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Abstract：
Numerous putative glycosyltransferases (GTs) have been identified using bioinformatic approaches. However, demonstrating the activity of these GTs remains a challenge. Here, we describe the development of a rapid in vitro GT-array screening platform for activity of GTs. GT-arrays are generated by cell-free in vitro protein synthesis and binding using microplates precoated with a N-terminal Halo- or a C-terminal GST-tagged GT-encoding plasmid DNA and a capture antibody. These arrays are then used for screening of transferase activities and the reactions are monitored by a luminescence GLO assay. The products formed by these reactions can be analyzed directly from the microplates by mass spectrometry. Using this platform, a total of 280 assays were performed to screen 22 putative fucosyltransferases (FUTs) from family GT37 (seven from Arabidopsis and 15 from rice) for activity toward five acceptors: non-fucosylated tamarind xyloglucan (TXyG), arabinotriose (Ara3), non-fucosylated rhamnogalacturonan I (RG-I), and RG-II from the mur1-1 Arabidopsis mutant, and the celery RG-II monomer lacking Arap and MeFuc of chain B and l-Gal of chain A. Our screen showed that AtFUT2, AtFUT5, and AtFUT10 have activity toward RG-I, while AtFUT8 was active on RG-II. Five rice OsFUTs have XyG-FUT activity and four rice OsFUTs have activity toward Ara3. None of the putative OsFUTs were active on the RG-I and RG-II. However, promiscuity toward acceptors was observed for several FUTs. These findings extend our knowledge of cell wall polysaccharide fucosylation in plants. We believe that in vitro GT-array platform provides a valuable tool for cell wall biochemistry and other research fields.
摘要：
已使用生物信息学方法鉴定了许多推定的糖基转移酶（GT）。然而,证明这些GTs的活动仍然是一个挑战。这里,我们描述了用于GTs活性的快速体外GT阵列筛选平台的开发。GT阵列是通过无细胞体外蛋白质合成和结合使用预涂有N末端Halo或C末端GST标记的GT编码质粒DNA和捕获抗体的微孔板产生的。然后将这些阵列用于转移酶活性的筛选,并通过发光GLO测定监测反应。由这些反应形成的产物可以通过质谱法从微板直接分析。利用这个平台,总共进行了280个测定，以筛选来自GT37家族的22个推定的岩藻糖基转移酶（FUTs）（来自拟南芥的7个和来自水稻的15个）对五个受体的活性：非岩藻糖基化的罗望子木葡聚糖（TXyG），阿拉伯三糖(Ara3),非岩藻糖基化鼠李糖半乳糖醛酸I（RG-I），和来自mur1-1拟南芥突变体的RG-II，以及缺乏链B的Arap和MeFuc和链A的l-Gal的芹菜RG-II单体。我们的屏幕显示AtFUT2，AtFUT5和AtFUT10对RG-I具有活性，而AtFUT8对RG-II有活性。五个水稻OsFUTs具有XyG-FUT活性，四个水稻OsFUTs具有Ara3活性。推定的OsFUT对RG-I和RG-II均无活性。然而,在几个FUTs中观察到对受体的滥交。这些发现扩展了我们对植物中细胞壁多糖岩藻糖基化的认识。我们认为体外GT-阵列平台为细胞壁生物化学和其他研究领域提供了有价值的工具。
