Galf-Specific Neolectins: Towards Promising Diagnostic Tools.

In the absence of naturally available galactofuranose-specific lectin, we report herein the bioengineering of GalfNeoLect, from the first cloned wild-type galactofuranosidase (Streptomyces sp. strain JHA19), which recognises and binds a single monosaccharide that is only related to nonmammalian species, usually pathogenic microorganisms. We kinetically characterised the GalfNeoLect to confirm attenuation of hydrolytic activity and used competitive inhibition assay, with close structural analogues of Galf, to show that it conserved interaction with its original substrate. We synthetised the bovine serum albumin-based neoglycoprotein (GalfNGP), carrying the multivalent Galf units, as a suitable ligand and high-avidity system for the recognition of GalfNeoLect which we successfully tested directly with the galactomannan spores of Aspergillus brasiliensis (ATCC 16404). Altogether, our results indicate that GalfNeoLect has the necessary versatility and plasticity to be used in both research and diagnostic lectin-based applications.

Expanding Glycomic Investigations through Thiol-Derivatized Glycans.

N-(2-thioethyl)-2-aminobenzamide (TEAB), a novel glycan auxiliary, was synthesized and its utility was evaluated. The auxiliary was conjugated to glycans by reductive amination with the water-stable reagent 2-picoline borane complex. Glycan products, which ranged from 1 to 7 linked hexoses, were all isolated in yields ranging from 60% to 90% after purification by reverse-phase chromatography. The novel conjugate introduces a convenient, shelf-stable thiol directly onto the desired free glycans with purification advantages and direct modification with efficient reactions through alkenes, halides, epoxides, disulfides, and carboxylates in yields of 49% to 93%. Subsequently, a thiol-selective modification of the BSA protein was used to generate a neoglycoprotein with a bifunctional PEG-maleimide linker. To further illustrate the utility of a thiol motif, 2-thiopyridine activation of a thiol-containing support facilitated the covalent chromatographic purification of labeled glycans in yields up to 63%. Finally, initial proof of concept of implementation in a light printed microarray was explored and validated through FITC-labeled concanavalin A binding. In conclusion, the thiol-functionalized glycans produced greatly expand the diversity of bioconjugation tools that can be developed with glycans and enable a variety of biological investigations.

A Branched and Double Alpha-Gal-Bearing Synthetic Neoglycoprotein as a Biomarker for Chagas Disease.

Chagas disease (CD) is caused by the parasite Trypanosoma cruzi and affects 6-7 million people worldwide. The diagnosis is still challenging, due to extensive parasite diversity encompassing seven genotypes (TcI-VI and Tcbat) with diverse ecoepidemiological, biological, and pathological traits. Chemotherapeutic intervention is usually effective but associated with severe adverse events. The development of safer, more effective therapies is hampered by the lack of biomarker(s) (BMKs) for the early assessment of therapeutic outcomes. The mammal-dwelling trypomastigote parasite stage expresses glycosylphosphatidylinositol-anchored mucins (tGPI-MUC), whose O-glycans are mostly branched with terminal, nonreducing α-galactopyranosyl (α-Gal) glycotopes. These are absent in humans, and thus highly immunogenic and inducers of specific CD anti-α-Gal antibodies. In search for α-Gal-based BMKs, here we describe the synthesis of neoglycoprotein NGP11b, comprised of a carrier protein decorated with the branched trisaccharide Galα(1,2)[Galα(1,6)]Galß. By chemiluminescent immunoassay using sera/plasma from chronic CD (CCD) patients from Venezuela and Mexico and healthy controls, NGP11b exhibited sensitivity and specificity similar to that of tGPI-MUC from genotype TcI, predominant in those countries. Preliminary evaluation of CCD patients subjected to chemotherapy showed a significant reduction in anti-α-Gal antibody reactivity to NGP11b. Our data indicated that NGP11b is a potential BMK for diagnosis and treatment assessment in CCD patients.

Synthesis of Galα(1,3)Galß(1,4)GlcNAcα-, Galß(1,4)GlcNAcα- and GlcNAc-containing neoglycoproteins and their immunological evaluation in the context of Chagas disease.

