Characterization of Recombinant Pig Enamelysin Activity and Cleavage of Recombinant Pig and Mouse Amelogenins

Ryu, Ok Hee; Fincham, Alan G.; Hu, C.-C.; Zhang, C.; Qin, Qian; Bartlett, John D.; Simmer, James P.

doi:10.1177/00220345990780030601

Cited by 182 publications

(212 citation statements)

References 36 publications

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“…Western blots have demonstrated that the early 41-and 45-kDa bands are MMP-20 . Digestion of amelogenin by MMP-20 in vitro generates the complete profile of amelogenins that accumulate in vivo, establishing conclusively that MMP-20 is the predominant secretory stage enzyme (Ryu et al, 1999). Furthermore, MMP-20 cleaves ameloblastin in vitro, generating cleavage products identical to those that can be isolated from secretory stage enamel (Iwata et al, 2007).…”

Section: Stages Of Enamel Formationmentioning

confidence: 72%

Functions of KLK4 and MMP-20 in dental enamel formation

Papagerakis²,

Yamakoshi

et al. 2008

BCHM

216

190

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Two proteases are secreted into the enamel matrix of developing teeth. The early protease is enamelysin . The late protease is kallikrein 4 (KLK4). Mutations in MMP20 and KLK4 both cause autosomal recessive amelogenesis imperfecta, a condition featuring soft, porous enamel containing residual protein. MMP-20 is secreted along with enamel proteins by secretory stage ameloblasts. Enamel protein cleavage products accumulate in the space between the crystal ribbons, helping to support them. MMP-20 steadily cleaves accumulated enamel proteins, so their concentration decreases with depth. Kallikrein 4 is secreted by transition and maturation stage ameloblasts. KLK4 aggressively degrades the retained organic matrix following the termination of enamel protein secretion. The principle functions of MMP-20 and KLK4 in dental enamel formation are to facilitate the orderly replacement of organic matrix with mineral, generating an enamel layer that is harder, less porous, and unstained by retained enamel proteins.

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Section: Stages Of Enamel Formationmentioning

confidence: 72%

Functions of KLK4 and MMP-20 in dental enamel formation

Papagerakis²,

Yamakoshi

et al. 2008

BCHM

216

190

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“…The generation of DSPP-derived proteins could serve both activation and degradation functions. The processing of DSP-DGP by MMP-20 and MMP-2 appears to us very similar to the processing of enamel proteins, such as amelogenin, by MMP-20 (39). MMP-20 processes amelogenins into a series of relatively stable cleavage products but continues to degrade those cleavage products, allowing the enamel to achieve higher degrees of mineralization.…”

Section: Discussionmentioning

confidence: 91%

Dentin Sialophosphoprotein Is Processed by MMP-2 and MMP-20 in Vitro and in Vivo

Yamakoshi

Iwata

et al. 2006

Journal of Biological Chemistry

116

135

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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 Dentin sialophosphoprotein (DSPP)2 is a multidomain extracellular matrix protein that is critical for proper dentin formation. The major DSPP domains are dentin sialoprotein (DSP), dentin glycoprotein (DGP), and dentin phosphoprotein (DPP) (1-3). In humans, nine disease-causing mutations in the DSPP gene on chromosome 4q21.3 have been reported in kindreds with isolated inherited dentin defects (4 -10). No other genes have been implicated in their etiology by mutation analyses. DSPP mutations result in a variety of dental phenotypes, including dentin dysplasia type II and dentinogenesis imperfecta types II and III. In mice, targeted knock-out of the Dspp gene (Dspp Ϫ/Ϫ ) resulted in tooth defects resembling dentinogenesis imperfecta type III (11). DSPP is expressed in other tissues besides dentin, such as in bone (12), but the protein levels in the secondary locations are hundreds of times lower than in dentin. Outside of a poorly understood association with progressive high frequency sensorineural hearing loss, DSPP mutations result in defects limited to the teeth. Perhaps because DSPP functions are so specialized, relatively little research has been focused on it, and many of its basic structural features are still unknown. DSPP itself has never been isolated or detected in dentin extracts. DSPP was discovered through its proteolytic cleavage products, starting with DPP (13). DPP is a highly acidic phosphoprotein with an isoelectric pH of 1.1 (14). Besides its distinctive amino acid composition, dominated by serine and aspartic acid (15), DPP has a conserved N-terminal sequence of AspAsp-Pro-Asn (1). DPP is difficult to study at the protein level, because it is resistant to digestion by proteases, such as trypsin (16). Cloning and characterization of the first DPP cDNA transcript revealed the extreme redundancy of its DNA and deduced amino acid sequences (17). Surprisingly, the cloned DPP transcript also encoded DSP, another major noncollagenous protein in dentin (18,19). DSP is a highly glycosylated proteoglycan (19 -21). Demonstration of a continuous open reading frame between the DSP and DPP coding regions disclosed that DSP and DPP are cleavage products of a larger protein, called DSPP (22). DGP is the most recent and smallest DSPP-derived pro...

