Processing of ameloblastin by MMP-20
Processing of ameloblastin by MMP-20
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DOI：
10.1177/154405910708600209
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发表时间：
2007-02-01
影响因子：
7.6
通讯作者：
Simmer, J. P.
中科院分区：
文献类型：
--
作者：
Iwata, T.;Yamakoshi, Y.;Simmer, J. P.
成釉蛋白（AMBN）裂解产物是发育中牙釉质基质中含量最多的非成釉蛋白。AMBN n端解理产物集中在釉质棒之间的鞘隙中，而AMBN c端解理产物集中在釉质棒内部。我们验证了MMP-20是切割AMBN的蛋白酶的假设。糖基化重组猪AMBN （rpAMBN）在人肾293F细胞中表达，重组猪漆溶素（rpMMP-20）在细菌中表达。纯化后的蛋白以酶与底物1:100的比例一起孵育。AMBN酶切产物的n端测序结果表明，rpMMP-20在Pro(2)、Gln（130）、Gln（139）、Arg（170）和Ala（222）之后裂解rpAMBN。这表明MMP-20产生始于Tyr（223）的23 kda AMBN，以及在分泌阶段集中在鞘间隙的17 kda (Val（1)Arg(170)）和15 kda (Val(1)-Gln(130)) AMBN切割产物。我们得出结论，MMP-20在体内和体外都能处理成釉酶。
Ameloblastin ( AMBN) cleavage products are the most abundant non-amelogenin proteins in the enamel matrix of developing teeth. AMBN N-terminal cleavage products accumulate in the sheath space between enamel rods, while AMBN C-terminal products localize within rods. We tested the hypothesis that MMP-20 is the protease that cleaves AMBN. Glycosylated recombinant porcine AMBN (rpAMBN) was expressed in human kidney 293F cells, and recombinant porcine enamelysin (rpMMP-20) was expressed in bacteria. The purified proteins were incubated together at an enzyme: substrate ratio of 1:100. N-terminal sequencing of AMBN digestion products determined that rpMMP-20 cleaved rpAMBN after Pro(2), Gln(130), Gln(139), Arg(170), and Ala(222). This shows that MMP-20 generates the 23-kDa AMBN starting at Tyr(223), as well as the 17-kDa (Val(1)Arg(170)) and 15-kDa (Val(1)-Gln(130)) AMBN cleavage products that concentrate in the sheath space during the secretory stage. We conclude that MMP-20 processes ameloblastin in vitro and in vivo.