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Background The separation of isomeric compounds from a mixture is a recurring problem in chemistry and phytochemistry research. The purification of pharmacologically active ginsenoside Rb3 from ginseng extracts is limited by the co-existence of its isomer Rb2. The aim of the present study was to develop an enzymatic elimination-combined purification method to obtain pure Rb3 from a mixture of isomers. Methods To isolate Rb3 from the isomeric mixture, a simple enzymatic selective elimination method was used. A ginsenoside-transforming glycoside hydrolase (Bgp2) was employed to selectively hydrolyze Rb2 into ginsenoside Rd. Ginsenoside Rb3 was then efficiently separated from the mixture using a traditional chromatographic method. Results Chromatographic purification of Rb3 was achieved using this novel enzymatic elimination-combined method, with 58.6-times higher yield and 13.1% less time than those of the traditional chromatographic method, with a lower minimum column length for purification. The novelty of this study was the use of a recombinant glycosidase for the selective elimination of the isomer. The isolated ginsenoside Rb3 can be used in further pharmaceutical studies. Conclusions Herein, we demonstrated a novel enzymatic elimination-combined purification method for the chromatographic purification of ginsenoside Rb3. This method can also be applied to purify other isomeric glycoconjugates in mixtures.

Graphical abstract, Highlights • The phylogenetic distribution of GT1 family enzymes showed domain-dependent pattern. • The domain-dependent characterized GTs showed distinct substrates spectrum. • Two regions that discriminate the GT1 enzymes from different domains were identified., Glycosyltransferases (GTs) are responsible for transferring glycosyl moieties from activated sugar donors to certain acceptors, among which the GT1 family enzymes have been known for their outstanding glycosylation capacities toward diverse natural products, such as glycolipids, flavonoids and macrolides etc. However, there still lacks a systematic collation of this important family members. In this minireview, all the GT1 family sequences were phylogenetically analyzed, and the grouping of GT1 proteins exhibited a taxonomic life domain-dependent pattern, revealing many untapped clades of GTs. The further phylogenetic analysis of the characterized GTs facilitated the classification of substrates coverage of GT1 family enzymes from different life domains, whereby the GTs from bacteria can tolerate a wider spectrum of chemical skeletons as substrates, showing higher promiscuity than those from other domains. Furthermore, the sequence sizes of GTs from different domains were compared to understand their different substrates selectivity. Based on the multiple sequence alignments of 28 representative GT1 enzymes with crystal structures, two critical regions located in the N-terminal of GTs were identified, which were most variable among sequences from different taxonomic domains and essential for substrates binding and/or catalysis. The key roles of these two regions were validated by enumerating the influential residues that interacted with substrates in the representative structures from bacteria and plants. The atlas for GT1 family in terms of phylogeny, substrates selectivity, sequence length, and critical motifs provides the clues for the exploration of unknown GT1s and rational engineering of known enzymes, synthesizing novel promising glycoconjugates for pharmaceutical application.

Background Ginsenoside Rh2 is well known for many pharmacological activities, such as anticancer, antidiabetes, antiinflammatory, and antiobesity properties. Glycosyltransferases (GTs) are ubiquitous enzymes present in nature and are widely used for the synthesis of oligosaccharides, polysaccharides, glycoconjugates, and novel derivatives. We aimed to synthesize new ginsenosides from Rh2 using the recombinant GT enzyme and investigate its cytotoxicity with diverse cell lines. Methods We have used a GT gene with 1,224-bp gene sequence cloned from Lactobacillus rhamnosus (LRGT) and then expressed in Escherichia coli BL21 (DE3). The recombinant GT protein was purified and demonstrated to transform Rh2 into two novel ginsenosides, and they were characterized by nuclear magnetic resonance (NMR) techniques and evaluated by 3-(4, 5-dimethylthiazol-2-yl)-2-5-diphenyltetrazolium bromide assay. Results Two novel ginsenosides with an additional glucopyranosyl (6→1) and two additional glucopyranosyl (6→1) linked with the C-3 position of the substrate Rh2 were synthesized, respectively. Cell viability assay in the lung cancer (A549) cell line showed that glucosyl ginsenoside Rh2 inhibited cell viability more potently than ginsenoside Rg3 and Rh2 at a concentration of 10 μM. Furthermore, glucosyl ginsenoside Rh2 did not exhibit any cytotoxic effect in murine macrophage cells (RAW264.7), mouse embryo fibroblasts cells (3T3-L1), and skin cells (B16BL6) at a concentration of 10 μM compared with ginsenoside Rh2 and Rg3. Conclusion This is the first report on the synthesis of two novel ginsenosides, namely, glucosyl ginsenoside Rh2 and diglucosyl ginsenoside Rh2 from Rh2 by using recombinant GT isolated from L. rhamnosus. Moreover, diglucosyl ginsenoside Rh2 might be a new candidate for treatment of inflammation, obesity, and skin whiting, and especially for anticancer.