The protozoan parasite, Trypanosoma cruzi, the etiologic agent of Chagas disease (ChD), has a cell surface covered by immunogenic glycoconjugates. One of the immunodominant glycotopes, the trisaccharide Galα(1,3)Galß(1,4)GlcNAcα, is expressed on glycosylphosphatidylinositol-anchored mucins of the infective trypomastigote stage of T. cruzi and triggers high levels of protective anti-α-Gal antibodies (Abs) in infected individuals. Here, we have efficiently synthesized the mercaptopropyl glycoside of that glycotope and conjugated it to maleimide-derivatized bovine serum albumin (BSA). Chemiluminescent-enzyme-linked immunosorbent assay revealed that Galα(1,3)Galß(1,4)GlcNAcα-BSA is recognized by purified anti-α-Gal Abs from chronic ChD patients ∼230-fold more strongly than by anti-α-Gal Abs from sera of healthy individuals (NHS anti-α-Gal). Similarly, the pooled sera of chronic Chagas disease patients (ChHSP) recognized Galα(1,3)Galß(1,4)GlcNAcα ∼20-fold more strongly than pooled NHS. In contrast, the underlying disaccharide Galß(1,4)GlcNAcα and the monosaccharide GlcNAcα or GlcNAcß conjugated to BSA are poorly or not recognized by purified anti-α-Gal Abs or sera from Chagasic patients or healthy individuals. Our results highlight the importance of the terminal Galα moiety for recognition by Ch anti-α-Gal Abs and the lack of Abs against nonself Galß(1,4)GlcNAcα and GlcNAcα glycotopes. The substantial difference in binding of Ch vs. NHS anti-α-Gal Abs to Galα(1,3)Galß(1,4)GlcNAcα-BSA suggests that this neoglycoprotein (NGP) might be suitable for experimental vaccination. To this end, the Galα(1,3)Galß(1,4)GlcNAcα-BSA NGP was then used to immunize α1,3-galactosyltransferase-knockout mice, which produced antibody titers 40-fold higher as compared with pre-immunization titers. Taken together, our results indicate that the synthetic Galα(1,3)Galß(1,4)GlcNAcα glycotope coupled to a carrier protein could be a potential diagnostic and vaccine candidate for ChD.

Exploring the glycan interaction in vivo: Future prospects of neo-glycoproteins for diagnostics.

Herein the biodistributions and in vivo kinetics of chemically prepared neoglycoproteins are reviewed. Chemical methods can be used to conjugate various mono- and oligosaccharides onto a protein surface. The kinetics and organ-specific accumulation profiles of these glycoconjugates, which are introduced through intravenous injections, have been analyzed using conventional dissection studies as well as noninvasive methods such as single photon emission computed tomography, positron emission tomography and fluorescence imaging. These studies suggest that glycan-dependent protein distribution kinetics may be useful for pharmacological and diagnostic applications.

Preparation and Characteristic of Dextran-BSA Antibody and Establishment of its ELISA Immunoassay.

The enzyme-linked immunosorbent assay (ELISA) is a potential tool for the determination of dextran. In this study, dextran neoglycoprotein antigens were prepared by Reductive Amination method, and were confirmed by SDS-PAGE and free amino detection. The impact factors such as different oxidation degree of dextran, the conjugate reaction time to BSA were investigated. The best preparation conditions were obtained (n(dextran)/n(oxidant) of NaIO4 = 1/120, the reaction time of 24 h), and the antigen with best combination with standard was obtained. The antigens interacted with standard antibody and were evaluated through ELISA. The immunogen was immunized with white rabbits to obtained antibody, respectively. A general and broad class-specific ELISA immunoassay was developed for dextran detection according to ELISA theory. The optimized conditions of assay used coating antigen at 10 µg/mL, reaction time of antibody and rabbit-anti-bovine IgG in 45 min, blocking reagents with 5% calf serum. The developed ELISA detection method with good linear and accuracy was put to use for quantitative analysis of dextran T40 in commercial sugarpractical for detection of dextran.

Big is not better: Comparing two alpha-Gal-bearing glycotopes in neoglycoproteins as biomarkers for Leishmania (Viannia) braziliensis infection.

