Substrate Specificity, Membrane Topology, and Activity Regulation of Human Alkaline Ceramidase 2 (ACER2)
Substrate Specificity, Membrane Topology, and Activity Regulation of Human Alkaline Ceramidase 2 (ACER2)
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DOI：
10.1074/jbc.m109.069203
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发表时间：
2010-03-19
影响因子：
4.8
通讯作者：
Mao, Cungui
中科院分区：
文献类型：
--
作者：
Sun, Wei;Jin, Junfei;Mao, Cungui
人碱性神经酰胺酶2(ACER2)通过调节细胞内神经酰胺的水解度，在细胞反应中发挥重要作用。在这里，我们报告了它的生化特性、膜拓扑结构和活性调节。在缺乏内源性神经酰胺酶活性的酵母突变细胞(Delta Ypc1 Delta Ydc1)中表达了重组ACER2，并利用表达ACER2的酵母细胞的微粒体对ACER2进行了生化鉴定。ACER2催化多种神经酰胺的水解，符合Michaelis-Menten动力学。ACER2的体外和细胞活性都需要钙离子。ACER2有7个跨膜结构域，其氨基(N)和羧基(C)末端分别定位在高尔基复合体的管腔和胞浆中。缺失N末端(前36个氨基酸残基)的ACER2突变体(ACER2 Delta N36)显示出无法检测到的活性，并被错误地定位到内质网，这表明N端尾巴对ACER2活性和高尔基体定位都是必需的。缺少前13个残基的ACER2突变体(ACER2 Delta N13)虽然保留了神经酰胺酶的活性，但也错误地定位在内质网中。ACER2、ACER2 Delta N13的过表达而不是ACER2 Delta N36的过表达增加了细胞1-磷酸鞘氨醇的释放，表明其错误定位并不影响ACER2调节1-磷酸鞘氨醇分泌的能力。然而，ACER2的过表达而不是ACER2 Delta N13或ACER2 Delta N36的过表达抑制了整合素β1亚基和LAMP1的糖基化，表明它的错误靶向取消了ACER2调节蛋白质糖基化的能力。这些数据表明，ACER2具有广泛的底物特异性，需要钙离子才能发挥其活性，并且ACER2具有胞浆C末端和管腔N末端，这对其活性、正确的细胞定位和蛋白质糖基化的调节是必不可少的。
Human alkaline ceramidase 2 (ACER2) plays an important role in cellular responses by regulating the hydrolysis of ceramides in cells. Here we report its biochemical characterization, membrane topology, and activity regulation. Recombinant ACER2 was expressed in yeast mutant cells (Delta ypc1 Delta ydc1) that lack endogenous ceramidase activity, and microsomes from ACER2-expressiong yeast cells were used to biochemically characterize ACER2. ACER2 catalyzed the hydrolysis of various ceramides and followed Michaelis-Menten kinetics. ACER2 required Ca2+ for both its in vitro and cellular activities. ACER2 has 7 putative transmembrane domains, and its amino (N) and carboxyl (C) termini were found to be oriented in the lumen of the Golgi complex and cytosol, respectively. ACER2 mutant (ACER2 Delta N36) lacking the N-terminal tail (the first 36 amino acid residues) exhibited undetectable activity and was mislocalized to the endoplasmic reticulum, suggesting that the N-terminal tail is necessary for both ACER2 activity and Golgi localization. ACER2 mutant (ACER2 Delta N13) lacking the first 13 residues was also mislocalized to the endoplasmic reticulum although it retained ceramidase activity. Overexpression of ACER2, ACER2 Delta N13, but not ACER2 Delta N36 increased the release of sphingosine 1-phosphate from cells, suggesting that its mislocalization does not affect the ability of ACER2 to regulate sphingosine 1-phosphate secretion. However, overexpression of ACER2 but not ACER2 Delta N13 or ACER2 Delta N36 inhibited the glycosylation of integrin beta 1 subunit and Lamp1, suggesting that its mistargeting abolishes the ability of ACER2 to regulation protein glycosylation. These data suggest that ACER2 has broad substrate specificity and requires Ca2+ for its activity and that ACER2 has the cytosolic C terminus and luminal N terminus, which are essential for its activity, correct cellular localization, and regulation for protein glycosylation.