino acid sequence.

Dentin sialoprotein (DSP) and phosphophoryns (DPP) are major dentin-specific non-collagenous proteins and are synthesized by odontoblasts. DPP are extremely acidic, rich in aspartic acid and serine, possess a high affinity for calcium and collagen, and are believed to function in dentin mineralization. Whereas DSP and DPP are the products of a single gene in mouse and rat, an analogous human gene has not been described. Using RT-PCR based cloning strategies, we have cloned human DPP cDNA from immature molar root total RNA. The open reading frame of this human DPP cDNA comprises 2364 bp encoding 788 amino acids rich in serine (58%), aspartic acid (26%) and asparagine (9%). These are mostly arranged as (DSS)n (n = 1-16), DS and NSS motifs. The N-terminal sequence (DDP) matches that obtained from human DPP extracted from the roots of immature teeth. The core protein of this human DPP was calculated to have a molecular weight of 76,906 Da and a net charge of -206 with an isoelectric point of 2.65. Of the serine residues, 53% can potentially be phosphorylated by casein kinases I and II. Thus, this newly cloned human cDNA, which encodes a protein with characteristics similar to rat and mouse DPP, is identified as a human DPP.