The protozoan parasite Leishmania (Viannia) braziliensis is among Latin America's most widespread Leishmania species and is responsible for tegumentary leishmaniasis (TL). This disease has multiple clinical presentations, with cutaneous leishmaniasis (CL) being the most frequent. It manifests as one or a few localized skin ulcers, which can spread to other body areas. Hence, early diagnosis and treatment, typically with pentavalent antimonials, is critical. Traditional diagnostic methods, like parasite culture, microscopy, or the polymerase chain reaction (PCR) for detection of the parasite DNA, have limitations due to the uneven distribution of parasites in biopsy samples. Nonetheless, studies have revealed high levels of parasite-specific anti-α-Gal antibodies in L. (V.) braziliensis-infected patients. Previously, we demonstrated that the neoglycoprotein NGP28b, consisting of the L. (Leishmania) major type-2 glycoinositolphospholipid (GIPL)-3-derived trisaccharide Galpα1,6Galpα1,3Galfß conjugated to bovine serum albumin (BSA) via a linker, acts as a reliable serological biomarker (BMK) for L. (V.) braziliensis infection in Brazil. This indicates the presence of GIPL-3 or a similar structure in this parasite, and its terminal trisaccharide either functions as or is part of an immunodominant glycotope. Here, we explored whether extending the trisaccharide with a mannose unit would enhance its efficacy as a biomarker for the serological detection of L. (V.) braziliensis. We synthesized the tetrasaccharide Galpα1,6Galpα1,3Galfß1,3Manpα(CH2)3SH (G31SH) and conjugated it to maleimide-functionalized BSA to afford NGP31b. When we assessed the efficacy of NGP28b and NGP31b by chemiluminescent enzyme-linked immunosorbent assay on a cohort of CL patients with L. (V.) braziliensis infection from Bolivia and Argentina against a healthy control group, both NGPs exhibited similar or identical sensitivity, specificity, and accuracy. This finding implies that the mannose moiety at the reducing end is not part of the glycotope recognized by the parasite-specific anti-α-Gal antibodies in patients' sera, nor does it exert a relevant influence on the terminal trisaccharide's conformation. Moreover, the mannose does not seem to inhibit glycan-antibody interactions. Therefore, NGP31b is a viable and dependable BMK for the serodiagnosis of CL caused by L. (V.) braziliensis.

Neoglycoproteins as carbohydrate antigens: synthesis, analysis, and polyclonal antibody response.

The analysis and polyclonal antibody response for newly synthesized maltose-BSA conjugate neoglycoproteins is described. In this first proof of concept study, a simple carbohydrate antigen, maltose, was linked to BSA by reductive amination. An aglycone spacer was utilized to conserve the intact annular maltose structure and to promote the accessibility of the carbohydrate immunogen hapten during immunization. The neoglycoproteins were investigated by CGE and the number of conjugated maltose residues was determined by MALDI-TOF MS. The neoglycoproteins were then evaluated by immunization of BALB/c mice and the polyclonal antibody response was tested by ELISA as evidence for the presence of sugar-containing epitope-specific antibodies. Selective antibody binding was demonstrated to the synthesized neoglycoproteins with different (low and high) glycosylation degrees suggesting the possible use of this approach to generate antibodies. Moreover, the polyclonal antibody response was not inhibited by maltose or other simple carbohydrates to confirm presence of the neoglycoprotein-specific antibodies.

Fucosyl neoglycoprotein binds to mouse epididymal spermatozoa and inhibits sperm binding to the egg zona pellucida.

Glycan epitopes of cellular glycoconjugates act as versatile biochemical signals, and this sugar coding plays an important role in cell-to-cell recognition processes. In this study, our aims were to determine the distribution of sperm receptors with activity for fucosyl- and galactosyl glycans and to address whether monosugar neoglycoproteins functionally mimic the binding between zona pellucida (ZP) glycoproteins and spermatozoa. In mouse epididymal spermatozoa with intact acrosomes, fucopyranosyl bovine serum albumin (BSA-Fuc) bound to the segment of the acrosome, the equatorial segment, and the postacrosome region of the sperm head. Galactosyl BSA (BSA-Gal) binding activity was similar to that of BSA-Fuc, but was weaker. In acrosome-reacted spermatozoa treated with the Ca(2+) ionophore A23187, BSA-zuc binding was lost in the apical segment of the acrosome but remained in the equatorial segment and postacrosome regions. BSA-Gal binding to the equatorial region was increased. In the presence of 2.5 µg ml(-1) BSA-Fuc, in vitro sperm-ZP binding was significantly decreased, indicating that fucosyl BSA functionally mimics ZP glycoproteins during sperm-egg ZP interactions. At the same concentration, BSA-Gal was not effective. Fucosyl BSA that efficiently inhibited the sperm-ZP binding can mimic the ZP glycoconjugate and has potential for use as a sperm fertility control agent in mouse.