Carbohydrate-containing biopolymers such as glycoproteins, proteoglycans, and glycolipids are an incredibly diverse and complex set of natural building blocks used by biological systems to endorse a wide range of molecular functions that are critical to life processes. Simplified synthetic analogs of these polymeric glycoconjugates can capture some of these functions and are increasingly exploited toward the development of supramolecular biomaterials. These carbohydrate amphiphiles can mimic structural aspects through cooperative molecular self-assembly or target distinct signaling pathways through engineered (multivalent) interactions with biological systems. Herein, we discuss the supramolecular principles that regulate the glycome function(s) and the translation of these in the design of supramolecular biomaterials in which carbohydrates are used as information-rich structural elements with huge potential as therapeutic supramolecular biomaterials., We acknowledge the EU’s H2020 program (Forecast 668983) and the Portuguese FCT (BD/113794/2015; PTDC/NAN-MAT/28468/2017 CANCER_CAGE; PTDC/ BTM-MAT/28327/2017 CardioHeal) for the financial support. A.B. is grateful to the Portuguese League Against Cancer for her fellowship

The synthesis of homogeneous and well-defined glycan is important for detailed structural and functional studies of glycoscience and glycotechnology. Chemo-enzymatic synthesis of glycan maximizes the benefits of both chemical and enzymatic syntheses. This method enables the efficient synthesis of glycans by capitalizing on the high regio- and stereo-selectivity of enzymes in a well-used chemical synthesis where the number of steps and the time are spent. In this article, we describe the efficient synthesis of oligosaccharides by enzymatically adding saccharides to the synthesized core glycan, and the efficient synthesis of glycopeptides and glycoproteins by enzymatically binding glycan chains to chemically synthesized peptides and proteins. In particular, we describe in detail an efficient chemo-enzymatic synthesis of homogeneous and well-defined glycopeptides and glycoproteins by using the transglycosylation activity of microbial endoglycosidases and the chemically synthesized intermediate as a substrate. Furthermore, the application of this chemo-enzymatic method to the synthesis of bioactive substances and biopharmaceuticals is discussed, as well as the chemo-enzymatic glycoengineering and remodeling of sugar chains of glycoproteins.

In this book chapter, the advances in the determination of carbohydrates and glycoconjugates by capillary and microchip electrophoresis with electrochemical detection (CE/ME-ED) will be studied, covering from 2002 to 2019. Amperometric detection, capacitively coupled contactless conductivity detection, and enzyme-based amperometric detection as well as electrochemical derivatization schemes will be critically discussed, showing their advantages and limitations. Furthermore, the most important applications in clinical, pharmaceutical, and food fields will also be highlighted. Future outlooks of CE/ME-ED for carbohydrate determination will also be shown, pointing out those strategies that may improve the sensitivity and selectivity of analytical methods.

In this chapter, different high-performance liquid chromatography (HPLC) and capillary electrophoresis (CE) methodologies applied in foods are discussed. Due to the complex structure of food matrices, the chapter is focused on those food categories, based on the Codex Alimentarius classification, where carbohydrates play an important technological or nutritional role. The chapter aims to inform readers about instrumental techniques and modifications of known HPLC and CE methods to improve the separation, quantification, and detection of sugars and their derivatives. In addition, it covers many applications to different types of foods and drinks to assist readers with a critical selection of HPLC and CE methods for analyzing carbohydrates based on the food sample and final purpose.

High-performance anion-exchange chromatography with pulsed amperometric detection (HPAEC-PAD) is a well-established technique for carbohydrate determinations. This chapter first reviews the basics of HPAEC-PAD and new developments in column and electrochemical detection technologies. The remainder of the chapter is dedicated to the application of HPAEC-PAD. The major applications of HPAEC-PAD: monosaccharide analysis, sialic acid analysis, and glycan/oligosaccharide profiling are updated. Then applications that have either increased in popularity or emerged in the last 20 years are described: profiling human milk oligosaccharides, analyzing glycoconjugate vaccines, carbohydrate determinations in biofuel research, determination of carbohydrates in urine and serum, determination of carbohydrates in atmospheric aerosols, and determination of carbohydrates in plants and food. The chapter concludes showing the role of HPAEC-PAD in regulatory and standard methods.

Establishment of neural circuits requires reproducible and precise interactions between growing axons, dendrites and their tissue environment. Cell adhesion molecules and guidance factors are involved in the process, but how specificity is achieved remains poorly understood. Glycans are the third major class of biopolymers besides nucleic acids and proteins, and are usually covalently linked to proteins to form glycoconjugates. Common to most glycans is an extraordinary level of molecular diversity, making them attractive candidates to contribute specificity during neural development. Indeed, many genes important for neural development encode glycoproteins, or enzymes involved in synthesizing or modifying glycans. Glycoconjugates are classified based on both the types of glycans and type of attachment that link them to proteins. Here I discuss progress in understanding the function of glycans, glycan modifications and glycoconjugates during neural development in Caenorhabditis elegans. I will also highlight relevance to human disease and known roles of glycoconjugates in regeneration.

The biosynthesis pathways of bacterial glycoconjugate molecules, especially of those molecules that traffic across the cytoplasmic membrane and reside extracytoplasmically has biologically conserved features including the saccharides are formed as lipid-linked glycans anchored to cell membranes, the lipid anchors are polyisoprenoid phosphates, and the assembly reactions occur at both sides of the cell membrane, typically commencing on the cytosolic side and continuing at the opposite side of the membrane, for which the transmembrane movement of the lipid-linked saccharides is an obligatory step. After membrane translocation, additional reactions at the external side of the cell membrane are essential to complete the biosynthesis of bacterial glycoconjugates. One of these reactions involves the glycosyl transfer of the saccharide portion (a monosaccharide, an oligosaccharide, or a polysaccharide chain) of polyisoprenyl-linked substrates to the appropriate acceptor molecules. This chapter describes the main structural and topological properties of prototypic bacterial posttranslocation glycosyltransferases or “glycan ligases.”

