Jan C.-C. Hu

Expression, structure, and function of enamel proteinases

Proteinases serve two important functions during dental enamel formation: They (a) process and (b) degrade enamel proteins. Different enzymes carry out these functions. Enamelysin (MMP-20) is the foremost enamel matrix-processing enzyme. Its expression initiates prior to the onset of dentin mineralization and continues throughout the secretory stage of amelogenesis. In vitro, enamelysin catalyzes all of the amelogenin cleavages that are known to occur during the secretory stage in vivo, and it is probably the enzyme responsible for the processing of all enamel proteins. There is evidence suggesting that enamelysin activity is critical for proper enamel formation. Uncleaved and processed enamel proteins often segregate into different compartments within the developing enamel layer, suggesting that they may have different functions. Intact ameloblastin and its C-terminal cleavage products localize in the superficial rod and interrod enamel, while its N-terminal cleavage products congregate in the sheath space. Intact enamelin is only present at the mineralization front within a micrometer of the enamel surface, while its cleavage products concentrate in the rod and interrod enamel. Processed enamel proteins accumulate during the secretory stage, but disappear early in the maturation stage. Enamel matrix serine proteinase 1 (EMSP1), now officially designated kallikrein 4 (KLK4), is believed to be the predominant degradative enzyme that clears enamel proteins from the matrix during maturation. KLK4 expression initiates during the transition stage and continues throughout maturation. KLK4 concentrates at the enamel surface when the enamel matrix disappears, and aggressively degrades amelogenin in vitro. During tooth development, proteinases are secreted by ameloblasts into the extracellular space, where they cleave enamel proteins by catalyzing the hydrolysis of peptide bonds. Enamel proteinases are present in low abundance and are not likely to participate directly in the mineralization process. Two major enamel proteinases have been identified: enamelysin (MMP20) and kallikrein 4 (KLK4). These proteinases are expressed at different times and have different functions. Their roles are to modify and/or to eliminate enamel matrix proteins, which affects the way enamel proteins interact with each other and with the developing enamel crystallites. A brief review of dental enamel formation is presented, followed by a more detailed analysis of enamelysin and KLK4 expression, structure, and function.

Characterization of the mouse and human PRSS17 genes, their relationship to other serine proteases, and the expression of PRSS17 in developing mouse incisors ☆

The human PRSS17 (serine protease 17) gene, which is located on chromosome 19q in a cluster of genes encoding serine proteases, has been variously designated enamel matrix serine proteinase 1 (EMSP1), prostase, KLK4, and KLK-L1. We have cloned and characterized the mouse and human PRSS17 genes. Both have six exons and five introns. The mouse PRSS17 gene sequence is 10134bp; the human sequence is 7115bp. Computer analysis of the mouse PRSS17 gene sequence upstream of the translation initiation codon identified two potential transcription initiation sites, at nucleotides 2878 and 2336. The first nucleotide of the reported mouse PRSS17 cDNA sequence corresponds to position 2352 on the gene, only 16 bases downstream from one of the putative transcription initiation sites. Repetitive DNA sequences from the MSR1 family are found in both the mouse and human PRSS17 genes. Additionally, the human PRSS17 gene contains Tigger2, MER8, and Alu repetitive sequences. Phylogenetic analyses of human and rodent proteases suggest that the PRSS17 protein is not a member of the kallikrein family of serine proteases but that the PRSS17 gene may have originated prior to the divergence of the kallikrein and trypsin families of proteases. To better characterize the timing of PRSS17 expression in developing teeth, we performed in-situ hybridization on postnatal day 3 developing mouse mandibular incisors. PRSS17 mRNA was not detected in secretory stage ameloblasts but could be detected in odontoblasts, while transition-stage and maturation-stage ameloblasts were strongly positive. This pattern supports a role for the PRSS17 protein in the degradation of enamel proteins.

Mmp-20 and Klk4 Cleavage Site Preferences for Amelogenin Sequences

Mmp-20 and Klk4 are the two key enamel proteases. Can both enzymes process amelogenin to generate the major cleavage products that accumulate during the secretory stage of amelogenesis? We isolated Mmp-20 and Klk4 from developing pig teeth and used them to digest the tyrosine-rich amelogenin polypeptide (TRAP), the leucine-rich amelogenin protein (LRAP), and 5 fluorescence peptides. We characterized the digestion products by LC-MSMS, SDS-PAGE, and C18 RP-HPLC monitored with fluorescence and UV detectors. Mmp-20 cleaves amelogenin sequences after Pro162, Ser148, His62, Ala63, and Trp45. These cleavages generate all of the major cleavage products that accumulate in porcine secretory-stage enamel: the 23-kDa, 20-kDa, 13-kDa, 11-kDa, and 6-kDa (TRAP) amelogenins. Mmp-20 cleaves LRAP after Pro45 and Pro40, producing the two LRAP products previously identified in tooth extracts. Among these key cleavage sites, Klk4 was able to cleave only after His62. We propose that Mmp-20 alone processes amelogenin during the secretory stage.