The myrosinase-glucosinolate system to generate neoglycoproteins: A case study targeting mannose binding lectins.

A convenient strategy for a 'one-pot' synthesis of neoglycoproteins (NGP) was developed using the myrosinase-glucosinolate couple, a natural enzyme-substrate system. This enzymatic reaction allowed us to generate an isothiocyanate in situ which then reacted with the lysine residues of bovine serum albumin protein (BSA) to produce multivalent neoglycoproteins. Using two models, glucomoringin which is a natural glucosinolate bearing a l-rhamnose unit, and an artificial glucosinolate specifically designed for mannose type lectins, an average of up to 17.8 and 28.7 carbohydrate residues could be respectively grafted onto the BSA protein. This process is comparable to commercial approaches using BSA-ManC without the disadvantage of handling harmful chemical reagents. Lectin binding screening (GLYcoPROFILE®) showed that among all NGPs synthesized, BSA-Man 16 gave similar and in some cases better affinities in comparison with commercial BSA-Manc towards various mannose-specific lectins.

Neoglycoprotein and Glycoprotein Printing on a Hydrogel Functionalized Microarray Surface and Incubation with Labeled Lectins.

Glycan microarrays are widely used to elucidate carbohydrate binding specificity and affinity of various analytes including proteins, microorganisms, cells, and tissues. Glycan microarrays comprise a wide variety of platforms, differing in surface chemistry, presentation of carbohydrates, carbohydrate valency, and detection strategies, all of which impact on analyte performance. This chapter describes detailed methods for printing neoglycoprotein and glycoprotein microarrays on hydrogel-coated slides and incubation of these glycan microarrays with fluorescently labeled lectins.

Homo- and Heterovalent Neoglycoproteins as Ligands for Bacterial Lectins.

Click chemistry gives access to unlimited set of multivalent glycoconjugates to explore carbohydrate-protein interactions and discover high affinity ligands. In this study, we have created supramolecular systems based on a carrier protein that was grafted by Cu(I)-catalyzed azide-alkyne cycloaddition with tetravalent glycodendrons presenting αGal, ßGal and/or αFuc. Binding studies of the homo- (4 a-c) and heterovalent (5) neoglycoproteins (neoGPs) with the LecA and LecB lectins from P. aeruginosa has first confirmed the interest of the multivalent presentation of glycodendrons by the carrier protein (IC50 up to 2.8 nM). Moreover, these studies have shown that the heterovalent display of glycans (5) allows the interaction with both lectins (IC50 of 10 nM) despite the presence of unspecific moieties, and even with similar efficiency for LecB. These results demonstrate the potential of multivalent and multispecific neoGPs as a promising strategy to fight against resistant pathogens.

Immunoprotective neo-glycoproteins: Chemoenzymatic synthesis of multivalent glycomimetics for inhibition of cancer-related galectin-3.

Galectin-3 plays a crucial role in cancerogenesis; its targeting is a prospective pathway in cancer diagnostics and therapy. Multivalent presentation of glycans was shown to strongly increase the affinity of glycoconjugates to galectin-3. Further strengthening of interaction with galectin-3 may be accomplished using artificial glycomimetics with apt aryl substitutions. We established a new, as yet undescribed chemoenzymatic method to produce selective C-3-substituted N,N'-diacetyllactosamine glycomimetics and coupled them to human serum albumin. From a library of enzymes, only ß-N-acetylhexosaminidase from Talaromyces flavus was able to efficiently synthesize the C-3-propargylated disaccharide. Various aryl residues were attached to the functionalized N,N'-diacetyllactosamine via click chemistry to assess the impact of the aromatic substitution. In ELISA-type assays with galectin-3, free glycomimetics exhibited up to 43-fold stronger inhibitory potency to Gal-3 than the lactose standard. Coupling to human serum albumin afforded multivalent neo-glycoproteins with up to 4209-fold increased inhibitory potency per glycan compared to the monovalent lactose standard. Surface plasmon resonance brought further information on the kinetics of galectin-3 inhibition. The potential of prepared neo-glycoproteins to target galectin-3 was demonstrated on colorectal adenocarcinoma DLD-1 cells. We investigated the uptake of neo-glycoproteins into cells and observed limited non-specific transport into the cytoplasm. Therefore, neo-glycoproteins primarily act as efficient scavengers of exogenous galectin-3 of cancer cells, inhibiting its interaction with the cell surface, and protecting T-lymphocytes against galectin-3-induced apoptosis. The present neo-glycoproteins combine the advantage of a straightforward synthesis, selectivity, non-toxicity, and high efficiency for targeting exogenous galectin-3, with possible application in the immunomodulatory treatment of galectin-3-overexpressing cancers.

