Department of Medicine, Medical University of South Carolina, Charleston, South Carolina 29425, USA.
J Biol Chem. 2010 Mar 19;285(12):8995-9007. doi: 10.1074/jbc.M109.069203. Epub 2010 Jan 20.
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 (Deltaypc1Deltaydc1) 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 Ca(2+) 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 (ACER2DeltaN36) 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 (ACER2DeltaN13) lacking the first 13 residues was also mislocalized to the endoplasmic reticulum although it retained ceramidase activity. Overexpression of ACER2, ACER2DeltaN13, but not ACER2DeltaN36 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 ACER2DeltaN13 or ACER2DeltaN36 inhibited the glycosylation of integrin beta1 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 Ca(2+) 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.
人类碱性鞘脂酶 2（ACER2）通过调节细胞内神经酰胺的水解，在细胞反应中发挥重要作用。本文报道了其生化特性、膜拓扑结构和活性调节。重组 ACER2 在缺乏内源性鞘脂酶活性的酵母突变细胞（Deltaypc1Deltaydc1）中表达，并使用来自 ACER2 表达酵母细胞的微粒体对 ACER2 进行生化特性分析。ACER2 催化各种神经酰胺的水解，遵循米氏动力学。ACER2 的体外和细胞活性均需要 Ca2+。ACER2 具有 7 个假定的跨膜结构域，其氨基（N）和羧基（C）末端分别定位于高尔基体腔和细胞质中。缺失 N 端尾巴（前 36 个氨基酸残基）的 ACER2 突变体（ACER2DeltaN36）几乎没有活性，且定位于内质网，表明 N 端尾巴对 ACER2 活性和高尔基体定位都是必需的。虽然缺失前 13 个残基的 ACER2 突变体（ACER2DeltaN13）仍保留鞘脂酶活性，但也定位于内质网。ACER2、ACER2DeltaN13 而非 ACER2DeltaN36 的过表达均增加了细胞中 1-磷酸鞘氨醇的释放，表明其错误定位不影响 ACER2 调节 1-磷酸鞘氨醇分泌的能力。然而，ACER2 的过表达而非 ACER2DeltaN13 或 ACER2DeltaN36 的过表达抑制了整合素β1 亚基和 Lamp1 的糖基化，表明其靶向错误消除了 ACER2 调节蛋白糖基化的能力。这些数据表明，ACER2 具有广泛的底物特异性，其活性需要 Ca2+，并且 ACER2 具有细胞质 C 末端和腔 N 末端，这对于其活性、正确的细胞定位和蛋白糖基化的调节都是必需的。
