Preferential and selective degradation and removal of amelogenin adsorbed on hydroxyapatites by MMP20 and KLK4 in vitro.
Preferential and selective degradation and removal of amelogenin adsorbed on hydroxyapatites by MMP20 and KLK4 in vitro.
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DOI：
10.3389/fphys.2014.00268
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发表时间：
2014
影响因子：
4
通讯作者：
Li W
中科院分区：
文献类型：
--
作者：
Zhu L;Liu H;Witkowska HE;Huang Y;Tanimoto K;Li W
最硬的牙釉质组织从富含蛋白质的基质的软层发育而来，在牙釉质发育的分泌阶段，由上皮成釉细胞分泌的釉原蛋白占主导地位。在釉质形成过程中，由常驻蛋白酶、基质金属蛋白酶20（MMP 20）和激肽释放酶4（KLK 4）进行的良好控制的基质蛋白的逐步去除将为磷灰石晶体生长提供空间。为了更好地了解釉原蛋白降解在釉质生物矿化中的作用，本研究探讨了釉原蛋白在羟基磷灰石（HAP）晶体上的吸附如何影响其被釉质蛋白酶、MMP 20和KLK 4降解。消化前通过蛋白质测定确认的等量釉原蛋白（吸附至HAP或溶液中）与MMP 20或KLK 4一起孵育。通过分光光度法、SDS-PAGE、高效液相色谱（HPLC）和LC-MALDI MS/MS对在不同时间点收集的消化样品进行分析。我们发现，通过酶处理，吸附在HAP上的大部分釉原蛋白被释放到周围溶液中（MMP 20为88%，KLK 4为98%）。结果表明，与溶液中的釉原蛋白相比，HAP结合的釉原蛋白被MMP 20和KLK 4以显著更高的速率水解。使用LC-MALDI MS/MS，与溶液中的釉原蛋白相比，鉴定了吸附在HAP晶体上的釉原蛋白的更易接近的切割位点和来自MMP 20/KLK 4消化的水解片段。这些结果表明，釉原蛋白的吸附HAP导致其优先和选择性降解和去除HAP的MMP 20和KLK 4在体外。基于这些发现，提出了一个与牙釉质晶体生长相关的新降解模型。
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.
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DOI：
10.1002/anie.201100181
发表时间：
2011-08-08
期刊：
Angewandte Chemie (International ed. in English)
影响因子：
--
作者：
Friddle RW;Battle K;Trubetskoy V;Tao J;Salter EA;Moradian-Oldak J;De Yoreo JJ;Wierzbicki A
通讯作者：
Wierzbicki A
影响因子：
3
作者：
FUKAE, M;SHIMIZU, M
通讯作者：
SHIMIZU, M
影响因子：
2.9
作者：
Bartlett, JD;Zhou, ZJ;Tryggvason, K
通讯作者：
Tryggvason, K
影响因子：
3
作者：
Hart, PS;Aldred, MJ;Wright, JT
通讯作者：
Wright, JT
影响因子：
4
作者：
FINCHAM, AG;BELCOURT, AB;COTHRAN, WC
通讯作者：
COTHRAN, WC