Surface Carbohydrate Research Articles

Multivalent Glycosylation of Fluorescent Gold Nanoclusters Promotes Increased Human Dendritic Cell Targeting via Multiple Endocytic Pathways.

We report the synthesis and characterization of gold nanoclusters (Au NCs) stabilized by a mixture of zwitterionic and multivalent mannose ligands. Characterization of this carbohydrated nanosystem confirms its small size (∼2 nm), intense red-NIR fluorescence, relatively high affinity to lectin (ConA), and stability in physiological media. Cell studies performed using human-monocyte-derived dendritic cells (DCs) show that Au NC uptake efficiency is greatly enhanced by the presence of surface carbohydrate (>250% compared to noncarbohydrated Au NCs), allowing their detection in cells by fluorescence following incubation with concentrations as low as 1 μg mL(-1). Investigation using electron microscopy and pharmacological inhibitors indicates that Au NC uptake is mediated by multiple endocytic pathways involving the engulfment of Au NCs into endosomes and partial transport to lysosomes. Results show that clathrin- and F-actin-dependent pathways play major roles in Au NC uptake by DCs, regardless of whether or not they are coated with carbohydrates. In contrast, a specific C-lectin inhibitor induces a 60% decrease in DC particle uptake only for the carbohydrate-coated Au NCs. This study demonstrates that the combination of ultrasmall gold NCs and functionalization with multivalent mannose ligands results in greatly enhanced human DC targeting, presumably due to increased diffusion and target cell binding, respectively.

Involvement of a caleosin in lipid storage, spore dispersal, and virulence in the entomopathogenic filamentous fungus, Beauveria bassiana.

Eukaryotic cells store lipids in membrane-encased droplets. The entomopathogenic fungus, Beauveria bassiana, initiates infection via attachment of its spores to the epicuticle or waxy layer of target insects, degrading and assimilating host surface hydrocarbons, carbohydrates and proteins. Caleosins are components of the proteinaceous coat of lipid droplets and a single B. bassiana caleosin homologue, Bbcal1, was identified and characterized. The BbCal1 sequence contained an EF-hand Ca(2+) binding domain and potential hydrophobic stretches similar to those found in plant caleosins, along with a proline knot motif defined by only two proline residues. Targeted gene inactivation of Bbcal1 did not appear to affect spore germination, growth on lipid substrates or stress response, but changes in lipid, vacuole and endoplasmic reticulum/multilamellar vesicle-like structures, and altered cellular lipid profiles were seen in conidia grown on a variety of substrates including potato dextrose agar, olive oil, glyceride trioleate, oleic acid and the alkane, C16 . The ΔBbcal1 mutant produced more compact assemblages of conidia, displayed a reduced and delayed spore dispersal phenotype, and showed decreased virulence in insect bioassays using the greater wax moth, Galleria mellonella. Our data indicate novel functions for caleosins in fungal virulence, spore development and the trafficking and/or turnover of lipid-related structures.

Bioactive Hybrid Particles from Poly(D,L-lactide-co-glycolide) Nanoparticle Stabilized Lipid Droplets.

Biodegradable and bioactive hybrid particles composed of poly(D,L-lactide-co-glycolide) (PLGA) nanoparticles and medium-chain triglycerides were prepared by spray drying lipid-in-water emulsions stabilized by PLGA nanoparticles, to form PLGA-lipid hybrid (PLH) microparticles approximately 5 μm in mean diameter. The nanoparticle stabilizer was varied and mannitol was also incorporated during the preparation to investigate the effect of stabilizer charge and cryoprotectant content on the particle microstructure. An in vitro lipolysis model was used to demonstrate the particles' bioactivity by manipulating the digestion kinetics of encapsulated lipid by pancreatic lipase in simulated gastrointestinal fluid. Lipid digestion kinetics were enhanced in PLH and PLGA-lipid-mannitol hybrid (PLMH) microparticles for both stabilizers, compared to a coarse emulsion, in biorelevant media. An optimal digestion rate was observed for the negatively charged PLMH system, evidenced by a 2-fold increase in the pseudo-first-order rate constant compared to a coarse emulsion. Improved microparticle redispersion, probed by dual dye confocal fluorescence microscopy, increased the available surface area of lipid for lipase adsorption, enhancing digestion kinetics. Thereby, lipase action was controlled in hybrid microparticles by altering the surface charge and carbohydrate content. Our results demonstrate that bioactive microparticles composed of versatile and biodegradable polymeric particles and oil droplets have great potential for use in smart food and nutrient delivery, as well as safer and more efficacious oral delivery of drugs and drug combinations.

