Engineering and Dissecting the Glycosylation Pathway of a Streptococcal Serine-rich Repeat Adhesin.
Engineering and Dissecting the Glycosylation Pathway of a Streptococcal Serine-rich Repeat Adhesin.
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DOI：
10.1074/jbc.m116.752998
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发表时间：
2016-12-30
期刊：
影响因子：
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通讯作者：
Wu H
中科院分区：
文献类型：
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作者：
Zhu F;Zhang H;Yang T;Haslam SM;Dell A;Wu H
富含丝氨酸的重复糖蛋白（SRRP）在革兰氏阳性细菌中是保守的。它们对于调节生物膜形成和细菌-宿主相互作用至关重要。 SRRP 的糖基化在此过程中起着关键作用；因此，了解所涉及的糖基转移酶是确定新治疗药物靶点的关键。 Fap1 是副血链球菌的 SRRP，其糖基化由 6 个基因组成的基因簇介导：gtf1、gtf2、gly、gtf3、dGT1 和 galT2。成熟的 Fap1 聚糖具有 Rha1–3Glc1-(Glc1–3GlcNAc1)-2,6-Glc1–6GlcNAc 序列。 Gtf12、Gtf3 和 dGT1 负责 Fap1 糖基化的前四个步骤，依次催化 GlcNAc、Glc、Glc 和 GlcNAc 残基转移至蛋白质骨架。 GalT2 和 Gly 在 Fap1 糖基化中的作用尚不清楚。在本研究中，我们通过整合簇中的所有六个基因，在大肠杆菌中合成了完全修饰的 Fap1 聚糖。这项研究代表了大肠杆菌中外源逐步 O-糖基化合成途径的首次重建。此外，我们还确定GalT2通过添加鼠李糖残基介导Fap1糖基化的第五步，而Gly通过转移葡萄糖残基介导最后的糖基化步骤。此外，每个糖基转移酶基因的失活都会导致副血链球菌生物膜的不同程度受损，这证明了 Fap1 糖基化在生物膜形成中的重要性。 Fap1 糖基化系统提供了一个出色的模型，可以使用糖基转移酶的不同排列来工程化聚糖，并研究 SRRP 的生物合成途径，因为 SRRP 基因位点高度保守。
Serine-rich repeat glycoproteins (SRRPs) are conserved in Gram-positive bacteria. They are crucial for modulating biofilm formation and bacterial-host interactions. Glycosylation of SRRPs plays a pivotal role in the process; thus understanding the glycosyltransferases involved is key to identifying new therapeutic drug targets. The glycosylation of Fap1, an SRRP of Streptococcus parasanguinis, is mediated by a gene cluster consisting of six genes: gtf1, gtf2, gly, gtf3, dGT1, and galT2. Mature Fap1 glycan possesses the sequence of Rha1–3Glc1-(Glc1–3GlcNAc1)-2,6-Glc1–6GlcNAc. Gtf12, Gtf3, and dGT1 are responsible for the first four steps of the Fap1 glycosylation, catalyzing the transfer of GlcNAc, Glc, Glc, and GlcNAc residues to the protein backbone sequentially. The role of GalT2 and Gly in the Fap1 glycosylation is unknown. In the present study, we synthesized the fully modified Fap1 glycan in Escherichia coli by incorporating all six genes from the cluster. This study represents the first reconstitution of an exogenous stepwise O-glycosylation synthetic pathway in E. coli. In addition, we have determined that GalT2 mediates the fifth step of the Fap1 glycosylation by adding a rhamnose residue, and Gly mediates the final glycosylation step by transferring glucosyl residues. Furthermore, inactivation of each glycosyltransferase gene resulted in differentially impaired biofilms of S. parasanguinis, demonstrating the importance of Fap1 glycosylation in the biofilm formation. The Fap1 glycosylation system offers an excellent model to engineer glycans using different permutations of glycosyltransferases and to investigate biosynthetic pathways of SRRPs because SRRP genetic loci are highly conserved.