Biomedical subjects

Yasuo Yamakoshi

Publications and source records attributed to Yasuo Yamakoshi.

14 recordsLinked to original sources

PubMed2006

Dentin sialophosphoprotein is processed by MMP-2 and MMP-20 in vitro and in vivo.

Dentin sialophosphoprotein (DSPP) is a major secretory product of odontoblasts and is critical for proper tooth dentin formation. During dentinogenesis, DSPP is proteolytically cleaved into smaller subunits. These cleavages are proposed activation steps, and failure to make these cleavages is a potential cause of developmental tooth defects. We tested the hypothesis that dentin-resident matrix metalloproteinases catalyze the cleavages that process DSPP. We defined the exact DSPP cleavages that are catalyzed by proteases during crown formation by isolating DSPP-derived proteins from developing porcine molars and characterizing their N-terminal sequences and apparent size on SDS-PAGE and Western blots. The in vivo DSPP cleavage sites were on the N-terminal sides of Thr(200), Ser(330), Val(353), Leu(360), Ile(362), Ser(377), Ser(408), and Asp(458). The initial DSPP cleavage is between dentin glycoprotein (DGP) and dentin phosphoprotein (DPP), generating dentin sialoprotein (DSP)/DGP and DPP. Gelatin and casein zymograms identified MMP-2, MMP-20, and KLK4 in the dentin extracts. MMP-2 and MMP-20 were purified from over 150 g of porcine dentin powder and incubated with DSP-DGP and DPP. These enzymes show no activity in further cleaving DPP. MMP-20 cleaves DSP-DGP to generate DSP and DGP. MMP-20 also cleaves DSP at multiple sites, releasing N-terminal DSP cleavage products ranging in size from 25 to 38 kDa. MMP-2 makes multiple cleavages near the DSP C terminus, releasing larger forms of DGP, or "extended DGPs." Exact correspondence between DSPP cleavage sites that occur in vivo and those generated in vitro demonstrates that MMP-2 and MMP-20 process DSPP into smaller subunits in the dentin matrix during odontogenesis.

Amino Acid Sequence↗

PubMed2006

MMP-20 is predominately a tooth-specific enzyme with a deep catalytic pocket that hydrolyzes type V collagen.

Matrix metalloproteinase-20 (MMP-20, enamelysin) has a highly restricted pattern of expression. In healthy tissues, MMP-20 is observed in the enamel organ and pulp organ of developing teeth and is present only as an activated enzyme. To identify other tissues that may express MMP-20, we performed a systematic mouse tissue expression screen. Among the non-tooth tissues assayed, MMP-20 transcripts were identified only in minute quantities within the large intestine. The murine Mmp20 promoter was cloned, sequenced, and assessed for potential tooth-specific regulatory elements. In silico analysis identified four promoter modules that were common to Mmp20 and at least two of three coregulated predominantly tooth-specific genes that encode ameloblastin, amelogenin, and enamelin. We asked if the highly restricted MMP-20 expression pattern was associated with a broad substrate specificity that might preclude its expression in other tissues. An iterative mixture-based random doedecamer peptide library screen with Edman sequencing of MMP-20 cleavage products revealed that, among MMPs previously screened, MMP-20 had unique substrate preferences. These preferences indicate that MMP-20 has a deep and wide catalytic pocket that can accommodate substrates with large aromatic residues in the P1' position. On the basis of matrices derived from the peptide library data, we identified and then confirmed that type V collagen is an MMP-20 substrate. Since type V collagen is not present in dental enamel but is an otherwise widely distributed collagen, and since only active MMP-20 has been observed in teeth, our data suggest that control of MMP-20 activity is primarily regulated by transcriptional means.

Animals↗

PubMed2006

Proteomic analysis of enamel matrix using a two-dimensional protein fractionation system.

