Fluorescence-Polarization-Based Assaying of Lysozyme with Chitooligosaccharide Tracers.

Lysozyme is a well-known enzyme found in many biological fluids which plays an important role in the antibacterial protection of humans and animals. Lysozyme assays are used for the diagnosis of a number of diseases and utilized in immunohistochemistry, genetic and cellular engineering studies. The assaying methods are divided into two categories measuring either the concentration of lysozyme as a protein or its activity as an enzyme. While the first category of methods traditionally uses an enzyme-linked immunosorbent assay (ELISA), the methods for the determination of the enzymatic activity of lysozyme use either live bacteria, which is rather inconvenient, or natural peptidoglycans of high heterogeneity and variability, which leads to the low reproducibility of the assay results. In this work, we propose the use of a chemically synthesized substrate of a strictly defined structure to measure in a single experiment both the concentration of lysozyme as a protein and its enzymatic activity by means of the fluorescence polarization (FP) method. Chito-oligosaccharides of different chain lengths were fluorescently labeled and tested leading to the selection of the pentasaccharide as the optimal size tracer and the further optimization of the assay conditions for the accurate (detection limit 0.3 µM) and rapid (<30 min) determination of human lysozyme. The proposed protocol was applied to assay human lysozyme in tear samples and resulted in good correlation with the reference assay. The use of synthetic fluorescently labeled tracer, in contrast to natural peptidoglycan, in FP analysis allows for the development of a reproducible method for the determination of lysozyme activity.

ASCA-related antibodies in the blood sera of healthy donors and patients with colorectal cancer: characterization with oligosaccharides related to Saccharomyces cerevisiae mannan.

Mannans are polysaccharide antigens expressed on the cell wall of different fungal species including Saccharomyces cerevisiae and Candida spp. These fungi are components of the normal intestinal microflora, and the presence of antibodies to fungal antigens is known to reflect the features of the patient's immune system. Thus, titers of IgG and IgA antibodies against Saccharomyces cerevisiae mannan (ASCA) are markers for clinical diagnostics of inflammatory bowel diseases. The complex organization and heterogeneity of cell-wall mannans may reduce the quality and reproducibility of ELISA results due to interference by different antigenic epitopes. In this research, we analyzed the levels of IgG antibodies in the sera of healthy donors and patients with colorectal cancer using an array of synthetic oligosaccharides related to distinct fragments of fungal mannan. This study aimed to establish the influence of oligosaccharide structure on their antigenicity. Variations in the structure of the previously established ASCA epitope (changing type of linkage, chain length, and the presence of branches) significantly modified the ability of ligands to bind to circulating antibodies in blood sera. The study showed that surface presentation density of the ligand critically affects the results of enzyme immunoassay. The transition from natural coating antigens to their corresponding synthetic mimetics with a defined structure opens new opportunities for improving existing ELISA test systems, as well as developing diagnostic kits with new properties.

Stereocontrolled Synthesis and Conformational Analysis of a Series of Disaccharides α,ß-d-GlcA-(1→3)-α-L-Fuc.

D-Glucuronic acid is a fundamental building block of many biologically important polysaccharides, either in its non-substituted form or bearing a variety of substituents, among them sulfates. We have previously performed a study of the effects of exhaustive sulfation on the conformational behavior of ß-gluronopyranosides. Herein, we report an investigation comparing α- and ß-derivatives of this monosaccharide within the title disaccharides using NMR and quantum chemistry approaches. It was found that for α-linked disaccharides, the introduction of sulfates did not greatly affect their conformational behavior. However, for ß-derivatives, considerable conformational changes were observed. In general, they resemble those that took place for the monosaccharides, except that NOESY experiments and calculations of intra-ring spin-spin coupling constants suggest the presence of a 1S5 conformer along with 3S1 in the fully sulfated disaccharide. During the synthesis of model compounds, hydrogen bond-mediated aglycone delivery was used as an α-directing stereocontrol approach in the glucuronidation reaction.

Synthesis of methylphosphorylated oligomannosides structurally related to lipopolysaccharide O-antigens of Klebsiella pneumoniae serotype O3 and their application for detection of specific antibodies in rabbit and human sera.

