Two bacterial glycosphingolipid synthases responsible for the synthesis of glucuronosylceramide and α-galactosylceramide
Two bacterial glycosphingolipid synthases responsible for the synthesis of glucuronosylceramide and α-galactosylceramide
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DOI：
10.1074/jbc.ra120.013796
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发表时间：
2020-07-31
影响因子：
4.8
通讯作者：
Ito, Makoto
中科院分区：
文献类型：
--
作者：
Okino, Nozomu;Li, Mengbai;Ito, Makoto
细菌鞘糖脂如葡萄糖醛酸神经酰胺和半乳糖神经酰胺已被鉴定为恒定的自然杀伤T细胞的配体，并在宿主防御中发挥重要作用。然而，生产这些神经酰胺所需的鞘糖脂脱氢酶尚未得到很好的表征。在这里，我们报告的葡萄糖醛酸神经酰胺合酶（神经酰胺UDP-葡萄糖醛酸转移酶[Cer-GlcAT]）在运动发酵单胞菌，革兰氏阴性菌的细胞膜含有葡萄糖醛酸神经酰胺的鉴定和表征。通过比较细菌和植物中编码甘油二酯GlcAT的基因序列，我们发现了一个广泛分布于鞘氨醇单胞菌中的同源基因。活动基因组我们首先克隆了该基因，并在大肠杆菌中表达，然后用镍-琼脂糖亲和层析和凝胶过滤层析纯化蛋白。使用高度富集的酶，我们观察到它具有高的糖基转移酶活性与UDP-葡萄糖醛酸和神经酰胺作为糖供体和受体底物，分别。Cer-GlcAT缺失导致葡萄糖醛酸神经酰胺的损失并增加神经酰胺磷酸甘油的水平，其在WT细胞中仅以非常低的水平表达。此外，我们还发现了与Sphingobium yanoikuyae和Bacteroides fragilis中的Cer-GlcAT同源的序列，已报道它们分别产生葡萄糖醛酸神经酰胺和α-半乳糖神经酰胺。我们在大肠杆菌中表达了cer-glcat基因的两个同源物。coliand发现每个基因分别编码Cer-GlcAT和Cer-galactosyltransferase。这些结果有助于理解细菌鞘糖脂在宿主-细菌相互作用中的作用以及细菌鞘糖脂在细菌生理学中的功能。
Bacterial glycosphingolipids such as glucuronosylceramide and galactosylceramide have been identified as ligands for invariant natural killer T cells and play important roles in host defense. However, the glycosphingolipid synthases required for production of these ceramides have not been well-characterized. Here, we report the identification and characterization of glucuronosylceramide synthase (ceramide UDP-glucuronosyltransferase [Cer-GlcAT]) in Zymomonas mobilis, a Gram-negative bacterium whose cellular membranes contain glucuronosylceramide. On comparing the gene sequences that encode the diacylglycerol GlcAT in bacteria and plants, we found a homologous gene that is widely distributed in the orderSphingomonadalesin theZ. mobilisgenome. We first cloned the gene and expressed it inEscherichia coli, followed by protein purification using nickel-Sepharose affinity and gel filtration chromatography. Using the highly enriched enzyme, we observed that it has high glycosyltransferase activity with UDP-glucuronic acid and ceramide as sugar donor and acceptor substrate, respectively. Cer-GlcAT deletion resulted in a loss of glucuronosylceramide and increased the levels of ceramide phosphoglycerol, which was expressed in WT cells only at very low levels. Furthermore, we found sequences homologous to Cer-GlcAT inSphingobium yanoikuyaeandBacteroides fragilis, which have been reported to produce glucuronosylceramide and alpha-galactosylceramide, respectively. We expressed the two homologs of thecer-glcatgene inE. coliand found that each gene encodes Cer-GlcAT and Cer-galactosyltransferase, respectively. These results contribute to the understanding of the roles of bacterial glycosphingolipids in host-bacteria interactions and the function of bacterial glycosphingolipids in bacterial physiology.