Cell Wall Glycans Research Articles

Towards the synthesis of UDP-apiofuranose

AbstractApiose is a rare sugar present in cell-wall glycans of all higher plants. Apiosyltransferases use uridine diphosphate-α-d-apiofuranose (UDP-Api) to biosynthesize apiose-containing pectic polysaccharides, namely rhamnogalacturonan-II (RG-II) and apiogalacturonan. However, the study of apiosyltransferases is hampered by the limited availability of UDP-Api. Here, we report the challenging chemical synthesis of α-apiosyl 1-phosphate as the key intermediate towards UDP-Api synthesis. Various protecting group patterns and synthetic routes, including phosphoglycosylation, phosphorylation of the hemiacetal, and phosphorylation of the anomeric bromide were screened. The latter strategy enabled the synthesis of apiosyl 1-phosphate with good α-selectivity (α:β = 5:1). Additionally, we describe a pyrophosphate coupling reaction of the synthesized glycosyl 1-phosphate with commercially available uridine-monophosphate morpholidate to obtain UDP-Api. This test reaction was monitored by 31P-NMR spectroscopy, which showed formation of the desired product and its partial hydrolysis due to an unwanted intramolecular cyclisation typical of 1,2-cis UDP-sugars. Graphical abstract

Structural characterization of arabinogalactan-II and pectin from Urtica cannabina

Comparative analysis of extracellular and cell wall glycans from Urtica cannabina leaves was performed using chemical methods, GC, GC–MS, 1D, and 2D NMR spectroscopy. The structures of extracellular AG-II and cell wall AG-II are similar. The units are typical for AG-IIs: β-GlcpA-4-OMe-(1→, Rhap-(1 → 4)-β-GlcpA-(1→, attached to β-Galp at O-6, as well as arabinan chains attached to β-Galp at O-3. A single Araf and a trisaccharide formed by 2,5-Araf and two terminal Araf form short arabinan side chains in AG-II. 1,5-arabinan with a backbone substituted by a single Araf at O-3 was identified only in the side chains of cell wall AG-II. The side chains can be attached to O-3 and O-6 of the same β-Galp to form a bifurcated AG side chain. The backbone of AG-II is formed by 1,6- rather than 1,3-linked Galp, although it does include some 1,3-Galp. The high content of 3,6-Galp shows the highly branched nature of the AG carbohydrate chains. From the cell wall, AGP was extracted together with pectin, the simultaneous elution of which from both DEAE-cellulose and Sepharose may indicate a link between them.

The Tissue Browning and Concomitant Toughening of Yellow Flammulina filiformis Stipes Is Caused by Oxidative Damage-Mediated Metabolic Disorder and Cell Wall Glycan Remodeling.

The browning and associated toughening of fruiting body stipes are the main causes of declines in the commercial production of yellow Flammulina filiformis. The dynamic metabolic changes from the top to bottom stipe sections of yellow F. filiformis fruiting bodies were investigated by integrating physiological, transcriptomic, and metabolomic analyses. The results indicated that oxidative stress levels gradually increased accompanying the degree of tissue browning and toughening from the top to bottom sections of F. filiformis stipes. In-depth analysis showed that there were remarkable changes in the expression of genes, and the content of metabolites correlated with the primary and secondary metabolism of F. filiformis stipes. Interestingly, the expression levels of genes participating in chitosan biosynthesis and the degree of deacetylation of chitosan increased from top to bottom in F. filiformis stipes, implying that cell wall glycan remodeling may contribute to concomitant toughening of the browning of F. filiformis stipes.

