Sino-Australia Plant Cell Wall Research Centre, The State Key Laboratory of Subtropical Silviculture, College of Forestry and Biotechnology, Zhejiang A & F University, Lin'an 311300, China.
School of BioSciences, The University of Melbourne, Parkville, VIC 3010, Australia.
Int J Mol Sci. 2021 Jan 29;22(3):1360. doi: 10.3390/ijms22031360.
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 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 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 () 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-Gal) and β-1,3-galactopentose (β-1,3-Gal) 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 GT clone collection and the biochemical analysis pipeline present an efficient means to identify and characterize novel GT catalytic activities.
糖基转移酶（GTs）催化糖苷键的合成，是糖链、糖缀合物（糖脂和糖蛋白）和糖苷合成的关键酶。由于细胞壁的合成和各种糖基化次生代谢产物的存在，植物基因组通常编码的 GT 比动物基因组多得多。该基因组预计编码超过 573 种 GT，目前分为 42 个不同的家族。这些 GT 的大多数生化功能仍然未知。在这项研究中，我们通过将另外 105 个 GT cDNA 克隆到 Gateway 兼容载体中，对 JBEI GT 克隆集进行了更新，总共克隆了 508 个（89%），用于下游特征分析。我们进一步建立了一个功能分析管道，使用烟草中的瞬时表达（），然后进行酶测定、通过反相高效液相色谱（RP-HPLC）对酶产物进行分级和通过质谱（MS）进行表征。以 GT14 家族为例，我们概述了一种鉴定 GT 酶有效底物的策略。通过向表达单个 GT14 酶的微粒体中添加 UDP-GlcA 作为供体和合成受体半乳糖-硝基苯并二氮杂（Gal-NBD）、β-1,6-半乳糖四糖（β-1,6-Gal）和β-1,3-半乳糖五糖（β-1,3-Gal），我们验证了该家族的三个成员（AtGlcAT14A、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（AT4G27480，248）表现出比其他 GT14 酶更高的活性。我们的数据表明，这些 GT14 成员可能在阿拉伯半乳聚糖蛋白（AGP）生物合成中发挥作用。总之，更新的 GT 克隆集和生化分析管道提供了一种有效的方法来鉴定和表征新型 GT 催化活性。
