Functional Relationships of the Genetic Locus Encoding the Glycosyltransferase Enzymes Involved in Expression of the Lacto-N-neotetraose Terminal Lipopolysaccharide Structure in Neisseria meningitidis*
Functional Relationships of the Genetic Locus Encoding the Glycosyltransferase Enzymes Involved in Expression of the Lacto-N-neotetraose Terminal Lipopolysaccharide Structure in Neisseria meningitidis*
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DOI：
10.1074/jbc.271.32.19166
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发表时间：
1996-08
期刊：
影响因子：
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通讯作者：
W. Wakarchuk;Adèle Martin;Michael P. Jennings;E. Moxon;James C. Richards
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文献类型：
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作者：
W. Wakarchuk;Adèle Martin;Michael P. Jennings;E. Moxon;James C. Richards
采用突变株脂多糖（LPS）结构分析和糖基转移酶活性测定，确定了脑膜炎奈瑟菌lgtABE基因位点的生物合成功能。通过在脑膜炎奈索菌免疫型L3株φ3 MC58的三个基因lgtA、lgtB、lgtE中分别插入抗生素耐药盒所产生的突变体，获得LPS。亲本菌株LPS表达末端乳酸-n -新四糖结构，Galβ1→4GlcNAcβ1→3Galβ1→4Glc。对脂多糖进行温和的肼处理，得到o脱酰化的样品，直接用电喷雾电离质谱（ESI-MS）在负离子模式下进行分析。结合糖分析的结果，ESI-MS揭示了lgt B， lgt A和lgt E LPS中Gal， GlcNAc和Gal的连续损失。通过二维同核和异核NMR方法，研究了用KOH水溶液处理lgt B LPS得到的O-和n -脱酰基LPS样品的结构。利用合成的β-GlcNAc受体和β-乳糖受体，明确了lgtB和lgtA基因编码的糖基转移酶活性。这些数据提供了第一个明确的证据，证明这三个基因编码了奈瑟菌LPS中乳-n -新四糖糖结构末端三糖部分生物合成所需的糖基转移酶。从ESI-MS数据中，还确定了lgt E突变体表达的gal缺陷LPS与免疫型L3 gale缺陷菌株表达的主要成分相同。编码udp -葡萄糖-4- epimase的galE基因在将Gal整合到脑膜炎球菌LPS中起重要作用。
The biosynthetic function of the lgtABE genetic locus of Neisseria meningitidis was determined by structural analysis of lipopolysaccharide (LPS) derived from mutant strains and enzymic assay for glycosyltransferase activity. LPS was obtained from mutants generated by insertion of antibiotic resistance cassets in each of the three genes lgtA, lgtB, lgtE of the N. meningitidis immunotype L3 strain φ3 MC58. LPS from the parent strain expresses the terminal lacto-N-neotetraose structure, Galβ1→4GlcNAcβ1→3Galβ1→4Glc. Mild hydrazine treatment of the LPS afforded O-deacylated samples that were analyzed directly by electrospray ionization mass spectrometry (ESI-MS) in the negative ion mode. In conjunction with results from sugar analysis, ESI-MS revealed successive loss of the sugars Gal, GlcNAc, and Gal in lgt B, lgt A, and lgt E LPS, respectively. The structure of a sample of O- and N-deacylated LPS derived by aqueous KOH treatment of lgt B LPS was determined in detail by two-dimensional homo- and heteronuclear NMR methods. Using a synthetic β-GlcNAc acceptor and a β-lactose acceptor, the glycosyltransferase activities encoded by the lgtB and lgtA genes were unambiguously established. These data provide the first definitive evidence that the three genes encode the respective glycosyltransferases required for biosynthesis of the terminal trisaccharide moiety of the lacto-N-neotetraose structure in Neisseria LPS. From ESI-MS data, it was also determined that the Gal-deficient LPS expressed by the lgt E mutant is identical to that of the major component expressed by immunotype L3 galE-deficient strains. The galE gene which encodes for UDP-glucose-4-epimerase plays an essential role in the incorporation of Gal into meningococcal LPS.