AtFUT4 and AtFUT6 Are Arabinofuranose-Specific Fucosyltransferases.

Maria J Soto,Maria J Peña,Ian M Black,Fabian Pfrengle,Digantkumar Chapla,Michael G Hahn,Breeanna R Urbanowicz,Parastoo Azadi,Jason Backe,Hsin-Tzu Wang,Max Bartetzko,Kelley W Moremen,Pradeep Kumar Prabhakar

doi:10.3389/fpls.2021.589518

Abstract

The bulk of plant biomass is comprised of plant cell walls, which are complex polymeric networks, composed of diverse polysaccharides, proteins, polyphenolics, and hydroxyproline-rich glycoproteins (HRGPs). Glycosyltransferases (GTs) work together to synthesize the saccharide components of the plant cell wall. The Arabidopsis thaliana fucosyltransferases (FUTs), AtFUT4, and AtFUT6, are members of the plant-specific GT family 37 (GT37). AtFUT4 and AtFUT6 transfer fucose (Fuc) onto arabinose (Ara) residues of arabinogalactan (AG) proteins (AGPs) and have been postulated to be non-redundant AGP-specific FUTs. AtFUT4 and AtFUT6 were recombinantly expressed in mammalian HEK293 cells and purified for biochemical analysis. We report an updated understanding on the specificities of AtFUT4 and AtFUT6 that are involved in the synthesis of wall localized AGPs. Our findings suggest that they are selective enzymes that can utilize various arabinogalactan (AG)-like and non-AG-like oligosaccharide acceptors, and only require a free, terminal arabinofuranose. We also report with GUS promoter-reporter gene studies that AtFUT4 and AtFUT6 gene expression is sub-localized in different parts of developing A. thaliana roots.

Highlights

The plant cell wall is a complex polymeric network composed of diverse polysaccharides, proteins, polyphenolics, and hydroxyproline-rich glycoproteins (HRGPs)
To determine if the recombinant GFP-AtFUT4 and GFP-AtFUT6 fusion proteins maintained the same acceptor substrate specificity as had been determined in a previous study (Wu et al, 2010), five structurally distinct AG-related oligosaccharides (55, 65, 68, 69, and 70), synthesized by automated glycan assembly, were evaluated for their ability to serve as acceptor substrates (Bartetzko et al, 2015; Ruprecht et al, 2017; Bartetzko and Pfrengle, 2019; Figure 2A)
In contrast to previous findings, our data show that AtFUT4 and AtFUT6 have the same donor and acceptor substrate specificity and identically recognize various AG-like and non-AG-like oligosaccharides and polysaccharides as acceptor substrates (Figures 3, 5, 6)

Summary

Introduction

The plant cell wall is a complex polymeric network composed of diverse polysaccharides, proteins, polyphenolics, and hydroxyproline-rich glycoproteins (HRGPs). The polysaccharide and glycoprotein components of the cell wall confer a range of important functions, from structural integrity to cell-cell communication (Darvill et al, 1985). These complex glycopolymers are comprised of numerous monosaccharide building blocks, such as glucose (Glc), galactose (Gal), arabinose (Ara), galacturonic acid (GalA), xylose (Xyl), rhamnose (Rha), and fucose (Fuc), AtFUT4 and AtFUT6 are Arabinofuranose-Specific Fucosyltransferases among others. XyG is a hemicellulosic polysaccharide composed of a β-(1,4)-linked Glc backbone with side-chains initiated by α-(1,6) linked Xyl residues; these are often further decorated with Gal and Fuc residues (Pauly and Keegstra, 2016). Primary cell wall polysaccharides, including pectins and hemicelluloses, are deeply implicated to be involved in plant growth, cell expansion, wall porosity, and several other functions (Ridley et al, 2001; Willats et al, 2001; Mohnen, 2008; Pauly and Keegstra, 2016)

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Conclusion