The YhhN protein of Legionella pneumophila is a Lysoplasmalogenase
The YhhN protein of Legionella pneumophila is a Lysoplasmalogenase
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DOI：
10.1016/j.bbamem.2014.11.011
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发表时间：
2015-02-01
影响因子：
3.4
通讯作者：
Bell, Charles E.
中科院分区：
文献类型：
--
作者：
Jurkowitz, Marianne S.;Patel, Aalapi;Bell, Charles E.
溶胞浆原酶催化溶胞浆原的乙烯基醚键的水解裂解，产生脂肪醛和甘油磷酸乙醇胺或甘油磷酸胆碱。我们最近从大鼠肝微粒体中纯化了溶浆原酶，并将该蛋白鉴定为 TMEM86B，这是一种内在膜蛋白，是在多种真核生物和细菌中发现的 YhhN 家族的成员。为了验证细菌 YhhN 蛋白也发挥溶浆原酶功能的假设，我们克隆了嗜肺军团菌的 Lpg1991 基因，该基因编码 216 个氨基酸的 YhhN 蛋白 (LpYhhN)，并在大肠杆菌中将其表达为 C 端-GFP-His8 融合体。溶解膜并通过镍亲和层析纯化融合蛋白，用烟草蚀纹病毒蛋白酶裂解，并经过反向镍柱纯化未标记的LpYhhN。融合蛋白和未标记的 LpYhhN 均表现出强大的溶浆原酶活性，以 12 mu mol/min/mg 蛋白的 V-max 和 45 mu M 的 K-m 裂解溶原浆原的乙烯基醚键。LpYhhN 对二拉基缩醛磷脂、1-烯基甘油和单酰基甘油磷酸乙醇胺没有活性或单酰基甘油磷酸胆碱；最适pH值为6.5-7.0。这些特性与哺乳动物 TMEM86B 非常相似。序列分析表明，YhhN 蛋白含有 8 个跨膜螺旋、N-in/C-in 拓扑结构以及约 5 个可能形成活性位点的高度保守的氨基酸残基。这项工作首次证明了细菌 YhhN 蛋白的功能，即作为溶胞浆原特异性的乙烯基醚键水解酶。由于嗜肺军团菌不含有内源性缩醛磷脂，我们推测 LpYhhN 可能有助于保护细菌免遭源自宿主缩醛磷脂的溶血醛原的裂解。由 Elsevier B.V. 出版
Lysoplasmalogenase catalyzes hydrolytic cleavage of the vinyl-ether bond of lysoplasmalogen to yield fatty aldehyde and glycerophospho-ethanolamine or glycerophospho-choline. We recently purified lysoplasmalogenase from rat liver microsomes and identified the protein as TMEM86B, an integral membrane protein that is a member of the YhhN family found in numerous species of eukaryotes and bacteria. To test the hypothesis that bacterial YhhN proteins also function as lysoplasmalogenase enzymes, we cloned the Lpg1991 gene of Legionella pneumophila, which encodes a 216 amino acid YhhN protein (LpYhhN), and expressed it in Escherichia coli as a C-terminal-GFP-His8-fusion. Membranes were solubilized and the fusion protein was purified by nickel-affinity chromatography, cleaved with Tobacco Etch Virus protease, and subjected to a reverse nickel column to purify the un-tagged LpYhhN. Both the fusion protein and un-tagged LpYhhN exhibit robust lysoplasmalogenase activity, cleaving the vinyl-ether bond of lysoplasmalogen with a V-max of 12 mu mol/min/mg protein and a K-m of 45 mu M. LpYhhN has no activity on diradyl plasmalogen, 1-alkenyl-glycerol, and monoacylglycerophospho-ethanolamine or monoacylglycerophospho-choline; the pH optimum is 6.5-7.0. These properties are very similar to mammalian TMEM86B. Sequence analysis suggests that YhhN proteins contain eight transmembrane helices, an N-in/C-in topology, and about 5 highly conserved amino acid residues that may form an active site. This work is the first to demonstrate a function for a bacterial YhhN protein, as a vinyl ether bond hydrolase specific for lysoplasmalogen. Since L. pneumophila does not contain endogenous plasmalogens, we hypothesize that LpYhhN may serve to protect the bacterium from lysis by lysoplasmalogen derived from plasmalogens of the host. Published by Elsevier B.V.