A Glycan Array-Based Assay for the Identification and Characterization of Plant Glycosyltransferases.

Growing plants with modified cell wall compositions is a promising strategy to improve resistance to pathogens, increase biomass digestibility, and tune other important properties. In order to alter biomass architecture, a detailed knowledge of cell wall structure and biosynthesis is a prerequisite. We report here a glycan array-based assay for the high-throughput identification and characterization of plant cell wall biosynthetic glycosyltransferases (GTs). We demonstrate that different heterologously expressed galactosyl-, fucosyl-, and xylosyltransferases can transfer azido-functionalized sugar nucleotide donors to selected synthetic plant cell wall oligosaccharides on the array and that the transferred monosaccharides can be visualized "on chip" by a 1,3-dipolar cycloaddition reaction with an alkynyl-modified dye. The opportunity to simultaneously screen thousands of combinations of putative GTs, nucleotide sugar donors, and oligosaccharide acceptors will dramatically accelerate plant cell wall biosynthesis research.

Automated Glycan Assembly of Plant Oligosaccharides and Their Application in Cell-Wall Biology.

The plant cell wall provides the richest available resource of fermentable carbohydrates and biobased materials. The main component of plant cell walls is cellulose, which is the most abundant biomolecule on earth. Apart from cellulose, which is constructed from relatively simple ß-1,4-glucan chains, plant cell walls also contain structurally more complex heteropolysaccharides (hemicellulose and pectin), as well as lignin and cell-wall proteins. A detailed understanding of the molecular structures, functions, and biosyntheses of cell-wall components is required to further promote their industrial use. Plant cell-wall research is, to a large degree, hampered by a lsack of available well-defined oligosaccharide samples that represent the structural features of cell-wall glycans. One technique to access these oligosaccharides is automated glycan assembly; a technique in which monosaccharide building blocks are, similarly to automated peptide and oligonucleotide chemistry, successively added to a linker-functionalized resin in a fully automated manner. Herein, recent research into the automated glycan assembly of different classes of cell-wall glycans used as molecular tools for cell-wall biology is discussed. More than 60 synthetic oligosaccharides were prepared and printed as microarrays for screening monoclonal antibodies that recognize plant cell-wall polysaccharides. The synthesized oligosaccharides have also been used to investigate glycosyltransferases and glycoside hydrolases, which are involved in synthesis and degradation of plant cell walls, as well as for the analysis of cell-wall-remodeling enzymes.

A Synthetic Glycan Microarray Enables Epitope Mapping of Plant Cell Wall Glycan-Directed Antibodies.

In the last three decades, more than 200 monoclonal antibodies have been raised against most classes of plant cell wall polysaccharides by different laboratories worldwide. These antibodies are widely used to identify differences in plant cell wall components in mutants, organ and tissue types, and developmental stages. Despite their importance and broad use, the precise binding epitope has been determined for only a few of these antibodies. Here, we use a plant glycan microarray equipped with 88 synthetic oligosaccharides to comprehensively map the epitopes of plant cell wall glycan-directed antibodies. Our results reveal the binding epitopes for 78 arabinogalactan-, rhamnogalacturonan-, xylan-, and xyloglucan-directed antibodies. We demonstrate that, with knowledge of the exact epitopes recognized by individual antibodies, specific glycosyl hydrolases can be implemented into immunological cell wall analyses, providing a framework to obtain structural information on plant cell wall glycans with unprecedented molecular precision.

Determining Substrate Specificities of ß1,4-Endogalactanases Using Plant Arabinogalactan Oligosaccharides Synthesized by Automated Glycan Assembly.

Pectin is a structurally complex plant polysaccharide with many industrial applications in food products. The structural elucidation of pectin is aided by digestion assays with glycosyl hydrolases. We report the automated glycan assembly of oligosaccharides related to the arabinogalactan side chains of pectin as novel biochemical tools to determine the substrate specificities of endogalactanases. Analysis of the digestion products revealed different requirements for the lengths and arabinose substitution pattern of the oligosaccharides to be recognized and hydrolyzed by the galactanases.

Direct Experimental Evidence for Halogen-Aryl π†Interactions in Solution from Molecular Torsion Balances.

We dissected halogen-aryl π interactions experimentally using a bicyclic N-arylimide based molecular torsion balances system, which is based on the influence of the non-bonded interaction on the equilibria between folded and unfolded states. Through comparison of balances modulated by higher halogens with fluorine balances, we determined the magnitude of the halogen-aryl π interactions in our unimolecular systems to be larger than -5.0 kJ mol-1 , which is comparable with the magnitude estimated in the biomolecular systems. Our study provides direct experimental evidence of halogen-aryl π interactions in solution, which until now have only been revealed in the solid state and evaluated theoretically by quantum-mechanical calculations.

Semisynthetic Glycoconjugate Vaccine Candidates against Escherichia coli O25B Induce Functional IgG Antibodies in Mice.

Extraintestinal pathogenic Escherichia coli (ExPEC) is a major health concern due to emerging antibiotic resistance. Along with O1A, O2, and O6A, E. coli O25B is a major serotype within the ExPEC group, which expresses a unique O-antigen. Clinical studies with a glycoconjugate vaccine of the above-mentioned O-types revealed O25B as the least immunogenic component, inducing relatively weak IgG titers. To evaluate the immunological properties of semisynthetic glycoconjugate vaccine candidates against E. coli O25B, we here report the chemical synthesis of an initial set of five O25B glycan antigens differing in length, from one to three repeat units, and frameshifts of the repeat unit. The oligosaccharide antigens were conjugated to the carrier protein CRM197. The resulting semisynthetic glycoconjugates induced functional IgG antibodies in mice with opsonophagocytic activity against E. coli O25B. Three of the oligosaccharide-CRM197 conjugates elicited functional IgGs in the same order of magnitude as a conventional CRM197 glycoconjugate prepared with native O25B O-antigen and therefore represent promising vaccine candidates for further investigation. Binding studies with two monoclonal antibodies (mAbs) revealed nanomolar anti-O25B IgG responses with nanomolar K D values and with varying binding epitopes. The immunogenicity and mAb binding data now allow for the rational design of additional synthetic antigens for future preclinical studies, with expected further improvements in the functional antibody responses. Moreover, acetylation of a rhamnose residue was shown to be likely dispensable for immunogenicity, as a deacylated antigen was able to elicit strong functional IgG responses. Our findings strongly support the feasibility of a semisynthetic glycoconjugate vaccine against E. coli O25B.

AtFUT4 and AtFUT6 Are Arabinofuranose-Specific Fucosyltransferases.

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.

Automated Glycan Assembly of Oligosaccharides Related to Arabinogalactan Proteins.

Arabinogalactan proteins are heavily glycosylated proteoglycans in plants. Their glycan portion consists of type-II arabinogalactan polysaccharides whose heterogeneity hampers the assignment of the arabinogalactan protein function. Synthetic chemistry is key to the procurement of molecular probes for plant biologists. Described is the automated glycan assembly of 14 oligosaccharides from four monosaccharide building blocks. These linear and branched glycans represent key structural features of natural type-II arabinogalactans and will serve as tools for arabinogalactan biology.