1. Streamlining assays of glycosyltransferases activity using in vitro GT-array (i-GT-ray) platform: Application to family GT37 fucosyltransferases.
- Author
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Bhattarai M, Wang Q, Javaid T, Venkataraghavan A, Al Hassan MT, O'Neill M, Tan L, Chen H, and Faik A
- Subjects
- Apium enzymology, Apium genetics, Arabidopsis enzymology, Arabidopsis genetics, Arabidopsis metabolism, Cell Wall chemistry, Cell Wall enzymology, Cell Wall metabolism, Mass Spectrometry, Oryza enzymology, Polysaccharides chemistry, Polysaccharides metabolism, Enzyme Assays instrumentation, Enzyme Assays methods, Fucosyltransferases analysis, Fucosyltransferases classification, Fucosyltransferases metabolism, Glycosyltransferases analysis, Glycosyltransferases metabolism, Plant Proteins analysis, Plant Proteins metabolism
- 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 (Ara
3 ), 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., Competing Interests: Conflict of interest The authors declare that they have no conflicts of interest with the contents of this article., (Copyright © 2024 The Authors. Published by Elsevier Inc. All rights reserved.)- Published
- 2024
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