Marta Busse-Wicher

The pattern of xylan acetylation suggests xylan may interact with cellulose microfibrils as a twofold helical screw in the secondary plant cell wall of Arabidopsis thaliana.

The interaction between xylan and cellulose microfibrils is important for secondary cell wall properties in vascular plants; however, the molecular arrangement of xylan in the cell wall and the nature of the molecular bonding between the polysaccharides are unknown. In dicots, the xylan backbone of β-(1,4)-linked xylosyl residues is decorated by occasional glucuronic acid, and approximately one-half of the xylosyl residues are O-acetylated at C-2 or C-3. We recently proposed that the even, periodic spacing of GlcA residues in the major domain of dicot xylan might allow the xylan backbone to fold as a twofold helical screw to facilitate alignment along, and stable interaction with, cellulose fibrils; however, such an interaction might be adversely impacted by random acetylation of the xylan backbone. Here, we investigated the arrangement of acetyl residues in Arabidopsis xylan using mass spectrometry and NMR. Alternate xylosyl residues along the backbone are acetylated. Using molecular dynamics simulation, we found that a twofold helical screw conformation of xylan is stable in interactions with both hydrophilic and hydrophobic cellulose faces. Tight docking of xylan on the hydrophilic faces is feasible only for xylan decorated on alternate residues and folded as a twofold helical screw. The findings suggest an explanation for the importance of acetylation for xylan–cellulose interactions, and also have implications for our understanding of cell wall molecular architecture and properties, and biological degradation by pathogens and fungi. They will also impact strategies to improve lignocellulose processing for biorefining and bioenergy.

GUX1 and GUX2 glucuronyltransferases decorate distinct domains of glucuronoxylan with different substitution patterns

Xylan comprises up to one-third of plant cell walls, and it influences the properties and processing of biomass. Glucuronoxylan in Arabidopsis is characterized by a linear β-(1,4)-linked backbone of xylosyl residues substituted by glucuronic acid and 4-O-methylglucuronic acid (collectively termed [Me]GlcA). The role of these substitutions remains unclear. GUX1 (glucuronic acid substitution of xylan 1) and GUX2, recently identified as glucuronyltransferases, are both required for substitution of the xylan backbone with [Me]GlcA. Here, we demonstrate clear differences in the pattern of [Me]GlcA substitution generated by each of these glucuronyltransferases. GUX1 decorates xylan with a preference for addition of [Me]GlcA at evenly spaced xylosyl residues. Intervals of eight or 10 residues dominate, but larger intervals are observed. GUX2, in contrast, produces more tightly clustered decorations with most frequent spacing of five, six or seven xylosyl residues, with no preference for odd or even spacing. Moreover, each of these GUX transferases substitutes a distinct domain of secondary cell wall xylan, which we call the major and minor domains. These major and minor xylan domains were not separable from each other by size or charge, a finding that suggests that they are tightly associated. The presence of both differently [Me]GlcA decorated domains may produce a xylan molecule that is heterogeneous in its properties. We speculate that the major and minor domains of xylan may be specialised, such as for interaction with cellulose or lignin. These findings have substantial implications for our understanding of xylan synthesis and structure, and for models of the molecular architecture of the lignocellulosic matrix of plant cell walls.

The extostosin family: Proteins with many functions☆

Heparan sulfates are complex sulfated molecules found in abundance at cell surfaces and in the extracellular matrix. They bind to and influence the activity of a variety of molecules like growth factors, proteases and morphogens and are thus involved in various cell-cell and cell-matrix interactions. The mammalian EXT proteins have glycosyltransferase activities relevant for HS chain polymerization, however their exact role in this process is still confusing. In this review, we summarize current knowledge about the biochemical activities and some proposed functions of the members of the EXT protein family and their roles in human disease.

An even pattern of xylan substitution is critical for interaction with cellulose in plant cell walls

Xylan and cellulose are abundant polysaccharides in vascular plants and essential for secondary cell wall strength. Acetate or glucuronic acid decorations are exclusively found on even-numbered residues in most of the glucuronoxylan polymer. It has been proposed that this even-specific positioning of the decorations might permit docking of xylan onto the hydrophilic face of a cellulose microfibril1–3. Consequently, xylan adopts a flattened ribbon-like twofold screw conformation when bound to cellulose in the cell wall4. Here we show that ESKIMO1/XOAT1/TBL29, a xylan-specific O-acetyltransferase, is necessary for generation of the even pattern of acetyl esters on xylan in Arabidopsis. The reduced acetylation in the esk1 mutant deregulates the position-specific activity of the xylan glucuronosyltransferase GUX1, and so the even pattern of glucuronic acid on the xylan is lost. Solid-state NMR of intact cell walls shows that, without the even-patterned xylan decorations, xylan does not interact normally with cellulose fibrils. We conclude that the even pattern of xylan substitutions seen across vascular plants enables the interaction of xylan with hydrophilic faces of cellulose fibrils, and is essential for development of normal plant secondary cell walls.Plant cell wall consists of multiple components and complex structure. Here, ssNMR was used to investigate the physical interactions between two principle cell wall components, cellulose and xylan, and demonstrate the mechanism for their interactions