Glycome profiling and immunohistochemistry uncover changes in cell walls of Arabidopsis thaliana roots during spaceflight

A large and diverse library of glycan-directed monoclonal antibodies (mAbs) was used to determine if plant cell walls are modified by low-gravity conditions encountered during spaceflight. This method called glycome profiling (glycomics) revealed global differences in non-cellulosic cell wall epitopes in Arabidopsis thaliana root extracts recovered from RNA purification columns between seedlings grown on the International Space Station-based Vegetable Production System and paired ground (1-g) controls. Immunohistochemistry on 11-day-old seedling primary root sections showed that ten of twenty-two mAbs that exhibited spaceflight-induced increases in binding through glycomics, labeled space-grown roots more intensely than those from the ground. The ten mAbs recognized xyloglucan, xylan, and arabinogalactan epitopes. Notably, three xylem-enriched unsubstituted xylan backbone epitopes were more intensely labeled in space-grown roots than in ground-grown roots, suggesting that the spaceflight environment accelerated root secondary cell wall formation. This study highlights the feasibility of glycomics for high-throughput evaluation of cell wall glycans using only root high alkaline extracts from RNA purification columns, and subsequent validation of these results by immunohistochemistry. This approach will benefit plant space biological studies because it extends the analyses possible from the limited amounts of samples returned from spaceflight and help uncover microgravity-induced tissue-specific changes in plant cell walls.

Chemo-Enzymatic Synthesis of Long-Chain Oligosaccharides for Studying Xylan-Modifying Enzymes.

Plant research is hampered in several aspects by a lack of pure oligosaccharide samples that closely represent structural features of cell wall glycans. An alternative to purely chemical synthesis to access these oligosaccharides is chemo-enzymatic synthesis using glycosynthases. These enzymes enable the ligation of oligosaccharide donors, when activated for example as α-glycosyl fluorides, with suitable acceptor oligosaccharides. Herein, the synthesis of xylan oligosaccharides up to dodecasaccharides is reported, with glycosynthase-mediated coupling reactions as key steps. The xylo-oligosaccharide donors were protected at the non-reducing end with a 4-O-tetrahydropyranyl (THP) group to prevent polymerization. Installation of an unnatural 3-O-methylether substituent at the reducing end xylose of the oligosaccharides ensured good water solubility. Biochemical assays demonstrated enzymatic activity for the xylan acetyltransferase XOAT1 from Arabidopsis thaliana, xylan arabinofuranosyl-transferase XAT3 enzymes from rice and switchgrass, and the xylan glucuronosyltransferase GUX3 from Arabidopsis thaliana. In case of the glucuronosyltransferase GUX3, MALDI-MS/MS analysis of the reaction product suggested that a single glucuronosyl substituent was installed primarily at the central xylose residues of the dodecasaccharide acceptor, demonstrating the value of long-chain acceptors for assaying biosynthetic glycosyltransferases.

A Staphylococcal Glucosaminidase Drives Inflammatory Responses by Processing Peptidoglycan Chains to Physiological Lengths.

The peptidoglycan of Staphylococcus aureus is a critical cell envelope constituent and virulence factor that subverts host immune defenses and provides protection against environmental stressors. Peptidoglycan chains of the S. aureus cell wall are processed to characteristically short lengths by the glucosaminidase SagB. It is well established that peptidoglycan is an important pathogen-associated molecular pattern (PAMP) that is recognized by the host innate immune system and promotes production of proinflammatory cytokines, including interleukin-1β (IL-1β). However, how bacterial processing of peptidoglycan drives IL-1β production is comparatively unexplored. Here, we tested the involvement of staphylococcal glucosaminidases in shaping innate immune responses and identified SagB as a mediator of IL-1β production. A ΔsagB mutant fails to promote IL-1β production by macrophages and dendritic cells, and processing of peptidoglycan by SagB is essential for this response. SagB-dependent IL-1β production by macrophages is independent of canonical pattern recognition receptor engagement and NLRP3 inflammasome-mediated caspase activity. Instead, treatment of macrophages with heat-killed cells from a ΔsagB mutant leads to reduced caspase-independent cleavage of pro-IL-1β, resulting in accumulation of the pro form in the macrophage cytosol. Furthermore, SagB is required for virulence in systemic infection and promotes IL-1β production in a skin and soft tissue infection model. Taken together, our results suggest that the length of S. aureus cell wall glycan chains can drive IL-1β production by innate immune cells through a previously undescribed mechanism related to IL-1β maturation.

