Analyzing Xyloglucan Endotransglycosylases by Incorporating Synthetic Oligosaccharides into Plant Cell Walls

Abstract

The plant cell wall is a cellular exoskeleton consisting predominantly of a complex polysaccharide network that defines the shape of cells. During growth, this network can be loosened through the action of xyloglucan endotransglycosylases (XETs), glycoside hydrolases that "cut and paste" xyloglucan polysaccharides through a transglycosylation process. We have analyzed cohorts of XETs in different plant species to evaluate the substrate specificities of xyloglucan acceptors by using a set of synthetic oligosaccharides obtained by automated glycan assembly. The ability of XETs to incorporate the oligosaccharides into polysaccharides printed as microarrays and into stem sections of Arabidopsis thaliana, beans, and peas was assessed. We found that single xylose substitutions are sufficient for transfer, and xylosylation of the terminal glucose residue is not required by XETs, independent of plant species. To obtain information on the potential xylosylation pattern of the natural acceptor of XETs, that is, the nonreducing end of xyloglucan, we further tested the activity of xyloglucan xylosyl transferase (XXT) 2 on the synthetic xyloglucan oligosaccharides. These data shed light on inconsistencies between previous studies towards determining the acceptor substrate specificities of XETs and have important implications for further understanding plant cell wall polysaccharide synthesis and remodeling.

Automated Glycan Assembly: A Perspective

“…In addition, the synthetic, conjugation-ready xyloglucans were coupled to fluorescein (FC) to evaluate the activity of plant xyloglucan endotransglycosylases on glycan arrays. 69 Synthetic FC-labeled xyloglucans were incorporated into plant sections.…”

Critical Review of Plant Cell Wall Matrix Polysaccharide Glycosyltransferase Activities Verified by Heterologous Protein Expression

“…Use of this cell type is desirable for X-ray crystallography screening because digestion with Endoglycosidase F1 results in a polypeptide with single GlcNAc residue at each N -glycosylation site (Urbanowicz et al, 2017). Xys1 from Arabidopsis and a green algal species ( Klebsormidium flaccidum ), XXT2, and the GAUT1:GAUT7 complex have also been expressed in HEK293F cells in sufficient quantities for detailed enzymatic characterization and kinetics studies (Urbanowicz et al, 2014; Amos et al, 2018; Jensen et al, 2018; Ruprecht et al, 2018; Tables 1, 2, 5).…”

Multivalent glycan arrays

“…40 AGA was exploited to determine the binding epitopes of many plant cell-wall glycan-directed mAbs. 45,46 A total of 88 synthetic oligosaccharides, including arabinogalactan-, rhamnogalacturonan-, xylan-, and xyloglucan were printed on a microarray aiming to comprehensively map the epitopes of plant cell-wall glycan-directed antibodies (see Fig. 2).…”