Biochemical Characterization of the O-Linked Glycosylation Pathway in Neisseria gonorrhoeae Responsible for Biosynthesis of Protein Glycans Containing N,N′-Diacetylbacillosamine
Biochemical Characterization of the O-Linked Glycosylation Pathway in Neisseria gonorrhoeae Responsible for Biosynthesis of Protein Glycans Containing N,N′-Diacetylbacillosamine
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DOI：
10.1021/bi2003372
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发表时间：
2011-06-07
期刊：
影响因子：
2.9
通讯作者：
Imperiali, Barbara
中科院分区：
文献类型：
--
作者：
Hartley, Meredith D.;Morrison, Michael J.;Imperiali, Barbara
淋病奈瑟菌的o -连接蛋白糖基化途径负责在二磷酸十一戊烯基上合成复合寡糖，随后将多糖整体转移到选定的质周蛋白的丝氨酸残基上。蛋白糖基化（pgl）基因已在生物信息学和自上而下的质谱分析pgl-null菌株蛋白修饰的基础上进行了注释[Aas， F. E.等（2007）Mol. Microbiol. 65, 607-624；Vik， A.，等（2009）Proc. Natl。学会科学。[美国文献106,4447-4452]，但迄今为止进行的生化分析相对较少。在本报告中，我们介绍了7种Pgl酶的表达、纯化和功能表征。具体来说，所研究的酶负责合成一种罕见的尿苷二磷酸（UDP）糖（pld、PglC和pglb -乙酰基转移酶结构域）、聚糖组装（pglb -磷酸糖基转移酶结构域、PglA、PglE和PglH）和最终的低聚糖转移（PglO）。通过酶法合成了甘聚糖生物合成中的第一个糖——udp -2,4-二乙酰氨基-2,4,6-三脱氧- α -d -己糖（DATDH），核磁共振表征其立体化学结构为尿苷二磷酸N′-二乙酰杆菌胺（UDP-diNAcBac）。此外，我们在体外分析了磷酸糖基转移酶、糖基转移酶和寡糖转移酶（OTase）的底物特异性，在大多数情况下，这些酶相对于密切相关的聚糖，对天然底物表现出强烈的偏好。特别是，来自空肠弯曲杆菌的o -连接的oase PglO和n -连接的oase PglB（Cj）分别偏爱天然的淋病奈索菌和空肠奈索菌底物。该研究首次对这一重要的0链糖基化途径进行了全面的生化表征，为进一步研究这些酶作为抗菌靶点提供了基础。
The O-linked protein glycosylation pathway in Neisseria gonorrhoeae is responsible for the synthesis of a complex oligosaccharide on undecaprenyl diphosphate and subsequent en bloc transfer of the glycan to serine residues of select periplasmic proteins. Protein glycosylation (pgl) genes have been annotated on the basis of bioinformatics and top-down mass spectrometry analysis of protein modifications in pgl-null strains [Aas, F. E., et al. (2007) Mol. Microbiol. 65, 607-624; Vik, A., et al. (2009) Proc. Natl. Acad. Sci. U.S.A. 106, 4447-4452], but relatively little biochemical analysis has been performed to date. In this report, we present the expression, purification, and functional characterization of seven Pgl enzymes. Specifically, the enzymes studied are responsible for synthesis of an uncommon uridine diphosphate (UDP)-sugar (PglD, PglC, and PglB-acetyltransferase domain), glycan assembly (PglB-phospho-glycosyltransferase domain, PglA, PglE, and PglH), and final oligosaccharide transfer (PglO). UDP-2,4-diacetamido-2,4,6-trideoxy-alpha-D-hexose (DATDH), which is the first sugar in glycan biosynthesis, was produced enzymatically, and the stereochemistry was assigned as uridine diphosphate N'-diacetylbacillosamine (UDP-diNAcBac) by nuclear magnetic resonance characterization. In addition, the substrate specificities of the phospho-glycosyltransferase, glycosyltransferases, and oligosaccharyltransferase (OTase) were analyzed in vitro, and in most cases, these enzymes exhibited strong preferences for the native substrates relative to closely related glycans. In particular, PglO, the O-linked OTase, and PglB(Cj), the N-linked OTase from Campylobacter jejuni, preferred the native N. gonorrhoeae and C. jejuni substrates, respectively. This study represents the first comprehensive biochemical characterization of this important 0-linked glycosylation pathway and provides the basis for further investigations of these enzymes as antibacterial targets.