Electrochemical Impedance Spectroscopy Biosensor Enabling Kinetic Monitoring of Fucosyltransferase Activity.

Monitoring glycosyltransferases on biosensors is of great interest for pathogen and cancer diagnostics. As a proof of concept, we here demonstrate the layer-by-layer immobilization of a multivalent neoglycoprotein (NGP) as a substrate for a bacterial fucosyltransferase (FucT) and the subsequent binding of the fucose-specific Aleuria aurantia lectin (AAL) on an electrochemical impedance spectroscopy (EIS) sensor. We report for the first time the binding kinetics of a glycosyltransferase in real-time. Highly stable EIS measurements are obtained by the modification of counter and reference electrodes with polypyrrole: polystyrene sulfonate (PPy:PSS). In detail, the N-acetyllactosamine (LacNAc)-carrying NGP was covalently immobilized on the gold working electrode and served as a substrate for the FucT-catalyzed reaction. The LacNAc epitopes were converted to Lewisx (Lex) and detected by AAL. AAL binding to the Lex epitope was further confirmed in a lectin displacement and a competitive lectin binding inhibition experiment. We monitored the individual kinetic processes via EIS. The time constant for covalent immobilization of the NGP was 653 s. The FucT kinetics was the slowest process with a time constant of 1121 s. In contrast, a short time constant of 11.8 s was determined for the interaction of AAL with the modified NGPs. When this process was competed by 400 mM fucose, the binding was significantly slowed down, as indicated by a time constant of 978 s. The kinetics for the displacement of bound AAL by free fucose was observed with a time constant of 424 s. We conclude that this novel EIS biosensor and the applied workflow has the potential to detect FucT and other GT activities in general and further monitor protein-glycan interactions, which may be useful for the detection of pathogenic bacteria and cancer cells in future biomedical applications.

Preparation and Detection of Glycan-Binding Activity of Influenza Virus.

We describe a method to detect influenza virus using an evanescent-field-activated fluorescence scanner type glycan array and ELISA system. Neoglycoprotein was prepared by combination of organic chemistry and biomaterial preparation. These ligands were spotted on a glass plate or plastic well to make a glycan array and ELISA plate. We detected cultured influenza virus using glycan array and ELISA. Then, we showed that the neoglycoprotein binds to Cy3-labeled hemagglutinins (H1 and H5), a NeuAcα2,6LacNAc or NeuAcα2,3LacNAc recognized protein, as detected.

Galectin-Carbohydrate Interactions in Biomedicine and Biotechnology.

Cellular communication events are mediated by interactions between cell-surface sugars and lectins, which are carbohydrate-binding proteins. Galectins are ß-galactosyl-binding lectins that bridge molecules by their sugar moieties, forming a signaling and adhesion network. Severe changes in glycosylation and galectin expression accompany major processes in oncogenesis, cardiovascular disorders, and other pathologies, making galectins attractive therapeutic targets. Here we discuss advanced strategies of chemo-enzymatic carbohydrate synthesis for creating lead glycomimetics and (neo-)glycoconjugates for galectin-1 and -3 targeting in biomedicine and biotechnology. We will describe the challenges and bottlenecks on the route into biomedical and biotechnological practice and present the first clinical candidates. The coming era will see an exciting translation of selective well-defined high-affinity galectin ligands from bench to bedside.

Biotinylated N-Acetyllactosamine- and N,N-Diacetyllactosamine-Based Oligosaccharides as Novel Ligands for Human Galectin-3.