Cell Wall Glycan Changes in Different Brachypodium Tissues Give Insights into Monocot Biomass

The annual temperate grass Brachypodium distachyon has become a model system for monocot biomass crops and for understanding lignocellulosic recalcitrance to employ better saccharification and fermentation approaches. It is a monocot plant used to study the grass cell walls that differ from the cell walls of dicot plants such as the eudicot model Arabidopsis. The B. distachyon cell wall is predominantly composed of cellulose, arabinoxylans, and mixed-linkage glucans, and it resembles the cell walls of other field grasses. It has a vascular bundle anatomy similar to C3 grasses. These features make Brachypodium an ideal model to study cell walls. Cell walls are composed of polymers with complex structures that vary between cell types and at different developmental stages. Antibodies that recognize specific cell wall components are currently one of the most effective and specific molecular probes to determine the location and distribution of polymers in plant cell walls in situ. Here, we investigated the glycan distribution in the cell walls of the root and leaf tissues of Brachypodium by employing cell-wall-directed antibodies against diverse glycan epitopes. There are distinct differences in the presence of the epitopes between the root and leaf tissues as well as in the cell type level, which gives insights into monocot biomass.

S8.3c Cell wall glycans as targets for the development of new antifungals

S8.3 How the fungal cell wall glycan can modulate the immune response?, September 23, 2022, 3:00 PM - 4:30 PMBeing an extracellular organelle, the cell wall plays a crucial role in fungal life, by protecting fungal cells from a hostile environments and providing cells with mechanical strength. Exploiting the essentiality of this cell wall, available antifungal drugs target cell wall biosynthesis directly or by altering the fungal cell membrane. However, the emergence of resistance to antifungal drugs, new at risk-cohorts, and drug-drug interaction issue with antifungals demand new/alternative therapeutic strategy. In this regard, we have identified that surfactant protein-D (SP-D; a host humoral immune component) has growth inhibitory activity on Aspergillus fumigatus, a ubiquitous airborne opportunistic pathogen. SP-D, a soluble pattern recognition receptor of the collection family, targets galactosaminogalactan and galactomannan, the cell wall glycans of A. fumigatus hyphae, as the ligands. Hyphae grown in presence of SP-D show a significant decrease in the growth and are hyperbranched compared to control hyphae. SP-D treatment alters surface exposure of hyphal cell wall polysaccharides, thereby modifying hyphal immunoreactivity. SP-D pre-treatment increases the efficacy of the echinocandin and azole classes of antifungals against A. fumigatus. Interestingly, SP-D show hyphal growth inhibitory activity against multi-/pan-azole-resistant isolates of A. fumigatus. Overall, SP-D seems to target A. fumigatus cell wall glycans to execute its fungistatic activity. We are currently investigating the exact mechanism of anti-A. fumigatus activity associated with SP-D.

S8.3d Characterization of glycosylphosphatidylinositol-linked aspartyl proteases in Candida glabrata: Role in pathogenicity

S8.3 How the Fungal Cell Wall Glycan Can Modulate the Immune Response?, September 23, 2022, 3:00 PM - 4:30 PM Candida glabrata is the second to fourth most common yeast pathogen found in Candida bloodstream infections, depending upon the geographical location. C. glabrata, which belongs to the Nakaseomyces clade, possesses a distinct set of virulence attributes which include the ability to survive and proliferate in macrophages, adhere to biotic and abiotic surfaces and survive a wide range of stresses. Our research is focused on unveiling the strategies that C. glabrata employs to survive the nutrient-poor hostile host environment and evade host immune response. Toward this end, we are delineating the cellular processes, that are regulated by the family of 11 putative glycosylphosphatidylinositol-linked, cell surface-associated aspartyl proteases (CgYapsins, CgYps1-11). We have recently characterized the secretome of C. glabrata wild-type and aspartyl protease-deficient mutant strains, and showed that the GPI-anchored aspartyl proteases are both bonafide constituents and key modulators of the C. glabrata secretome. Further, besides elucidating the role of CgYapsins in the suppression of the host pro-inflammatory immune response, we have identified the flavodoxin-like protein CgPst2 as a substrate of the CgYps1 protease and demonstrated that the CgYps1-mediated cleavage of CgPst2 is pivotal to oligomerization and activity, and functions of CgPst2 in quinone detoxification. These findings underscoring the importance of multifunctional CgYapsins in the physiology and pathogenesis of C. glabrata will be presented.

