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Protonated ions of fucose-containing oligosaccharides are prone to undergo internal glycan rearrangement which results in chimeric fragments that obfuscate mass-spectrometric analysis. Lack of accessible tools that would facilitate systematic analysis of glycans in the gas phase limits our understanding of this phenomenon. In this work, we use density functional theory modeling to interpret cryogenic IR spectra of Lewis a and blood group type H1 trisaccharides and to establish whether these trisaccharides undergo the rearrangement during gas-phase analysis. Structurally unconstrained search reveals that none of the parent ions constitute a thermodynamic global minimum. In contrast, predicted collision cross sections and anharmonic IR spectra provide a good match to available experimental data which allowed us to conclude that fucose migration does not occur in these antigens. By comparing the predicted structures with those obtained for Lewis x and blood group type H2 epitopes, we demonstrate that the availability of the mobile proton and a large difference in the relative stability of the parent ions and rearrangement products constitute the prerequisites for the rearrangement reaction.

The enzymatic synthesis of hybrid Lewis antigens including KH-1 (Lewis y-Lewis x-Lactose, Le y -Le x -Lac), Lewis a-Lewis x-Lactose (Le a -Le x -Lac), and Lewis b-Lewis x-Lactose (Le b -Le x -Lac) has been achieved using a facile enzymatic modular assembly strategy. Starting from a readily available tetrasaccharide, 3 complex hybrid Lewis antigens were achieved in over 40% total yields in less than 5 linear steps of sequential enzymatic glycosylation using 6 enzyme modules. The regio-selective fucosylation was achieved by simply controlling the donor-acceptor ratio. This strategy provides an easy access to these biologically important complex hybrid Lewis antigens at preparative scales.

Immunotherapy has been successful in treating many tumour types. The development of additional tumour-antigen binding monoclonal antibodies (mAbs) will help expand the range of immunotherapeutic targets. Lewis histo-blood group and related glycans are overexpressed on many carcinomas, including those of the colon, lung, breast, prostate and ovary, and can therefore be selectively targeted by mAbs. Here we examine the molecular and structural basis for recognition of extended Lea and Lex containing glycans by a chimeric mAb. Both the murine (FG88.2) IgG3 and a chimeric (ch88.2) IgG1 mAb variants showed reactivity to colorectal cancer cells leading to significantly reduced cell viability. We determined the X-ray structure of the unliganded ch88.2 fragment antigen-binding (Fab) containing two Fabs in the unit cell. A combination of molecular docking, glycan grafting and molecular dynamics simulations predicts two distinct subsites for recognition of Lea and Lex trisaccharides. While light chain residues were exclusively used for Lea binding, recognition of Lex involved both light and heavy chain residues. An extended groove is predicted to accommodate the Lea-Lex hexasaccharide with adjoining subsites for each trisaccharide. The molecular and structural details of the ch88.2 mAb presented here provide insight into its cross-reactivity for various Lea and Lex containing glycans. Furthermore, the predicted interactions with extended epitopes likely explains the selectivity of this antibody for targeting Lewis-positive tumours., (© 2020 The Author(s). Published by Portland Press Limited on behalf of the Biochemical Society.)

Initial cell attachment of rotavirus (RV) to specific cell surface glycan receptors, which is the essential first step in RV infection, is mediated by the VP8* domain of the spike protein VP4. Recently, human histo-blood group antigens (HBGAs) have been identified as receptors or attachment factors for human RV strains. RV strains in the P[4] and P[8] genotypes of the P[II] genogroup share common recognition of the Lewis b (Leb) and H type 1 antigens, however, the molecular basis of receptor recognition by the major human P[8] RVs remains unknown due to lack of experimental structural information. Here, we used nuclear magnetic resonance (NMR) spectroscopy-based titration experiments and NMR-derived high ambiguity driven docking (HADDOCK) methods to elucidate the molecular basis for P[8] VP8* recognition of the Leb (LNDFH I) and type 1 HBGAs. We also used X-ray crystallography to determine the molecular details underlying P[6] recognition of H type 1 HBGAs. Unlike P[6]/P[19] VP8*s that recognize H type 1 HBGAs in a binding surface composed of an α-helix and a β-sheet, referred as the "βα binding site", the P[8] and P[4] VP8*s bind Leb HBGAs in a previously undescribed pocket formed by the edges of two β-sheets, referred to as the "ββ binding site". Importantly, the P[8] and P[4] VP8*s retain binding capability to non-Leb type 1 HBGAs using the βα binding site. The presence of two distinct binding sites for Leb and non-Leb HBGA glycans in the P[8] and P[4] VP8* domains suggests host-pathogen co-evolution under structural and functional adaptation of RV pathogens to host glycan polymorphisms. Assessment and understanding of the precise impact of this co-evolutionary process in determining RV host ranges and cross-species RV transmission should facilitate improved RV vaccine development and prediction of future RV strain emergence and epidemics., Competing Interests: The authors have declared that no competing interests exist.

Carbohydrate sequences are important for various biological processes. It has recently been estimated to have 100,000-500,000 carbohydrate structures in mammalian glycome. However, the peripheral carbohydrate determinants on N- and O-glycoproteins, glycolipids, polysaccharides and secreted free sugars are limited in numbers. Among these blood-group-related antigens the ABO(H)- and Lewis-types are particularly important. Negative-ion MS/MS has been successfully used in assignment of these epitopes on free reducing sugars but cannot be applied to reduced sugars, e.g. O-glycans typically released from mucins as alditols, or in positive-ion detection of either reducing or reduced oligosaccharides. In the present study, we investigate the fragmentation features of permethylated reducing and reduced sugars under positive-ion conditions of multi-stage MALDI-MS, and propose the concept of epitope ion and epitope spectrum for determination of peripheral blood-group related epitopes on secreted human milk oligosaccharides and N-glycans as reducing sugars and O-glycans as reduced alditols in conjunction with MALDI-MS glycan profiling., (Copyright © 2019 Elsevier Ltd. All rights reserved.)

Human ovarian cyst glycoproteins (HOC, cyst gps) isolated from pseudomucinous type of human ovarian cyst fluids is one of the richest and pioneer sources for studying biosynthesis, structures and functional roles of blood group ABH, Le a,b,x,y , sLe a and sLe x active glycoproteins. After 70 + years of exploration, four top highlights are shared. (i) an updated concept of glycotopes and their internal structures in cyst gps was composited; (ii) the unknown codes of new genes in secreted cyst gps were unlocked as Le x and Le y ; (iii) recognition profiles of cyst glycans and a sialic acid-rich (18%) glycan with lectins and antibodies were shown. (iv) Co-expression of Blood Group A/ A-Le b/y and B/B-Le b/y active Glycotopes in the same glycan chains were isolated and illustrated. These are the most advanced achievements since 1980.

Analogues of cancer-associated Lewis Y (Le y ) antigen with varying structures at the reducing end were synthesized by a highly efficient strategy involving one-pot preactivation-based iterative glycosylation to obtain the key tetra-/pentasaccharide intermediates, which was followed by stereoselective fucosylation. After global deprotection, these oligosaccharides were coupled with carrier protein keyhole limpet hemocyanin. The resultant glycan-protein conjugates were subjected to immunological studies in mice. It was disclosed that the conjugate of the pentasaccharide analogue of Lewis Y antigen was more immunogenic than that of the hexasaccharide analogue, but the antisera of both conjugates could indiscriminately recognize each carbohydrate hapten. These results suggested that the short Lewis Y analogue may be utilized to develop functional conjugate cancer vaccines. More importantly, the results also proved that the reducing-end glucose residue in the hexasaccharide analogue of Lewis Y was probably not involved in its interaction with the immune system, whose discovery can have a broad impact on the design of new cancer vaccines.