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“…Mmp-20 is secreted along with amelogenin, ameloblastin, and enamelin by secretory stage ameloblasts (35)(36)(37). Mmp-20 activity can account for the range of cleavages observed in secretory stage enamel proteins (38) and appears to be the only protease secreted by ameloblasts during the secretory stage. Mmp20-null mice have enamel that is thinner and softer than normal, lacks enamel rod organization, and tends to chip off the crown surface (39,40).…”

mentioning

confidence: 99%

Hypomaturation Enamel Defects in Klk4 Knockout/LacZ Knockin Mice

Simmer

Lertlam

et al. 2009

Journal of Biological Chemistry

132

184

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Kallikrein 4 (Klk4) is believed to play an essential role in enamel biomineralization, because defects in KLK4 cause hypomaturation amelogenesis imperfecta. We used gene targeting to generate a knockin mouse that replaces the Klk4 gene sequence, starting at the translation initiation site, with a lacZ reporter gene. Correct targeting of the transgene was confirmed by Southern blot and PCR analyses. Histochemical X-gal (5-bromo-4-chloro-3-indolyl-␤-D-galactopyranoside) staining demonstrated expression of ␤-galactosidase in maturation stage ameloblasts. No X-gal staining was observed in secretory stage ameloblasts or in odontoblasts. Retained enamel proteins were observed in the maturation stage enamel of the Klk4 null mouse, but not in the Klk4 heterozygous or wild-type mice. The enamel layer in the Klk4 null mouse was normal in thickness and contained decussating enamel rods but was rapidly abraded following weaning, despite the mice being maintained on soft chow. In function the enamel readily fractured within the initial rod and interrod enamel above the parallel enamel covering the dentinoenamel junction. Despite the lack of Klk4 and the retention of enamel proteins, significant levels of crystal maturation occurred (although delayed), and the enamel achieved a mineral density in some places greater than that detected in bone and dentin. An important finding was that individual enamel crystallites of erupted teeth failed to grow together, interlock, and function as a unit. Instead, individual crystallites seemed to spill out of the enamel when fractured. These results demonstrate that Klk4 is essential for the removal of enamel proteins and the proper maturation of enamel crystals.Dental enamel is composed of highly ordered, very long crystals of calcium hydroxyapatite (Ca 10 (PO 4 ) 6 (OH) 2 ). Mature enamel crystallites are about 70 nm wide and 30 nm thick, but are of unmeasurable length (1), probably extending all the way from the dentin layer to the surface of the tooth (2). Enamel crystallites are organized into bundles called rods, with about 10,000 parallel crystals in a rod (3). Each enamel rod is the product of a single ameloblast, the cell type that forms a continuous sheet over the developing enamel and orchestrates its formation. Dental enamel of erupted teeth is ϳ95% mineral (by weight) (4), with most of the non-mineral component being water. Protein comprises Ͻ1% of its weight. Forming enamel, however, is over 30% protein (5). Much of the protein is reabsorbed by ameloblasts and degraded in lysosomes (6, 7), but extracellular proteases also play a role in matrix protein removal (8 -10).Dental enamel formation is divided into secretory, transition, and maturation stages (11,12). During the secretory stage, enamel crystals grow primarily in length. As the crystals extend, the enamel layer expands. Enamel crystallites lengthen along a mineralization front at the secretory surface of the ameloblast cell membrane. There, mineral deposits rapidly on the crystallite tips, and very slowly on their sides...

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Characterization of Recombinant Pig Enamelysin Activity and Cleavage of Recombinant Pig and Mouse Amelogenins

Cited by 182 publications

References 36 publications

Functions of KLK4 and MMP-20 in dental enamel formation

Functions of KLK4 and MMP-20 in dental enamel formation

Dentin Sialophosphoprotein Is Processed by MMP-2 and MMP-20 in Vitro and in Vivo

Hypomaturation Enamel Defects in Klk4 Knockout/LacZ Knockin Mice

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