Methylphosphorylated mono-, di- and trimannosides structurally related to the lipopolysaccharide (LPS) O-antigens of Klebsiella pneumoniae of serotype O3 were synthesized and conjugated with a biotin tag. The stereo- and regioselective assembly of target carbohydrate chains was conducted using uniform monosaccharide synthetic blocks. After that, a methylphosphate group was introduced by coupling with a methyl-H-phosphonate reagent followed by oxidation and deprotection to give the target oligosaccharides. The 1H and 13C NMR spectra of the obtained compounds showed a good fit with the spectrum of the corresponding natural polysaccharide. The newly prepared biotinylated oligosaccharides along with the previously reported biotinylated glycoconjugates related to galactan I and galactan II of K. pneumoniae LPS were used for the ELISA detection of antibodies in anti-K. pneumoniae rabbit sera. Anti-O3 serum antibodies specifically recognized the synthesized oligosaccharide ligands with terminal methylphosphomannosyl residues, whereas anti-O1 serum antibodies recognized the oligosaccharide related to K. pneumoniae galactan II. The analysis of human sera from patients with confirmed Klebsiella infection also revealed the presence of antibodies against the synthesized oligosaccharides in clinical cases. Thus, the described compounds together with other Klebsiella related antigenic oligosaccharides could be potentially used as molecular probes for K. pneumoniae serological diagnostics development and strain serotyping.

Identification of a new DC-SIGN binding pentamannoside epitope within the complex structure of Candida albicans mannan.

The dendritic cell-specific intercellular adhesion molecule-3-grabbing non-integrin (DC-SIGN) is an innate immune C-type lectin receptor that recognizes carbohydrate-based pathogen associated with molecular patterns of various bacteria, fungi, viruses and protozoa. Although a range of highly mannosylated glycoproteins have been shown to induce signaling via DC-SIGN, precise structure of the recognized oligosaccharide epitope is still unclear. Using the array of oligosaccharides related to selected fragments of main fungal antigenic polysaccharides we revealed a highly specific pentamannoside ligand of DC-SIGN, consisting of α-(1 â†’ 2)-linked mannose chains with one inner α-(1 â†’ 3)-linked unit. This structural motif is present in Candida albicans cell wall mannan and corresponds to its antigenic factors 4 and 13b. This epitope is not ubiquitous in other yeast species and may account for the species-specific nature of fungal recognition via DC-SIGN. The discovered highly specific oligosaccharide ligands of DC-SIGN are tractable tools for interdisciplinary investigations of mechanisms of fungal innate immunity and anti-Candida defense. Ligand- and receptor-based NMR data demonstrated the pentasaccharide-to-DC-SIGN interaction in solution and enabled the deciphering of the interaction topology.

Biorecognition Layer Based On Biotin-Containing [1]Benzothieno[3,2-b][1]benzothiophene Derivative for Biosensing by Electrolyte-Gated Organic Field-Effect Transistors.

Requirements of speed and simplicity in testing stimulate the development of modern biosensors. Electrolyte-gated organic field-effect transistors (EGOFETs) are a promising platform for ultrasensitive, fast, and reliable detection of biological molecules for low-cost, point-of-care bioelectronic sensing. Biosensitivity of the EGOFET devices can be achieved by modification with receptors of one of the electronic active interfaces of the transistor gate or organic semiconductor surface. Functionalization of the latter gives the advantage in the creation of a planar architecture and compact devices for lab-on-chip design. Herein, we propose a universal, fast, and simple technique based on doctor blading and Langmuir-Schaefer methods for functionalization of the semiconducting surface of C8-BTBT-C8, allowing the fabrication of a large-scale biorecognition layer based on the novel functional derivative of BTBT-containing biotin fragments as a foundation for further biomodification. The fabricated devices are very efficient and operate stably in phosphate-buffered saline solution with high reproducibility of electrical properties in the EGOFET regime. The development of biorecognition properties of the proposed biolayer is based on the streptavidin-biotin interactions between the consecutive layers and can be used for a wide variety of receptors. As a proof-of-concept, we demonstrate the specific response of the BTBT-based biorecognition layer in EGOFETs to influenza A virus (H7N1 strain). The elaborated approach to biorecognition layer formation is appropriate but not limited to aptamer-based receptor molecules and can be further applied for fabricating several biosensors for various analytes on one substrate and paves the way for "electronic tongue" creation.

Computational and NMR Conformational Analysis of Galactofuranoside Cycles Presented in Bacterial and Fungal Polysaccharide Antigens.

Unlike pyranoside cycles which are generally characterized by strictly defined conformational preferences, furanosides are flexible and may adopt a wide range of available conformations. During our previous studies, conformational changes of galactofuranoside cycles upon total sulfation were described computationally, using a simple Hartree-Fock (HF) method, and principal conformers of the 5-membered galactose ring were revealed. However, in the case of more complex disaccharide structures, it was found that this method and the widely applied DFT-B3LYP produced results that deviated from experimental evidence. In this study, other DFT functionals (PBE0 and double hybrid B2PLYP) along with RI-MP2 are employed to study the conformational behavior of the galactofuranoside ring. Reinvestigation of galactofuranosides with a lactic acid substituent at O-3 revealed that changes in the orientation of lactic acid residue at O-3 might induce conformational changes of the furanoside cycle. Such findings are important for further modeling of carbohydrate-protein interaction.