Multipotent SSEA1 Positive Cells Population Differentiation into Primordial Germ Cells and Subsequently Progress into Oocyte-like Cells.

Stage-specific embryonic antigen-1 (SSEA1) is a cell surface carbohydrate that its pattern expression is changed during induction of mouse embryonic stem cell differentiation. In this study, the spatial distribution of SSEA1 on primordial germ cells differentiation and subsequent progression into oocyte-like cells from mouse embryonic stem cells in vitro was evaluated. Embryoid bodies from mouse embryonic stem cells were cultured for two days with 5 ng/mL BMP4. SSEA1 positive and negative cells were separated using the MACS system and cultured separately in a conditioned medium consist of in vitro maturation medium diluted in DMEM [1:1] for 10 days. We assayed viability, colony formation and alkaline phosphatase activity (ALP) of sorted cells. Also, germ cell markers were analyzed by flow cytometry, immunocytochemistry and RT-PCR. Viability percent SSEA1 positive cells were more than SSEA1 negative cells. SSEA1 positive cells and SSEA1 negative cells formed compact and flat colonies respectively. Unlike the SSEA1 positive population, the SSEA1 negative colonies showed a weak ALP activity. SSEA1 positive cells expressed Oct4, Stella, Mvh, c-kit, Scp3, Desmin, GFAP and Albumin. Interestingly, SSEA1 negative cells expressed Desmin and GFAP. The population of Mvh-positive cells in SSEA1 positive was 17.74%. All specific oocyte mentioned genes were detected in the SSEA1 positive. Also, oocyte specific proteins GDF9 and ZP3 were detected using immunocytochemistry. Our results suggest that conditioned medium provides a suitable niche to differentiation and progression putative primordial germ cells derived from the SSEA1 positive toward oocyte-like cells.

Structural investigation of cell wall polysaccharides of Lactobacillus delbrueckii subsp. bulgaricus 17

Lactobacilli are valuable strains for commercial (functional) food fermentations. Their cell surface-associated polysaccharides (sPSs) possess important functional properties, such as acting as receptors for bacteriophages (bacterial viruses), influencing autolytic characteristics and providing protection against antimicrobial peptides. The current report provides an elaborate molecular description of several surface carbohydrates of Lactobacillus delbrueckii subsp. bulgaricus strain 17. The cell surface of this strain was shown to contain short chain poly(glycerophosphate) teichoic acids and at least two different sPSs, designated here as sPS1 and sPS2, whose chemical structures were examined by 2D nuclear magnetic resonance spectroscopy and methylation analysis. Neutral branched sPS1, extracted with n-butanol, was shown to be composed of hexasaccharide repeating units (-[α-d-Glcp-(1-3)-]-4-β-l-Rhap2OAc-4-β-d-Glcp-[α-d-Galp-(1-3)]-4-α-Rhap-3-α-d-Galp-), while the major component of the TCA-extracted sPS2 was demonstrated to be a linear d-galactan with the repeating unit structure being (-[Gro-3P-(1-6)-]-3-β-Galf-3-α-Galp-2-β-Galf-6-β-Galf-3-β-Galp-).

Multi-dimensional glycan microarrays with glyco-macroligands.

Glycan microarray has become a powerful high-throughput tool for examining binding interactions of carbohydrates with the carbohydrate binding biomolecules like proteins, enzymes, antibodies etc. It has shown great potential for biomedical research and applications, such as antibody detection and profiling, vaccine development, biomarker discovery, and drug screening. Most glycan microarrays were made with monovalent glycans immobilized directly onto the array surface via either covalent or non-covalent bond, which afford a multivalent glycans in two dimensional (2D) displaying. A variety of glyco-macroligands have been developed to mimic multivalent carbohydrate-protein interactions for studying carbohydrate-protein interactions and biomedical research and applications. Recently, a number of glyco-macroligands have been explored for glycan microarray fabrication, in particular to mimick the three dimensional (3D) multivalent display of cell surface carbohydrates. This review highlights these recent developments of glyco-macroligand-based microarrays, predominantly, novel glycan microarrays with glyco-macroligands like glycodendrimers, glycopolymers, glycoliposomes, neoglycoproteins, and glyconanoparticles with the effort in controlling the density and orientation of glycans on the array surface, which facilitate both their binding specificity and affinity and thus the high performance of glycan microarrays.