Our objectives in this study were to perform separate proteomic analyses of porcine soft and hard enamel matrices, using the ProteomeLab PF-2D System, to compare the contents of the hard and soft enamel and to identify matrix constituents that are absent from the early maturation stage. Developing first permanent molars were dissected from 6-month-old pigs. Both immature and mature enamel samples were obtained by scraping the secretory-stage (soft) and maturation-stage (hard) enamel, respectively. Enamel matrix samples were sequentially extracted and fractionated with 50 mM phosphate buffer (pH 7.4) and then with 50 mM carbonate buffer (pH 10.8). The neutral enamel extract was separated into four fractions by successive ammonium sulfate precipitations. The alkaline enamel extract was separated into four fractions by ion-exchange chromatography. These eight extracts from both the soft and hard enamel were injected for chromatofocusing. Soft enamel fractions containing constituents absent from the hard enamel were further separated by reverse-phase high-performance liquid chromatography. The major soft enamel constituents absent from the hard enamel were acidic glycoproteins, corresponding to the 32-kDa enamelin, and the 29-, 27-, 15-, 13-, 8- and 6-kDa C-terminal fragments of ameloblastin. Loss of these glycoproteins is associated with a post-transition increase in enamel mineralization.

Amelogenesis↗

PubMed2006

Porcine SPARC: isolation from dentin, cDNA sequence, and computer model.

Genes encoding the major enamel matrix proteins and non-collagenous proteins of bone and dentin are members of the secretory calcium-binding phosphoprotein (SCPP) family, which originated from ancestral SPARC (secreted protein, acidic and rich in cysteine; BM-40/osteonectin). To better understand the role of SPARC in mineralizing systems, we isolated SPARC from developing pig teeth, deduced its primary structure from the cDNA sequence, and determined its quaternary structure by homology modelling with reference to human SPARC crystal structures. The guanidine/EDTA extract from porcine dentin was fractionated by anion-exchange and size-exclusion chromatography. Stains-all positive bands at 38 and 35 kDa gave the N-terminal sequences APQQEALPDETEV and DFEKNYNMYIFPV, which corresponded to the SPARC N terminus and an internal region of the protein. Porcine SPARC contains 300 amino acids, including the 17-amino acid signal peptide, and shares 96.2% amino acid sequence identity with human SPARC. Without post-translational modifications, the 283-amino acid secreted protein has a molecular mass of 32.3 kDa. The three-dimensional model revealed that porcine SPARC contains a single N-linked glycosylation at N113, seven intramolecular disulfide bridges, and assembles into dimers. SPARC is composed of three structural/functional domains: an acidic Ca2+-binding, a follistatin-like, and an extracellular calcium-binding domain.

Amino Acid Sequence↗

PubMed2006

How do enamelysin and kallikrein 4 process the 32-kDa enamelin?

The activities of two proteases--enamelysin (MMP-20) and kallikrein 4 (KLK4)--are necessary for dental enamel to achieve its high degree of mineralization. We hypothesize that the selected enamel protein cleavage products which accumulate in the secretory-stage enamel matrix do so because they are resistant to further cleavage by MMP-20. Later, they are degraded by KLK4. The 32-kDa enamelin is the only domain of the parent protein that accumulates in the deeper enamel. Our objective was to identify the cleavage sites of 32-kDa enamelin that are generated by proteolysis with MMP-20 and KLK4. Enamelysin, KLK4, the major amelogenin isoform (P173), and the 32-kDa enamelin were isolated from developing porcine enamel. P173 and the 32-kDa enamelin were incubated with MMP-20 or KLK4 for up to 48 h. Then, the 32-kDa enamelin digestion products were fractionated by reverse-phase high-performance liquid chromatography (RP-HPLC) and characterized by Edman sequencing, amino acid analysis, and mass spectrometry. Enamelysin cleaved the 32-kDa enamelin only after it was deglycosylated. Kallikrein 4 digestion of the 32-kDa enamelin generated nine major cleavage products, six of which were successfully characterized. After 12 h of digestion with KLK4, all of the 32-kDa enamelin had been cleaved, but some cleavage products persisted after 48 h of digestion.

Amelogenesis↗

PubMed2006

Porcine dentin matrix protein 1: gene structure, cDNA sequence, and expression in teeth.

Dentin matrix protein 1 (DMP1) is an acidic non-collagenous protein that is necessary for the proper biomineralization of bone, cartilage, cementum, dentin, and enamel. Dentin matrix protein 1 is highly phosphorylated and potentially glycosylated, but there is no experimental data identifying which specific amino acids are modified. For the purpose of facilitating the characterization of DMP1 from pig, which has the advantage of large developing teeth for obtaining protein in quantity and extensive structural information concerning other tooth matrix proteins, we characterized the porcine DMP1 cDNA and gene structure, raised anti-peptide immunoglobulins that are specific for porcine DMP1, and detected DMP1 protein in porcine tooth extracts and histological sections. Porcine DMP1 has 510 amino acids, including a 16-amino acid signal peptide. The deduced molecular weight of the secreted, unmodified protein is 53.5 kDa. The protein has 93 serines and 12 threonines in the appropriate context for phosphorylation, and four asparagines in a context suitable for glycosylation. Dentin matrix protein 1 protein bands with apparent molecular weights between 30 and 45 kDa were observed in partially purified dentin extracts. In developing teeth, immunohistochemistry localized DMP1 in odontoblasts and the dentinal tubules of mineralized dentin and in ameloblasts, but not in the enamel matrix.

