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Preferential and selective degradation and removal of amelogenin adsorbed on hydroxyapatites by MMP20 and KLK4 in vitro.

Zhu L, Liu H, Witkowska HE, Huang Y, Tanimoto K, Li W - Front Physiol (2014)

Bottom Line: We found that majority of amelogenin adsorbed on HAP was released into the surrounding solution by enzymatic processing (88% for MMP20 and 98% for KLK4).The results show that as compared with amelogenin in solution, the HAP-bound amelogenin was hydrolyzed by both MMP20 and KLK4 at significantly higher rates.These results suggest that the adsorption of amelogenin to HAP results in their preferential and selective degradation and removal from HAP by MMP20 and KLK4 in vitro.

View Article: PubMed Central - PubMed

Affiliation: Department of Orofacial Sciences, School of Dentistry, University of California, San Francisco San Francisco, CA, USA.

ABSTRACT
The hardest tooth enamel tissue develops from a soft layer of protein-rich matrix, predominated by amelogenin that is secreted by epithelial ameloblasts in the secretory stage of tooth enamel development. During enamel formation, a well-controlled progressive removal of matrix proteins by resident proteases, Matrix metalloproteinase 20 (MMP20), and kallikrein 4 (KLK4), will provide space for the apatite crystals to grow. To better understand the role of amelogenin degradation in enamel biomineralization, the present study was conducted to investigate how the adsorption of amelogenin to hydroxyapatite (HAP) crystals affects its degradation by enamel proteinases, MMP20 and KLK4. Equal quantities of amelogenins confirmed by protein assays before digestions, either adsorbed to HAP or in solution, were incubated with MMP20 or KLK4. The digested samples collected at different time points were analyzed by spectrophotometry, SDS-PAGE, high performance liquid chromatography (HPLC), and LC-MALDI MS/MS. We found that majority of amelogenin adsorbed on HAP was released into the surrounding solution by enzymatic processing (88% for MMP20 and 98% for KLK4). The results show that as compared with amelogenin in solution, the HAP-bound amelogenin was hydrolyzed by both MMP20 and KLK4 at significantly higher rates. Using LC-MALDI MS/MS, more accessible cleavage sites and hydrolytic fragments from MMP20/KLK4 digestion were identified for the amelogenin adsorbed on HAP crystals as compared to the amelogenin in solution. These results suggest that the adsorption of amelogenin to HAP results in their preferential and selective degradation and removal from HAP by MMP20 and KLK4 in vitro. Based on these findings, a new degradation model related to enamel crystal growth is proposed.

No MeSH data available.


Related in: MedlinePlus

Proposed model for enamel crystal growth guided by crystal-amelogenin-proteinase interactions. (A) HAP crystal interacts with amelogenins and causes their structural changes; (B) The structural changes of adsorbed amelogenins result in preferential amelogenin degradation on crystal surface; (C) Removal of the amelogenin on the crystal surface releases the space for HAP crystal growth; (D) Summary of the proposed model of enamel crystal growth guided by crystal-amelogenin-proteinase interactions. Amelogenin interactions with crystal surface change its conformation, which increases its susceptibility to proteinase. The direct effect of proteinase-crystal interactions will change the structure of proteinase, showing inhibitory effects.
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Figure 6: Proposed model for enamel crystal growth guided by crystal-amelogenin-proteinase interactions. (A) HAP crystal interacts with amelogenins and causes their structural changes; (B) The structural changes of adsorbed amelogenins result in preferential amelogenin degradation on crystal surface; (C) Removal of the amelogenin on the crystal surface releases the space for HAP crystal growth; (D) Summary of the proposed model of enamel crystal growth guided by crystal-amelogenin-proteinase interactions. Amelogenin interactions with crystal surface change its conformation, which increases its susceptibility to proteinase. The direct effect of proteinase-crystal interactions will change the structure of proteinase, showing inhibitory effects.

Mentions: Based on the results obtained from this study, we propose a model to explain how the enamel crystal growth is mediated by interactions among crystal, amelogenin, and proteinases. As shown in Figure 6A, the amelogenins secreted into enamel matrix first bind to the nascent HAP crystals and the adsorption of amelogenins onto the crystal surface results in conformational changes of the bound proteins. The conformational changes may expose more cleavage sites to proteinases. As a result, cleavage of amelogenins by MMP20 or KLK4 is enhanced (Figure 6B). The preferential removal of amelogenins from the crystal surface opens up the space surrounding the crystals for their further growth (Figures 6C,D). When the nascent crystals growing in this new space come into contact with the next layer of amelogenin nanospheres, yet another cycle of the interaction-mediated preferential removal of bound amelogenin and crystal growth is initiated. The cycle of binding-growth-digestion repeats until the HAP crystals grow to fill up the entire enamel space during tooth enamel development.


Preferential and selective degradation and removal of amelogenin adsorbed on hydroxyapatites by MMP20 and KLK4 in vitro.