Mass spectrometry (MS) has become the primary method for high-sensitivity structural determination of oligosaccharides. Fragmentation in the negative-ion MS can provide a wealth of structural information and these can be used for sequence determination. However, although negative-ion MS of neutral oligosaccharide using the deprotonated molecule [M-H] - as the precursor has been very successful for electrospray ionization (ESI), it has only limited success for matrix-assisted laser desorption/ionization (MALDI). In the present study, the features of negative-ion MALDI primary spectra were investigated in detail and the product-ion spectra using [M-H] - and [M+Cl] - as the precursors were carefully compared. The formation of [M-H] - was the main difficulty for MALDI while [M+Cl] - was proved to be useful as alternative precursor anion for MALDI-MS/MS to produce similar fragmentation for sequencing of neutral oligosaccharides. N-(1-naphthyl)ethylenediamine dihydrochloride was then used as both the matrix and the Cl - dopant to evaluate the extent of structural information that can be obtained by negative-ion fragmentation from [M+Cl] - using laser-induced dissociation (LID)-MS/MS for linkage assignment of gluco-oligosaccharides and for typing of blood-group ABO(H) and Lewis antigens on either type 1 or type 2 backbone-chains., (Copyright © 2019 Elsevier B.V. All rights reserved.)

Mumps virus (MuV) is an important aerosol-transmitted human pathogen causing epidemic parotitis, meningitis, encephalitis, and deafness. MuV preferentially uses a trisaccharide containing α2,3-linked sialic acid as a receptor. However, given the MuV tropism toward glandular tissues and the central nervous system, an additional glycan motif(s) may also serve as a receptor. Here, we performed a large-scale glycan array screen with MuV hemagglutinin-neuraminidase (MuV-HN) attachment proteins by using 600 types of glycans from The Consortium for Functional Glycomics Protein-Glycan Interaction Core in an effort to find new glycan receptor motif(s). According to the results of the glycan array, we successfully determined the crystal structures of MuV-HN proteins bound to newly identified glycan motifs, sialyl Lewis X (SLe X ) and the oligosaccharide portion of the GM2 ganglioside (GM2-glycan). Interestingly, the complex structures showed that SLe X and GM2-glycan share the same configuration with the reported trisaccharide motif, 3'-sialyllactose (3'-SL), at the binding site of MuV-HN, while SLe X and GM2-glycan have several unique interactions compared with those of 3'-SL. Thus, MuV-HN protein can allow an additional spatial modification in GM2-glycan and SLe X at the second and third carbohydrates from the nonreducing terminus of the core trisaccharide structure, respectively. Importantly, MuV entry was efficiently inhibited in the presence of 3'-SL, SLe X , or GM2-glycan derivatives, which indicates that these motifs can serve as MuV receptors. The α2,3-sialylated oligosaccharides, such as SLe X and 3'-sialyllactosamine, are broadly expressed in various tissues, and GM2 exists mainly in neural tissues and the adrenal gland. The distribution of these glycan motifs in human tissues/organs may have bearing on MuV tropism. IMPORTANCE Mumps virus (MuV) infection is characterized by parotid gland swelling and can cause pancreatitis, orchitis, meningitis, and encephalitis. MuV-related hearing loss is also a serious complication because it is usually irreversible. MuV outbreaks have been reported in many countries, even in high-vaccine-coverage areas. MuV has tropism toward glandular tissues and the central nervous system. To understand the unique MuV tropism, revealing the mechanism of receptor recognition by MuV is very important. Here, using a large-scale glycan array and X-ray crystallography, we show that MuV recognizes sialyl Lewis X and GM2 ganglioside as receptors, in addition to a previously reported MuV receptor, a trisaccharide containing an α2,3-linked sialic acid. The flexible recognition of these glycan receptors by MuV may explain the unique tropism and pathogenesis of MuV. Structures will also provide a template for the development of effective entry inhibitors targeting the receptor-binding site of MuV., (Copyright © 2019 American Society for Microbiology.)

The caudal-related homeobox protein 1 (CDX1) is a transcription factor, which is important in the development, differentiation, and homeostasis of the gut. Although the involvement of CDX genes in the regulation of the expression levels of a few glycosyltransferases has been shown, associations between glycosylation phenotypes and CDX1 mRNA expression have hitherto not been well studied. Triggered by our previous study, we here characterized the N -glycomic phenotype of 16 colon cancer cell lines, selected for their differential CDX1 mRNA expression levels. We found that high CDX1 mRNA expression associated with a higher degree of multi-fucosylation on N -glycans, which is in line with our previous results and was supported by up-regulated gene expression of fucosyltransferases involved in antenna fucosylation. Interestingly, hepatocyte nuclear factors (HNF)4A and HNF1A were, among others, positively associated with high CDX1 mRNA expression and have been previously proven to regulate antenna fucosylation. Besides fucosylation, we found that high CDX1 mRNA expression in cancer cell lines also associated with low levels of sialylation and galactosylation and high levels of bisection on N -glycans. Altogether, our data highlight a possible role of CDX1 in altering the N -glycosylation of colorectal cancer cells, which is a hallmark of tumor development.

An efficient streamlined chemoenzymatic approach has been developed for gram-scale synthesis of Lewis a angtigen (Le a βProN 3 ) and a library of sialyl Lewis a antigens (sLe a βProN 3 ) containing different sialic acid forms. Intially, commercially available inexpensive N-acetylglucosamine (GlcNAc) was converted to its N'-glycosyl p-toluenesulfonohydrazide in one step. Followed by chemical glycosylation, GlcNAcβProN 3 was synthesized using this protecting group-free method in high yield (82%). Sequential one-pot multienzyme (OPME) β1-3-galactosylation of GlcNAcβProN 3 followed by OPME α1-4-fucosylation reactions produced target Le a βProN 3 in gram-scale. Structurally diverse sialic acid forms was successfully introduced using a OPME sialylation reation containing a CMP-sialic acid synthetase and Pasteurella multocida α2-3-sialyltransferase 1 (PmST1) mutant PmST1 M144D with or without a sialic acid aldolase to form sLe a βProN 3 containing naturally occurring or non-natural sialic acid forms in preparative scales., (Copyright © 2018 Elsevier Ltd. All rights reserved.)

There is considerable potential for the use of ion mobility mass spectrometry in structural glycobiology due in large part to the gas-phase separation attributes not typically observed by orthogonal methods. Here, we evaluate the capability of traveling wave ion mobility combined with negative ion collision-induced dissociation to provide structural information on N-linked glycans containing multiple fucose residues forming the Lewis x and Lewis y epitopes. These epitopes are involved in processes such as cell-cell recognition and are important as cancer biomarkers. Specific information that could be obtained from the intact N-glycans by negative ion CID included the general topology of the glycan such as the presence or absence of a bisecting GlcNAc residue and the branching pattern of the triantennary glycans. Information on the location of the fucose residues was also readily obtainable from ions specific to each antenna. Some isobaric fragment ions produced prior to ion mobility could subsequently be separated and, in some cases, provided additional valuable structural information that was missing from the CID spectra alone. Graphical abstract ᅟ.