S8.3b How fungal cell wall glycans modulate the activation of platelets ?

S8.3 How the fungal cell wall glycan can modulate the immune response?, September 23, 2022, 3:00 PM - 4:30 PM Platelets are the first circulating blood cells that interact and adhere to vascular lesions. They play an important role in vascular repair and maintenance of blood homeostasis. Platelets are involved in the immune defense of the host against many infections caused by bacteria, viruses, and fungi. Following infection, these microorganisms can alter platelet function leading to platelet activation. During fungal infection, β-glucans, mannan, and chitin, are critical components of Candida albicans, an opportunistic pathogenic yeast of humans. These fungal glycans play an important role in the modulation of the host response. They are released into the bloodstream and can be detected up to 10 days before the onset of clinical signs of invasive candidiasis. However, their role in the modulation of platelet activities is unknown. In our studies, we observed that β -glucans and chitin decrease platelet activation and modulate the production of inflammatory mediators, mediated by TLR4 or TLR8 respectively. These glycans also reduce platelet adherence to C. albicans and to polynuclear neutrophils, suggesting a process by which C. albicans can escape from cells of innate immunity by releasing cell wall polysaccharides into the close environment or the blood circulation. This study provides evidence that fungal glycans play a role in the modulation of the immune response and how C. albicans can escape from the innate immune response.

Synthesis of Fungal Cell Wall Oligosaccharides and Their Ability to Trigger Plant Immune Responses.

Oligosaccharide fragments of fungal cell wall glycans are important molecular probes for studying both the biology of fungi and fungal infections of humans, animals, and plants. The fungal cell wall contains large amounts of various polysaccharides that are ligands for pattern recognition receptors (PRRs), eliciting an immune response upon recognition. Towards the establishment of a glycan array platform for the identification of new ligands of plant PRRs, tri‐, penta‐, and heptasaccharide fragments of different cell wall polysaccharides were prepared. Chito‐ and β‐(1→6)‐gluco‐oligosaccharides were synthesized by automated glycan assembly (AGA), and α‐(1→3)‐ and α‐(1→4)‐gluco‐oligosaccharides were synthesized in solution using a recently reported highly α‐selective glycosylation methodology. Incubation of plants with the synthesized oligosaccharides revealed i) length dependence for plant activation by chito‐oligosaccharides and ii) β‐1,6‐glucan oligosaccharides as a new class of glycans capable of triggering plant activation.

Glycome Profiling and Bioprospecting Potential of the Himalayan Buddhist Handmade Paper of Tawang Region of Arunachal Pradesh.

