Four proteins encoded in the gspB-secY2A2 operon of Streptococcus gordonii mediate the intracellular glycosylation of the platelet-binding protein GspB
Four proteins encoded in the gspB-secY2A2 operon of Streptococcus gordonii mediate the intracellular glycosylation of the platelet-binding protein GspB
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DOI：
10.1128/jb.186.21.7100-7111.2004
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发表时间：
2004-11-01
影响因子：
3.2
通讯作者：
Sullam, PM
中科院分区：
文献类型：
--
作者：
Takamatsu, D;Bensing, BA;Sullam, PM
戈登链球菌M99株与血小板的结合主要是由细胞表面糖蛋白GspB介导的。该粘附素由一个N端信号肽、两个富含丝氨酸的区域(SRR1和SRR2)、一个介于SRR1和SRR2之间的碱性区域以及一个C端的细胞壁锚定结构域组成。GspB的糖基化至少部分是由Gly和Nss介导的，它们编码在gspB下游的secY2A2基因座上。该区域还编码两种蛋白质(GTF和Orf4)，它们是GspB表达所必需的，但其功能尚未被描述。在这项研究中，我们通过研究一系列谷胱甘肽S-转移酶-GspB融合蛋白在M99以及在Gly、Nss、GTf和orf4突变体中的表达和糖基化来进一步研究Gly、Nss、GTf和orn的作用。与野生型背景中表达的融合蛋白相比，突变株背景中表达的融合蛋白的电泳率发生了变化。此外，融合蛋白在gtf和orf4突变体的原生质体中形成了不溶的聚集体。糖链检测和凝集素印迹分析表明，SRR1和SRR2是糖基化的，但碱性区域没有修饰。当融合蛋白在大肠杆菌中表达时，只有在GTF和orf4存在的情况下才能观察到该蛋白的糖基化。这些结果表明，Gly、NSS、GTF和Orf4都参与了SRRs的细胞内糖基化。此外，GTF和Orf4是糖基化所必需的，而糖基化又对GspB的溶解性起着重要作用。
Platelet binding by Streptococcus gordonii strain M99 is mediated predominantly by the cell surface glycoprotein GspB. This adhesin consists of a putative N-terminal signal peptide, two serine-rich regions (SRR1 and SRR2), a basic region between SRR1 and SRR2, and a C-terminal cell wall anchoring domain. The glycosylation of GspB is mediated at least in part by Gly and Nss, which are encoded in the secY2A2 locus immediately downstream of gspB. This region also encodes two proteins (Gtf and Orf4) that are required for the expression of GspB but whose functions have not been delineated. In this study, we further characterized the roles of Gly, Nss, Gtf, and OrN by investigating the expression and glycosylation of a series of glutathione S-transferase-GspB fusion proteins in M99 and in gly, nss, gtf, and orf4 mutants. Compared with fusion proteins expressed in the wild-type background, fusion proteins expressed in the mutant strain backgrounds showed altered electrophoretic mobility. In addition, the fusion proteins formed insoluble aggregates in protoplasts of the gtf and orf4 mutants. Glycan detection and lectin blot analysis revealed that SRR1 and SRR2 were glycosylated but that the basic region was unmodified. When the fusion protein was expressed in Escherichia coli, glycosylation of this protein was observed only in the presence of both gtf and orf4. These results demonstrate that Gly, Nss, Gtf, and Orf4 are all involved in the intracellular glycosylation of SRRs. Moreover, Gtf and Orf4 are essential for glycosylation, which in turn is important for the solubility of GspB.