Work

Streamlining assays of glycosyltransferases activity using in vitro GT-array (i-GT-ray) platform: Application to family GT37 fucosyltransferases

Year: 2024

Type: article

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 ( i -GT-ray) screening platform for activity of GTs. GT-arrays are generated by cell-free in vitro protein synthesis and binding using microplates pre-coated with a N-terminal Halo- or 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 TM 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 Ara p and MeFuc of chain B and l-Gal of chain A. Our screen showed that At FUT2, At FUT5 and At FUT10 have activity toward RG-I, while At FUT8 was active on RG-II. Five rice Os FUTs have XyG-FUT activity and four rice Os FUTs have activity toward Ara 3 . None of the putative Os FUTs 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 i -GT-ray platform provides a valuable tool for cell wall biochemistry and other research fields.

Source: Journal of Biological Chemistry

DOI: 10.1016/j.jbc.2024.105734