Prolactin-inducible protein (PIP) is a glycoprotein found in body secretions from exocrine glands like saliva and seminal plasma. Important biological functions of PIP concentrations have been demonstrated, e.g. in tumor diagnosis and progression. PIP quantity has been also found useful to determine the success of chemotherapy of mammary carcinoma. Here, we present the analysis of the N-glycosylation of PIP isolated from different sources by LC-MS(/MS) and 1 H-NMR. We found a very uncommon N-type glycosylation of PIP in healthy individuals from both, seminal fluid and saliva. PIP carries unusual highly fucosylated N-linked glycans with multiple Lewis y (Le y ) epitopes on bi-, tri- and tetraantennary structures resulting in up to nine fucosyl residues on a tetraantennary glycan. In most organs, Le y epitopes are not present on N-glycans except in case of a tumor when it is highly up-regulated and important for prognosis. Here, for the first time on a specific glycoprotein Le y antigens are unambiguously characterized on an N-type glycan by NMR spectroscopy. So far, for specific glycoproteins Le y epitopes had only been reported on O-glycans. Furthermore, a correlation between a nonsynonymous single nucleotide polymorphism (SNP) and glycosylation pattern was detected: individuals heterozygous for the SNP causing the amino acid exchange 51 Gln to 51 His have glycan structures with a higher degree of sialylation compared to individuals lacking the SNP.

Helicobacter pylori α1-3/4-fucosyltransferase (Hp3/4FT) was expressed in Escherichia coli at a level of 30 mg L -1 culture and used as a diverse catalyst in a one-pot multienzyme (OPME) system for high-yield production of l-fucose-containing carbohydrates including Lewis antigens such as Lewis a, b, and x, O-sulfated Lewis x, and sialyl Lewis x and human milk fucosides such as 3-fucosyllactose (3-FL), lacto-N-fucopentaose (LNFP) III, and lacto-N-difuco-hexaose (LNDFH) II and III. Noticeably, while difucosylation of tetrasaccharides was readily achieved using an excess amount of donor, the synthesis of LNFP III was achieved by Hp3/4FT-catalyzed selective fucosylation of the N-acetyllactosamine (LacNAc) component in lacto-N-neotetraose (LNnT).

Helicobacter pylori infect millions of people around the world. It occupies a niche in the human gastrointestinal tract characterized by high expression of a repertoire of carbohydrates. ABO and Lewis histo-blood group systems are controlled by genes coding for functional glycosyltransferases which synthesize great diversity of related fucosylated carbohydrate in different tissues, including gastrointestinal mucosa, and exocrine secretions. The structural diversity of histo-blood group carbohydrates is highly complex and depends on epistatic interactions among gene-encoding glycosyltransferases. The histo-blood group glycosyltransferases act in the glycosylation of proteins and lipids in the human gastrointestinal tract allowing the expression of a variety of potential receptors in which H. pylori can adhere. These oligosaccharide molecules are part of the gastrointestinal repertoire of carbohydrates which act as potential receptors for microorganisms, including H. pylori. This Gram-negative bacillus is one of the main causes of the gastrointestinal diseases such as chronic active gastritis, peptic ulcer, and cancer of stomach. Previous reports showed that some H. pylori strains use carbohydrates as receptors to adhere to the gastric and duodenal mucosa. Since some histo-blood group carbohydrates are highly expressed in one but not in others histo-blood group phenotypes it has pointed out that quantitative differences among them influence the susceptibility to diseases caused by H. pylori. Additionally, some experiments using animal model are helping us to understand how this bacillus explore histo-blood group carbohydrates as potential receptors, offering possibility to explore new strategies of management of infection, disease treatment, and prevention. This text highlights the importance of structural diversity of ABO and Lewis histo-blood group carbohydrates as facilitators for H. pylori infection., (Copyright © 2017 Elsevier B.V. All rights reserved.)

Molecular assemblies varying morphologies in a wide range from spherical micelle, nanosheet, curved sheet, nanotube and vesicle were prepared and loaded with Lewis y (Le y ) tumor-associated carbohydrate antigen on the assembly surface. The molecular assemblies were composed of poly(sarcosine) m -block-poly(L-lactic acid) 30 (m = 15 or 50, Lactosome), poly(sarcosine) m -block-(D/L-Leu-Aib) n (m = 22 or 30, n = 6 or 8) and their combinations. The molecular assemblies carrying Le y on the surface were administered in BALB/c nu/nu mice. The major epitopes of the molecular assemblies are commonly Le y and poly(sarcosine). IgM productions upon administrations of the molecular assemblies were assayed by ELISA, showing that anti-poly(sarcosine) IgM was highly produced by Lactosome of spherical micelle but with a negligible amount of anti-Le y IgM. On the other hand, the nanosheet of the interdigitated monolayer triggered the production of anti-Le y IgM but with less anti-poly(sarcosine) IgM production. Taken together, IgM specificity differs according to the molecular environment of the epitopes in the molecular assemblies. The antigenicity of poly(sarcosine) was augmented in polymeric micelle providing loose environment for B cells to penetrate in, whereas a high density of Le y on the molecular assembly was required for anti-Le y IgM production. The antigenicity of Le y is therefore dependent on the molecular assemblies on which Le y is displayed on the surface. Copyright © 2016 European Peptide Society and John Wiley & Sons, Ltd., (Copyright © 2016 European Peptide Society and John Wiley & Sons, Ltd.)

The Japanese cedar pollen allergen (Cry j1) and the mountain cedar pollen allergen (Jun a1) are glycosylated with plant complex type N-glycans bearing Lewis a epitope(s) (Galβ1-3[Fucα1-4]GlcNAc-). The biological significance of Lewis a type plant N-glycans and their effects on the human immune system remain to be elucidated. Since a substantial amount of such plant specific N-glycans are required to evaluate immunological activity, we have searched for good plant-glycan sources to characterize Lewis a epitope-containing plant N-glycans. In this study, we have found that three water plants, Elodea nuttallii, Egeria densa, and Ceratophyllum demersum, produce glycoproteins bearing Lewis a units. Structural analysis of the N-glycans revealed that almost all glycoproteins expressed in these three water plants predominantly carry plant complex type N-glycans including the Lewis a type, suggesting that these water plants are good sources for preparation of Lewis a type plant N-glycans in substantial amounts., (Copyright © 2016 Elsevier Ltd. All rights reserved.)

Seven lipophilic constructs containing Lewis (Le a , Le b , Le y ) or chimeric Lewis/ABH (ALe b , BLe b , ALe y , BLe y ) glycans were obtained starting from corresponding oligosaccharides in form of 3-aminopropyl glycosides. ALe b and BLe b pentasaccharides were synthesized via [3 + 1] blockwise approach. The constructs (neoglycolipids, or FSLs) were inserted in erythrocyte membrane, and obtained "kodecytes" were used to map the immunochemical specificity of historical and contemporary monoclonal and polyclonal blood group system Lewis reagents., (Copyright © 2016. Published by Elsevier Ltd.)