Immune sensing of fungal cell wall carbohydrates programs immunity to A. fumigatus infection: role of γδ T cells. (MPF6P.662)

Abstract The human fungal pathogen Aspergillus fumigatus is associated with allergic sensitization and invasive infection in susceptible individuals. How the combined recognition of fungal molecular patterns programs host adaptive immunity is not well understood. Previously, we observed that fungal chitin promotes Th2-skewed immunity and eosinophil recruitment that proved detrimental to fungal clearance in invasive infection. In this study, we further characterized the role of early cytokine and effector cells in chitin derived eosinophil activation. We observed that single aspiration of a high-chitin isolate (Af5517) resulted in a marked increase in early transcription of IL-17A. Furthermore, eosinophil recruitment was not increased in RAG1-/- mice, suggesting that γδ T cells are required for early recognition of conidia-associated chitin. Although IL-17A was not required, the absence of γδ T cells (known to be potent sources of innate IL-17A) resulted in a significant decrease in eosinophil recruitment after single and multiple aspirations of Af5517. Also, fungal strains displaying chitin increased exposure or manipulating cell surface carbohydrate ratio by blockage of β-glucan recognition on receptor level resulted in increased airway eosinophil recruitment suggesting that relative high chitin/low β-glucan recognition leads to increased eosinophil recruitment and Th2 responses that are promoted by early responses of γδ T cells.

Synthesis and Characterization of Ribonucleic Acid Aptamers Targeted at Aspergillus Fungus Cell Surface Carbohydrates

Aspergillosis is a disease characterized by the degradation of the respiratory system due to Aspergillus fumigatus fungal hyphae. The slow establishment of the infection yields low rates of detection and high mortality rates in immune‐suppressed patients in Intensive Care Units (ICU) and Internal Medical Wards (IMW). RNA aptamers may can aid in the detection of aspergillosis using the cell surface carbohydrate, beta‐D‐glucan. The aptamers would be selected to bind selectively to cell surface carbohydrates of the fungus, where positive signals would be useful in the identification of infection. This work will describe isolation of RNA aptamers from a randomized sequence population of 1020 to 1024 variants. RNA aptamer pools were synthesized using PCR amplification reactions to combine the DNA template with primers. In vitro transcription was conducted to form the RNA molecules, where selection was conducted in rounds against beta‐D‐glucan. Sequencing and binding assays will be used to characterize the RNA aptamer pool.

Possible involvement of Tachylectin-2-like lectin from Acropora tenuis in the process of Symbiodinium acquisition

Most reef-building corals in tropical and subtropical areas symbiose with microalgae from the genus Symbiodinium (dinoflagellate) and depend on the photosynthate produced by the microalgae. The majority of corals acquire Symbiodinium from the surrounding environment through horizontal transfer, but the molecular mechanisms involved in the acquisition of Symbiodinium remain unknown. It has been hypothesized that carbohydrate-binding proteins, or lectins, of the host coral recognize cell surface carbohydrates of Symbiodinium in the process of acquiring symbionts. Thus, we examined the molecular mechanisms involving lectins and carbohydrates using model organism Acropora tenuis, a common reef-building coral, and Symbiodinium culture strains. Juvenile polyps acquire more cells of Symbiodinium strain NBRC102920 at 72–96 h of metamorphosis induction than in any other period. Glycosidase treatment of Symbiodinium inhibited the acquisition of Symbiodinium by juvenile coral polyps. The presence of carbohydrates D-galactose, N-acetyl-D-galactosamine, and N-acetyl-D-glucosamine at 10 mM also tended to decrease Symbiodinium acquisition. We isolated two N-acetyl-D-galactosamine binding lectins with apparent molecular masses of 14.6 and 29.0 kDa from A. tenuis, and de novo sequencing and cDNA cloning showed that the 29.0 kDa protein is Tachylectin-2-like lectin (AtTL-2). The anti-Tachylectin-2 antibody is suggested to bind specifically to AtTL-2. The antibody also inhibited binding of AtTL-2 to N-acetyl-D-galactosamine-resin and the acquisition of Symbiodinium by juvenile A. tenuis polyps. Based on these results, AtTL-2 is likely involved in the process of Symbiodinium acquisition.

Single cell profiling of surface carbohydrates on Bacillus cereus.

