Department of Pharmaceutical Chemistry and Pharmaceutical Sciences, University of California, San Francisco, CA 94143, USA.
Glycobiology. 2010 Jun;20(6):763-74. doi: 10.1093/glycob/cwq033. Epub 2010 Mar 5.
In previous work, our laboratory generated novel chimeric lipopolysaccharides (LPS) in Escherichia coli transformed with a plasmid containing exogenous lipooligosaccharide synthesis genes (lsg) from Haemophilus influenzae. Analysis of these novel oligosaccharide-LPS chimeras allowed characterization of the carbohydrate structures generated by several putative glycosyltransferase genes within the lsg locus. Here, we adapted this strategy to construct a modular approach to study the synthetic properties of individual glycosyltransferases expressed alone and in combinations. To this end, a set of expression vectors containing one to four putative glycosyltransferase genes from the lsg locus, lsgC-F, were transformed into E. coli K12 (XL-1) which is defective in LPS O-antigen biosynthesis. This strategy relied on the inclusion of the H. influenzae gene product lsgG in every plasmid construct, which partially rescues the E. coli LPS biosynthesis defect by priming uridine diphosphate-undecaprenyl in the WecA-dependent O-antigen synthetic pathway with N-acetyl-glucosamine (GlcNAc). This GlcNAc-undecaprenyl then served as an acceptor substrate for further carbohydrate extension by transformed glycosyltransferases. The resultant LPS-linked chimeric glycans were isolated from their E. coli constructs and characterized by mass spectrometry, methylation analysis and enzyme-linked immunosorbent assays. These structural data allowed the specificity of various glycosyltransferases to be unambiguously assigned to individual genes. LsgF was found to transfer a galactose (Gal) to terminal GlcNAc. LsgE was found to transfer GlcNAc to Gal-GlcNAc, and both LsgF and LsgD were found to transfer Gal to GlcNAc-Gal-GlcNAc but with differing linkage specificities. This method can be generalized and readily adapted to study the substrate specificity of other putative or uncharacterized glycosyltransferases.
在以前的工作中，我们的实验室在含有流感嗜血杆菌外源脂寡糖合成基因（lsg）的质粒转化的大肠杆菌中生成了新型嵌合脂多糖（LPS）。这些新型寡糖-LPS 嵌合体的分析允许对 lsg 基因座内的几个假定糖基转移酶基因产生的碳水化合物结构进行特征描述。在这里，我们采用了这种策略来构建一种模块化方法来研究单独和组合表达的单个糖基转移酶的合成性质。为此，一组含有 lsg 基因座中的一个至四个假定糖基转移酶基因（lsgC-F）的表达载体被转化到 LPS O-抗原生物合成缺陷的大肠杆菌 K12（XL-1）中。这种策略依赖于每个质粒构建物中包含流感嗜血杆菌基因产物 lsgG，它通过在 WecA 依赖性 O-抗原合成途径中用 N-乙酰葡萄糖胺（GlcNAc）引发尿苷二磷酸-十一碳烯基来部分挽救大肠杆菌 LPS 生物合成缺陷。这种 GlcNAc-十一碳烯基然后作为转化糖基转移酶进一步碳水化合物延伸的受体底物。从其大肠杆菌构建物中分离出 LPS 连接的嵌合聚糖，并通过质谱、甲基化分析和酶联免疫吸附测定进行表征。这些结构数据允许将各种糖基转移酶的特异性明确分配给单个基因。LsgF 被发现将半乳糖（Gal）转移到末端 GlcNAc 上。LsgE 被发现将 GlcNAc 转移到 Gal-GlcNAc 上，并且发现 LsgF 和 LsgD 都将 Gal 转移到 GlcNAc-Gal-GlcNAc 上，但具有不同的连接特异性。这种方法可以推广并易于适应研究其他假定的或未表征的糖基转移酶的底物特异性。