Zhu L, Liu H, Witkowska HE, Huang Y, Tanimoto K, Li W - Front Physiol (2014)

Proposed model for enamel crystal growth guided by crystal-amelogenin-proteinase interactions. (A) HAP crystal interacts with amelogenins and causes their structural changes; (B) The structural changes of adsorbed amelogenins result in preferential amelogenin degradation on crystal surface; (C) Removal of the amelogenin on the crystal surface releases the space for HAP crystal growth; (D) Summary of the proposed model of enamel crystal growth guided by crystal-amelogenin-proteinase interactions. Amelogenin interactions with crystal surface change its conformation, which increases its susceptibility to proteinase. The direct effect of proteinase-crystal interactions will change the structure of proteinase, showing inhibitory effects.
© Copyright Policy - open-access
Related In: Results  -  Collection

License
Show All Figures
getmorefigures.php?uid=PMC4109566&req=5

Figure 6: Proposed model for enamel crystal growth guided by crystal-amelogenin-proteinase interactions. (A) HAP crystal interacts with amelogenins and causes their structural changes; (B) The structural changes of adsorbed amelogenins result in preferential amelogenin degradation on crystal surface; (C) Removal of the amelogenin on the crystal surface releases the space for HAP crystal growth; (D) Summary of the proposed model of enamel crystal growth guided by crystal-amelogenin-proteinase interactions. Amelogenin interactions with crystal surface change its conformation, which increases its susceptibility to proteinase. The direct effect of proteinase-crystal interactions will change the structure of proteinase, showing inhibitory effects.
Mentions: Based on the results obtained from this study, we propose a model to explain how the enamel crystal growth is mediated by interactions among crystal, amelogenin, and proteinases. As shown in Figure 6A, the amelogenins secreted into enamel matrix first bind to the nascent HAP crystals and the adsorption of amelogenins onto the crystal surface results in conformational changes of the bound proteins. The conformational changes may expose more cleavage sites to proteinases. As a result, cleavage of amelogenins by MMP20 or KLK4 is enhanced (Figure 6B). The preferential removal of amelogenins from the crystal surface opens up the space surrounding the crystals for their further growth (Figures 6C,D). When the nascent crystals growing in this new space come into contact with the next layer of amelogenin nanospheres, yet another cycle of the interaction-mediated preferential removal of bound amelogenin and crystal growth is initiated. The cycle of binding-growth-digestion repeats until the HAP crystals grow to fill up the entire enamel space during tooth enamel development.

Bottom Line: We found that majority of amelogenin adsorbed on HAP was released into the surrounding solution by enzymatic processing (88% for MMP20 and 98% for KLK4).The results show that as compared with amelogenin in solution, the HAP-bound amelogenin was hydrolyzed by both MMP20 and KLK4 at significantly higher rates.These results suggest that the adsorption of amelogenin to HAP results in their preferential and selective degradation and removal from HAP by MMP20 and KLK4 in vitro.

View Article: PubMed Central - PubMed

Affiliation: Department of Orofacial Sciences, School of Dentistry, University of California, San Francisco San Francisco, CA, USA.

ABSTRACT
The hardest tooth enamel tissue develops from a soft layer of protein-rich matrix, predominated by amelogenin that is secreted by epithelial ameloblasts in the secretory stage of tooth enamel development. During enamel formation, a well-controlled progressive removal of matrix proteins by resident proteases, Matrix metalloproteinase 20 (MMP20), and kallikrein 4 (KLK4), will provide space for the apatite crystals to grow. To better understand the role of amelogenin degradation in enamel biomineralization, the present study was conducted to investigate how the adsorption of amelogenin to hydroxyapatite (HAP) crystals affects its degradation by enamel proteinases, MMP20 and KLK4. Equal quantities of amelogenins confirmed by protein assays before digestions, either adsorbed to HAP or in solution, were incubated with MMP20 or KLK4. The digested samples collected at different time points were analyzed by spectrophotometry, SDS-PAGE, high performance liquid chromatography (HPLC), and LC-MALDI MS/MS. We found that majority of amelogenin adsorbed on HAP was released into the surrounding solution by enzymatic processing (88% for MMP20 and 98% for KLK4). The results show that as compared with amelogenin in solution, the HAP-bound amelogenin was hydrolyzed by both MMP20 and KLK4 at significantly higher rates. Using LC-MALDI MS/MS, more accessible cleavage sites and hydrolytic fragments from MMP20/KLK4 digestion were identified for the amelogenin adsorbed on HAP crystals as compared to the amelogenin in solution. These results suggest that the adsorption of amelogenin to HAP results in their preferential and selective degradation and removal from HAP by MMP20 and KLK4 in vitro. Based on these findings, a new degradation model related to enamel crystal growth is proposed.

No MeSH data available.


Related in: MedlinePlus