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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

KLK4 digestion of amelogenin in solution and on HAP shown by SDS-PAGE. (A) Amelogenin digestion in solution. (B) KLK4 hydrolysis of amelogenin adsorbed on HAP. M, standard molecular weight.
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Figure 4: KLK4 digestion of amelogenin in solution and on HAP shown by SDS-PAGE. (A) Amelogenin digestion in solution. (B) KLK4 hydrolysis of amelogenin adsorbed on HAP. M, standard molecular weight.

Mentions: Similarly, SDS-PAGE data showed that KLK4 hydrolyzed amelogenin much faster on HAP than that in solution (Figure 4). At the end of 180 min of KLK4 hydrolysis, there is much less HAP-bound 25 kDa amelogenin remained as compared with amelogenin digested in solution.


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)

KLK4 digestion of amelogenin in solution and on HAP shown by SDS-PAGE. (A) Amelogenin digestion in solution. (B) KLK4 hydrolysis of amelogenin adsorbed on HAP. M, standard molecular weight.
© Copyright Policy - open-access
Related In: Results  -  Collection

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

Figure 4: KLK4 digestion of amelogenin in solution and on HAP shown by SDS-PAGE. (A) Amelogenin digestion in solution. (B) KLK4 hydrolysis of amelogenin adsorbed on HAP. M, standard molecular weight.
Mentions: Similarly, SDS-PAGE data showed that KLK4 hydrolyzed amelogenin much faster on HAP than that in solution (Figure 4). At the end of 180 min of KLK4 hydrolysis, there is much less HAP-bound 25 kDa amelogenin remained as compared with amelogenin digested in solution.

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