The paper and pulp industry (PPI) is one of the largest industries that contribute to the growing economy of the world. While wood remains the primary raw material of the PPIs, the demand for paper has also grown alongside the expanding global population, leading to deforestation and ecological imbalance. Wood-based paper production is associated with enormous utilization of water resources and the release of different wastes and untreated sludge that degrades the quality of the environment and makes it unsafe for living creatures. In line with this, the indigenous handmade paper making from the bark of Daphne papyracea, Wall. ex G. Don by the Monpa tribe of Arunachal Pradesh, India is considered as a potential alternative to non-wood fiber. This study discusses the species distribution modeling of D. papyracea, community-based production of the paper, and glycome profiling of the paper by plant cell wall glycan-directed monoclonal antibodies. The algorithms used for ecological and geographical modeling indicated the maximum predictive distribution of the plant toward the western parts of Arunachal Pradesh. It was also found that the suitable distribution of D. papyracea was largely affected by the precipitation and temperature variables. Plant cell walls are primarily made up of cellulose, hemicellulose, lignin, pectin, and glycoproteins. Non-cellulosic cell wall glycans contribute significantly to various physical properties such as density, crystallinity, and tensile strength of plant cell walls. Therefore, a detailed analysis of non-cellulosic cell wall glycan through glycome profiling and glycosyl residue composition analysis is important for the polymeric composition and commercial processing of D. papyracea paper. ELISA-based glycome profiling results demonstrated that major classes of cell wall glycans such as xylan, arabinogalactans, and rhamnogalacturonan-I were present on D. papyracea paper. The presence of these polymers in the Himalayan Buddhist handmade paper of Arunachal Pradesh is correlated with its high tensile strength. The results of this study imply that non-cellulosic cell wall glycans are required for the production of high-quality paper. To summarize, immediate action is required to strengthen the centuries-old practice of handmade paper, which can be achieved through education, workshops, technical know-how, and effective marketing aid to entrepreneurs.

Overexpression of UDP-sugar pyrophosphorylase leads to higher sensitivity towards galactose, providing new insights into the mechanisms of galactose toxicity in plants.

SUMMARYGalactose toxicity (Gal‐Tox) is a widespread phenomenon ranging from Escherichia coli to mammals and plants. In plants, the predominant pathway for the conversion of galactose into UDP‐galactose (UDP‐Gal) and UDP‐glucose is catalyzed by the enzymes galactokinase, UDP‐sugar pyrophosphorylase (USP) and UDP‐galactose 4‐epimerase. Galactose is a major component of cell wall polymers, glycolipids and glycoproteins; therefore, it becomes surprising that exogenous addition of galactose leads to drastic root phenotypes including cessation of primary root growth and induction of lateral root formation. Currently, little is known about galactose‐mediated toxicity in plants. In this study, we investigated the role of galactose‐containing metabolites like galactose‐1‐phosphate (Gal‐1P) and UDP‐Gal in Gal‐Tox. Recently published data from mouse models suggest that a reduction of the Gal‐1P level via an mRNA‐based therapy helps to overcome Gal‐Tox. To test this hypothesis in plants, we created Arabidopsis thaliana lines overexpressing USP from Pisum sativum. USP enzyme assays confirmed a threefold higher enzyme activity in the overexpression lines leading to a significant reduction of the Gal‐1P level in roots. Interestingly, the overexpression lines are phenotypically more sensitive to the exogenous addition of galactose (0.5 mmol L−1 Gal). Nucleotide sugar analysis via high‐performance liquid chromatography‐mass spectrometry revealed highly elevated UDP‐Gal levels in roots of seedlings grown on 1.5 mmol L−1 galactose versus 1.5 mmol L−1 sucrose. Analysis of plant cell wall glycans by comprehensive microarray polymer profiling showed a high abundance of antibody binding recognizing arabinogalactanproteins and extensins under Gal‐feeding conditions, indicating that glycoproteins are a major target for elevated UDP‐Gal levels in plants.

Mechanistic insights into the digestion of complex dietary fibre by the rumen microbiota using combinatorial high-resolution glycomics and transcriptomic analyses

There is a knowledge gap regarding the factors that impede the ruminal digestion of plant cell walls or if rumen microbiota possess the functional activities to overcome these constraints. Innovative experimental methods were adopted to provide a high-resolution understanding of plant cell wall chemistries, identify higher-order structures that resist microbial digestion, and determine how they interact with the functional activities of the rumen microbiota. We characterized the total tract indigestible residue (TTIR) from cattle fed a low-quality straw diet using two comparative glycomic approaches: ELISA-based glycome profiling and total cell wall glycosidic linkage analysis. We successfully detected numerous and diverse cell wall glycan epitopes in barley straw (BS) and TTIR and determined their relative abundance pre- and post-total tract digestion. Of these, xyloglucans and heteroxylans were of higher abundance in TTIR. To determine if the rumen microbiota can further saccharify the residual plant polysaccharides within TTIR, rumen microbiota from cattle fed a diet containing BS were incubated with BS and TTIR ex vivo in batch cultures. Transcripts coding for carbohydrate-active enzymes (CAZymes) were identified and characterized for their contribution to cell wall digestion based on glycomic analyses, comparative gene expression profiles, and associated CAZyme families. High-resolution phylogenetic fingerprinting of these sequences encoded CAZymes with activities predicted to cleave the primary linkages within heteroxylan and arabinan.This experimental platform provides unprecedented precision in the understanding of forage structure and digestibility, which can be extended to other feed-host systems and inform next-generation solutions to improve the performance of ruminants fed low-quality forages.