Reinvestigation of Carbohydrate Specificity of EBCA-1 Monoclonal Antibody Used for the Detection of Candida Mannan.

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 ß-Man-(1→2)-α-Man-(1→2)-α-Man and not homo-α-(1→2)-linked pentamannoside, as was reported previously.

Synthesis and conformational analysis of vicinally branched trisaccharide ß-d-Galf-(1 → 2)-[ß-d-Galf-(1 → 3)-]-α-Galp from Cryptococcus neoformans galactoxylomannan.

The synthesis of a vicinally branched trisaccharide composed of two d-galactofuranoside residues attached viaß-(1 → 2)- and ß-(1 → 3)-linkages to the α-d-galactopyranoside unit has been performed for the first time. The reported trisaccharide represents the galactoxylomannan moiety first described in 2017, which is the capsular polysaccharide of the opportunistic fungal pathogen Cryptococcus neoformans responsible for life-threatening infections in immunocompromised patients. The NMR-data reported here for the synthetic model trisaccharide are in good agreement with the previously assessed structure of galactoxylomannan and are useful for structural analysis of related polysaccharides. The target trisaccharide as well as the constituent disaccharides were analyzed by a combination of computational and NMR methods to demonstrate good convergence of the theoretical and experimental results. The results suggest that the furanoside ring conformation may strongly depend on the aglycon structure. The reported conformational tendencies are important for further analysis of carbohydrate-protein interaction, which is critical for the host response toward C. neoformans infection.

Potential of Chemically Synthesized Oligosaccharides To Define the Carbohydrate Moieties of the Fungal Cell Wall Responsible for the Human Immune Response, Using Aspergillus fumigatus Galactomannan as a Model.

Methodologies to identify epitopes or ligands of the fungal cell wall polysaccharides influencing the immune response of human pathogens have to date been imperfect. Using the galactomannan (GM) of Aspergillus fumigatus as a model, we have shown that synthetic oligosaccharides of distinct structures representing key fragments of cell wall polysaccharides are the most precise tools to study the serological and immunomodulatory properties of a fungal polysaccharide.

Biotinylated Oligo-α-(1 → 4)-d-galactosamines and Their N-Acetylated Derivatives: α-Stereoselective Synthesis and Immunology Application.

Using 3-O-benzoyl-4,6-O-di-tert-butylsilylidene-2-azido-2-deoxy-selenogalactoside, biotinylated oligo-α-(1 → 4)-d-galactosamines comprising from two to six GalN units were prepared for the first time together with their N-acetylated derivatives. The combination of blocking groups used herein provided stereocontrol for the α-stereospecific glycosylation, to show also high efficiency of phenyl 2-azido-2-deoxy-selenogalactosides as glycosyl donors. The obtained glycoconjugates are related to fragments of exopolysaccharide galactosaminogalactan (GG) found in Aspergillus fumigatus, which is the most important airborne human fungal pathogen in industrialized countries. The synthesized glycoconjugates were arrayed on streptavidin-coated plates and used to investigate the GG epitopes recognized by mouse monoclonal antibodies against GG and by human antibodies in the sera of patients with aspergillosis. The obtained data showed that the oligo-α-(1 → 4)-d-galactosamines and their N-acetylated derivatives allowed the first precise analysis of the specificity of the antibody responses to this extremely complex fungal polysaccharide.

Synthetic Oligosaccharides Mimicking Fungal Cell Wall Polysaccharides.

The cell wall of pathogenic fungi is highly important for the development of fungal infections and is the first cellular component to interact with the host immune system. The fungal cell wall is mainly built up of different polysaccharides representing ligands for pattern recognition receptors (PRRs) on immune cells and antibodies. Purified fungal polysaccharides are not easily available; in addition, they are structurally heterogenic and have wide molecular weight distribution that limits the possibility to use natural polysaccharides to assess the structure of their active determinants. The synthetic oligosaccharides of definite structure representing distinct polysaccharide fragments are indispensable tools for a variety of biological investigations and represent an advantageous alternative to natural polysaccharides. The attachment of a spacer group to these oligosaccharides permits their efficient transformation into immunogenic glycoconjugates as well as their immobilization on plates or microbeads. Herein, we summarize current information on synthetic availability of the variety of oligosaccharides related to main types of fungal cell wall components: galactomannan, α- and ß-mannan, α- and ß-(1 → 3)-glucan, chitin, chitosan, and others. These data are supplemented with published results of biochemical and immunological applications of synthetic oligosaccharides as molecular probes especially as the components of thematic glycoarrays suitable for characterization of anti-polysaccharide antibodies and cellular lectins or PRRs.

