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Expansins are small proteins that play a key role in modifying the structure of the cell wall during various physiological processes, in particular cell growth by elongation. Expansins are encoded by a large multigene family and are divided into four subfamilies, the main ones being alpha- and beta-expansins; the latter have received special development in cereals. Expansins are thought to modify the interactions of cellulose with xyloglucan (alpha-expansins) or with arabinoxylan (beta-expansins). At the same time, expansins do not have catalytic activity, the specific mechanism of their action is unclear, same as the physiological significance of such a wide variety of isoforms. To study the expression patterns of individual expansins, we conducted a transcriptomic analysis of all expansin genes identified in the maize genome using a convenient model system—zones of the maize growing primary root that differ in the stage of cell development and the composition of their cell walls. Of the 91 maize expansin genes, 67 were expressed in the root, and most expansin genes were characterized by a narrow range of zones with maximum transcript levels. Using a glycoarray containing 183 polysaccharides from the cell walls of plants of various species, we demonstrated that recombinant expansins AtEXPA1 and AtEXPB1 are able to bind to arabinogalactans and rhamnogalacturonans I and a number of other cell wall polysaccharides, which expands the list of their potential carbohydrate targets. Differences in the specificity of the interaction of alpha- and beta-expansins with various polysaccharides, both quantitatively and qualitatively, have been demonstrated. It has been hypothesized that the abundance of expansins in one plant organism and the fine regulation of their expression can be explained, at least in part, by the specific binding of individual expansins to specific cell wall polysaccharides. [ABSTRACT FROM AUTHOR]

The specificity of the most plant carbohydrate-binding proteins (CBP), many of which are known only through bioinformatic analysis of the genome, has either not been studied at all or characterized to a limited extent. The task of deciphering the carbohydrate specificity of the proteins can be solved using glycoarrays composed of many tens or even hundreds of glycans immobilized on a glass surface. Plant carbohydrates are the most significant natural ligands for plant proteins; this work shows that plant polysaccharides without additional modification can be immobilized on the surface, bearing N-hydroxysuccinimide activated carboxyl groups. As a result, an array of 113 well-characterized polysaccharides isolated from various plant cell walls, 23 mono- and oligosaccharides – components of polysaccharides, and glycans – ligands for widely known plant lectins was designed. Upon chemical immobilization of polysaccharides, their functional activity was preserved, which was confirmed by the results of interaction with antibodies and the plant lectin ricin. Using the constructed array, a previously unknown ability of ricin to bind polysaccharides was found, which significantly expands the knowledge of its specificity, and it was also found that a large variety of antibodies to plant polysaccharides are present in human peripheral blood. [ABSTRACT FROM AUTHOR]

Monoclonal antibody EBCA-1 is used in the sandwich immune assay for the detection of circulating Candida mannan in blood sera samples for the diagnosis of invasive candidiasis. To reinvestigate carbohydrate specificity of EBCA-1, a panel of biotinylated oligosaccharides structurally related to distinct fragments of Candida mannan were loaded onto a streptavidin-coated plate to form a glycoarray. Its use demonstrated that EBCA-1 recognizes the trisaccharide β-Man-(1→2)-α-Man- (1→2)-α-Man and not homo-α-(1→2)-linked pentamannoside, as was reported previously. [ABSTRACT FROM AUTHOR]

Klebsiella pneumoniae Is a Gram‐negative pathogenic bacterium that emerges a challenge for modern medicine due to intensively rising multi‐drug resistance. Specific LPS O‐chains represent promising targets for development of vaccines for immunoprophylaxis or immunotherapy of K. pneumoniae‐caused diseases. Herein, the synthesis of di‐, tetra‐, and hexasaccharides which represent structural fragments of both galactan I and galactan II of K. pneumoniae LPS O‐chains is described. The galactofuranoside building block used for assembling the galactan I chains was prepared by pyranoside‐into‐furanoside (PIF) rearrangement of the corresponding galactopyranoside precursor bearing an acid‐labile temporary p‐methoxybenzyl protecting group at O(3). The synthesized set of K. pneumoniae antigenic oligosaccharides in the form of their biotinylated derivatives was used to create a glycoarray which was applied for the screening of antibodies to galactan I and galactan II in anti‐K. pneumoniae sera. The obtained results demonstrate the applicability of such a glycoarray‐based assay for typing antibodies to Klebsiella strains belonging to most frequent O1 serogroup. [ABSTRACT FROM AUTHOR]

