The prosequence of human lactase-phlorizin hydrolase modulates the folding of the mature enzyme
The prosequence of human lactase-phlorizin hydrolase modulates the folding of the mature enzyme
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DOI：
10.1074/jbc.m111500200
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发表时间：
2002-03-08
影响因子：
4.8
通讯作者：
Naim, HY
中科院分区：
文献类型：
--
作者：
Jacob, R;Peters, K;Naim, HY
蛋白质沿着分泌途径的有效转运要求多肽采用稳定折叠的构象以排出内质网。（ER）。刷状缘酶LPH的可转运前体pro-LPH在Are（734）和LeU（735）之间的反式高尔基体网络中经历细胞内裂解过程，产生LPH β（初始）。通过在COS-1细胞中两种形式的单独表达，分析了包含前LPH（LPH α）的N-末端734个氨基酸的前结构域在LPH β（初始）折叠事件中的作用。在37 ℃下合成后，LPH β（初始）获得错误折叠和无酶活性的构象，可被胰蛋白酶降解。温度变化至20 ℃产生稳定的、胰蛋白酶抗性的和酶活性的LPHbeta（初始），表明LPHbeta（初始）的个体表达导致温度敏感性构象。这种形式在非允许温度下依次与ER分子伴侣免疫球蛋白结合蛋白和钙连接蛋白相互作用，导致ER滞留。当单独表达时，LH α前结构域存在于ER中。它揭示了由二硫桥稳定的紧凑结构特征。LPHalpha和LPHbeta（初始）在共表达时容易相互作用，并且这种相互作用似乎触发胰蛋白酶抗性、正确折叠、具有酶活性和转运能力的LPHbeta（初始）多肽的形成。这些数据清楚地表明，pro-LPH的前区是一种分子内伴侣，在pro-LPH多肽的背景下，其在促进中间形式LPH β（初始）的折叠方面至关重要。
The efficient transport of proteins along the secretory pathway requires that the polypeptide adopts a stably folded conformation to egress the endoplasmic reticulum. (ER). The transport-competent precursor of the brush border enzyme LPH, pro-LPH, undergoes an intracellular cleavage process in the trans-Golgi network between Are(734) and LeU(735) to yield LPHbeta(initial). The role of the prodomain comprising the N-terminally located 734 amino acids of pro-LPH, LPHalpha, in the folding events of LPHbeta(initial) has been analyzed by the individual expression of both forms in COS-1 cells. Following synthesis at 37 degreesC LPHbeta(initial) acquires a misfolded and enzymatically inactive conformation that is degraded by trypsin. A temperature shift to 20 degreesC generates a stable, trypsin-resistant, and enzymatically active LPHbeta(initial) indicating that the individual expression of LPHbeta(initial) results in a temperature-sensitive conformation. This form interacts at non-permissive temperatures sequentially with the ER chaperones immunoglobulin-binding protein and calnexin resulting in an ER retention. The LPHalpha prodomain resides in the ER when individually expressed. It reveals compact structural features that are stabilized by disulfide bridges. LPHalpha and LPHbeta(initial) readily interact with each other upon coexpression, and this interaction appears to trigger the formation of a trypsin-resistant, correctly folded, enzymatically active, and transport-competent LPHbeta(initial) polypeptide. These data clearly demonstrate that the proregion of pro-LPH is an intramolecular chaperone that is critically essential in facilitating the folding of the intermediate form LPHbeta(initial) in the context of the pro-LPH polypeptide.