Importance of Candida Antigenic Factors: Structure-Driven Immunomodulation Properties of Synthetically Prepared Mannooligosaccharides in RAW264.7 Macrophages.

The incidence and prevalence of serious fungal infections is rising, especially in immunosuppressed individuals. Moreover, co-administration of antibiotics and immunosuppressants has driven the emergence of new multidrug-resistant pathogens. The significant increase of multidrug-resistant pathogens, together with their ability to form biofilms, is associated with morbidity and mortality. Research on novel synthetically prepared immunomodulators as potential antifungal immunotherapeutics is of serious interest. Our study demonstrated the immunobiological activity of synthetically prepared biotinylated mannooligosaccharides mimicking Candida antigenic factors using RAW264.7 macrophages. Macrophage exposure to the set of eight structurally different mannooligosaccharides induced a release of Th1, Th2, Th17, and Treg cytokine signature patterns. The observed immune responses were tightly associated with structure, dose, exposure time, and selected signature cytokines. The viability/cytotoxicity of the mannooligosaccharide formulas was assessed based on cell proliferation. The structure-based immunomodulatory activity of the formulas was evaluated with respect to the length, branching and conformation of the various formulas. Glycoconjugate formulas with terminal ß-mannosyl-units tended to be more potent in terms of Candida relevant cytokines IL-12 p70, IL-17, GM-CSF, IL-6, and TNFα induction and cell proliferation, and this tendency was associated with structural differences between the studied glycoconjugate formulas. The eight tested mannooligosaccharide conjugates can be considered potential in vitro immunomodulative agents suitable for in vitro Candida diagnostics or prospectively for subcellular anti-Candida vaccine design.

Driving Force of the Pyranoside-into-Furanoside Rearrangement.

Ab initio calculations of fully O-sulfated model monosaccharides, including common hexoses (glucose, galactose, fucose, and mannose) and pentoses (arabinose and xylose), were performed to study the energetic properties of the recently discovered pyranoside-into-furanoside (PIF) rearrangement. It was shown that the per-O-sulfated derivatives of furanoside isomers generally had lower energies than the corresponding per-O-sulfated pyranosides, while nonsulfated furanosides were always less favored than nonsulfated pyranosides. Mannose, which is known to be unreactive in PIF rearrangement, was the only exception. The results of the theoretical calculations were confirmed by experimental studies of monosaccharide models and explained the driving force of such unusual ring contraction process as PIF rearrangement. The conclusions of performed investigation can be used for prediction of new substrates applicability for PIF rearrangement.

Monoclonal Antibody AP3 Binds Galactomannan Antigens Displayed by the Pathogens Aspergillus flavus, A. fumigatus, and A. parasiticus.

Aspergillus fumigatus and A. flavus are the fungal pathogens responsible for most cases of invasive aspergillosis (IA). Early detection of the circulating antigen galactomannan (GM) in serum allows the prompt application of effective antifungal therapy, thus improving the survival rate of IA patients. However, the use of monoclonal antibodies (mAbs) for the diagnosis of IA is often associated with false positives due to cross-reaction with bacterial polysaccharides. More specific antibodies are therefore needed. Here we describe the characterization of the Aspergillus-specific mAb AP3 (IgG1κ), including the precise identification of its corresponding antigen. The antibody was generated using A. parasiticus cell wall fragments and was shown to bind several Aspergillus species. Immunofluorescence microscopy revealed that AP3 binds a cell wall antigen, but immunoprecipitation and enzyme-linked immunosorbent assays showed that the antigen is also secreted into the culture medium. The inability of AP3 to bind the A. fumigatus galactofuranose (Galf )-deficient mutant ΔglfA confirmed that Galf residues are part of the epitope. Several lines of evidence strongly indicated that AP3 recognizes the Galf residues of O-linked glycans on Aspergillus proteins. Glycoarray analysis revealed that AP3 recognizes oligo-[ß-D-Galf-1,5] sequences containing four or more residues with longer chains more efficiently. We also showed that AP3 captures GM in serum, suggesting it may be useful as a diagnostic tool for patients with IA.

Influence of per-O-sulfation upon the conformational behaviour of common furanosides.