Carbohydrates or glycans and their conjugates are involved in a wide range of biological processes and play an important role in various diseases, including inflammation, viral and bacterial infections, and tumor progression and metastasis. Studying the biological significances of carbohydrates has been challenging due in part to their structural diversity and the limited access to complex carbohydrate-containing molecules. Conventional methods such as isothermal titration calorimetry and enzyme-linked lectin assay can be laborious and require significant amounts of time and materials. The emerging of glycan microarrays as high-throughput technology for studying carbohydrate interactions has overcome some of these challenges, and has greatly contributed to our understanding of the biological roles of carbohydrates and their glycoconjugates. In addition, glycan microarrays offer new applications in biomedical research, drug discovery and development. This chapter will focus on the biomedical applications of glycan microarrays and their potential role in drug discovery and development.

This chapter highlights the most relevant examples of selective protein modification strategies featuring their application for the development of synthetic, homogeneous carbohydrate-based vaccine candidates able to induce specific and selective immune responses. Key strategies for glycoprotein synthesis will be recapitulated. It starts by presenting the transition from classical, random bioconjugation methods to more selective, modern protocols. Particularly, bioorthogonal reactions targeting noncanonical amino acids, which represent a truly chemo and regioselective glycan-to-protein reacting point. It subsequently describes selected examples for the preparation of broadly useful glycoprotein vaccines. Finally, a comparison of methods together with the future perspectives in the field is described highlighting the power of selective protein-modification chemistry for the preparation of complex glycoprotein substrates not only for the development of clinically effective, potent therapeutic vaccines against cancer, bacterial, and/or viral infections but also for basic research within the glycoimmunology field.

This chapter summarizes the implemented synthetic strategies aimed at generating molecular diversity in a new family of stable glycoconjugate mimetics, namely sp2-iminosugar glycolipids and glycopeptides. It further illustrates the potential of these compounds as antitumor, antileishmanial, and anti-inflammatory agents. The presence of a pseudoamide functionality at the place of the endocyclic oxygen atom typical of monosaccharides makes sp2-iminosugars very good substrates for glycosylation reactions. Structure-activity relationship studies have revealed strong effects of the nature of glycosidic linkage (N, S, C, SO, SO2) and the aglycone moiety in the biological properties. The mechanisms of action are also discussed, pointing at p38α mitogen-activated protein kinase as a main pharmacological target.

Fungal infections are a serious threat for human beings, especially for those who are considered at risk due to underline diseases or medical treatments that weaken their immunological status or facilitate tissues invasion by fungal pathogens. The available antifungal drugs are characterized by severe adverse effects; in addition, new and antimicrobial-resistant strains are emerging making their effective treatment more problematic. Alternative strategies like effective immunoprophylaxis are increasingly needed. Carbohydrates dominate the fungal cell wall (CW) are exposed on their surface and, therefore, represent a logical target for antifungal vaccines development. Since they are T-cell independent, they are poorly immunogenic in infants and do not induce immunological memory and high affinity antibodies which limits their applicability in all classes of age. Conjugation to protein carriers has solved this drawback. Accordingly, carbohydrate-based antifungal vaccines approached are based on conjugation of selected glycan antigens to protein carriers. Antifungal glycoconjugate vaccines are being explored at preclinical level for at least three of the major fungal pathogens: Candida albicans, Aspergillus fumigatus, and Cryptococcus neoformans. Glycoconjugates against C. albicans are the more advanced in terms of preclinical evidence, and different glycans have been tested (e.g., α- and β-mannosides, β-glucans, poly-N-acetyl-(1 → 6)-α-glucosamine). Efforts to develop a glycoconjugate vaccine against C. neoformans are also ongoing and deserve a lot of attention. One of the main challenges is to identify and reproduce, by chemical synthesis, those epitopes located in the structure of the capsular polysaccharide glucuronoxylomannan (GXM) able to elicit protective antibodies. More effort is needed to investigate the protective role of carbohydrate antigens from Aspergillus CW although some preliminary evidence has been reported for β-(1 → 3) and increasing interest is being given to α-(1 → 3)-glucans. Since besides glycans, fungal pathogens display also other virulence and potentially protective factors like proteins, it seems reasonable that an optimal vaccine should simultaneously target both class of antigens. In this regard, glycoconjugate vaccines of selected fungal glycans and protein antigens appear an attractive perspective.

Licensed carbohydrate-based vaccines are obtained by conjugation of native or sized polysaccharides to an appropriate carrier protein. They are part of routine vaccinations in many countries. Advances in the organic and chemo-enzymatic synthesis of carbohydrate antigens combined with novel technologies (e.g., glycoengineering), new conjugation approaches (site-selective protein glycoconjugation), alternative carrier systems and development in glycan structure analysis methods offer new perspectives in the development of carbohydrate-protein conjugate vaccines. This review presents a status of chemistries and analytical methods used in the licensed vaccines as the development of new technologies and new tools that could be used to produce and characterize the future glycoconjugates vaccines.