Cell surface carbohydrates are important to various bacterial activities and functions. It is well known that different types of Bacillus display heterogeneity of surface carbohydrate compositions, but detection of their presence, quantitation and estimation of variation at the single cell level have not been previously solved. Here, using atomic force microscopy (AFM)-based recognition force mapping coupled with lectin probes, the specific carbohydrate distributions of N-acetylglucosamine and mannose/glucose were detected, mapped and quantified on single B. cereus surfaces at the nanoscale across the entire cell. Further, the changes of the surface carbohydrate compositions from the vegetative cell to spore were shown. These results demonstrate AFM-based 'recognition force mapping' as a versatile platform to quantitatively detect and spatially map key bacterial surface biomarkers (such as carbohydrate compositions), and monitor in situ changes in surface biochemical properties during intracellular activities at the single cell level.

Examination of whole cell galectin binding by solid phase and flow cytometric analysis.

We have utilized simple flow cytometric and fluorescence-based solid phase assays to study the interaction of glycan-binding proteins (GBP) to cell surface glycoconjugates. These methods utilize commonly employed flow cytometry techniques and commercially available streptavidin-coated microplates to immobilize various biotinylated ligands, such as glycopeptides, oligosaccharides, and whole cells. Using this approach, fluorescently labeled GBPs, in particular, members of the galectin family, can be interrogated for potential interactions with cell surface carbohydrates, including elucidation of the potential impact of alterations in glycosylation on carbohydrate recognition. Using these approaches, we present examples of flow cytometric and fluorescence-based solid phase assays to study galectin-carbohydrate interactions.

Structural analysis of Pore-Forming CEL-III

CEL-III is a hemolytic lectin isolated from the sea cucumber Cucumaria echinata. This lectin is composed of two carbohydrate-binding domains (domains 1-2) and one oligomerization domain (domain 3). After binding to the cell surface carbohydrate chains through domains 1-2, domain 3 self-associates to form transmembrane pores, leading to cell lysis or death, which resembles other pore-forming toxins of diverse organisms. To elucidate the pore-formation mechanism of CEL-III, the crystal structure of the CEL-III oligomer was determined. The CEL-III oligomer has a heptameric structure with a long β-barrel as a transmembrane pore. This β-barrel is composed of 14 β-strands resulting from a large structural transition of α-helices accommodated in the interface between domains 1-2 and domain 3 in the monomeric structure, suggesting that the dissociation of these α-helices triggered their structural transition into a β-barrel. After heptamerization, domains 1-2 form a flat ring, in which all carbohydrate- binding sites remain bound to cell surface carbohydrate chains, stabilizing the transmembrane β-barrel in a position perpendicular to the plane of the lipid bilayer.

Crystal Structure of the Chlamydomonas Starch Debranching Enzyme Isoamylase ISA1 Reveals Insights into the Mechanism of Branch Trimming and Complex Assembly

The starch debranching enzymes isoamylase 1 and 2 (ISA1 and ISA2) are known to exist in a large complex and are involved in the biosynthesis and crystallization of starch. It is suggested that the function of the complex is to remove misplaced branches of growing amylopectin molecules, which would otherwise prevent the association and crystallization of adjacent linear chains. Here, we investigate the function of ISA1 and ISA2 from starch producing alga Chlamydomonas. Through complementation studies, we confirm that the STA8 locus encodes for ISA2 and sta8 mutants lack the ISA1·ISA2 heteromeric complex. However, mutants retain a functional dimeric ISA1 that is able to partly sustain starch synthesis in vivo. To better characterize ISA1, we have overexpressed and purified ISA1 from Chlamydomonas reinhardtii (CrISA1) and solved the crystal structure to 2.3 Å and in complex with maltoheptaose to 2.4 Å. Analysis of the homodimeric CrISA1 structure reveals a unique elongated structure with monomers connected end-to-end. The crystal complex reveals details about the mechanism of branch binding that explains the low activity of CrISA1 toward tightly spaced branches and reveals the presence of additional secondary surface carbohydrate binding sites.

Genogroup IV and VI canine noroviruses interact with histo-blood group antigens.

