A Pipeline towards the Biochemical Characterization of the Arabidopsis GT14 Family.
A Pipeline towards the Biochemical Characterization of the Arabidopsis GT14 Family.
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DOI：
10.3390/ijms22031360
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发表时间：
2021-01-29
影响因子：
5.6
通讯作者：
Zeng W
中科院分区：
文献类型：
--
作者：
Xuan L;Zhang J;Lu W;Gluza P;Ebert B;Kotake T;Lu M;Zhang Y;Clausen MH;Johnson KL;Doblin MS;Heazlewood JL;Bacic A;Song L;Zeng W
糖基转移酶（GT）催化糖苷键的合成，并且在聚糖、糖缀合物（糖脂和糖蛋白）和糖苷的生物合成中是必需的。植物基因组通常编码比动物基因组多得多的GT，这是由于细胞壁和各种糖基化次级代谢产物的合成。拟南芥基因组预计编码超过573个GT，目前被分为42个不同的家庭。大多数这些GT的生化功能仍然是未知的。在这项研究中，我们更新了JBEI拟南芥GT克隆收集的额外的105 GT cDNA，共508（89%），到网关兼容载体的下游表征。我们进一步建立了一个功能分析管道，该管道使用烟草（Nicotiana benthamiana）中的瞬时表达，然后进行酶测定、通过反相高效液相色谱（RP-HPLC）对酶产物进行分级分离并通过质谱（MS）进行表征。使用GT 14家族作为范例，我们概述了一个战略，确定有效的底物GT酶。通过将UDP-GlcA作为供体和合成受体半乳糖-硝基苯并二唑（Gal-NBD）、β-1，6-半乳糖四糖（β-1，6-Gal 4）和β-1，3-半乳糖戊糖（β-1，3-Gal 5）添加到表达单个GT 14酶的微粒体中，我们验证了该家族的三个成员（AtGlcAT 14 A、B和E）的β-葡萄糖醛酸基转移酶（GlcAT）活性。此外，一个新的家族成员（AT 4G 27480，248）显示具有比其他GT 14酶显著更高的活性。我们的数据表明，阿拉伯半乳聚糖蛋白（AGP）的生物合成这些GT 14成员可能的作用。总之，更新的拟南芥GT克隆收集和生化分析管道提出了一个有效的手段来识别和表征新的GT催化活性。
Glycosyltransferases (GTs) catalyze the synthesis of glycosidic linkages and are essential in the biosynthesis of glycans, glycoconjugates (glycolipids and glycoproteins), and glycosides. Plant genomes generally encode many more GTs than animal genomes due to the synthesis of a cell wall and a wide variety of glycosylated secondary metabolites. The Arabidopsis thaliana genome is predicted to encode over 573 GTs that are currently classified into 42 diverse families. The biochemical functions of most of these GTs are still unknown. In this study, we updated the JBEI Arabidopsis GT clone collection by cloning an additional 105 GT cDNAs, 508 in total (89%), into Gateway-compatible vectors for downstream characterization. We further established a functional analysis pipeline using transient expression in tobacco (Nicotiana benthamiana) followed by enzymatic assays, fractionation of enzymatic products by reversed-phase HPLC (RP-HPLC) and characterization by mass spectrometry (MS). Using the GT14 family as an exemplar, we outline a strategy for identifying effective substrates of GT enzymes. By addition of UDP-GlcA as donor and the synthetic acceptors galactose-nitrobenzodiazole (Gal-NBD), β-1,6-galactotetraose (β-1,6-Gal4) and β-1,3-galactopentose (β-1,3-Gal5) to microsomes expressing individual GT14 enzymes, we verified the β-glucuronosyltransferase (GlcAT) activity of three members of this family (AtGlcAT14A, B, and E). In addition, a new family member (AT4G27480, 248) was shown to possess significantly higher activity than other GT14 enzymes. Our data indicate a likely role in arabinogalactan-protein (AGP) biosynthesis for these GT14 members. Together, the updated Arabidopsis GT clone collection and the biochemical analysis pipeline present an efficient means to identify and characterize novel GT catalytic activities.
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