Cleavage site specificity of MMP-20 for secretory-stage ameloblastin.
Cleavage site specificity of MMP-20 for secretory-stage ameloblastin.
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DOI：
10.1177/0022034510366903
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发表时间：
2010-08
影响因子：
7.6
通讯作者：
Simmer JP
中科院分区：
文献类型：
--
作者：
Chun YH;Yamakoshi Y;Yamakoshi F;Fukae M;Hu JC;Bartlett JD;Simmer JP
成釉蛋白是一种分泌的磷酸化糖蛋白，在釉质形成过程中被蛋白酶加工。我们测试的假设，Mmp-20催化裂解产生的Ambn裂解产物，积累在发展中的釉质。我们从发育中的釉质中分离出一个23 kDa的Ambn裂解产物，并确定其N-末端序列始于Tyr 223。成釉蛋白从HEK 293-H细胞稳定表达和分泌，纯化并用Mmp-20或Klk 4消化。通过SDS-PAGE和蛋白质印迹法分析酶切产物，并通过N-末端测序表征切割产物。用Mmp-20和Klk 4消化六种荧光肽，并通过RP-HPLC和质谱法分析。Mmp-20在与体内催化的Ambn裂解相对应的位点精确地裂解每个肽：在Met 32、Gln 131、Leu 171、Tyr 223、Leu 301和Tyr 343的N-末端侧。Klk 4在体内未观察到的许多位点切割Ambn和荧光肽，并且仅能够在单个正确位点切割：在Leu 171之前。我们的结论是，Mmp-20是酶的过程中Ambn在分泌阶段的釉质形成。
Ameloblastin is a secreted phosphorylated glycoprotein that is processed by protease(s) during enamel formation. We test the hypothesis that Mmp-20 catalyzes the cleavages that generate the Ambn cleavage products that accumulate in developing enamel. We isolated a 23-kDa Ambn cleavage product from developing enamel and determined its N-terminus sequence started at Tyr223. Ameloblastin was stably expressed and secreted from HEK293-H cells, purified and digested with Mmp-20 or Klk4. The digests were analysed by SDS-PAGE and Western blotting, and the cleavage products were characterized by N-terminal sequencing. Six fluorescent peptides were digested with Mmp-20 and Klk4 and analyzed by RP-HPLC and by mass spectrometry. Mmp-20 cleaved each peptide exactly at the sites correspsonding to Ambn cleavages catalyzed in vivo: on the N-terminal sides of Met32, Gln131, Leu171, Tyr223, Leu301, and Tyr343. Klk4 cleaved Ambn and the fluorescent peptides at many sites not observed in vivo, and was only able to cleave at a single correct site: before Leu171. We conclude that Mmp-20 is the enzyme that processes Ambn during the secretory stage of amelogenesis.
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