Biosynthesis of ganglioside mimics in Campylobacter jejuni OH4384 -: Identification of the glycosyltransferase genes, enzymatic synthesis of model compounds, and characterization of nanomole amounts by 600-MHz 1H and 13C NMR analysis
Biosynthesis of ganglioside mimics in Campylobacter jejuni OH4384 -: Identification of the glycosyltransferase genes, enzymatic synthesis of model compounds, and characterization of nanomole amounts by 600-MHz 1H and 13C NMR analysis
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DOI：
10.1074/jbc.275.6.3896
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发表时间：
2000-02-11
影响因子：
4.8
通讯作者：
Wakarchuk, WW
中科院分区：
文献类型：
--
作者：
Gilbert, M;Brisson, JR;Wakarchuk, WW
我们已经应用了两种策略克隆的四个基因的生物合成的GT 1a神经节苷脂模拟的脂寡糖（LOS）的细菌病原体，空肠弯曲杆菌OH 4384，这已与格林-巴利综合征。我们首先使用活性筛选策略克隆了编码α-2，3-唾液酸转移酶（cst-1）的基因。然后，我们使用来自空肠弯曲菌NCTC 11168的最近完成的序列的核苷酸序列信息来扩增参与空肠弯曲菌OH 4384的LOS生物合成的区域。来自空肠弯曲菌OH 4384的LOS生物合成基因座是11.47个酶对，并且编码13个部分或完全开放的阅读框，而在空肠弯曲菌OH 4384中的相应基因座是11.47个酶对，jejuni NCTC 11168跨越13.49个内切酶对，包含15个开放阅读框，表明这两个菌株之间存在不同的组织。潜在的糖基转移酶基因被单独克隆，在大肠杆菌中表达，并使用合成的荧光寡糖作为受体进行测定。我们鉴定了编码β-1，4-N-乙酰半乳糖胺基转移酶（cgtA）、β-1，3-半乳糖基转移酶（cgtB）和双功能唾液酸转移酶（cst-II）的基因，所述双功能唾液酸转移酶将唾液酸转移到0-3的半乳糖和0-8的唾液酸，所述唾液酸连接α-2，3至半乳糖，通过在600 MHz下对纳摩尔量的体外合成的模型化合物进行NMR分析，证实了每个鉴定的糖基转移酶的连接特异性。使用梯度反向宽带纳米NMR探针，可以通过检测跨糖苷键的（3）J（C，H）相关性来获得序列信息。cgtA和csf-II在空肠弯曲杆菌OH 4384中合成GT 1a模拟物中的作用通过将它们的序列和活性与在其LOS中表达较短神经节苷脂模拟物的两种相关空肠弯曲杆菌菌株中的相应同源物进行比较来证实。
We have applied two strategies for the cloning of four genes responsible for the biosynthesis of the GT1a ganglioside mimic in the lipooligosaccharide (LOS) of a bacterial pathogen, Campylobacter jejuni OH4384, which has been associated with Guillain-Barre syndrome. We first cloned a gene encoding an alpha-2,3-sialyltransferase (cst-I) using an activity screening strategy. We then used nucleotide sequence information from the recently completed sequence from C.jejuni NCTC 11168 to amplify a region involved in LOS biosynthesis from C, jejuni OH4384, The LOS biosynthesis locus from C, jejuni OH4384 is 11.47 kilobase pairs and encodes 13 partial or complete open reading frames, while the corresponding locus in C,jejuni NCTC 11168 spans 13.49 kilobase pairs and contains 15 open reading frames, indicating a different organization between these two strains. Potential glycosyltransferase genes were cloned individually, expressed in Escherichia coli, and assayed using synthetic fluorescent oligosaccharides as accepters. We identified genes encoding a beta-1,4-N-acetylgalactosaminyl-transferase (cgtA), a beta-1,3-galactosyltransferase (cgtB), and a bifunctional sialyltransferase (cst-II), which transfers sialic acid to 0-3 of galactose and to 0-8 of a sialic acid that is linked alpha-2,3 to a galactose, The linkage specificity of each identified glycosyltransferase was confirmed by NMR analysis at 600 MHz on nanomole amounts of model compounds synthesized in vitro. Using a gradient inverse broadband nano-NMR probe, sequence information could be obtained by detection of (3)J(C,H) correlations across the glycosidic bond. The role of cgtA and csf-II in the synthesis of the GT1a mimic in C,jejuni OH4384 were confirmed by comparing their sequence and activity with corresponding homologues in two related C,jejuni strains that express shorter ganglioside mimics in their LOS.