The novel and efficient synthesis of type 2 Lewis antigens is reported in this study. The rationally designed lactosamine-3,2'-diol derivative with an orthogonal set of protecting groups is efficiently glycosylated with a benzyl protected 1-thio-l-fucoside donor in a unique regioselective manner to produce Lewis x (Le(x)) and Lewis y (Le(y)) derivatives in good yields. These derivatives can be prepared not only exclusively but also synchronously by choosing the appropriate reaction temperature and donor-acceptor molar ratio. The Le(x) derivatives are easily converted into sulfated or non-sulfated Le(x) bearing a terminal azido functionalized oligo-(ethyleneoxide) linker; the Le(y) derivative having the same linker can also be prepared, all of which can be further used for the chemical modification of other compounds and materials., (Copyright © 2016 Elsevier Ltd. All rights reserved.)

Blood group oligosaccharides are one of the most clinically important antigen families and they may also act as secondary ligands for bacterial toxins from Escherichia coli and Vibrio cholerae. Herein we report the synthesis of spacered (sp = CH2CH2CH2NH2) glycosides of A antigen {α-D-GalNAc-(l→3)-[α-L-Fuc-(l→2)]-β-D-Gal-}, B antigen{α-D-Gal-(l→3)-[α-L-Fuc-(l→2)]-β-D-Gal-}, LewisX{α-D-Gal-(l→4)-[α-L-Fuc-(l→3)]-β-D-GlcNAc-}, A type-II {α-D-GalNAc-(l→3)-[α-L-Fuc-(l→2)]-β-D-Gal-(1→4)-β-D-GlcNAc-}, B type-II {α-D-Gal-(l→3)-[α-L-Fuc-(l→2)]-β-D-Gal-(1→4)-β-D-GlcNAc-}, H type-II{α-L-Fuc-(l→2)-β-D-Gal-(1→4)-β-D-GlcNAc-}, xenoantigen {α-D-Gal-(l→3)-β-D-Gal-(1→4)-[α-L-Fuc-(l→2)]-β-D-GlcNAc-} and Linear B Type II {α-D-Gal-(l→3)-β-D-Gal-(1→4)-β-D-GlcNAc-} useful for a range of biochemical investigations. This linker was chosen so as to facilitate the future conjugation of the antigens to proteins or other molecules. We also measured the affinities of some synthesized oligosaccharides against El Tor CTB strain from V. cholera.

Background: Non-secretor individuals lack ABO blood group antigens in their secretions like saliva; these carbohydrate structures play an important role in protection of the oral cavity from exogenous pathogens; therefore these individuals are more susceptible to mucous membrane damages. The aim was to assess the secretory state of patients with oral lichen planus (OLP) in comparison with healthy controls., Methods: Fifty patients and 100 age-gender matched control subjects were recruited to the study. Patients were visited in the outpatient clinic of dermatology at Shohada-e-Tajrish Hospital, Shahid Beheshti University of Medical Sciences from 2012 - 2014. Two-milliliter (mL) blood was collected from each subject to detect Lewis phenotypes. According to Lewis phenotype of each subject, secretory state was determined except in subjects with Le (a-b-) phenotype, in whom saliva was collected to determine the secretor status., Results: Non-secretor status in patients with OLP was more frequent compared with healthy controls (37 out of 50 patients (74%) vs. 24 out of 100 healthy controls (24%), (P < 0.001)). There was no association between secretory state, and type of OLP and disease duration (P > 0.05)., Conclusion: This study supported the possible role of cell surface histo-blood group antigens in protection of mucosal surface from exogenous pathogens. Therefore, it appears that non-secretor individuals are more prone to oral lichen planus.

Monoclonal antibodies represent the most successful class of biopharmaceuticals for the treatment of cancer. Mechanisms of action of therapeutic antibodies are very diverse and reflect their ability to engage in antibody-dependent effector mechanisms, internalize to deliver cytotoxic payloads, and display direct effects on cells by lysis or by modulating the biological pathways of their target antigens. Importantly, one of the universal changes in cancer is glycosylation and carbohydrate-binding antibodies can be produced to selectively recognize tumor cells over normal tissues. A promising group of cell surface antibody targets consists of carbohydrates presented as glycolipids or glycoproteins. In this review, we outline the basic principles of antibody-based targeting of carbohydrate antigens in cancer. We also present a detailed structural view of antibody recognition and the conformational properties of a series of related tissue-blood group (Lewis) carbohydrates that are being pursued as potential targets of cancer immunotherapy., (Copyright © 2015 Elsevier Ltd. All rights reserved.)

Background: Angiogenesis is a fundamental process underlying cancer progression and autoimmune disease. Lewis Y is known as a regulated glycan-structure supporting human endothelial function and angiogenesis., Objectives: We hypothesize that Lewis Y based analogues interfere with Lewis Y mediated endothelial functions and angiogenesis. We therefore evaluated the ability of 3, 4-bis [(b-D-galactopyranosyl)osy]-methyl-furan (BGF) a furan-based Lewis-Y saccharide mimetic to inhibit human endothelial adhesion, migration and in vitro angiogenesis., Material and Methods: The ability of BGF and additional furan-based saccharide-mimetics was investigated to inhibit adhesion and migration of human bone marrow endothelial cells (HBMEC). Influence of BGF was tested on a multicelluar in vitro - angiogenesis assay in the presence of VEGF., Results: BGF significantly inhibited HBMEC adhesion and migration stimulated by TNF-alpha by up to 70%. The anti-adhesive effect of BGF was particularly evident when HBMEC adhesion and migration was tested on collagen as extracellular matrix with weaker effect when laminin and fibronectin were used as an extracellular matrix. BGF was ineffective when HBMEC were stimulated with VEGF. The inhibition of endothelial function translated into a significant inhibitory effect of BGF in the multicellular in vitro angiogenesis-assay. BGF reduced the angiogenesis index compared to the positive controls by 32%., Conclusions: We identified the ability of the furan-based Lewis Y saccharide mimetic BGF as a specific modulator of TNF-alpha activated endothelial function and in vitro angiogenesis. BGF and other related glycan analogues should further be explored for their ability to down modulate endothelial activation in TNF-alpha driven pathophysiologic conditions in autoimmune disease and cancer indications.

Human noroviruses (huNoVs) recognize histo-blood group antigens (HBGAs) as attachment factors, in which genogroup (G) I and GII huNoVs use distinct binding interfaces. The genetic and evolutionary relationships of GII huNoVs under selection by the host HBGAs have been well elucidated via a number of structural studies; however, such relationships among GI NoVs remain less clear due to the fact that the structures of HBGA-binding interfaces of only three GI NoVs with similar binding profiles are known. In this study the crystal structures of the P dimers of a Lewis-binding strain, the GI.8 Boxer virus (BV) that does not bind the A and H antigens, in complex with the Lewis b (Le(b)) and Le(y) antigens, respectively, were determined and compared with those of the three previously known GI huNoVs, i.e. GI.1 Norwalk virus (NV), GI.2 FUV258 (FUV) and GI.7 TCH060 (TCH) that bind the A/H/Le antigens. The HBGA binding interface of BV is composed of a conserved central binding pocket (CBP) that interacts with the β-galactose of the precursor, and a well-developed Le epitope-binding site formed by five amino acids, including three consecutive residues from the long P-loop and one from the S-loop of the P1 subdomain, a feature that was not seen in the other GI NoVs. On the other hand, the H epitope/acetamido binding site observed in the other GI NoVs is greatly degenerated in BV. These data explain the evolutionary path of GI NoVs selected by the polymorphic human HBGAs. While the CBP is conserved, the regions surrounding the CBP are flexible, providing freedom for changes. The loss or degeneration of the H epitope/acetamido binding site and the reinforcement of the Le binding site of the GI.8 BV is a typical example of such change selected by the host Lewis epitope.

