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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

MMP-20 hydrolysis of amelogenin in solution (AMG-S) and bound on HAP (AMG-HAP) were digested and quantified by HPLC. (A) HPLC peaks of 25 and 23 kDa amelgenins after 1 and 4 h of digestion. (B) Quantified UV areas of 25 kDa amelogenin substrate after digestion. (C) Quantified UV areas of 23 kDa amelogenin derivative during digestion.
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Figure 3: MMP-20 hydrolysis of amelogenin in solution (AMG-S) and bound on HAP (AMG-HAP) were digested and quantified by HPLC. (A) HPLC peaks of 25 and 23 kDa amelgenins after 1 and 4 h of digestion. (B) Quantified UV areas of 25 kDa amelogenin substrate after digestion. (C) Quantified UV areas of 23 kDa amelogenin derivative during digestion.

Mentions: Further validation and comparison were achieved through HPLC analysis to observe the loss of the 25 kDa protein peak and concomitant generation of peptide peaks. The results of HPLC demonstrated that the digestion of amelogenin on HAP or in solution immediately produced one product peak (labeled “23 kDa” in Figure 3A). The corresponding HPLC fraction of this peak was collected and analyzed by MALDI TOF MS. The measured mass (m/z = 18,616) suggested that the 23 kDa band seen on SDS-PAGE corresponded to amelogenin truncated at P164/S (theoretical m/z = 18,615). The areas of individual peaks measured on HPLC traces before and after digestion were used to analyze the rate of hydrolysis. As shown in Figure 3B, the full-length amelogenin in solution was hydrolyzed significantly slower than the protein attached to the crystal surface. The same trend was observed for the 23 kDa proteolytic fragment, which was digested much slower in solution than in its HAP-bound form (Figure 3C). Interestingly, we note that the 23 kDa amelogenin fragment rapidly increases in the first hour of the digestion. This result indicates that MMP20 rapidly cleaves off amelogenin C-terminus, causing the accumulation of the truncated hydrophobic peptide 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)

MMP-20 hydrolysis of amelogenin in solution (AMG-S) and bound on HAP (AMG-HAP) were digested and quantified by HPLC. (A) HPLC peaks of 25 and 23 kDa amelgenins after 1 and 4 h of digestion. (B) Quantified UV areas of 25 kDa amelogenin substrate after digestion. (C) Quantified UV areas of 23 kDa amelogenin derivative during digestion.
© Copyright Policy - open-access
Related In: Results  -  Collection

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

Figure 3: MMP-20 hydrolysis of amelogenin in solution (AMG-S) and bound on HAP (AMG-HAP) were digested and quantified by HPLC. (A) HPLC peaks of 25 and 23 kDa amelgenins after 1 and 4 h of digestion. (B) Quantified UV areas of 25 kDa amelogenin substrate after digestion. (C) Quantified UV areas of 23 kDa amelogenin derivative during digestion.
Mentions: Further validation and comparison were achieved through HPLC analysis to observe the loss of the 25 kDa protein peak and concomitant generation of peptide peaks. The results of HPLC demonstrated that the digestion of amelogenin on HAP or in solution immediately produced one product peak (labeled “23 kDa” in Figure 3A). The corresponding HPLC fraction of this peak was collected and analyzed by MALDI TOF MS. The measured mass (m/z = 18,616) suggested that the 23 kDa band seen on SDS-PAGE corresponded to amelogenin truncated at P164/S (theoretical m/z = 18,615). The areas of individual peaks measured on HPLC traces before and after digestion were used to analyze the rate of hydrolysis. As shown in Figure 3B, the full-length amelogenin in solution was hydrolyzed significantly slower than the protein attached to the crystal surface. The same trend was observed for the 23 kDa proteolytic fragment, which was digested much slower in solution than in its HAP-bound form (Figure 3C). Interestingly, we note that the 23 kDa amelogenin fragment rapidly increases in the first hour of the digestion. This result indicates that MMP20 rapidly cleaves off amelogenin C-terminus, causing the accumulation of the truncated hydrophobic peptide 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