Adaptation of Arabidopsis halleri to extreme metal pollution through limited metal accumulation involves changes in cell wall composition and metal homeostasis.

Metallophytes constitute powerful models for the study of metal homeostasis, adaptation to extreme environments and the evolution of naturally selected traits. Arabidopsis halleri is a pseudometallophyte which shows constitutive zinc/cadmium (Zn/Cd) tolerance and Zn hyperaccumulation but high intraspecific variability in Cd accumulation. To examine the molecular basis of the variation in metal tolerance and accumulation, ionome, transcriptome and cell wall glycan array profiles were compared in two genetically close A.halleri populations from metalliferous and nonmetalliferous sites in Northern Italy. The metallicolous population displayed increased tolerance to and reduced hyperaccumulation of Zn, and limited accumulation of Cd, as well as altered metal homeostasis, compared to the nonmetallicolous population. This correlated well with the differential expression of transporter genes involved in trace metal entry and in Cd/Zn vacuolar sequestration in roots. Many cell wall-related genes were also more highly expressed in roots of the metallicolous population. Glycan array and histological staining analyses demonstrated that there were major differences between the two populations in terms of the accumulation of specific root pectin and hemicellulose epitopes. Our results support the idea that both specific cell wall components and regulation of transporter genes play a role in limiting accumulation of metals in A. halleri at contaminated sites.

Commensal fungi and their cell-wall β-glucans direct differential responses in human intestinal epithelial cells.

Intestinal epithelial cells (IECs) are the first to encounter luminal antigens and play an active role in intestinal immune responses. We previously reported that β-glucans, major fungal cell-wall glycans, induced chemokine secretion by IEC lines in a Dectin-1- and Syk-dependent manner. Here, we show that in contrast to β-glucans, stimulation of IEC lines with Candida albicans and Saccharomyces cerevisiae did not induce secretion of any of the proinflammatory cytokines IL-8, CCL2, CXCL1, and GM-CSF. Commensal fungi and β-glucans activated Syk and ERK in IEC lines. However, only β-glucans activated p38, JNK, and the transcription factors NF-κB p65 and c-JUN, which were necessary for cytokine secretion. Furthermore, costimulation of IEC lines with β-glucans and C. albicans yielded decreased cytokine secretion compared to stimulation with β-glucans alone. Finally, exvivo stimulation of human colonic mucosal explants with zymosan and C. albicans, leads to epithelial Syk and ERK phosphorylation, implying recognition of fungi and similar initial signaling pathways as in IEC lines. Lack of cytokine secretion in response to commensal fungi may reflect IECs' response to fungal glycans, other than β-glucans, that contribute to mucosal tolerance. Skewed epithelial response to commensal fungi may impair homeostasis and contribute to intestinal inflammation.