Oligosaccharides, polysaccharides, and glycoconjugates are abundant in all living organisms, taking part in a multitude of biological processes. The application of natural carbohydrates in glycobiological studies and drug development is limited due to weak binding properties and sensitivity to enzymatic hydrolysis. These issues made it necessary to design new, synthetic carbohydrate mimetics via structural modifications. This chapter discusses linkage modifications, where the glycosidic oxygen is replaced by sulfur, selenium, nitrogen, carbon, or longer interglycosidic connections comprising two or three heteroatoms, increasing the enzymatic stability of the product while retaining the biological properties. Beyond the naturally occurring head-to-head and head-to-tail linkages involving at least one anomeric center, diglycose derivatives consisting of two monosaccharides linked at nonanomeric positions by various two- and three-bond linkages are also discussed. Due to the numerous literature results and review articles available, we focus on the synthetic methods published in the last 15 years.

The structures of bacterial cell surface carbohydrates such as capsular polysaccharides, lipopolysaccharides, and teichoic acids can be determined using nuclear magnetic resonance (NMR) spectroscopy. The one-dimensional (1D) proton spectrum constitutes a structural fingerprint of the glycan present which can be assigned using an array of 1D and two-dimensional (2D) NMR homo- and heteronuclear techniques to establish through-bond and space connectivities and thus determine the structure of the repeating unit. The surface carbohydrates can be profiled by high-resolution magic angle spinning (HR-MAS); they play a key role in protecting bacteria from host defenses and so many of these antigens are vaccine targets. Polysaccharide vaccines are being replaced by the more immunogenic glycoconjugate vaccines in which the carbohydrate is covalently attached to an appropriate carrier protein. NMR establishes the identity of the polysaccharide vaccine and can be used to monitor the structural integrity of the attached glycan chains in the conjugate and identify key epitopes.

Pathogenic micro-organisms utilize protein receptors in adhesion to host tissues, a process that in some cases relies on the interaction between lectin and human glycoconjugates. Oligosaccharide epitopes are recognized through their three-dimensional structure and their flexibility is a key issue in specificity. In this paper, we analyse by X-ray crystallography the structures of the lectin LecB from two strains of Pseudomonas aeruginosa in complex with Lewis x oligosaccharide present on cell surfaces of human tissues. An unusual conformation of the glycan was observed in all binding sites with a non-canonical syn orientation of the N-acetyl group of N-acetyl-glucosamine. A PDB-wide search revealed that such an orientation occurs only in 2% of protein/carbohydrate complexes. Theoretical chemistry calculations showed that the observed conformation is unstable in solution but stabilised by the lectin. A reliable description of LecB/Lewis x complex by force field-based methods had proven as especially challenging due to the special feature of the binding site, two closely apposed Ca2+ ions which induce strong charge delocalisation. By comparing various force-field parametrisations, we design general protocols which will be useful in near future for designing carbohydrate-based ligands (glycodrugs) against other calcium-dependent protein receptors.

Sialic acid-based glycoconjugates cover the surfaces of many different cell types, defining key properties of the cell surface such as overall charge or likely interaction partners. Because of this prominence, sialic acids play prominent roles in mediating attachment and entry to viruses belonging to many different families. In this review, we first describe how interactions between viruses and sialic acid-based glycan structures can be identified and characterized using a range of techniques. We then highlight interactions between sialic acids and virus capsid proteins in four different viruses, and discuss what these interactions have taught us about sialic acid engagement and opportunities to interfere with binding.

Sialic acid (Sia) is involved in many biological activities and commonly occurs as a monosialyl residue at the nonreducing terminal end of glycoconjugates. The loss of activity of UDP-GlcNAc2-epimerase/ManNAc kinase, which is a key enzyme in Sia biosynthesis, is lethal to the embryo, which clearly indicates the importance of Sia in embryogenesis. Occasionally, oligo/polymeric Sia structures such as disialic acid (diSia), oligosialic acid (oligoSia), and polysialic acid (polySia) occur in glycoconjugates. In particular, polySia, a well-known epitope that commonly occurs in neuroinvasive bacteria and vertebrate brains, is one of the most well-known and biologically/neurologically important glycotopes in vertebrates. The biological effects of polySia, especially on neural cell-adhesion molecules, have been well studied, and in-depth knowledge regarding polySia has been accumulated. In addition, the importance of diSia and oligoSia epitopes has been reported. In this chapter, the recent advances in the study of diSia, oligoSia, and polySia residues in glycoproteins in neurology, and their history, definition, occurrence, analytical methods, biosynthesis, and biological functions evaluated by phenotypes of gene-targeted mice, biochemical features, and related diseases are described.

