A pipeline towards the biochemical characterization of the arabidopsis gt14 family

Lingling Xuan, Jie Zhang, Weitai Lu, Pawel Gluza, Berit Ebert, Toshihisa Kotake, Mengzhu Lu, Yuan Zhang, Mads Hartvig Clausen, Kim L. Johnson, Monika S. Doblin, Joshua L. Heazlewood, Antony Bacic, Lili Song*, Wei Zeng

*Corresponding author for this work

Research output: Contribution to journalJournal articleResearchpeer-review

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Abstract

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-com-patible 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 identify-ing effective substrates of GT enzymes. By addition of UDP-GlcA as donor and the synthetic accep-tors galactose-nitrobenzodiazole (Gal-NBD), β-1,6-galactotetraose (β-1,6-Gal4) and β-1,3-galacto-pentose (β-1,3-Gal5) to microsomes expressing individual GT14 enzymes, we verified the β-glucu-ronosyltransferase (GlcAT) activity of three members of this family (AtGlcAT14A, B, and E). In ad-dition, 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) biosyn-thesis 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.

Original languageEnglish
Article number1360
JournalInternational Journal of Molecular Sciences
Volume22
Issue number3
Number of pages22
ISSN1661-6596
DOIs
Publication statusPublished - 2021

Keywords

  • AGP
  • Arabidopsis
  • CAZy
  • Glycosylation
  • Glycosyltransferase
  • Plant cell wall

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