ABSTRACTHuman noroviruses (HuNV) are a significant cause of viral gastroenteritis in humans worldwide. HuNV attaches to cell surface carbohydrate structures known as histo-blood group antigens (HBGAs) prior to internalization, and HBGA polymorphism among human populations is closely linked to susceptibility to HuNV. Noroviruses are divided into 6 genogroups, with human strains grouped into genogroups I (GI), II, and IV. Canine norovirus (CNV) is a recently discovered pathogen in dogs, with strains classified into genogroups IV and VI. Whereas it is known that GI to GIII noroviruses bind to HBGAs and GV noroviruses recognize terminal sialic acid residues, the attachment factors for GIV and GVI noroviruses have not been reported. This study sought to determine the carbohydrate binding specificity of CNV and to compare it to the binding specificities of noroviruses from other genogroups. A panel of synthetic oligosaccharides were used to assess the binding specificity of CNV virus-like particles (VLPs) and identified α1,2-fucose as a key attachment factor. CNV VLP binding to canine saliva and tissue samples using enzyme-linked immunosorbent assays (ELISAs) and immunohistochemistry confirmed that α1,2-fucose-containing H and A antigens of the HBGA family were recognized by CNV. Phenotyping studies demonstrated expression of these antigens in a population of dogs. The virus-ligand interaction was further characterized using blockade studies, cell lines expressing HBGAs, and enzymatic removal of candidate carbohydrates from tissue sections. Recognition of HBGAs by CNV provides new insights into the evolution of noroviruses and raises concerns regarding the potential for zoonotic transmission of CNV to humans.IMPORTANCE Infections with human norovirus cause acute gastroenteritis in millions of people each year worldwide. Noroviruses can also affect nonhuman species and are divided into 6 different groups based on their capsid sequences. Human noroviruses in genogroups I and II interact with histo-blood group antigen carbohydrates, bovine noroviruses (genogroup III) interact with alpha-galactosidase (α-Gal) carbohydrates, and murine norovirus (genogroup V) recognizes sialic acids. The canine-specific strains of norovirus are grouped into genogroups IV and VI, and this study is the first to characterize which carbohydrate structures they can recognize. Using canine norovirus virus-like particles, this work shows that representative genogroup IV and VI viruses can interact with histo-blood group antigens. The binding specificity of canine noroviruses is therefore very similar to that of the human norovirus strains classified into genogroups I and II. This raises interesting questions about the evolution of noroviruses and suggests it may be possible for canine norovirus to infect humans.

English

Our previous studies showed that living and heat-killed cells of the Rhizobium leguminosarum strain P.SOM induce in pea roots systemic resistance to infection by Orobanche crenata. To better understand the mechanisms of induced resistance, we focused on identifying the inducing agent. Since heat stable bacterial surface carbohydrates such as exopolysaccharides (EPS) and lipopolysaccharides (LPS) are essential for recognition in the symbiotic interaction between Rhizobium and legumes, their role in the R. leguminosarum-pea interaction was studied. EPS and LPS were extracted from bacterial cultures, applied to pea roots, and tested for activity as an inducer of plant resistance to broomrape. Whereas EPS did not affect O. crenata infection, LPS reduced Orobanche infection significantly in concentrations as low as 1 and 0.5 mg ml-1. In contrast these compounds did not influence parasite germination induced by the artificial stimulant, GR24. Our results show that soil treatments with LPS resulted in a highly significant reduction of O. crenata infection of pea roots. The results clearly showed that LPS of R. leguminosarum P.SOM act as the inducing agent of systemic resistance in pea roots against Orobanche. Key words: Rhizobium leguminosarum, Lipopolysaccharides, Exopolysaccharides, Induced resistance, Orobanche crenata, Pea.

Techniques and applications: Self-like antigens under fire.

This study describes the use of a new microbial glycan microarray to characterize how host immune molecules bind to bacterial cell surface carbohydrates.

Hemolytic Lectin CEL-III Heptamerizes via a Large Structural Transition from α-Helices to a β-Barrel during the Transmembrane Pore Formation Process

CEL-III is a hemolytic lectin isolated from the sea cucumber Cucumaria echinata. This lectin is composed of two carbohydrate-binding domains (domains 1 and 2) and one oligomerization domain (domain 3). After binding to the cell surface carbohydrate chains through domains 1 and 2, domain 3 self-associates to form transmembrane pores, leading to cell lysis or death, which resembles other pore-forming toxins of diverse organisms. To elucidate the pore formation mechanism of CEL-III, the crystal structure of the CEL-III oligomer was determined. The CEL-III oligomer has a heptameric structure with a long β-barrel as a transmembrane pore. This β-barrel is composed of 14 β-strands resulting from a large structural transition of α-helices accommodated in the interface between domains 1 and 2 and domain 3 in the monomeric structure, suggesting that the dissociation of these α-helices triggered their structural transition into a β-barrel. After heptamerization, domains 1 and 2 form a flat ring, in which all carbohydrate-binding sites remain bound to cell surface carbohydrate chains, stabilizing the transmembrane β-barrel in a position perpendicular to the plane of the lipid bilayer.