Processing of Ameloblastin by MMP-20

Ameloblastin (AMBN) cleavage products are the most abundant non-amelogenin proteins in the enamel matrix of developing teeth. AMBN N-terminal cleavage products accumulate in the sheath space between enamel rods, while AMBN C-terminal products localize within rods. We tested the hypothesis that MMP-20 is the protease that cleaves AMBN. Glycosylated recombinant porcine AMBN (rpAMBN) was expressed in human kidney 293F cells, and recombinant porcine enamelysin (rpMMP-20) was expressed in bacteria. The purified proteins were incubated together at an enzyme:substrate ratio of 1:100. N-terminal sequencing of AMBN digestion products determined that rpMMP-20 cleaved rpAMBN after Pro2, Gln130, Gln139, Arg170, and Ala222. This shows that MMP-20 generates the 23-kDa AMBN starting at Tyr223, as well as the 17-kDa (Val1-Arg170) and 15-kDa (Val1-Gln130) AMBN cleavage products that concentrate in the sheath space during the secretory stage. We conclude that MMP-20 processes ameloblastin in vitro and in vivo.

Cleavage Site Specificity of MMP-20 for Secretory-stage Ameloblastin

Ameloblastin is processed by protease(s) during enamel formation. We tested the hypothesis that MMP-20 (enamelysin) catalyzes the cleavages that generate secretory-stage ameloblastin cleavage products. We isolated a 23-kDa ameloblastin cleavage product from developing enamel and determined its N-terminus sequence. Ameloblastin was stably expressed and secreted from HEK293-H cells, purified, and digested with MMP-20 or Klk4 (kallikrein 4). The digests were analyzed by SDS-PAGE and Western blotting, and cleavage products were characterized by N-terminal sequencing. Six fluorescent peptides were digested with MMP-20 and Klk4 and analyzed by RP-HPLC and by mass spectrometry. MMP-20 cleaved each peptide exactly at the sites corresponding to ameloblastin cleavages catalyzed in vivo. Klk4 cleaved ameloblastin and the fluorescent peptides at sites not observed in vivo, and cleaved at only a single correct site: before Leu171. We conclude that MMP-20 is the enzyme that processes ameloblastin during the secretory stage of amelogenesis, and we present a hypothesis about the sequence of ameloblastin cleavages.

Cloning and Characterization of the Mouse and Human Enamelin Genes

Enamelin is likely to be essential for proper dental enamel formation. It is secreted by ameloblasts throughout the secretory stage and can readily be isolated from the enamel matrix of developing teeth. The gene encoding human enamelin is located on the long arm of chromosome 4, in a region previously linked to an autosomal-dominant form of amelogenesis imperfecta (AI). To gain information on the structure of the enamelin gene and to facilitate the future assessment of the role of enamelin in normal and diseased enamel formation, we have cloned and characterized the mouse and human enamelin genes. Both genes are about 25 kilobases long. The enamelin gene has 10 exons interrupted by 9 introns. Translation initiates in exon 3 and terminates in exon 10. All of the intron/exon junctions within the mouse and human enamelin coding regions are between codons, so there are no partial codons in any exon, and deletion of one or more coding exons by alternative RNA splicing would not shift the downstream reading frame.

Splicing Determines the Glycosylation State of Ameloblastin

In developing porcine enamel, the space between enamel rods selectively binds lectins and ameloblastin (Ambn) N-terminal antibodies. We tested the hypothesis that ameloblastin N-terminal cleavage products are glycosylated. Assorted Ambn cleavage products showed positive lectin staining by peanut agglutinin (PNA), Maclura pomifera agglutinin (MPA), and Limulus polyphemus agglutinin (LPA), suggesting the presence of an O-linked glycosylation containing galactose (Gal), N-acetylgalactosamine (GalNAc), and sialic acid. Edman sequencing of the lectin-positive bands gave the Ambn N-terminal sequence: VPAFPRQPGT X GVASL X LE. The blank cycles for Pro11 and Ser17 confirmed that these residues are hydroxylated and phosphorylated, respectively. The O-glycosylation site was determined by Edman sequencing of pronase-digested Ambn, which gave HPPPLP X QPS, indicating that Ser86 is the site of the O-linked glycosylation. This modification is within the 15-amino-acid segment (73-YEYSLPVHPPPLPSQ-87) deleted by splicing in the mRNA encoding the 380-amino-acid Ambn isoform. We conclude that only the N-terminal Ambn products derived from the 395-Ambn isoform are glycosylated.

Transgenic Rescue of Enamel Phenotype in Ambn Null Mice

Ameloblastin null mice fail to make an enamel layer, but the defects could be due to an absence of functional ameloblastin or to the secretion of a potentially toxic mutant ameloblastin. We hypothesized that the enamel phenotype could be rescued by the transgenic expression of normal ameloblastin in Ambn mutant mice. We established and analyzed 5 transgenic lines that expressed ameloblastin from the amelogenin (AmelX) promoter and identified transgenic lines that express virtually no transgene, slightly less than normal (Tg+), somewhat higher than normal (Tg++), and much higher than normal (Tg+++) levels of ameloblastin. All lines expressing detectable levels of ameloblastin at least partially recovered the enamel phenotype. When ameloblastin expression was only somewhat higher than normal, the enamel covering the molars and incisors was of normal thickness, had clearly defined rod and interrod enamel, and held up well in function. We conclude that ameloblastin is essential for dental enamel formation.

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.

Developmental Disorders of Dentin

Tooth morphogenesis, dentin, enamel, and cementum formation are highly regulated developmental processes. Dentin is less mineralized than enamel, but withstands frequent masticatory forces, provides support for dental enamel, and protects the dental pulp. Developmental dentin defects result from genetic or environmental factors. Mutations affecting genes regulating odontoblast differentiation, dentin matrix protein production and/or mineralization result in hereditary dentin defects (HDDs). Genetic studies have determined the etiologies of many forms of hereditary dentin defects, providing fresh insights into dentin formation and have enabled the use of efficient genetic tests to ensure correct diagnoses, appropriate treatment, and improved management of patients with HDDs.