Suppression of xylan endotransglycosylase PtxtXyn10A affects cellulose microfibril angle in secondary wall in aspen wood

Certain xylanases from family GH10 are highly expressed during secondary wall deposition, but their function is unknown. We carried out functional analyses of the secondary-wall specific PtxtXyn10A in hybrid aspen (Populus tremula × tremuloides). PtxtXyn10A function was analysed by expression studies, overexpression in Arabidopsis protoplasts and by downregulation in aspen. PtxtXyn10A overexpression in Arabidopsis protoplasts resulted in increased xylan endotransglycosylation rather than hydrolysis. In aspen, the enzyme was found to be proteolytically processed to a 68 kDa peptide and residing in cell walls. Its downregulation resulted in a corresponding decrease in xylan endotransglycosylase activity and no change in xylanase activity. This did not alter xylan molecular weight or its branching pattern but affected the cellulose-microfibril angle in wood fibres, increased primary growth (stem elongation, leaf formation and enlargement) and reduced the tendency to form tension wood. Transcriptomes of transgenic plants showed downregulation of tension wood related genes and changes in stress-responsive genes. The data indicate that PtxtXyn10A acts as a xylan endotransglycosylase and its main function is to release tensional stresses arising during secondary wall deposition. Furthermore, they suggest that regulation of stresses in secondary walls plays a vital role in plant development.

Evolution of xylan substitution patterns in gymnosperms and angiosperms: implications for xylan interaction with cellulose

The xylan decoration pattern in all lineages of gymnosperms permits hydrogen bonding of xylan to cellulose. The interaction between cellulose and xylan is important for the load-bearing secondary cell wall of flowering plants. Based on the precise, evenly spaced pattern of acetyl and glucuronosyl (MeGlcA) xylan substitutions in eudicots, we recently proposed that an unsubstituted face of xylan in a 2-fold helical screw can hydrogen bond to the hydrophilic surfaces of cellulose microfibrils. In gymnosperm cell walls, any role for xylan is unclear, and glucomannan is thought to be the important cellulose-binding polysaccharide. Here, we analyzed xylan from the secondary cell walls of the four gymnosperm lineages (Conifer, Gingko, Cycad, and Gnetophyta). Conifer, Gingko, and Cycad xylan lacks acetylation but is modified by arabinose and MeGlcA. Interestingly, the arabinosyl substitutions are located two xylosyl residues from MeGlcA, which is itself placed precisely on every sixth xylosyl residue. Notably, the Gnetophyta xylan is more akin to early-branching angiosperms and eudicot xylan, lacking arabinose but possessing acetylation on alternate xylosyl residues. All these precise substitution patterns are compatible with gymnosperm xylan binding to hydrophilic surfaces of cellulose. Molecular dynamics simulations support the stable binding of 2-fold screw conifer xylan to the hydrophilic face of cellulose microfibrils. Moreover, the binding of multiple xylan chains to adjacent planes of the cellulose fibril stabilizes the interaction further. Our results show that the type of xylan substitution varies, but an even pattern of xylan substitution is maintained among vascular plants. This suggests that 2-fold screw xylan binds hydrophilic faces of cellulose in eudicots, early-branching angiosperm, and gymnosperm cell walls.

Xylan decoration patterns and the plant secondary cell wall molecular architecture

The molecular architecture of plant secondary cell walls is still not resolved. There are several proposed structures for cellulose fibrils, the main component of plant cell walls and the conformation of other molecules is even less well known. Glucuronic acid (GlcA) substitution of xylan (GUX) enzymes, in CAZy family glycosyl transferase (GT)8, decorate the xylan backbone with various specific patterns of GlcA. It was recently discovered that dicot xylan has a domain with the side chain decorations distributed on every second unit of the backbone (xylose). If the xylan backbone folds in a similar way to glucan chains in cellulose (2-fold helix), this kind of arrangement may allow the undecorated side of the xylan chain to hydrogen bond with the hydrophilic surface of cellulose microfibrils. MD simulations suggest that such interactions are energetically stable. We discuss the possible role of this xylan decoration pattern in building of the plant cell wall.