Sialidases are glycosidases responsible for the removal of α-glycosidically linked sialic acid residues from carbohydrate portions of glycoproteins and glycolipids, this process being the initial step in the degradation of such glycoconjugates. Sialic acids are considered to play important roles in various biological processes largely in two ways, one related to their hydrophilic and acidic properties exerting physicochemical effects on the glycoconjugates to which they are attached, and the other as recognition sites or in an opposing fashion as masking sites. The removal of sialic acids catalyzed by a sialidase, therefore greatly influences many biological processes through changing the conformation of glycoproteins and through recognition and masking of biological sites of functional molecules. Sialidases are found widely distributed in metazoan animals, from echinoderms to mammals, and are also present in viruses and other microorganisms, including fungi, protozoa, and bacteria even mostly lacking sialic acids. In mammals, there are four forms of sialidase (Neu1, Neu2, Neu3, and Neu4), differing in their major subcellular localization and enzymatic properties. They have been implicated in regulation of various cellular activities, such as cell differentiation, cell growth, and cell adhesion and motility, depending on their particular properties. In contrast, in microorganisms the enzymes appear to play roles limited to nutrition and pathogenesis. In this chapter, the focus is on mammalian sialidases preferentially hydrolyzing gangliosides, mostly Neu3 and Neu4, with an attempt to provide a brief overview of their physiological and pathological roles.

In the three domains of life, lipid-linked glycans contribute to various cellular processes ranging from protein glycosylation to glycosylphosphatidylinositol anchor biosynthesis to peptidoglycan assembly. In generating many of these glycoconjugates, phosphorylated polyprenol-based lipids are charged with single sugars by polyprenol phosphate glycosyltransferases. The resultant substrates serve as glycosyltransferase donors, complementing the more common nucleoside diphosphate sugars. It had been accepted that these polyprenol phosphate glycosyltransferases acted similarly, given their considerable sequence homology. Recent findings, however, suggest that matters may not be so simple. In this Opinion we propose that the stereochemistry of sugar addition by polyprenol phosphate glycosyltransferases is not conserved across evolution, even though the GT-A fold that characterizes such enzymes is omnipresent. Keywords: dolichol phosphate, dolichol phosphate glucose synthase, dolichol phosphate mannose synthase, polyprenol phosphate, protein glycosylation, stereochemistry, Israel Science Foundation (Grant 109/16), National Institutes of Health (U.S.) (Grant GM-039334), Israel Science Foundation (Grant 2253/15), National Natural Science Foundation (China) (Grant 2193/16), German-Israeli Foundation for Scientific Research and Development (Grant I-1290-416.13/2015)

Glycosidases mediate the fragmentation of glycoconjugates in the body, including the vital recycling of endogenous molecules. Several inherited diseases in man concern deficiencies in lysosomal glycosidases degrading glycosphingolipids. Prominent is Gaucher disease caused by an impaired lysosomal β-glucosidase (glucocerebrosidase, GBA) and resulting in pathological lysosomal storage of glucosylceramide (glucocerebroside) in tissue macrophages. GBA is a retaining glucosidase with a characteristic glycosyl–enzyme intermediate formed during catalysis. Using the natural suicide inhibitor cyclophellitol as a lead, we developed mechanism-based irreversible inhibitors of GBA equipped with a fluorescent reporter. These reagents covalently link to the catalytic nucleophile residue of GBA and permit specific and sensitive visualization of active enzyme molecules. The amphiphilic activity-based probes (ABPs) allow in situ detection of active GBA in cells and organisms. Furthermore, they may be used to biochemically confirm the diagnosis of Gaucher disease and they might assist in screening for small compounds interacting with the catalytic pocket. While the focus of this chapter is ABPs for β-glucosidases and Gaucher disease, the described concept has meanwhile been extended to other retaining glycosidases and related disease conditions as well.

As a group of very important components in herbal drugs, carbohydrates analyses have gained increasing interest in the past decade. In this chapter, a brief description of herbal carbohydrates is presented, including the classification and pharmacological activities of mono-, oligo- and polysaccharides, and glycoconjugates such as glycopeptides and glycoproteins, glycosaminoglycans, and glycolipids. Next, sample preparation techniques such as extraction, purification, hydrolysis, and derivatization are discussed. Subsequently, separation and detection by capillary electrophoresis (CE) is reviewed. Finally, the applications of CE to the analysis of herbal carbohydrates are summarized. This chapter provides a reference for further applications of CE in this field.

Investigations of methodologies aimed on improving the stereoselective synthesis of sialosides and the efficient assembly of sialic acid glycoconjugates has been the mission of dedicated research groups from the late 1960s. This review presents major accomplishments in the field, with the emphasis on significant breakthroughs and influential synthetic strategies of the last decade.

Structural characterization of the lipopolysaccharide (LPS) from a nontypeable Helicobacter pylori strain PJ1 and two corresponding mutants, PJ1 HP1283:cam and PJ1 HP1284:cam, was performed using a combination of NMR and mass spectrometric techniques. It resulted in the core structure that differed significantly from the one proposed previously. Overall architecture of PJ1 LPS was found to be consistent with a structural model described for several other H. pylori strains. It contained a polymer of d -glycero- d -manno-heptose ( dd -Hep) as the O-chain component, linked to α-1,6-glucan through a dd -Hep oligosaccharide. H. pylori PJ1 HP1283:cam LPS was missing dd -heptan, terminating with an α-1,6-glucan chain containing 5–13 glucose residues. LPS of strain PJ1 HP1284:cam was missing dd -Hep from the core and had β-GlcNAc attached directly to O-3 of the inner-core ld -Hep residue. To investigate the role of dd -heptan in protective immunity, delipidated LPS (dLPS) from strain PJ1 was conjugated to tetanus toxoid (TT) and immunological properties of the resultant glycoconjugate dLPS(PJ1)-TT determined. The dLPS(PJ1)-TT conjugate was immunogenic in mice and rabbits and induced specific and cross-reactive functional antibodies against homologous and heterologous strains of H. pylori. Whole cell indirect ELISA performed on a selected number of H. pylori isolates confirmed that the immune response correlated with the presence of α-1,6-glucan and was not augmented by the dd -Hep content of these strains.