Carbohydrate-protein recognition is vital to many processes in health and disease. In particular, elucidation of the structural basis of carbohydrate binding is important to the development of oligosaccharides and oligosaccharide mimetics as vaccines for infectious diseases and cancer. Computational structural techniques are valuable for the study of carbohydrate-protein recognition due to the challenges associated with experimental determination of carbohydrate-protein complexes. AutoMap is a computer program that we have developed to study protein-ligand recognition. AutoMap determines the interactions taking place in a set of highly ranked poses obtained from molecular docking and processes these to identify the protein residues most likely to be involved in interactions. In this protocol, we describe the use of AutoMap and illustrate its suitability for studying antibody recognition of the Lewis Y tetrasaccharide, which is a potential cancer vaccine antigen.

Antibody response to carbohydrate antigens is often independent of T cells and the process of affinity/specificity improvement is considered strictly dependent on the germinal centers. Antibodies induced during a T cell-independent type 2 (TI-2) response are less variable and less functionally versatile than those induced with T cell help. The antigen specificity consequences of accumulation of somatic mutations in antibodies during TI-2 responses of Marginal Zone (MZ) B cells is a fact that still needs explanation. Germline genes that define carbohydrate-reactive antibodies are known to sculpt antibody-combining sites containing innate, key side-chain contacts that define the antigen recognition step. However, substitutions associated with MZ B cell derived antibodies might affect the mobility and polyspecificity of the antibody. To examine this hypothesis, we analyzed antibodies reactive with the neolactoseries antigen Lewis Y (LeY) to define the residue subset required for the reactive repertoire for the LeY antigen. Our molecular simulation studies of crystallographically determined and modeled antibody-LeY complexes suggests that the heavy-chain germline gene VH7183.a13.20 and the light-chain Vκ cr1 germline gene are sufficient to account for the recognition of the trisaccharide-H determinant Types 1-4, while the specificity for LeY is driven by the CDR3 backbone conformation of the heavy chain and not the side chain interactions. These results confirm that these monoclonals use germline-encoded amino acids to recognize simple carbohydrate determinants like trisaccharide-H but relies on somatic mutations in the periphery of the combining site to modify affinity for LeY through electrostatic interactions that leads to their optimized binding. These observations bring further attention to the role of mutations in T-cell independent antibodies to distinguish self from non-self carbohydrate antigens.

Human milk oligosaccharides (HMOs) are a major constituent of human breast milk and play an important role in reducing the risk of infections in infants. The structures of these HMOs show similarities with blood group antigens in protein glycosylation, in particular in relation to fucosylation in Lewis blood group-type epitopes, matching the maternal pattern. Previously, based on the Secretor and Lewis blood group system, four milk groups have been defined, i.e. Lewis-positive Secretors, Lewis-positive non-Secretors, Lewis-negative Secretors and Lewis-negative non-Secretors. Here, a rapid one-dimensional (1)H nuclear magnetic resonance (NMR) analysis method is presented that identifies the presence/absence of (α1-2)-, (α1-3)- and (α1-4)-linked fucose residues in HMO samples, affording the essential information to attribute different HMO samples to a specific milk group. The developed method is based on the NMR structural-reporter-group concept earlier established for glycoprotein glycans. Further evaluation of the data obtained from the analysis of 36 HMO samples shows that within each of the four milk groups the relative levels of the different fucosylation epitopes can greatly vary. The data also allow a separation of the Lewis-positive Secretor milk group into two sub-groups., (© The Author 2014. Published by Oxford University Press. All rights reserved. For permissions, please e-mail: journals.permissions@oup.com.)

Recent advances in glycobiology revealed the essential role of lectins for deciphering the glycocode by specific recognition of carbohydrates. Integrated multiscale approaches are needed for characterizing lectin specificity: combining on one hand high-throughput analysis by glycan array experiments and systematic molecular docking of oligosaccharide libraries and on the other hand detailed analysis of the lectin/oligosaccharide interaction by x-ray crystallography, microcalorimetry and free energy calculations. The lectins LecB from Pseudomonas aeruginosa and BambL from Burkholderia ambifaria are part of the virulence factors used by the pathogenic bacteria to invade the targeted host. These two lectins are not related but both recognize fucosylated oligosaccharides such as the histo-blood group oligosaccharides of the ABH(O) and Lewis epitopes. The specificities were characterized using semi-quantitative data from glycan array and analyzed by molecular docking with the Glide software. Reliable prediction of protein/oligosaccharide structures could be obtained as validated by existing crystal structures of complexes. Additionally, the crystal structure of BambL/Lewis x was determined at 1.6 Å resolution, which confirms that Lewis x has to adopt a high-energy conformation so as to bind to this lectin. Free energies of binding were calculated using a procedure combining the Glide docking protocol followed by free energy rescoring with the Prime/Molecular Mechanics Generalized Born Surface Area (MM-GBSA) method. The calculated data were in reasonable agreement with experimental free energies of binding obtained by titration microcalorimetry. The established predictive protocol is proposed to rationalize large sets of data such as glycan arrays and to help in lead discovery projects based on such high throughput technology.

Background: Helicobacter pylori is responsible for gastroduodenal diseases such as chronic gastritis, gastric and duodenal ulcers and also gastric malignances. During an infection, gastric mucins, especially secretory MUC5AC, are said to participate in interactions with bacterial adhesins. Recently, epithelial MUC1 mucin has also been proposed to be engaged in infection development. H. pylori surface possesses lipopolisaccharides with Lewis antigens whose role in interactions with gastric mucins has not been elucidated thoroughly so far., Objectives: To check the involvement of Lewis b and H type 1 structures of H. pylori in adhesion of bacteria to MUC1 mucin., Material and Methods: The study was performed on gastric juices taken from 10 clinical patients and 4 H. pylori strains. Bacteria were assessed for the presence of Lewis b and H type 1 structures by ELISA test. Interactions of H. pylori with MUC1 were analyzed by sandwich ELISA method with bacteria pretreated or not with Lewis b - HSA or H type 1 - HSA glycoconjugates., Results: In the majority of the patients, the slight increase of adhesion of H. pylori to MUC1, after pretreatment of bacteria with H type 1 glycoconjugate was observed. A statistically significant difference was revealed in one strain with a dose of conjugate 50µg/mL (P = 0.05). The influence of Lewis b on adhesion is considered to be contradictory., Conclusions: H type 1 antigens are suggested to be involved in carbohydrate - carbohydrate interactions of H. pylori with MUC1 mucin.

