Human acid sphingomyelinase - Assignment of the disulfide bond pattern
Human acid sphingomyelinase - Assignment of the disulfide bond pattern
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人酸性鞘磷脂酶 - 二硫键模式的分配
DOI：
10.1046/j.1432-1033.2003.03435.x
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发表时间：
2003
期刊：
影响因子：
5.4
通讯作者：
K. Sandhoff
中科院分区：
文献类型：
--
作者：
S. Lansmann;C. Schuette;O. Bartelsen;Joerg Hoernschemeyer;T. Linke;J. Weisgerber;K. Sandhoff
人酸性鞘磷脂酶（haSMase，EC 3.1.4.12）催化鞘磷脂溶酶体降解为神经酰胺和磷酰胆碱。遗传性 haSMase 缺陷会导致尼曼-皮克病，这是一种严重的鞘脂储存障碍。该酶在十多年前就被纯化和克隆。从那时起，只有少数 haSMase 的结构特性被阐明。为了了解其复杂功能，包括其在某些信号传导和细胞凋亡事件中的作用，需要有关该酶的完整结构信息。在此，首次报道了haSMase二硫键模式的鉴定。使用杆状病毒表达系统在SF21细胞中表达的功能性重组酶被纯化并用胰蛋白酶消化。对所得肽的 MALDI-MS 分析揭示了四个二硫键 Cys120-Cys131、Cys385-Cys431、Cys584-Cys588 和 Cys594-Cys607。通过结合部分还原和 MALDI-PSD 分析的方法，鉴定了两个无法通过胰蛋白酶裂解直接连接的另外的二硫键（Cys221-Cys226 和 Cys227-Cys250）。在 haSMase 的鞘脂激活蛋白 (SAP) 同源 N 端结构域中，一个二硫键被指定为 Cys120-Cys131。正如 SAP 型结构域的已知二硫键模式所预期的那样，该区域中存在两个额外的二硫键。这些结果支持这样的假设：haSMase 拥有分子内 SAP 型激活剂结构域，如序列比较预测的那样 [Ponting, C.P. （1994）蛋白质科学，3，359-361]。对从人胎盘中分离的 haSMase 进行的额外分析表明，重组蛋白和天然人蛋白具有相同的二硫键结构。
Human acid sphingomyelinase (haSMase, EC 3.1.4.12) catalyzes the lysosomal degradation of sphingomyelin to ceramide and phosphorylcholine. An inherited haSMase deficiency leads to Niemann-Pick disease, a severe sphingolipid storage disorder. The enzyme was purified and cloned over 10 years ago. Since then, only a few structural properties of haSMase have been elucidated. For understanding of its complex functions including its role in certain signaling and apoptosis events, complete structural information about the enzyme is necessary. Here, the identification of the disulfide bond pattern of haSMase is reported for the first time. Functional recombinant enzyme expressed in SF21 cells using the baculovirus expression system was purified and digested by trypsin. MALDI-MS analysis of the resulting peptides revealed the four disulfide bonds Cys120-Cys131, Cys385-Cys431, Cys584-Cys588 and Cys594-Cys607. Two additional disulfide bonds (Cys221-Cys226 and Cys227-Cys250) which were not directly accessible by tryptic cleavage, were identified by a combination of a method of partial reduction and MALDI-PSD analysis. In the sphingolipid activator protein (SAP)-homologous N-terminal domain of haSMase, one disulfide bond was assigned as Cys120-Cys131. The existence of two additional disulfide bridges in this region was proved, as was expected for the known disulfide bond pattern of SAP-type domains. These results support the hypothesis that haSMase possesses an intramolecular SAP-type activator domain as predicted by sequence comparison [Ponting, C.P. (1994) Protein Sci., 3, 359-361]. An additional analysis of haSMase isolated from human placenta shows that the recombinant and the native human protein possess an identical disulfide structure.
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DOI：
10.1074/jbc.m002957200
发表时间：
2000-09
期刊：
The Journal of biological chemistry
影响因子：
--
作者：
Susanne Kirschnek;Francois Paris;Michael Weller;H. Grassmé;K. Ferlinz;Andrea Riehle;Zvi Fuks;
通讯作者：
Susanne Kirschnek;Francois Paris;Michael Weller;H. Grassmé;K. Ferlinz;Andrea Riehle;Zvi Fuks;
DOI：
--
发表时间：
1991
期刊：
The Journal of biological chemistry
影响因子：
--
作者：
Schuchman,EH;Suchi,M;Takahashi,T;Sandhoff,K;Desnick,RJ
通讯作者：
Desnick,RJ
影响因子：
7.5
作者：
Spiegel, S;Foster, D;Kolesnick, R
通讯作者：
Kolesnick, R
克隆人类酸性鞘磷脂酶 cDNA，其中包含使该酶失活的新突变。
影响因子：
2.7
作者：
Ida,H;Rennert,OM;Eto,Y;Chan,WY
通讯作者：
Chan,WY