Background CEL-I is a galactose/N-acetylgalactosamine-specific C-type lectin isolated from the sea cucumber Cucumaria echinata. Its carbohydrate-binding site contains a QPD (Gln-Pro-Asp) motif, which is generally recognized as the galactose specificity-determining motif in the C-type lectins. In our previous study, replacement of the QPD motif by an EPN (Glu-Pro-Asn) motif led to a weak binding affinity for mannose. Therefore, we examined the effects of an additional mutation in the carbohydrate-binding site on the specificity of the lectin. Methods Trp105 of EPN-CEL-I was replaced by a histidine residue using site-directed mutagenesis, and the binding affinity of the resulting mutant, EPNH-CEL-I, was examined by sugar-polyamidoamine dendrimer assay, isothermal titration calorimetry, and glycoconjugate microarray analysis. Tertiary structure of the EPNH-CEL-I/mannose complex was determined by X-ray crystallographic analysis. Results Sugar-polyamidoamine dendrimer assay and glycoconjugate microarray analysis revealed a drastic change in the specificity of EPNH-CEL-I from galactose/N-acetylgalactosamine to mannose. The association constant of EPNH-CEL-I for mannose was determined to be 3.17 × 103 M- 1 at 25 °C. Mannose specificity of EPNH-CEL-I was achieved by stabilization of the binding of mannose in a correct orientation, in which the EPN motif can form proper hydrogen bonds with 3- and 4-hydroxy groups of the bound mannose. Conclusions Specificity of CEL-I can be engineered by mutating a limited number of amino acid residues in addition to the QPD/EPN motifs. General significance Versatility of the C-type carbohydrate-recognition domain structure in the recognition of various carbohydrate chains could become a promising platform to develop novel molecular recognition proteins., Biochimica et Biophysica Acta (BBA) - General Subjects, 1850(7), pp.1457-1465; 2015

Trypanosoma cruzi, the etiologic agent of Chagas disease, is covered by a dense glycocalix mainly composed by glycoproteins called mucins which are also the acceptors of sialic acid in a reaction catalyzed by a trans-sialidase (TcTS). Sialylation of trypomastigote mucins protects the parasite from lysis by the anti α-Galp antibodies from serum. The TcTS is essential for the infection process since T. cruzi is unable to biosynthesize sialic acid. The enzyme specifically transfers it from a terminal β-D-Galp unit in the host glycoconjugate to terminal β-D-Galp units in the parasite mucins to construct the D-NeuNAc(α2→3)β-D-Galp motif. On the other hand, although galactose is the most abundant sugar in mucins of both, the infective trypomastigotes and the insect stage epimastigotes, α-D-Galp is only present in the infective stage whereas β-D-Galf is characteristic of the epimastigote stage of the less virulent strains. Neither α-D-Galp nor D-Galf is acceptor of sialic acid. In the mucins, some of the oligosaccharides are branched with terminal β-D-Galp units to be able to accept sialic acid in the TcTS reaction. Based on previous reports showing that anti α-Galp antibodies only partially colocalize with sialic acid, we have undertaken the synthesis of the trisaccharide α-D-Galp(1→3)-[β-D-Galp(1→6)]-D-Galp, the smallest structure containing both, the antigenic D-Galp(α1→3)-D-Galp unit and the sialic acid-acceptor β-D-Galp unit. The trisaccharide was obtained as the 6-aminohexyl glycoside to facilitate further conjugation for biochemical studies. The synthetic approach involved the α-galactosylation at O-4 of a suitable precursor of the reducing end, followed by β-galactosylation at O-6 of the same precursor and introduction of the 6-aminohexyl aglycone. The fully deprotected trisaccharide was successfully sialylated by TcTS using either 3′-sialyllactose or fetuin as donors. The product, 6-aminohexyl α-D-NeuNAc(2→3)-β-D-Galp(1→6)-[α-D-Galp(1→3)]-β-D-Galp, was purified and characterized. Fil: Giorgi, María Eugenia. Consejo Nacional de Investigaciones Científicas y Técnicas. Oficina de Coordinación Administrativa Ciudad Universitaria. Centro de Investigaciones en Hidratos de Carbono. Universidad de Buenos Aires. Facultad de Ciencias Exactas y Naturales. Centro de Investigaciones en Hidratos de Carbono; Argentina Fil: Lopez, Laura Rosana. Consejo Nacional de Investigaciones Científicas y Técnicas. Oficina de Coordinación Administrativa Ciudad Universitaria. Centro de Investigaciones en Hidratos de Carbono. Universidad de Buenos Aires. Facultad de Ciencias Exactas y Naturales. Centro de Investigaciones en Hidratos de Carbono; Argentina Fil: Agusti, Rosalia. Consejo Nacional de Investigaciones Científicas y Técnicas. Oficina de Coordinación Administrativa Ciudad Universitaria. Centro de Investigaciones en Hidratos de Carbono. Universidad de Buenos Aires. Facultad de Ciencias Exactas y Naturales. Centro de Investigaciones en Hidratos de Carbono; Argentina Fil: Marino, María Carla. Consejo Nacional de Investigaciones Científicas y Técnicas. Oficina de Coordinación Administrativa Ciudad Universitaria. Centro de Investigaciones en Hidratos de Carbono. Universidad de Buenos Aires. Facultad de Ciencias Exactas y Naturales. Centro de Investigaciones en Hidratos de Carbono; Argentina Fil: Muchnik, Rosa. Consejo Nacional de Investigaciones Científicas y Técnicas. Oficina de Coordinación Administrativa Ciudad Universitaria. Centro de Investigaciones en Hidratos de Carbono. Universidad de Buenos Aires. Facultad de Ciencias Exactas y Naturales. Centro de Investigaciones en Hidratos de Carbono; Argentina