Dendritic cells (DCs) are antigen-presenting cells efficient in capturing pathogens, and processing their antigenic determinants for presentation to antigen-specific T cells to induce robust immune responses. Their location at peripheral tissues and the expression of pattern-recognition receptors, among them DC-specific intercellular adhesion molecule-3-grabbing non-integrin (DC-SIGN), facilitates the capture of pathogens before spreading. However, some pathogens have developed strategies to escape the immune system. One of the most successful is HIV-1, which targets DC-SIGN for transport to the lymph node where the virus infects CD4(+) T cells. Contact of HIV-1 with DC-SIGN is thus the first event in the pathogenic cascade and, therefore, it is the primary target point for therapies aimed at HIV infection prevention. DC-SIGN recognizes specific glycans on HIV-1 and this interaction can be blocked by competitive inhibition through glycans. Although the affinity of glycans is relatively low, multivalency may increase avidity and the strength to compete with HIV-1 virions. We have designed multivalent dendrimeric compounds based on Lewis-type antigens that bind DC-SIGN with high selectivity and avidity and that effectively block gp120 binding to DC-SIGN and, consequently, HIV transmission to CD4(+) T cells. Binding to DC-SIGN and gp120 inhibition was higher on glycodendrimers with larger molecular diameter, indicating that the geometry of the compounds is an important factor determining their functionality. Our compounds elicited DC-SIGN internalization, a property of the receptor upon triggering, but did not affect the maturation status of DCs. Thus, Le(X) glycodendrimers could be incorporated into topic prophylactic approaches for the prevention of HIV-1 transmission.

Human noroviruses cause recurrent epidemics of gastroenteritis known to be dominated by the clinically important GII.4 genotype which recognizes human Secretor gene-dependent ABH histo-blood group antigens (HBGAs) as attachment factors. There is increasing evidence that GII.4 noroviruses have undergone evolutionary changes to recognize Lewis antigens and non-Secretor saliva. In this study, we have investigated the possibilities of the Lewis α1,3/α1,4 fucoses as mediators of binding of GII.4 noroviruses to Lewis antigens. The study was carried out using molecular dynamics simulations of Lewis type-1 and type-2 chain HBGAs in complex with VA387 P domain dimers in explicit water. Based on the computer simulations, we suggest the possibility of two receptor binding modes for Lewis HBGAs: the "Secretor pose" with the Secretor Fucα1,2 in the binding site and the "Lewis pose" with the Lewis Fucα1,3/α1,4 residues in the binding site. This was further supported by an extensive GlyVicinity analysis of the Protein Data Bank with respect to the occurrence of the Lewis and Secretor poses in complexes of Lewis antigens with lectins and antibodies as well as GII norovirus strains. The Lewis pose can also explain the interactions of GII.4 norovirus strains with Le(x) and SLe(x) structures. Moreover, the present model suggests binding of complex branched polysaccharides, with the Lewis antigens at the nonreducing end, to P domain dimers of GII.4 strains. Our results are relevant for understanding the evolution of norovirus binding specificities and for in silico design of future antiviral therapeutics.

Helicobacter pylori infects more than half of the world's population. Although most patients are asymptomatic, persistent infection may cause chronic gastritis and gastric cancer. Adhesion of the bacteria to the gastric mucosa is a necessary prerequisite for the pathogenesis of H. pylori-related diseases and is mediated by mucin O-glycans. In order to define which glycans may be implicated in the binding of the bacteria to the gastric mucosa in humans, we have characterized the exact pattern of glycosylation of gastric mucins. We have identified that the major component was always a core 2-based glycan carrying two blood group H antigens, whatever was the blood group of individuals. We have also demonstrated that around 80% of O-glycans carried blood group A, B or H antigens, suggesting that the variation of gastric mucin glycosylation between individuals is partly due to the blood group status. This study will help better understanding the role of O-glycans in the physiology and homeostasis of gastric mucosa. Overall, the results reported here give us the necessary background information to begin studies to determine whether individuals who express certain carbohydrate epitopes on specific mucins are predisposed to certain gastric diseases.

Since the discovery of human milk oligosaccharides (HMO) >60 y ago, research has faced major challenges including (i) the development of methods to identify and characterize these components, (ii) the need to use HMO fractions for functional studies because single HMO were not available, (iii) the uncertainty of the purity of HMO fractions that were often "contaminated" by remainders of lactose, proteins, or glycoproteins, and (iv) the low availability of large quantities of a single HMO for animal and human studies. In the past 10 years, there has been tremendous progress in all of these areas, particularly in the development of methods for detailed structural analysis in extremely low milk volumes. The greatest success, however, is that biotechnological means are available today to produce large amounts even of a single HMO in a purity that allows human studies to be performed in the future. In this review, we summarize the current knowledge about the metabolic aspects of HMO in infants starting with the first studies by Lundblad and co-workers in the early 1980s. After discussing newer observations in recent years, the review closes with a perspective on some important questions regarding metabolic and functional aspects of HMO.

An exposed F-type lectin domain fused to the N-terminus of a cholesterol-dependent cytolysin scaffold allows Streptococcus mitis lectinolysin to cluster at fucose-rich sites on target cell membranes, thereby leading to increased pore-forming toxin activity. In this issue of Structure, Feil and coworkers define the structural basis for lectinolysin glycan-binding specificity., (Copyright © 2012 Elsevier Ltd. All rights reserved.)

The cholesterol-dependent cytolysins (CDCs) punch holes in target cell membranes through a highly regulated process. Streptococcus mitis lectinolysin (LLY) exhibits another layer of regulation with a lectin domain that enhances the pore-forming activity of the toxin. We have determined the crystal structures of the lectin domain by itself and in complex with various glycans that reveal the molecular basis for the Lewis antigen specificity of LLY. A small-angle X-ray scattering study of intact LLY reveals the molecule is flat and elongated with the lectin domain oriented so that the Lewis antigen-binding site is exposed. We suggest that the lectin domain enhances the pore-forming activity of LLY by concentrating toxin molecules at fucose-rich sites on membranes, thus promoting the formation of prepore oligomers on the surface of susceptible cells., (Copyright © 2012 Elsevier Ltd. All rights reserved.)

In this study we asked whether Helicobacter pylori whole cells and lipopolysaccharide (LPS) utilize sugar moieties of Lewis (Le) antigenic determinants to interact with DC-SIGN (dendritic cell specific ICAM grabbing nonintegrin) receptor on dendritic cells (DCs). For this purpose the soluble DC-SIGN/Fc adhesion assay and the THP-1 leukemia cells with induced expression of DC-SIGN were used. We showed that the binding specificity of DC-SIGN with H. pylori Le(X/Y) positive whole cells and H. pylori LPS of Le(X/Y) type was fucose dependent, whereas in Le(XY) negative H. pylori strains and LPS preparations without Lewis determinants, this binding was galactose dependent. The binding of soluble synthetic Le(X) and Le(Y) to the DC-SIGN-like receptor on THP-1 cells was also observed. In conclusion, the Le(XY) dependent as well as independent binding of H. pylori whole cells and H. pylori LPS to DC-SIGN was described. Moreover, we demonstrated that THP-1 cells may serve as an in vitro model for the assessment of H. pylori-DC-SIGN interactions mediated by Le(X) and Le(Y) determinants.