Toxoplasma gondii rhoptry neck protein 4 (TgRON4) is a component of the moving junction, a key structure for host cell invasion. We previously showed that host cellular β-tubulin is a binding partner of TgRON4 in the invasion process. Here, to identify other binding partners of TgRON4 in the host cell, we examined the binding of TgRON4 to components of the host cell surface. TgRON4 binds to various mammalian cells, but this binding disappeared in glycosaminoglycan- and heparan sulfate-deficient CHO cells and after heparitinase treatment of mammalian cells. The C-terminal half of TgRON4 showed relatively strong binding to cells and heparin agarose. A glycoarray assay indicated that TgRON4 binds to heparin and modified heparin derivatives. Immunoprecipitation of T. gondii -infected CHO cell lysates showed that TgRON4 interacts with glypican 1 during Toxoplasma invasion. This interaction suggests a role for heparan sulfate in parasite invasion. [ABSTRACT FROM AUTHOR]

Abstract: Due to the ability of Pseudomonas aeruginosa (PA) to develop antibiotic resistances, alternative therapeutic strategies have been proposed. Among others, carbohydrate multivalent molecules targeting lectin‐based virulent factors have been widely reported in particular those targeting LecA. LecA is a tetravalent galactose specific lectin involved in biofilm formation and cell internalization. Herein, we report the synthesis of 36 galactoclusters built from galactosides with aromatic and non‐aromatic aglycons and with an additional chain. The chains were either neutral or positively charged. Only the galactoclusters with naphthyl or tyrosine aglycon showed a moderate increase of binding for the positively charged 3‐dimethylammonium propyl chain. In contrast, the non‐aromatic galactoclusters display typically poorer binding properties towards LecA. The introduction of these side chains led to improved affinities up to becoming comparable to the high‐affinity aromatic galactoclusters. [ABSTRACT FROM AUTHOR]

We performed a serological investigation using glycoarray in Guillain-Barré syndrome (GBS), chronic inflammatory demyelinating polyradiculoneuropathy (CIDP), and multifocal motor neuropathy (MMN). Antibodies to 10 glycolipids and 45 glycolipid complexes were tested. Anti-GM1/sulfatide and anti-GA1/sulfatide IgG antibodies were common in GBS (20.0% and 19.0%, respectively). Anti-GQ1b/sulfatide IgG antibody was detected in 14.0% of GBS patients. IgG antibodies to antigens containing GQ1b were significantly correlated with ophthalmoplegia in GBS (p < 0.01). IgM antibodies to antigens containing GM1 or GalNAc-GD1a were in 50% and 37.5% of MMN patients, respectively. Glycoarray is efficient for detecting antibodies against numerous glycolipid complexes in immune-mediated neuropathies. [ABSTRACT FROM AUTHOR]

Abstract: Nowadays, there is a great interest for understanding the structure/function relationship governing recognition of carbohydrates by their receptors for the design of new treatments. Indeed, carbohydrates and glycoconjugates play a major role in key biological events such as cell–cell recognition, pathogenesis inflammation, and host pathogen interactions. Pseudomonas aeruginosa (PA) is one of the predominant bacterium encountered in nosocomial infections. PA infections often lead to chronic inflammation and eventually to death despite aggressive antibiotic therapy: the emergence of resistant strains and biofilm formation seems to give a selective advantage to the bacterium. A promising approach is to inhibit the virulence factors of PA such as PA-IL which is a galactose specific lectin. Herein, we develop a microarray to probe the binding of six galacto-conjugates to PA-IL differing by their spatial configuration and geometry. This microsystem is made of 40 independent microwells in which 64 spots of glycoconjugates probes are arrayed by using DNA Directed Immobilization (DDI). This microsystem allows, in a multiplex fashion, qualitative information on the binding by direct fluorescence readout as well as quantitative information by the determination of IC50 values in a competition assay and surface dissociation constants (K d). According to our data, direct fluorescent signals (FI635), IC50 and K d values provided similar ranking for glycoconjugates with respect to PA-IL binding thus affording a general tool for the selection of galacto-conjugates displaying the best affinities toward PA-IL. [Copyright &y& Elsevier]