Efficacious typhoid vaccines for young children will significantly reduce the disease burden in developing world. The Vi polysaccharide based conjugate vaccines (Vi-rEPA) against Salmonella Typhi Vi positive strains has shown high efficacy but may be ineffective against Vi negative S. Typhi. In this study, for the first time, we report the synthesis and evaluation of polysaccharide-protein conjugates of Vi negative S. Typhi as potential vaccine candidates. Four different conjugates were synthesized using recombinant exoprotein A of Pseudomonas aeruginosa (rEPA) and human serum albumin (HSA) as the carrier proteins, using either direct reductive amination or an intermediate linker molecule, adipic acid dihydrazide (ADH). Upon injection into mice, a significantly higher antibody titer was observed in mice administrated with conjugate-1 (OSP-HSA) (P = 0.0001) and conjugate 2 (OSP-rEPA) (P ≤ 0.0001) as compared to OSP alone. In contrast, the antibody titer elicited by conjugate 3 (OSPADH-HSA) and conjugate 4 (OSPADH-rEPA) were insignificant (P = 0.1684 and P = 0.3794, respectively). We conclude that reductive amination is the superior method to prepare the S. Typhi OSP glycoconjugate. Moreover, rEPA was a better carrier protein than HSA. Thus OSP-rEPA conjugate seems to be efficacious typhoid vaccines candidate, it may be evaluated further and recommended for the clinical trials.

As we enter into the postantibiotic era, vaccines to prevent bacterial infections previously treatable with antibiotics are urgently needed. Most successful antibacterial vaccines are glycoconjugates, composed of cell surface carbohydrates chemically attached to a carrier protein. Glycoconjugate vaccines provide a safe and consistent strategy against polysaccharide-encapsulated pathogens. The best examples are the conjugate vaccines against Haemophilus influenzae type b, Streptococcus pneumoniae, and Neisseria meningitidis, all based on capsular polysaccharides. Although these types of vaccines are effective, their current manufacturing process presents multiple drawbacks, such as biosafety risks and batch-to-batch variability. Furthermore, inclusion of additional serotypes is extremely slow, mainly due to the intricate chemical methods of conjugation. Thus, novel platforms for antibacterial vaccines are required. Gram-negative bacteria are able to produce outer membrane vesicles (OMVs). OMVs are mainly composed of lipopolysaccharide (LPS), outer membrane and periplasmic proteins, and phospholipids. Although their biogenesis is poorly understood, it is known that OMVs are formed by blebbing of the outer membrane. OMVs are attractive candidates for novel vaccine delivery platforms due to their immunogenic properties, self-adjuvanticity, and capacity for enhancement by recombinant engineering. We have shown that OMVs can be engineered to display surface glycans from different bacteria and that these glycoengineered OMVs (geOMVs) are effective in diverse animal models of infection. Here we provide a detailed method for the design and preparation of geOMV displaying the O-antigen from a prominent uropathogenic Escherichia coli (UPEC) serotype, O25b, as a proof of concept for the use of geOMVs as vaccine candidates.

Legionaminic acids are analogs of sialic acid that occur in cell surface glycoconjugates of several bacteria. Because legionaminic acids share the same stereochemistry as sialic acid but differ at C7 and C9, they are interesting analogs to probe the impact of varying exocyclic moieties (C7–C9) on biological activities such as susceptibilities to sialidases, interactions with Siglecs and immunogenicity. There are currently no reports on the bacterial enzymes that transfer legionaminic acids to these cell surface glycoconjugates, but some mammalian and bacterial sialyltransferases display donor promiscuity and can use CMP-Leg5,7Ac2 efficiently enough to transfer Leg5,7Ac2 to their natural acceptor glycans. When the natural activity with CMP-Leg5,7Ac2 is significant but relatively low, an alternate strategy has been to engineer versions with improved activity to transfer Leg5,7Ac2. Importantly, we have found that some bacterial sialyltransferases are very efficient for transferring Leg5,7Ac2 to small synthetic glycans with various aglycones. The two mammalian sialyltransferases that have been tested so far (porcine ST3Gal1 and human ST6Gal1) were found to be more efficient than the bacterial sialyltransferases for the modification of glycoproteins. We provide a review of the sialyltransferases selected to modify different types of glycoconjugates with Leg5,7Ac2, including small synthetic acceptors, glycolipids, and glycoproteins. In the first part, we also propose an optimized biosynthetic pathway for in vitro preparation of the donor CMP-Leg5,7Ac2, based on enzymes selected from two bacteria that naturally produce legionaminic acid., Series: Methods in Enzymology; no. 597