Lewis-y histo-blood group oligosaccharides are tumour-associated antigens prevalent in several different types of cancer, and they may also be secondary ligands for bacterial toxins from Escherichia coli and Vibrio cholerae. The key step in the synthesis of these sterically congested oligosaccharides involves difucosylation of partially protected lactosamine derivatives. Existing methods require either prolonged reaction times or elaborate glycosyl donors to ensure high stereoselectivity. Herein we report an optimised procedure for using a simple thioglycoside donor that leads to the desired products in high yield and excellent stereoselectivity. It is found that initial glycosylation of the 3'-hydroxy group of lactosamine derivatives in dichloromethane solution can inhibit subsequent glycosylation at the 2-position; however, reaction in toluene solution leads to Lewis-y oligosaccharides in high yield., (Copyright © 2011 Elsevier Ltd. All rights reserved.)

The Euonymus lectin (EUL) domain was recognized as the structural motif for a novel class of putative carbohydrate binding proteins. Confocal microscopy demonstrated that the lectin from Euonymus europaeus (EEA) as well as the EUL protein from Arabidopsis thaliana (ArathEULS3) are located in the nucleocytoplasmic compartment of the plant cell. ArathEULS3 as well as its EUL domain were successfully expressed in Pichia pastoris and purified. The EUL domain from Arabidopsis interacts with glycan structures containing Lewis Y, Lewis X and lactosamine, indicating that it can be considered a true lectin domain. Despite the high sequence identity between the EUL domains in EEA and ArathEULS3, both domains recognize different carbohydrate structures., (Copyright © 2011 Elsevier Inc. All rights reserved.)

Noroviruses, an important cause of acute gastroenteritis in humans, recognize the histo-blood group antigens (HBGAs) as host susceptible factors in a strain-specific manner. The crystal structures of the HBGA-binding interfaces of two A/B/H-binding noroviruses, the prototype Norwalk virus (GI.1) and a predominant GII.4 strain (VA387), have been elucidated. In this study we determined the crystal structures of the P domain protein of the first Lewis-binding norovirus (VA207, GII.9) that has a distinct binding property from those of Norwalk virus and VA387. Co-crystallization of the VA207 P dimer with Le(y) or sialyl Le(x) tetrasaccharides showed that VA207 interacts with these antigens through a common site found on the VA387 P protein which is highly conserved among most GII noroviruses. However, the HBGA-binding site of VA207 targeted at the Lewis antigens through the α-1, 3 fucose (the Lewis epitope) as major and the β-N-acetyl glucosamine of the precursor as minor interacting sites. This completely differs from the binding mode of VA387 and Norwalk virus that target at the secretor epitopes. Binding pocket of VA207 is formed by seven amino acids, of which five residues build up the core structure that is essential for the basic binding function, while the other two are involved in strain-specificity. Our results elucidate for the first time the genetic and structural basis of strain-specificity by a direct comparison of two genetically related noroviruses in their interaction with different HBGAs. The results provide insight into the complex interaction between the diverse noroviruses and the polymorphic HBGAs and highlight the role of human HBGA as a critical factor in norovirus evolution.

Lung cancer has a poor prognosis and a 5-year survival rate of 15%. Therefore, early detection is vital. Diagnostic testing of serum for cancer-associated biomarkers is a noninvasive detection method. Glycosylation is the most frequent post-translational modification of proteins and it has been shown to be altered in cancer. In this paper, high-throughput HILIC technology was applied to serum samples from 100 lung cancer patients, alongside 84 age-matched controls and significant alterations in N-linked glycosylation were identified. Increases were detected in glycans containing Sialyl Lewis X, monoantennary glycans, highly sialylated glycans and decreases were observed in core-fucosylated biantennary glycans, with some being detectable as early as in Stage I. The N-linked glycan profile of haptoglobin demonstrated similar alterations to those elucidated in the total serum glycome. The most significantly altered HILIC peak in lung cancer samples includes predominantly disialylated and tri- and tetra-antennary glycans. This potential disease marker is significantly increased across all disease groups compared to controls and a strong disease effect is visible even after the effect of smoking is accounted for. The combination of all glyco-biomarkers had the highest sensitivity and specificity. This study identifies candidates for further study as potential biomarkers for the disease.

Objective: To investigate whether Lewis Y (LeY) carried by integrin αvβ3 influences integrin αvβ3-mediated adhesion in an in vitro implantation model., Design: Laboratory research., Setting: Reproduction and glycobiology research laboratory., Intervention(s): Specific antibody blockage of LeY or integrin αvβ3 and knockdown of FUT4 expression in RL95-2 cells by transient transfection of FUT4 siRNA., Main Outcome Measure(s): The expression of integrin αvβ3 and LeY in both endometrial tissues and RL95-2 cells was measured. LeY carried by integrin αvβ3 was identified by examining the immunoprecipitated integrin αvβ3. The effect of knocking down FUT4 on the expression of integrin αvβ3 and LeY and their impact on the adhesion of JAR cells to the RL95-2 cells were assessed., Result(s): Integrin αvβ3 and LeY are expressed in both secretory-stage human endometrial tissue and in RL95-2 cells. Although integrin αvβ3 carries LeY, knocking down FUT4 expression only reduces the expression of LeY but not of integrin αvβ3. Knocking down FUT4, antibody blockade of LeY or integrin αvβ3 consistently decreases the adhesion of JAR cells to the RL95-2 cells and prevents focal adhesion kinase (FAK) phosphorylation., Conclusion(s): LeY carried by integrin αvβ3 plays a critical role on the attachment of JAR cells to the RL95-2 cells and activates integrin αvβ3/FAK signaling., (Copyright © 2011 American Society for Reproductive Medicine. Published by Elsevier Inc. All rights reserved.)

Helicobacter pylori is the primary cause of peptic ulcer disease and is estimated to account for about 60% of all cases of gastric cancer, the second most common cause of cancer death worldwide. Among the H. pylori virulence factors associated with disease, in addition to the well-known cag pathogenicity island, is the BabA adhesin, an outer membrane protein that binds with high affinity to fucosylated glycans on the gastric epithelium, such as Lewis B (Le(b)) and related terminal fucose residues found on the blood group O (H antigen), A and B antigens. BabA-mediated attachment to the gastric mucosa promotes chronic inflammation and gastric pathology, which from the bacterial perspective carries both risks and benefits. We recently described modulation in expression of BabA and related outer membrane proteins that occurs during colonization of experimental animals. Here we put these findings into a broader context, and speculate on their implications for the host-pathogen relationship., (© 2011 Landes Bioscience)

Salivary agglutinin plays a vital biological role modulating the protective effect in the oral cavity by interacting with a broad range of oral pathogens. Here, we describe the first characterization of the O-linked oligosaccharides of salivary agglutinin identified by negative ion liquid chromatography-mass spectrometry. The dominating structures were neutral or monosialylated core 1 (Galbeta1-3GalNAcalpha1-Ser/Thr) and core 2 (Galbeta1-3(GlcNAcbeta1-6)GalNAcalpha1-Ser/Thr) structures extended by fucosylated oligo-N-acetyllactosamine units. Oligosaccharides detected as [M-H](-) or [M-2H](2)(-) ions ranged from the disaccharide Galbeta1-3GalNAcol up to structures of almost 4000 Da, corresponding to core 1/2 structures with five N-acetyllactosamine units and 11 fucoses. Fucose was found either as terminal or internal blood group H structures in type 1 (Galbeta1-3GlcNAcbeta1-R), type 2 (Galbeta1-4GlcNAcbeta1-R) and type 3 (Galbeta1-3GalNAcalpha1-Ser/Thr) units, where the chains also could be fucosylated on GlcNAc yielding repeated Lewis a/b or Lewis x/y structures. Sialylation was located either at the non-reducing end of the N-acetyllactosamine chains as sialyl-Lewis x or as sialyl-T (NeuAcalpha2-3Galbeta1-3GalNAcalpha1-Ser/Thr) type structures with or without further extension of the C-6 branch of GalNAc with neutral fucosylated N-acetyllactosamine chains. The data indicated that sialylation, fucosylation and type 1 N-acetyllactosamine termination are important regulatory elements for controlling the oligosaccharide chain length. Furthermore, it was shown that these regulatory oligosaccharide elements could be utilized by the pathogen Helicobacter pylori to colonize the oral cavity, reside in dental plaque and serve as a reservoir for reinfection after successful clearance of H. pylori gastric infection.