Amino Acid Sequence↗

PubMed2006

Co-operative mineralization and protein self-assembly in amelogenesis: silica mineralization and assembly of recombinant amelogenins in vitro.

An amorphous silica mineralization technique was used to produce inorganic/protein composites to elucidate the structure and mechanism of formation of amelogenin assemblies, which may play an important role in regulating enamel structure during the initial stages of amelogenesis. Full-length recombinant amelogenins from mouse (rM179) and pig (rP172) were investigated along with key degradation products (rM166 and native P148) lacking the hydrophilic C terminus found in parent molecules. The resulting products were examined using transmission electron microscopy and/or small-angle X-ray scattering. Using protein concentrations of 0.1-3 mg ml-1, large monodisperse spheres of remarkably similar mean diameters were observed using rM179 (124+/-4 nm) and rP172 (126+/-7 nm). These spheres also exhibited 'internal structure', comprising nearly spherical monodisperse particles of approximately 20 nm in diameter. In the presence of rM166, P148, and bovine serum albumin (control), large unstructured and randomly shaped particles (250-1000 nm) were observed. Without added protein, large dense spherical particles of silica (mean approximately 500 nm) lacking internal structure were produced. These findings demonstrate that full-length amelogenins have the ability to form higher-order structures, whereas amelogenins that lack the hydrophilic C terminus do not. The results also suggest that full-length amelogenin can guide the formation of organized mineralized structures through co-operative interactions between assembling protein and forming mineral.

Amelogenesis↗

PubMed2006

Porcine enamel protein fractions contain transforming growth factor-beta1.

BACKGROUND: Enamel extracts are biologically active and capable of inducing osteogenesis and cementogenesis, but the specific molecules carrying these activities have not been ascertained. The purpose of this study was to identify osteogenic factors in porcine enamel extracts. METHODS: Enamel proteins were separated by size-exclusion chromatography into four fractions, which were tested for their osteogenic activity on osteoblast-like cells (ST2) and human periodontal ligament (HPDL) cells. RESULTS: Fraction 3 (Fr.3) and a transforming growth factor-beta 1 (TGF-beta1) control reduced alkaline phosphatase (ALP) activity in ST2 but enhanced ALP activity in HPDL cells. The enhanced ALP activity was blocked by anti-TGF-beta antibodies. Furthermore, using a dual-luciferase reporter assay, we demonstrated that Fr.3 can induce the promoter activity of the plasminogen activator inhibitor type 1 (PAI-1) gene. CONCLUSION: These results show that the osteoinductive activity of enamel extracts on HPDL cells is mediated by TGF-beta1.

Alkaline Phosphatase↗

PubMed2005

Dentin glycoprotein: the protein in the middle of the dentin sialophosphoprotein chimera.

Dentin sialophosphoprotein (DSPP) is a major secretory product of odontoblasts and is critical for proper dentin formation. DSPP is believed to be processed into only two structural/functional domains: dentin sialoprotein (DSP) and dentin phosphoprotein (DPP). Here we report the isolation and characterization of a third domain of DSPP, designated dentin glycoprotein (DGP). DGP was isolated from a guanidine/EDTA extract of porcine tooth dentin by ion exchange, hydroxyapatite affinity, size exclusion, and RP-HPL chromatography. Endoproteinase lysine C digestion products of DGP were characterized by Edman sequencing and mass spectrometry. The porcine DGP backbone is the 81-amino acid segment of DSPP (Ser392 to Gly472) between the DSP and DPP domains. DGP has four phosphorylated serine residues (Ser453, Ser455, Ser457, and Ser462) and one glycosylated asparagine (Asn397). There are no other post-translational modifications. DGP is a stains-all positive protein with an apparent molecular mass on SDS-PAGE of 19 kDa, which is reduced by glycopeptidase A digestion to 16 kDa. A variety of glycans can be linked to Asn397. All are complex biantennary structures with a common N-linked pentasaccharide core (mannose3-N-acetylglucosamine2), most with a fucosyl residue on the innermost N-acetylglucosamine. The alpha1-3 and alpha1-6 arms are always galactose beta1-4 N-acetylglucosamine beta1-2 mannose, and either or both arms can be unsialidated or monosialidated. The calculated monoisotopic molecular masses of the different glycosylated forms of the DGP phosphoprotein are: unsialidated 10,523 and 10,670, monosialidated 10,815 and 10,961, and disialidated 11,106, and 11,252 Da, with the disialidated forms being the most abundant.