Identification of proteins responsive to heterologous protein production in thermotolerant methylotrophic yeast Ogataea thermomethanolica TBRC656

The thermotolerant methylotrophic yeast Ogataea thermomethanolica TBRC656 is a potential host for heterologous protein production. However, overproduction of heterologous protein can induce cellular stress and limit the level of its secretion. To improve the secretion of heterologous protein, we identified the candidate proteins with altered production during production of heterologous protein in O. thermomethanolica by using a label-free comparative proteomic approach. Four hundred sixty-four proteins with various biological functions showed differential abundance between O. thermomethanolica expressing fungal xylanase (OT + Xyl) and a control strain. The induction of proteins in transport and proteasomal proteolysis was prominently observed. Eight candidate proteins involved in cell wall biosynthesis (Chs3, Gas4), chaperone (Sgt2, Pex19), glycan metabolism (Csf1), protein transport (Ypt35), and vacuole and protein sorting (Cof1, Npr2) were mutated by a CRISPR/Cas9 approach. An Sgt2 mutant showed higher phytase and xylanase activity compared with the control strain (13%-20%), whereas mutants of other genes including Cof1, Pex19, Gas4, and Ypt35 showed lower xylanase activity compared with the control strain (15%-25%). In addition, an Npr2 mutant showed defective growth, while overproduction of Npr2 enhanced xylanase activity. These results reveal genes that can be mutated to modulate heterologous protein production and growth of O. thermomethanolica TBRC656.

Plant terpenoid metabolism co-opts a component of the cell wall biosynthesis machinery.

Glycosylation is one of the most prevalent molecular modifications in nature. Single or multiple sugars can decorate a wide range of acceptors from proteins to lipids, cell wall glycans and small molecules, dramatically affecting their activity. Here, we discovered that by 'hijacking' an enzyme of the cellulose synthesis machinery involved in cell wall assembly, plants evolved cellulose synthase-like enzymes (Csls) and acquired the capacity to glucuronidate specialized metabolites, that is, triterpenoid saponins. Apparently, endoplasmic reticulum-membrane localization of Csls and of other pathway proteins was part of evolving a new glycosyltransferase function, as plant metabolite glycosyltransferases typically act in the cytosol. Discovery of glucuronic acid transferases across several plant orders uncovered the long-pursued enzymatic reaction in the production of a low-calorie sweetener from licorice roots. Our work opens the way for engineering potent saponins through microbial fermentation and plant-based systems.

Partial purification and immunodetection of cell surface glycoproteins from plants.

Cell surface glycoproteins in plants were first described more than 50 years ago, and yet, the precise mechanisms by which they operate remain elusive to this day. Studying glycoproteins is often challenging due to their subcellular localization (many secreted or membrane associated) and the extent of glycosylation present on the protein backbone, which can have profound effects on protein structure and behavior. In plants, additional layers of complexity exist as cell surface glycoproteins are in close contact, and in some cases, establish direct linkages with the polysaccharide networks present in the cell wall. In this chapter, we guide the reader through a protocol aimed to address the glycosylation status of a presumed cell surface glycoprotein. First, we discuss the advantages and disadvantages of using plants as homologous expression systems for recombinant glycoprotein production. Next, we describe a protocol for microsomal enrichment, followed by partial purification by affinity chromatography and finally glycodetection by immunoblotting using monoclonal antibodies targeting cell wall glycans. We particularly focus on the hydroxyproline-rich glycoprotein (HRGP) family, the most abundant family of glycoproteins in the plant cell wall. We provide examples of two putative HRGP chimeric proteins, one akin to extensins and the second an arabinogalactan protein (AGP)-like protein. For the latter, we provide an AGP-specific protocol to ensure enrichment of members of this group, which can be used independently or in conjunction with the described protocol. Throughout the chapter, we provide recommendations for the handling of plant glycoproteins and highlight special considerations for experimental design, along with troubleshooting suggestions.

Automated Glycan Assembly of Plant Cell Wall Oligosaccharides.

Synthetic cell wall oligosaccharides are promising molecular tools for investigating the structure and function of plant cell walls. Their well-defined structure and high purity prevents misinterpretations of experimental data, and the possibility to introduce chemical handles provides means for easier localization and detection. Automated glycan assembly as emerged has a powerful new method for the efficient preparation of oligosaccharide libraries. We recently made use of this technology to prepare a collection of plant cell wall glycans for cell wall research. In this chapter, detailed experimental procedures for the automated synthesis of oligosaccharides that are ready for use in biological assays are described.