Controlled proteolysis of amelogenins reveals exposure of both carboxy- and amino-terminal regions
Controlled proteolysis of amelogenins reveals exposure of both carboxy- and amino-terminal regions
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DOI：
10.1002/1097-0282(200106)58:7
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发表时间：
2001-06-01
期刊：
影响因子：
2.9
通讯作者：
Fincham, AG
中科院分区：
文献类型：
--
作者：
Moradian-Oldak, J;Jimenez, I;Fincham, AG
基质介导的釉质生物矿化涉及釉质特异性釉原蛋白的分泌，其通过自组装成纳米球结构提供框架，在该框架内形成初始釉质微晶。在釉质矿化过程中，釉原蛋白被牙齿特异性蛋白酶加工。本研究旨在探讨影响釉质蛋白酶加工釉原蛋白活性的因素。考虑了釉原蛋白自组装和酶特异性两个因素。我们采用了有限的蛋白水解方法，结合质谱，以确定表面可及性的釉原蛋白组件的保守结构域。使用一系列市售蛋白酶以及重组釉质溶解素，并在受控和有限的条件下检查其对重组釉原蛋白的蛋白水解作用。发现重组小鼠釉原蛋白rM 179的N-末端区域比C-末端区域更容易被隐蔽消化。内切蛋白酶Glu-C在N-末端（E-18/V）和C-末端（E-178/V）位点切割釉原蛋白。胰凝乳蛋白酶在羧基端（F-151/S）和氨基端（W-25/Y）区域切割釉原蛋白。有趣的是，肽键F/S-152也被重组小鼠釉原蛋白上的釉质溶解素的作用所识别，而嗜热菌蛋白酶除了切割T-63/L-64和I-159/L-160和M-29/I-30键之外，还切割S-152/M-153肽键。然后得出结论，当N-末端17个氨基酸残基被提议保护免受蛋白水解时，羧基和氨基末端的区域暴露在釉原蛋白纳米球的表面上，推测是由于它们参与直接的蛋白质-蛋白质相互作用。在有限的条件下，在小鼠（F-151/S-152）和猪釉原蛋白（S-148/M）中，重组釉质溶素均发生FSM位点周围的切割。我们在体外对釉原蛋白BL釉溶素的有限蛋白水解的观察表明，釉溶素在C-末端区域切割釉原蛋白，显示出酶切割S/M和F/S键的偏好。目前有限的蛋白水解研究提供了深入了解釉原蛋白降解的机制在成釉过程中。(C)John Wiley & Sons，Inc.
The matrix-mediated enamel biomineralization involves secretion of the enamel specific amelogenin proteins that through self-assembly into nanosphere structures provide the framework within which the initial enamel crystallites are formed. During enamel mineralization, amelogenin proteins are processed by tooth-specific proteinases. The aim of this study was to explore the factors that affect the activity of enamel proteases to process amelogenins. Two factors including amelogenin self-assembly and enzyme specificity are considered. We applied a limited proteolysis approach, combined with mass spectrometry, in order to determine the surface accessibility of conserved domains of amelogenin assemblies. A series of commercially available proteinases as well as a recombinant enamelysin were used, and their proteolytic actions on recombinant amelogenin were examined under controlled and limited conditions. The N-terminal region of the recombinant mouse amelogenin rM179 was found to be more accessible to cryptic digest than the C-terminal region. The endoproteinase Glu-C cleaved amelogenin at both the N-terminal (E-18/V) and C-terminal (E-178/V) sires. Chymotrypsin cleaved amelogenin at both the carboxy- (F-151/S) and amino-terminal (W-25/Y) regions. Interestingly, the peptide bond F/S-152 was also recognized by the action of enamelysin on recombinant mouse amelogenin whereas thermolysin cleaved the S-152/M-153 peptide bond in addition to T-63/L-64 and I-159/L-160 and M-29/I-30 bonds. It was then concluded that regions at both the carboxy - and amino-terminal were exposed on the surface of amelogenin nanospheres when the N-terminal 17 amino acid residues were proposed to be protected from proteolysis, presumably as the result of their involvement in direct protein-protein interaction. Cleavage around the FSM locus occurred by recombinant enamelysin under limited conditions, in both mouse (F-151/S-152) and pig amelogenins (S-148/M). Our in vitro observations on the limited proteolysis of amelogenin bl enamelysin suggest that enamelysin cleaved amelogenin at the C-terminal region showing a preference of the enzyme to cleave the S/M and F/S bonds. The present limited proteolysis studies provided insight into the mechanisms of amelogenin degradation during amelogenesis. (C) 2001 John Wiley & Sons, Inc.