O-Glycosylation of the N-terminal Region of the Serine-rich Adhesin Srr1 of Streptococcus agalactiae Explored by Mass Spectrometry
O-Glycosylation of the N-terminal Region of the Serine-rich Adhesin Srr1 of Streptococcus agalactiae Explored by Mass Spectrometry
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DOI：
10.1074/mcp.m114.038075
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发表时间：
2014-09-01
影响因子：
7
通讯作者：
Mistou, Michel-Yves
中科院分区：
文献类型：
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作者：
Chaze, Thibault;Guillot, Alain;Mistou, Michel-Yves
革兰氏阳性菌表面的富丝氨酸(SRR)蛋白是一类粘附素家族，与致病性葡萄球菌和链球菌的毒力有关。此前的凝集素结合实验表明，SRR蛋白高度糖基化。我们在这里首次报道了无乳链球菌Srr1糖基化的质谱分析。经Srr1浓缩和胰酶消化后，在碰撞诱导解离光谱中用X！双人行。然后使用更高能量的碰撞解离碎裂来改进该方法，这会导致糖残留物的同时损失，诊断氧离子的产生和糖肽的主干碎裂。为这项工作开发的一个新的开源软件工具(SpectrumFinder)利用了这一功能。通过结合这些方法，鉴定了27个糖肽，分别对应于Srr1[93-639]N-末端的6个不同片段。我们的数据明确地表明，相同的蛋白质残基可以被不同的多糖组合修饰，包括N-乙酰己糖胺、己糖和一种新的修饰，被鉴定为O-乙酰化-N-乙酰己糖胺。凝集素结合和单糖组成分析表明，HexNAc和Hex分别对应于N-乙酰氨基葡萄糖和葡萄糖。相同的蛋白质片段可以被多种糖链修饰，从而产生广泛的结构多样性的sr1。使用电子转移解离来指定糖基化位点，从而明确地鉴定了6个丝氨酸和1个苏氨酸残基。对缺乏辅助糖基转移酶编码基因的突变株所产生的纯化的Srr1的分析表明，O-GlcN酰化是Srr1糖基化的第一步，这可能是随后用Hex修饰所必需的。综上所述，我们通过与新软件工具相关的碎片质谱学技术相结合获得的数据，显示了Srr1的糖基化异质性，表征了一种新的蛋白质修饰，并确定了位于蛋白质N末端区域的六个糖基化位点。
Serine-rich (Srr) proteins exposed at the surface of Gram-positive bacteria are a family of adhesins that contribute to the virulence of pathogenic staphylococci and streptococci. Lectin-binding experiments have previously shown that Srr proteins are heavily glycosylated. We report here the first mass-spectrometry analysis of the glycosylation of Streptococcus agalactiae Srr1. After Srr1 enrichment and trypsin digestion, potential glycopeptides were identified in collision induced dissociation spectra using X! Tandem. The approach was then refined using higher energy collisional dissociation fragmentation which led to the simultaneous loss of sugar residues, production of diagnostic oxonium ions and backbone fragmentation for glycopeptides. This feature was exploited in a new open source software tool (SpectrumFinder) developed for this work. By combining these approaches, 27 glycopeptides corresponding to six different segments of the N-terminal region of Srr1 [93-639] were identified. Our data unambiguously indicate that the same protein residue can be modified with different glycan combinations including N-acetylhexosamine, hexose, and a novel modification that was identified as O-acetylated-N-acetylhexosamine. Lectin binding and monosaccharide composition analysis strongly suggested that HexNAc and Hex correspond to N-acetylglucosamine and glucose, respectively. The same protein segment can be modified with a variety of glycans generating a wide structural diversity of Srr1. Electron transfer dissociation was used to assign glycosylation sites leading to the unambiguous identification of six serines and one threonine residues. Analysis of purified Srr1 produced in mutant strains lacking accessory glycosyltransferase encoding genes demonstrates that O-GlcNAcylation is an initial step in Srr1 glycosylation that is likely required for subsequent decoration with Hex. In summary, our data obtained by a combination of fragmentation mass spectrometry techniques associated to a new software tool, demonstrate glycosylation heterogeneity of Srr1, characterize a new protein modification, and identify six glycosylation sites located in the N-terminal region of the protein.