Acetylglucosamine↗

PubMed2005

Proteomics and genetics of dental enamel.

The initiation of enamel crystals at the dentino-enamel junction is associated with the expression of dentin sialophosphoprotein (DSPP, a gene normally linked with dentin formation), three 'structural' enamel proteins--amelogenin (AMELX), enamelin (ENAM), and ameloblastin (AMBN)--and a matrix metalloproteinase, enamelysin (MMP20). Enamel formation proceeds with the steady elongation of the enamel crystals at a mineralization front just beneath the ameloblast distal membrane, where these proteins are secreted. As the crystal ribbons lengthen, enamelysin processes the secreted proteins. Some of the cleavage products accumulate in the matrix, others are reabsorbed back into the ameloblast. Once crystal elongation is complete and the enamel layer reaches its final thickness, kallikrein 4 (KLK4) facilitates the breakdown and reabsorption of accumulated enamel matrix proteins. The importance of the extracellular matrix proteins to proper tooth development is best illustrated by the dramatic dental phenotypes observed in the targeted knockouts of enamel matrix genes in mice (Dspp, Amelx, Ambn, Mmp20) and in human kindreds with defined mutations in the genes (DSPP, AMELX, ENAM, MMP20, KLK4) encoding these matrix proteins. However, ablation studies alone cannot give specific mechanistic information on how enamel matrix proteins combine to catalyze the formation of enamel crystals. The best approach for determining the molecular mechanism of dental enamel formation is to reconstitute the matrix and synthesize enamel crystals in vitro. Here, we report refinements to the procedures used to isolate porcine enamel and dentin proteins, recent advances in the characterization of enamel matrix protein posttranslational modifications, and summarize the results of human genetic studies that associate specific mutations in the genes encoding matrix proteins with a range of dental phenotypes.

Amelogenesis↗

PubMed2004

Porcine dentin sialoprotein is a proteoglycan with glycosaminoglycan chains containing chondroitin 6-sulfate.

Dentin sialoprotein (DSP) is a glycoprotein that is critical for proper tooth dentin formation, but little is known about the nature of its carbohydrate attachments and other post-translational modifications. We have isolated DSP from pig dentin and demonstrate that it is a proteoglycan. Polyclonal antibodies were raised in chicken against recombinant pig DSP, and used to identify native DSP in fractions of tooth dentin proteins extracted from developing pig molars. Amino acid analyses and characterization of lysylendopeptidase cleavage products confirmed that the purified protein was DSP, and that Arg391 is at the DSP C terminus. On SDS-PAGE and on urea gels, DSP appeared as a smear extending from 280 to 100 kDa, but in the presence of beta-mercaptoethanol the top of the DSP smear disappeared. The high molecular weight material was likely comprised of covalent DSP dimers connected by a disulfide bridge at Cys205. Oligosaccharides were released from DSP following N- and O-linked glycosidase digestions, but these digestions had little effect on the apparent molecular weight of DSP on SDS-PAGE, when compared with the significant reduction following chondroitinase ABC digestion. Glycosaminoglycanases with assorted glycosaminoglycan (GAG) cleavage specificities coupled with Western analyses of the cleaved GAG "stubs" demonstrated that the DSP GAG attachments contain chondroitin 6-sulfate, but not keratan sulfate, heparan sulfate, chondroitin, or chondroitin 4-sulfate. DSP binds biotin-labeled hyaluronic acid, and such binding is inhibited by the addition of unlabeled hyaluronic acid. We conclude that DSP is a proteoglycan and that GAG attachments are the predominant structural feature of porcine DSP.