A gene cluster for the synthesis of serotype g-specific polysaccharide antigen in Aggregatibacter actinomycetemcomitans

Aggregatibacter actinomycetemcomitans is an important pathogen related to aggressively progressive periodontal breakdown in adolescents and adults. The species can be divided into six serotypes (a-f) according to their surface carbohydrate antigens. Recently, a new serotype g of A. actinomycetemcomitans was proposed. The aim of the present study was to sequence the gene cluster associated with the biosynthesis of the serotype g-specific polysaccharide antigen and develop serotype-specific primers for PCR assay to identify serotype g strains of A. actinomycetemcomitans. The serotype-specific polysaccharide (SSPS) gene cluster of the NUM-Aa 4039 strain contained 21 genes in 21,842-bp nucleotides. The similarity of the SSPS gene cluster sequence was 96.7% compared with that of the serotype e strain. Seventeen serotype g genes showed more than 90% homology both in nucleotide and amino acids to the serotype e strain. Three additional genes with 1,579bp in NUM-Aa 4039 were inserted into the corresponding ORF13 of the serotype e strain. The serotype g-specific primers were designed from the insertion region of NUM-Aa 4039. Serotypes of the a-f strains were not amplified by serotype-specific g primers; only NUM-Aa 4039 showed an amplicon band. The NUM-Aa 4039 strain was three genes in the SSPS gene cluster different from those of serotype e strain. The specific primers derived from these different regions are useful for identification and distribution of serotype g strain among A. actinomycetemcomitans from clinical samples.

Campylobacter jejuni strain discrimination and temperature-dependent glycome expression profiling by lectin microarray

Gram-negative Campylobacter jejuni is the leading cause of bacterial gastroenteritis in humans worldwide and the most frequently identified infectious trigger in patients developing Guillain–Barré syndrome (GBS). While C. jejuni is pathogenic in humans, it is a commensal in avian hosts. Bacterial cell surface carbohydrates are important virulence factors and play roles in adherence, colonisation and infection. The mechanisms leading to infection or persistent colonisation of C. jejuni are not well understood but host temperature may provide an important stimulus for specific adaptation. Thus, examination of the modulation of the total surface glycome of C. jejuni in response to temperature may help shed light on commensal and pathogenic mechanisms for this species. C. jejuni strains 81116 and 81-176 were cultured at 37 and 42°C to simulate human and avian host conditions, respectively, and whole cells were profiled on lectin microarrays constructed to include a wide range of binding specificities. C. jejuni 81116 profiles indicated that the previously characterised lipopolysaccharide (LPS)-like molecule and N-linked glycans were the predominantly recognised cell surface structures while capsular polysaccharide (CPS), lipooligosaccharides (LOS) and N-linked glycosylation were best recognised for strain 81-176 at 37°C. The profiles of both strains varied and were distinguishable at both temperatures. At the higher temperature, reduced dominance of the LPS-like structure was associated with strain 81116 and a change in the relative distribution of CPS and LOS structures was indicated for strain 81-176. This change in LOS molecular mass species distribution between temperatures was confirmed by SDS–PAGE analysis. Additionally, opposite behaviour of certain lectins was noted between the plate agglutination assay and the microarray platform. Insights into the important glycosylation involved in C. jejuni host cell tropism at different growth temperatures were gained using the lectin microarray platform.

Lectin-microarray technique for glycomic profiling of fungal cell surfaces.

Lectin microarrays are rows of lectins with different carbohydrate-binding specificities spotted on surfaces of glass slides. Lectin microarray technique enables glycomic analyses of carbohydrate composition of fungal cell walls. We will describe an application of the technique in analyzing cell surface glycome of yeast-form fungal cells in the living state. The analysis reveals genus- and species-dependent complex cell surface carbohydrate structures of fungi, and enabled us, therefore, to suggest that cell walls of yeast cells, which have been considered to have relatively simple structures, actually have a more complex structure containing galactose and fucose. This shows that the technique can be used to find new insights into the study of phylogenetic relations and into the classification of cells in the fungal kingdom based on cell wall glycome.