Lewis histo-blood group antigens are one of the major classes of biologically active oligosaccharides. In this work, underivatized Lewis blood groups were studied by electrospray tandem mass spectrometry (ESI-MS(n)) in the positive mode with three different mass analyzers: Q-TOF (quadrupole time-of-flight), QqQ (triple quadrupole), and LIT (linear ion trap). It was observed that, under collision-induced fragmentations, type 1 Lewis antigens (Le(a) and Le(b)) could be distinguished from type 2 (Le(x) and Le(y)) on the basis of specific fragmentations of the GlcNAc unit. Whereas O-4-linked sugars of the GlcNAc are lost as residues, the O-3-linked sugars undergo fragmentation both as sugar units and as sugar residues (unit -18Da). Type 2 Lewis antigens also showed a characteristic cross-ring cleavage (0,2)A(2) of the GlcNAc. As a result, the product ions at m/z 388 and 305, characteristic of Le(x), and m/z 372, characteristic of Le(a), are proposed to distinguish the trisaccharide isomers Le(x)/Le(a). Also, the product ions at m/z 534 and 305, characteristic of Le(y), and m/z 372, characteristic of Le(b), are proposed to distinguish the tetrasaccharide isomers Le(b)/Le(y). These diagnostic fragment ions were further applied in the identification of Lewis type 2 antigens (Le(x) and Le(y)) in the lipopolysaccharide of the human gastric pathogen, Helicobacter pylori., (Copyright (c) 2009 Elsevier Inc. All rights reserved.)

Both Helicobacter pylori and Campylobacter jejuni are highly prevalent Gram-negative microaerophilic bacteria which are gastrointestinal pathogens of humans; H. pylori colonizes the gastroduodenal compartment and C. jejuni the intestinal mucosa. Although H. pylori causes chronic gastric infection leading to gastritis, peptic ulcers and eventually gastric cancer while C. jejuni causes acute infection inducing diarrhoeal disease, the endotoxin molecules of both bacterial species contrastingly contribute to their pathogenesis and the autoimmune sequelae each induces. Compared with enterobacterial endotoxin, that of H. pylori has significantly lower endotoxic and immuno-activities, the molecular basis for which is the underphosphorylation and underacylation of the lipid A component that interacts with immune receptors. This induction of low immunological responsiveness by endotoxin may aid the prolongation of H. pylori infection and therefore infection chronicity. On the other hand, this contrasts with acute infection-causing C. jejuni where overt inflammation contributes to pathology and diarrhoea production, and whose endotoxin is immunologically and endotoxically active. Futhermore, both H. pylori and C. jejuni exhibit molecular mimicry in the saccharide components of their endotoxins which can induce autoreactive antibodies; H. pylori expresses mimicry of Lewis and some ABO blood group antigens, C. jejuni mimicry of gangliosides. The former has been implicated in influencing the development of inflammation and gastric atrophy (a precursor of gastic cancer), the latter is central to the development of the neurological disorder Guillain-Barré syndrome. Both diseases raise important questions concerning infection-induced autoimmunity awaiting to be addressed.

Background: Lewis Y (Le(y)) is a blood group-related carbohydrate that is expressed at high surface densities on the majority of epithelial carcinomas and is a promising target for antibody-based immunotherapy. A humanized Le(y)-specific antibody (hu3S193) has shown encouraging safety, pharmacokinetic and tumor-targeting properties in recently completed Phase I clinical trials., Methodology/principal Findings: We report the three-dimensional structures for both the free (unliganded) and bound (Le(y) tetrasaccharide) hu3S193 Fab from the same crystal grown in the presence of divalent zinc ions. There is no evidence of significant conformational changes occurring in either the Le(y) carbohydrate antigen or the hu3S193 binding site, which suggests a rigid fit binding mechanism. In the crystal, the hu3S193 Fab molecules are coordinated at their protein-protein interface by two zinc ions and in solution aggregation of Fab can be initiated by zinc, but not magnesium ions. Dynamic light scattering revealed that zinc ions could initiate a sharp transition from hu3S193 Fab monomers to large multimeric aggregates in solution., Conclusions/significance: Zinc ions can mediate interactions between hu3S193 Fab in crystals and in solution. Whether metallic ion mediated aggregation of antibody occurs in vivo is not known, but the present results suggest that similar clustering mechanisms could occur when hu3S193 binds to Le(y) on cells, particularly given the high surface densities of antigen on the target tumor cells.

The presence of a fucose utilization operon in the Streptococcus pneumoniae genome and its established importance in virulence indicates a reliance of this bacterium on the harvesting of host fucose-containing glycans. The identities of these glycans, however, and how they are harvested is presently unknown. The biochemical and high resolution x-ray crystallographic analysis of two family 98 glycoside hydrolases (GH98s) from distinctive forms of the fucose utilization operon that originate from different S. pneumoniae strains reveal that one enzyme, the predominant type among pneumococcal isolates, has a unique endo-beta-galactosidase activity on the LewisY antigen. Altered active site topography in the other species of GH98 enzyme tune its endo-beta-galactosidase activity to the blood group A and B antigens. Despite their different specificities, these enzymes, and by extension all family 98 glycoside hydrolases, use an inverting catalytic mechanism. Many bacterial and viral pathogens exploit host carbohydrate antigens for adherence as a precursor to colonization or infection. However, this is the first evidence of bacterial endoglycosidase enzymes that are known to play a role in virulence and are specific for distinct host carbohydrate antigens. The strain-specific distribution of two distinct types of GH98 enzymes further suggests that S. pneumoniae strains may specialize to exploit host-specific antigens that vary from host to host, a factor that may feature in whether a strain is capable of colonizing a host or establishing an invasive infection.

The Lewis(x) (Le(x)) epitope (Gal(beta1-4)[Fuc(alpha1-3)]GlcNAc-R) has been associated with the development of the central nervous system of diverse species including human and rodents. In this work, Le(x) has been found in the tetanus neurotoxin insensitive vesicle-associated membrane protein (TI-VAMP) compartment of rat hippocampus neurons in culture, at 7 days in vitro (DIV), when neurite extension is abundant. The TI-VAMP compartment is known to be associated with neurite outgrowth. Le(x) was found predominantly in neurites but also in somata and in growth cones. Abundant Le(x)-carrier glycoproteins specific to neurons have been identified at this stage of differentiation. At a later stage of differentiation, at 14 DIV, Le(x) appeared in extrasynaptic sites of GABAergic neurons, and in synaptic sites of glutamatergic neurons.