Amino Acid Sequence↗

PubMed2003

Characterization of porcine dentin sialoprotein (DSP) and dentin sialophosphoprotein (DSPP) cDNA clones.

Dentin sialophosphoprotein (DSPP) is a chimeric glycoprotein with dentin sialoprotein (DSP) on its N-terminus and dentin phosphoprotein (DPP) on its C-terminus. We have constructed and screened a unidirectional cDNA library derived from the pulp organ of developing pig teeth, and isolated cDNA clones encoding DSP-only, as well as two DSPP clones with alternative sequences in their 3' coding regions. The DSP-only transcript has an open reading frame of 386 codons, and is generated through the use of a polyadenylation signal within intron 4, immediately following the DSP coding region. the use of this polyadenylation signal deletes the DPP coding region and places a TGA translation termination signal as the fourth codon following the exon 4-encoded segment. The DSPP cDNAs contain open reading frames of 593 and 600 codons. Northern blots hybridized to radiolabeled DSP probes showed bands at 1.4, 2.5, 4.4, and 4.8 kb. Cloning and characterization of reverse transcriptase polymerase chain reaction products confirmed the existence of mRNA encoding pDSP386, pDSPP593, and pDSPP600in vivo, but also suggested that DNA sequence redundancies in the DSPP coding region make it prone to cloning artifacts.

Amino Acid Sequence↗

PubMed2002

Porcine kallikrein-4 activation, glycosylation, activity, and expression in prokaryotic and eukaryotic hosts.

Kallikrein-4 (KLK4) is a serine proteinase believed to be important in the normal development of dental enamel. We isolated native KLK4 from developing pig enamel and expressed four recombinant forms. Pig KLK4 was expressed in bacteria with and without the propeptide, and in two eukaryotic systems. Recombinant pig KLK4 was secreted as a zymogen by '293' cells and purified. The proKLK4 was activated in vitro by thermolysin and recombinant pig enamelysin, but not by native KLK4. These results were confirmed using a fluorescent peptide analog of the KLK4 propeptide-enzyme junction. Native KLK4 appears as a doublet at 37 kDa and 34 kDa on sodium dodecyl sulfate-polyacrylamide gel electrophoresis. Removal of N-linked oligosaccharides by digestion with deglycosidase-F reduced the doublet to a single band at approximately 28 kDa, demonstrating that the active enzyme is glycosylated, and that the 37 kDa and 34 kDa forms differ only in their number of glycosylations. Deglycosylation was also associated with a loss of proteolytic activity. We digested recombinant pig amelogenin with native KLK4 and characterized the cleavage products by N-terminal sequencing and mass spectrometry. Eleven cleavage sites in the amelogenin protein were identified, demonstrating that KLK4 degrades amelogenin and is likely to participate in the degradation of enamel proteins in vivo.

Amelogenin↗

PubMed2002

Porcine N-acetylgalactosamine 6-sulfatase (GALNS) cDNA sequence and expression in developing teeth.

Mucopolysaccharidosis type IVA (Morquio A syndrome, MPS IVA) is a rare, autosomal recessive disorder with a prevalence of 1 in 170,000 live births. It is caused by a deficiency of N-acetylgalactosamine 6-sulfatase (GALNS), a lysosomal hydrolase encoded by a gene on human chromosome 16q24.3. Mucopolysaccharidosis type IVA is the only known MPS that is associated with structural defects in dental enamel. GALNS cleaves the sulfate group from N-acetylgalactosamine 6-sulfate and galactose 6-sulfate, which are specifically found in keratan sulfate and chondroitin 6-sulfate. A pathologic absence of GALNS activity results in the accumulation of these glycosaminoaglycans in the urine and in the lysosomes of tissues that turn them over. There is currently no animal model for MPS IVA. To learn more about how a GALNS deficit could lead to enamel defects, we have cloned and characterized a full-length pig GALNS cDNA. GALNS mRNA was localized in developing teeth by in situ hybridization, Northern blot, and reverse-transcription polymerase chain reaction analyses, while GALNS substrates were localized using immunohistochemistry. We report that secretory ameloblasts were positive for GALNS mRNA, as well as for keratan sulfate and chondroitin 6-sulfate. We conclude that enamel defects associated with the loss of GALNS activity in persons with MPS IVA are likely to result from the pathological accumulation of keratan sulfate and chondroitin 6-sulfate in the lysosomes of secretory stage ameloblasts.

Amino Acid Sequence↗