Studies of Enzymatic Cleavage of Cellulose Using Polysaccharide Analysis by Carbohydrate gel Electrophoresis (PACE)

With the advent of fast genome analysis, many genes encoding novel putative cellulolytic enzymes are being identified in diverse bacterial and fungal genomes. The discovery of these genes calls for quick, robust, and reliable methods for qualitative and quantitative characterization of the enzymatic activities of the encoded proteins. Here, we describe the use of the polysaccharide analysis by carbohydrate gel electrophoresis (PACE) method, which was previously used, among other applications, to characterize various hemicellulose degrading enzymes; for structural elucidation of these carbohydrates; and for analysis of products resulting from enzymatic cleavage of cellulose. PACE relies on fluorescent labeling of mono-, oligo-, and polysaccharides at their reducing end and separation of the labeled carbohydrates by polyacrylamide gel electrophoresis. Labeling can be carried out before or after enzymatic digestion. PACE is very sensitive and allows analysis of both substrate specificities and kinetic properties of cellulolytic enzymes.

Reduced Expression of EXTL2, a Member of the Exostosin (EXT) Family of Glycosyltransferases, in Human Embryonic Kidney 293 Cells Results in Longer Heparan Sulfate Chains

Background: EXTL2 belongs to the exostosin family of glycosyltransferases involved in heparan sulfate biosynthesis. Results: We determined the effect of reduced or increased amounts of EXTL2 on heparan sulfate structure in a human cell line. Conclusion: EXTL2 levels influence heparan sulfate chain elongation. Significance: Knowledge about how EXTL2 influences heparan sulfate structure is crucial for understanding heparan sulfate dependent processes. Heparan sulfate proteoglycans are ubiquitously located on cell surfaces and in the extracellular matrices. The negatively charged heparan sulfate chains interact with a multitude of different proteins, thereby influencing a variety of cellular and developmental processes, for example cell adhesion, migration, tissue morphogenesis, and differentiation. The human exostosin (EXT) family of genes contains five members: the heparan sulfate polymerizing enzymes, EXT1 and EXT2, and three EXT-like genes, EXTL1, EXTL2, and EXTL3. EXTL2 has been ascribed activities related to the initiation and termination of heparan sulfate chains. Here we further investigated the role of EXTL2 in heparan sulfate chain elongation by gene silencing and overexpression strategies. We found that siRNA-mediated knockdown of EXTL2 in human embryonic kidney 293 cells resulted in increased chain length, whereas overexpression of EXTL2 in the same cell line had little or no effect on heparan sulfate chain length. To study in more detail the role of EXTL2 in heparan sulfate chain elongation, we tested the ability of the overexpressed protein to catalyze the in vitro incorporation of N-acetylglucosamine and N-acetylgalactosamine to oligosaccharide acceptors resembling unmodified heparan sulfate and chondroitin sulfate precursor molecules. Analysis of the generated products revealed that recombinant EXTL2 showed weak ability to transfer N-acetylgalactosamine to heparan sulfate precursor molecules but also, that EXTL2 exhibited much stronger in vitro N-acetylglucosamine-transferase activity related to elongation of heparan sulfate chains.

Identification of an algal xylan synthase indicates that there is functional orthology between algal and plant cell wall biosynthesis

Summary Insights into the evolution of plant cell walls have important implications for comprehending these diverse and abundant biological structures. In order to understand the evolving structure–function relationships of the plant cell wall, it is imperative to trace the origin of its different components. The present study is focused on plant 1,4‐β‐xylan, tracing its evolutionary origin by genome and transcriptome mining followed by phylogenetic analysis, utilizing a large selection of plants and algae. It substantiates the findings by heterologous expression and biochemical characterization of a charophyte alga xylan synthase. Of the 12 known gene classes involved in 1,4‐β‐xylan formation, XYS1/IRX10 in plants, IRX7, IRX8, IRX9, IRX14 and GUX occurred for the first time in charophyte algae. An XYS1/IRX10 ortholog from Klebsormidium flaccidum, designated K. flaccidum XYLAN SYNTHASE‐1 (Kf XYS1), possesses 1,4‐β‐xylan synthase activity, and 1,4‐β‐xylan occurs in the K. flaccidum cell wall. These data suggest that plant 1,4‐β‐xylan originated in charophytes and shed light on the origin of one of the key cell wall innovations to occur in charophyte algae, facilitating terrestrialization and emergence of polysaccharide‐based plant cell walls.