Galectin inhibitor design is an emerging research field due to the involvement of galectins in cancer. Galectin-3, in particular, plays an important role in tumor progression. To generate inhibitors, modifications of the glycan structure can be introduced. Conjugation of hydrophobic compounds to saccharides has proven to be promising as increased binding of galectin-3 can be observed. In the present study, we report on neo-glycans carrying hydrophobic biotin as novel ligands for human galectin-3. We modified N-acetyllactosamine- and N,N-diacetyllactosamine-based tetrasaccharides at the C6-position of the terminal saccharide unit using selective enzymatic oxidation and subsequent chemical conjugation of biotinamidohexanoic acid hydrazide. These neo-glycans were much better bound by galectin-3 than the unmodified counterparts. High selectivity for galectin-3 over galectin-1 was also proven. We generated multivalent neo-glycoproteins by conjugation of neo-glycans to bovine serum albumin showing high affinity for galectin-3. Compared to non-biotinylated neo-glycoproteins, we achieved high binding levels of galectin-3 with a lesser amount of conjugated neo-glycans. Multivalent ligand presentation of neo-glycoproteins significantly increased the inhibitory potency towards galectin-3 binding to asialofetuin when compared to free monovalent glycans. Our findings show the positive impact of 6-biotinylation of tetrasaccharides on galectin-3 binding, which broadens the recent design approaches for producing high-affinity ligands.

Preparation of Glycan Arrays Using Pyridylaminated Glycans.

We describe the method to prepare neoglycoproteins from the conjugation of bovine serum albumin and pyridylaminated glycans. Large quantities of glycans (>1 mg) can be pyridylaminated and then converted to their 1-amino-1-deoxy derivatives by reaction with hydrogen followed by hydrazine. These pyridylaminated glycans can then be conjugated to bovine serum albumin via esterification with N-( m-maleimidobenzoyloxy)succinimide to form a neoglycoprotein, e.g., glycosylated bovine serum albumin. As a demonstration, we prepared High-mannose bovine serum albumin, which was immobilized on an activated glass slide. Then, we showed that the neoglycoprotein bind to Cy3-labeled Lens culinaris agglutinin, a mannose-specific plant lectin, as detected using an evanescent-field-activated fluorescence scanner system.

The surface coat of plant-parasitic nematodes: chemical composition, origin, and biological role-a review.

Chemical composition, origin, and biological role of the surface coat (SC) of plant-parasitic nematodes are described and compared with those of animal-parasitic and free-living nematodes. The SC of the plant-parasitic nematodes is 5-30 nm thick and is characterized by a net negative charge. It consists, at least in part, of glycoproteins and proteins with various molecular weights, depending upon the nematode species. The lability of its components and the binding of human red blood cells to the surface of many tylenchid plant-parasitic nematodes, as well as the binding of several neoglycoproteins to the root-knot nematode Meloidogyne, suggest the presence of carbohydrate-recognition-domains for host plants and parasitic or predatory soil microorganisms (Pasteuria penetrans and Dactylaria spp., for example). These features may also assist in nematode adaptations to soil environments and to plant hosts with defense mechanisms that depend on reactions to nematode surfaces. Surface coat proteins can be species and race specific, a characteristic with promising diagnostic potential.

Attachment of Pasteuria penetrans Endospores to the Surface of Meloidogyne javanica Second-stage Juveniles.

Pasteuria penetrans spore adhesion to Meloidogyne javanica second-stage juveniles (J2) was examined following several different pretreatments of the latter. The detergents sodium dodecyl sulfate and Triton X-100, the carbohydrates fucose and alpha-methyl-D-mannoside, and the lectins concanavalin A and wheat germ agglutinin reduced spore attachment. Spores exposed to M. javanica surface coat (SC) extract exhibited decreased adherence to the J2 surface. Second-stage juveniles that had been treated with antibodies recognizing a 250-kDa antigen of J2 SC extract had fewer spores attached to their surfaces, as compared to nontreated J2, except in the head region. This inhibition pattern was similar to that of antibody-labelling on M. javanica J2 as observed by electron microscopy. It is suggested that several SC components, such as carbohydrate residues, carbohydrate-recognition domains, and a 250-kDa antigen, are involved in P. penetrans spore attachment to the surface of M. javanica.