A microarray enables high-throughput interaction screening of numerous biomolecules; however, fabrication of a microarray composed of cellular membrane components has proven difficult. We report fabrication of a liposomal glyco-microarray by using an azide-reactive liposome that carries synthetic and natural glycolipids via chemically selective and biocompatible liposome immobilization chemistry. Briefly, liposomes carrying anchor lipid dipalmitoylphosphatidylethanolamine (DPPE)-PEG-triphenylphosphine and ganglioside (GM1 or GM3) were prepared first and were then printed onto an azide-modified glass slide so as to afford a liposomal glyco-microarray via Staudinger ligation. Fluorescent dye release kinetics and fluorescence imaging confirmed successful liposome immobilization and specific protein binding to the intact arrayed glycoliposomes. The liposomal glyco-microarray with different gangliosides showed their specific lectin and toxin binding with different binding affinity. The azide-reactive liposome provides a facile strategy for fabrication of either a natural or a synthetic glycolipid-based membrane-mimetic glycoarray. This liposomal glyco-microarray is simple and broadly applicable and thus will find important biomedical applications, such as studying glycolipid-protein interactions and toxin screening applications. [Figure not available: see fulltext.] [ABSTRACT FROM AUTHOR]

Porphyromonas gingivalis is a major pathogen in adult periodontitis. Fimbriae play an important role in the initial interaction between the bacteria and the host. Our earlier studies suggested that the oligosaccharide moiety of lactoferrin is involved in the interaction with fimbriae. Porphyromonas gingivalis fimbriae bound strongly to albumin-fucosylamide (albumin-1-amido-1-deoxy- l-fucose) and to a lesser extent to albumin- N-acetyl- d-galactosamine (albumin-p-aminophenyl- N-acetyl-β- d-galactosaminide, but failed to bind bovine serum albumin. In this study we explored the glycan array to determine the carbohydrate-binding specificity of P. gingivalis fimbriae. Purified fimbriae bind to glycans which have a Lewisx, Galβ1-4(Fucα1-3) GlcNAcβ structure in common. Interestingly, all glycans have a terminal fucose moiety and if fucose is removed, the fimbriae fail to bind. This is the first study that suggests that fucose is important for P. gingivalis fimbriae binding. [ABSTRACT FROM AUTHOR]

Abstract: A biotinylated mannotriose (Man3-bio) was dispersively immobilized in the matrix of biotinylated lactose (Gal-Glc-bio) on a streptavidin-covered, 27-MHz quartz crystal microbalance (QCM), and binding kinetics of concanavalin A (Con A) to Man3-bio in the Gal-Glc-bio matrix could be obtained from frequency decreases (mass increases) of the QCM. Association constants (K a) and binding and dissociation rate constants (k on and k off) could be determined separately as the 1:1 and 1:2 bindings of Con A to Man3-bio on the surface. When Man3-bio was immobilized with content of 1 to 5 mol% in the matrix, the 1:1 binding of Con A to Man3-bio was obtained as K a =(4±1)×106 M−1, k on =(4±1)×104 M−1 s−1, and k off =(12±2)×10–3 s−1. On the contrary, when Man3-bio was immobilized with content of 20 to 100 mol% in the matrix, the 1:2 binding of Con A to Man3-bio was obtained as K a =(14±2)×106 M−1, k on =(14±2)×104 M−1 s−1, and k off =(7±2)×10–3 s−1. Thus, K a for the 1:2 binding was 10 times larger than that for the 1:1 binding, with a three times larger binding rate constant (k on) and a three times smaller dissociation rate constant (k off). This is the first example to obtain separate kinetic parameters for the 1:1 and 1:2 bindings of lectins to carbohydrates on the surface. [Copyright &y& Elsevier]

Abstract: Glycoarrays are powerful tools for the understanding of protein/carbohydrate interactions and should find applications in the diagnosis of diseases involving these interactions. Immobilisation of the carbohydrate probe is a key issue in the elaboration of high performance devices. In the present study, we have compared the fluorescent signal intensity and determined the lower detection limit of glycoconjugates immobilised at two concentrations (0.5 and 25μM) by DNA-directed immobilisation (DDI), to glycoconjugates covalently immobilised on the solid support (borosilicate glass slide). At 0.5μM, DDI led to a stronger fluorescence signal (by a factor of 4.5) and to a lower detection limit (20nM) than covalent immobilisation (higher than 200nM). We also report the development of an IC50 measurement assay of DDI immobilised glycoconjugates. We found that the relative affinity per galactose residue of RCA 120 for glycoconjugates bearing one or three galactose residues was different by a factor of 23 when measured under IC50 conditions or by direct fluorescence reading. [Copyright &y& Elsevier]

