DPPI May Activate KLK4 during Enamel Formation

Kallikrein-4 (KLK4) is a serine protease expressed during enamel maturation, and proteolytic processing of the enamel matrix by KLK4 is critical for proper enamel formation. KLK4 is secreted as an inactive zymogen (pro-KLK4), and identification of its activator remains elusive. Dipeptidyl peptidase I (DPPI) is a cysteine aminopeptidase that can activate several serine proteases. In this study, we sought to examine DPPI expression in mouse enamel organ and determine if DPPI could activate KLK4. Real-time PCR showed DPPI expression throughout amelogenesis, with highest expression at maturation, and immunohistochemical staining of mouse incisors confirmed DPPI expression by ameloblasts. We demonstrate in vitro that DPPI activates pro-KLK4 to cleave a fluorogenic peptide containing a KLK4 cleavage site. Examination of mature enamel from DPPI null mice by FTIR showed no significant accumulation of protein; however, microhardness testing revealed that loss of DPPI expression significantly reduced enamel hardness

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

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

Fam83h is Associated with Intracellular Vesicles and ADHCAI

Defects in FAM83H on human chromosome 8q24.3 cause autosomal-dominant hypocalcified amelogenesis imperfecta (ADHCAI). FAM83H does not encode a recognizable signal peptide, so we predicted that the Fam83h protein functions within the cell. We tested this hypothesis by constitutively expressing mouse Fam83h with green fluorescent protein (GFP) fused to its C-terminus in HEK293 and HeLa cell lines. Green fluorescent signal from the Fam83h-GFP fusion protein was associated with perinuclear vesicles, usually in the vicinity of the Golgi apparatus. No signal was observed within the nucleus. In addition, we identified FAM83H nonsense mutations in Hispanic (C1330C>T; p.Q444X) and Caucasian (c.1192C>T; p.Q398X) families with ADHCAI. We conclude that Fam83h localizes in the intracellular environment, is associated with vesicles, and plays an important role in dental enamel formation. FAM83H is the first gene involved in the etiology of amelogenesis imperfecta (AI) that does not encode a secreted protein

Altered Enamelin Phosphorylation Site Causes Amelogenesis Imperfecta

Defects in the enamelin gene (ENAM) cause amelogenesis imperfecta (AI). Our objective was to identify the genetic etiology of enamel hypoplasia in a Caucasian proband. Our hypothesis was that ENAM was defective. The proband and his father have an AG insertion (g.13185_13186insAG; p.422FsX448) in ENAM previously identified in AI kindreds from Slovenia and Turkey. The proband, his brother, and his mother have a novel missense mutation (g.12573C>T) that substitutes leucine for a phosphorylated serine (p.S216L) in the 32-kDa enamelin cleavage product. In this family, a defect in one ENAM allele caused minor pitting or localized enamel hypoplasia, whereas defects in both alleles caused severe enamel malformations, with little or no mineral covering dentin. Ser216 is one of two serines on the 32-kDa enamelin that is phosphorylated by Golgi casein kinase and is thought to mediate calcium binding. We propose that phosphorylation of enamelin is critical for its function