Mimics of natural carbohydrates (glycomimetics) and unnatural glycoconjugates are under intense scrutiny for the development of anti-infective therapies alternative or complementary to classical antibiotics. Three current approaches are reviewed in this article: (1) antiadhesive therapies, that focus on disrupting carbohydrate-protein complexes involved in microbial adhesion to host cells; (2) vaccines that use as antigens either polysaccharide fragments from bacterial and viruses surface or bacterial and viral lectins; (3) small molecule glycomimetic inhibitors of bacterial enzymes and antivirals.

Lectin-glycoconjugate interactions are essential for numerous cellular and extracellular events. Binding stoichiometry is one of several factors that regulate the initiation and outcome of lectin-mediated biological functions. Therefore, the knowledge of accurate stoichiometry of glycoconjugate binding is pivotal for delineating the molecular mechanism of lectin-dependent processes. Quantitative precipitation and isothermal titration calorimetry can reliably determine the stoichiometry of glycoconjugate recognition by lectins. Stoichiometry plays important roles in affinity enhancement and it influences the structures of lectin-glycoconjugate cross-linked complexes. Correct stoichiometry helped reveal that the binding epitopes (glycans) on multi- and polyvalent glycoconjugates possess decreasing microscopic binding affinities for lectins and the binding of lectin involves an internal diffusion process called “bind and jump.” Interestingly, in certain cases, stoichiometry is unable to regulate affinity enhancement, binding thermodynamics, kinetics of cross-linking, and the lattice structures of the cross-linked complexes.

Synthetic oligosaccharide-based vaccines are promising alternatives to conventional antibacterial carbohydrate vaccines prepared with isolated polysaccharides. Unlike polysaccharides, synthetic glycans are well defined, contaminant-free, and accessible even for pathogens that cannot be fermented or show limited carbohydrate biosynthesis in vitro. However, identifying synthetic glycan B cell epitopes that induce protective immunity has traditionally been a time-consuming trial-and-error process, as predicting the immunogenicity of an oligosaccharide by means of structure alone is not straightforward. We here describe how synthetic oligosaccharide epitopes for candidate vaccines can be rationally identified prior to preclinical immunogenicity studies. Epitopes are selected on the basis of their recognition by antibodies associated with protection from disease in humans or small animals. In addition, we show how murine antibody responses to a large oligosaccharide can inform the identification of a minimal B cell epitope that may help designing easy to synthesize vaccine candidates. The procedures, exemplified with a surface carbohydrate of Clostridium difficile, may serve as a guideline for selecting protective oligosaccharide epitopes for vaccines against infectious and malignant diseases.

Comparative study of the fingerprinting patterns of oligosaccharides in individual human milk revealed the presence of three different patterns. Four oligosaccharides containing the Fucα1-2Gal group were missing in the milk of nonsecretor mothers, and three oligosaccharides containing the Fucα1-4GlcNAc group were missing in the milk of Lewis-negative individuals. Disappearance of some oligosaccharides in these samples led to the finding of five novel minor oligosaccharides, which were hidden under the missing oligosaccharides. Structures of many novel milk oligosaccharides were then elucidated. At least 13 core oligosaccharides were found among these oligosaccharides. By adding α-fucosyl residues and sialic acid residues to these core oligosaccharides, more than 100 oligosaccharides were formed. All these oligosaccharides contain lactose at their reducing termini. This evidence, together with the deletion phenomena found in the milk oligosaccharides of nonsecretor and Lewis negative individuals, suggested that the oligosaccharides are derived from lactose via the concerted action of glycosyltransferases, which are responsible for elongation and branching of the Galβ1-4GlcNAc group in the sugar chains of glycoconjugates on the surface of epithelial cells. Therefore, oligosaccharides in human milk could include many structures, starting from the Galβ1-4GlcNAc group in the sugar chains of various glycoconjugates produced by epithelial cells of the mammary gland, and play roles as various recognition signals.

Objectives Sulfated glycoconjugates (S-GCs) are essential components of dentinogenesis and osteogenesis, and are involved in the regulation of the mineralization process. The scales of Polypterus senegalus , which are homologous to teeth and phylogenetically derived from ancestral dermal skeleton, comprise true enamel, dentine, and bone. As part of the phylogenic evolutional studies on mesenchyme-derived hard tissues, we investigated the ultrastructural distribution patterns and histochemical properties of S-GCs in the dentine and bone of scales. Methods For detection of S-GCs, a high iron diamine–thiocarbohydrazide–silver proteinate (HID–TCH–SP) staining technique was used. Results S-GCs were observed in unmineralized predentine and osteoid, mineralized dentine, osteocyte pericellular matrix, but not in mineralized bone and isopedine. Most S-GCs in the dentine and osteoid were susceptible to testicular hyaluronidase, indicating that they are chondroitin sulfates. Conclusion Distinct differences in the S-GC distribution pattern between the dentine and bone were observed. The S-GC distribution pattern associated with the dentinogenesis of scales is very similar to that of mammalian mantle dentine.