Influenza A virus specificity for the host is mediated by the viral surface glycoprotein hemagglutinin (HA), which binds to receptors containing glycans with terminal sialic acids. Avian viruses preferentially bind to α2-3-linked sialic acids on receptors of intestinal epithelial cells, whereas human viruses are specific for the α2-6 linkage on epithelial cells of the lungs and upper respiratory tract. To define the receptor preferences of a number of human and avian H1 and H3 viruses, including the 1918 H1N1 pandemic strains, their hemagglutinins were analyzed using a recently described glycan array. The array, which contains 200 carbohydrates and glycoproteins, not only revealed clear differentiation of receptor preferences for α2-3 and/or α2-6 sialic acid linkage, but could also detect fine differences in HA specificity, such as preferences for fucosylation, sulfation and sialylation at positions 2 (Gal) and 3 (GlcNAc, GalNAc) of the terminal trisaccharide. For the two 1918 HA variants, the South Carolina (SC) HA (with Asp190, Asp225) bound exclusively α2-6 receptors, while the New York (NY) variant, which differed only by one residue (Gly225), had mixed α2-6/α2-3 specificity, especially for sulfated oligosaccharides. Only one mutation of the NY variant (Asp190Glu) was sufficient to revert the HA receptor preference to that of classical avian strains. Thus, the species barrier, as defined by the receptor specificity preferences of 1918 human viruses compared to likely avian virus progenitors, can be circumvented by changes at only two positions in the HA receptor binding site. The glycan array thus provides highly detailed profiles of influenza receptor specificity that can be used to map the evolution of new human pathogenic strains, such as the H5N1 avian influenza. [Copyright &y& Elsevier]

Understanding the specificity of cell-surface carbohydrates interaction with antibodies and receptors is important for the development of new therapeutics and high-sensitivity diagnostics. This approach is, however, limited to the availability of natural and truncated sequences of the oligosaccharides and the sensitivity of the assay system. Reported here is the synthesis of the cancer antigen Globo H hexasaccharide, an epitope found on the cell surface of breast, prostate, and ovarian cancers, and its truncated sequences by using the programmable one-pot synthesis strategy. The saccharides were then arrayed covalently on glass slides with different density and used for the fluorencense-based binding analysis of two monoclonal antibodies against Globo H and the serum from breast cancer patients, to define the specificity of these antibodies. It was shown that the terminal tetrasaccharide binds the monoclonal antibodies equally well as does the hexasaccharide and the fucose residue is required for effective binding. The serum binds both the defucosylated pentasaccharide and the fucosylated hexasaccharide without a significant difference, perhaps because of the polyclonal nature of the serum or the presence of diverse immune responses to different sugar epitopes at various stages. This method requires very small amounts of materials and is more effective and sensitive than the traditional ELISA method, and thus provides another platform to monitor the immune response to carbohydrate epitopes at different stages during differentiation, metastasis, or treatment. [ABSTRACT FROM AUTHOR]

Abstract: A sensitive, specific, and rapid method for the detection of carbohydrate–protein interactions was demonstrated using quantum dots (QDs) as a fluorescence label coupled with protein. 1,3-Dipolar cycloaddition between azide and alkyne was exploited to attach α-d-glucopyranoside to a C14 hydrocarbon chain that noncovalently binds to the microtiter well surface, and the product formation was detected by both electrospray ionization–mass spectrometry (ESI–MS) and QD- (or fluorescein isothiocyanate (FITC))-conjugated lectin binding. It indicated that the peak intensity of the fluorescence emission was proportional to the initial concanavalin A (Con A) concentration in the range of 2×10−3μmol/L to 2×10−2mmol/L with a detection limit at least 100 times lower than that of the FITC-based method. [Copyright &y& Elsevier]

A noncovalent carbohydrate microarray was used to screen for possible inhibitors to fucosyltransferases, which are critical to the synthesis of inflammation mediators like sialyl Lewis x (SLex). Inhibition was followed by observation of the transferred fucose on the carbohydrate array with the lectin Tetragonolobus purpureas. Of these compounds, four inhibitors with nanomolar Kis were discovered, with three of the top five inhibitors exhibiting a common architecture. [Copyright &y& Elsevier]

Abstract: The work presented herein is a new noncovalent glycoarray assembly method for microplates created by simply mixing together a carbohydrate and a tetradecylamine. α-Mannose was utilized in the model study and product formation was detected by lectin binding. The method can be further extended to array complex carbohydrates. [Copyright &y& Elsevier]