The studies on the recently discovered pyranoside-into-furanoside rearrangement have led us to conformational investigations of furanosides upon their total sulfation. Experimental NMR data showed that in some cases drastic changes of the ring conformation occurred while sometimes only the conformation of the exocyclic C4-C5 linkage changed. Herein we describe a combined quantum chemical and NMR conformational investigation of three common monosaccharide furanosides as their propyl glycosides: α-mannose, ß-glucose and ß-galactose. Full exploration of the furanoside ring by means of ab initio calculations was performed and coupling constants were calculated for each of the low-energy conformers. The results demonstrated preferred trans-orientation of H4-H5 protons in the non-sulfated molecules which changed to gauche-orientation upon sulfation. The effect is less pronounced in the galactosides. For all the studied structures changes in the conformational distribution were revealed by quantum mechanical calculations, that explained the observed changes in intraring coupling constants occurring upon introduction of sulfates.

Novel mouse monoclonal antibodies specifically recognizing ß-(1→3)-D-glucan antigen.

ß-(1→3)-D-Glucan is an essential component of the fungal cell wall. Mouse monoclonal antibodies (mAbs) against synthetic nona-ß-(1→3)-D-glucoside conjugated with bovine serum albumin (BSA) were generated using hybridoma technology. The affinity constants of two selected mAbs, 3G11 and 5H5, measured by a surface plasmon resonance biosensor assay using biotinylated nona-ß-(1→3)-D-glucan as the ligand, were approximately 11 nM and 1.9 nM, respectively. The glycoarray, which included a series of synthetic oligosaccharide derivatives representing ß-glucans with different lengths of oligo-ß-(1→3)-D-glucoside chains, demonstrated that linear tri-, penta- and nonaglucoside, as well as a ß-(1→6)-branched octasaccharide, were recognized by mAb 5H5. By contrast, only linear oligo-ß-(1→3)-D-glucoside chains that were not shorter than pentaglucosides (but not the branched octaglucoside) were ligands for mAb 3G11. Immunolabelling indicated that 3G11 and 5H5 interact with both yeasts and filamentous fungi, including species from Aspergillus, Candida, Penicillium genera and Saccharomyces cerevisiae, but not bacteria. Both mAbs could inhibit the germination of Aspergillus fumigatus conidia during the initial hours and demonstrated synergy with the antifungal fluconazole in killing C. albicans in vitro. In addition, mAbs 3G11 and 5H5 demonstrated protective activity in in vivo experiments, suggesting that these ß-glucan-specific mAbs could be useful in combinatorial antifungal therapy.

Nanomedical Relevance of the Intermolecular Interaction Dynamics-Examples from Lysozymes and Insulins.

Insulin and lysozyme share the common features of being prone to aggregate and having biomedical importance. Encapsulating lysozyme and insulin in micellar nanoparticles probably would prevent aggregation and facilitate oral drug delivery. Despite the vivid structural knowledge of lysozyme and insulin, the environment-dependent oligomerization (dimer, trimer, and multimer) and associated structural dynamics remain elusive. The knowledge of the intra- and intermolecular interaction profiles has cardinal importance for the design of encapsulation protocols. We have employed various biophysical methods such as NMR spectroscopy, X-ray crystallography, Thioflavin T fluorescence, and atomic force microscopy in conjugation with molecular modeling to improve the understanding of interaction dynamics during homo-oligomerization of lysozyme (human and hen egg) and insulin (porcine, human, and glargine). The results obtained depict the atomistic intra- and intermolecular interaction details of the homo-oligomerization and confirm the propensity to form fibrils. Taken together, the data accumulated and knowledge gained will further facilitate nanoparticle design and production with insulin or lysozyme-related protein encapsulation.

Reinvestigation of carbohydrate specificity of EB-A2 monoclonal antibody used in the immune detection of Aspergillus fumigatus galactomannan.

Great progresses have been made in the recent years in the detection of circulating galactofuranose-bearing molecules for the diagnosis of aspergillosis. However, the test used in the clinical practice is hampered by the occurrence of false positives. A glycoarray with dozens of oligosaccharides structurally related to the Aspergillus fumigatus galactomannan has allowed us to reinvestigate the carbohydrate specificity of the EB-A2 monoclonal antibody used in the PlateliaTM Aspergillus sandwich immune assay. We have now demonstrated that the mAb can recognize shorter oligosaccharides than the previously reported tetrasaccharide Galf-ß-(1→5)-Galf-ß-(1→5)-Galf-ß-(1→5)-Galf-ß and oligosaccharides which contains alternating ß-(1→5)/ß-(1→6)-linkages. This result could explain the occurrence of false-positive signals due to the presence of the abovementioned epitopes not only in A. fumigatus galactomannan but also in other bacteria and fungi.