The structure of a Bacteroides thetaiotaomicron carbohydrate-binding module provides new insight into the recognition of complex pectic polysaccharides by the human microbiome.

The Bacteroides thetaiotaomicron has developed a consortium of enzymes capable of overcoming steric constraints and degrading, in a sequential manner, the complex rhamnogalacturonan II (RG-II) polysaccharide. BT0996 protein acts in the initial stages of the RG-II depolymerisation, where its two catalytic modules remove the terminal monosaccharides from RG-II side chains A and B. BT0996 is modular and has three putative carbohydrate-binding modules (CBMs) for which the roles in the RG-II degradation are unknown. Here, we present the characterisation of the module at the C-terminal domain, which we designated BT0996-C. The high-resolution structure obtained by X-ray crystallography reveals that the protein displays a typical ß-sandwich fold with structural similarity to CBMs assigned to families 6 and 35. The distinctive features are: 1) the presence of several charged residues at the BT0996-C surface creating a large, broad positive lysine-rich patch that encompasses the putative binding site; and 2) the absence of the highly conserved binding-site signatures observed in CBMs from families 6 and 35, such as region A tryptophan and region C asparagine. These findings hint at a binding mode of BT0996-C not yet observed in its homologues. In line with this, carbohydrate microarrays and microscale thermophoresis show the ability of BT0996-C to bind α1-4-linked polygalacturonic acid, and that electrostatic interactions are essential for the recognition of the anionic polysaccharide. The results support the hypothesis that BT0996-C may have evolved to potentiate the action of BT0996 catalytic modules on the complex structure of RG-II by binding to the polygalacturonic acid backbone sequence.

Feasibility of Brewer's Spent Yeast Microcapsules as Targeted Oral Carriers.

Brewer's spent yeast (BSY) microcapsules have a complex network of cell-wall polysaccharides that are induced by brewing when compared to the baker's yeast (Saccharomyces cerevisiae) microcapsules. These are rich in (ß1→3)-glucans and covalently linked to (α1→4)- and (ß1→4)-glucans in addition to residual mannoproteins. S. cerevisiae is often used as a drug delivery system due to its immunostimulatory potential conferred by the presence of (ß1→3)-glucans. Similarly, BSY microcapsules could also be used in the encapsulation of compounds or drug delivery systems with the advantage of resisting digestion conferred by (ß1→4)-glucans and promoting a broader immunomodulatory response. This work aims to study the feasibility of BSY microcapsules that are the result of alkali and subcritical water extraction processes, as oral carriers for food and biomedical applications by (1) evaluating the resistance of BSY microcapsules to in vitro digestion (IVD), (2) their recognition by the human Dectin-1 immune receptor after IVD, and (3) the recognition of IVD-solubilized material by different mammalian immune receptors. IVD digested 44-63% of the material, depending on the extraction process. The non-digested material, despite some visible agglutination and deformation of the microcapsules, preserved their spherical shape and was enriched in (ß1→3)-glucans. These microcapsules were all recognized by the human Dectin-1 immune receptor. The digested material was differentially recognized by a variety of lectins of the immune system related to (ß1→3)-glucans, glycogen, and mannans. These results show the potential of BSY microcapsules to be used as oral carriers for food and biomedical applications.

Structural differences on cell wall polysaccharides of brewer's spent Saccharomyces and microarray binding profiles with immune receptors.

Brewing practice uses the same yeast to inoculate the following fermentation (repitching). Saccharomyces pastorianus, used to produce Lager beer, is widely reused, not changing its fermentation performance. However, S. cerevisiae, used to produce Ale beer, is partial or not even reused, due to its poor performance. It is hypothesized that cells modulate their wall polysaccharides to increase the cell-wall strength. In this work industrial S. cerevisiae and S. pastorianus brewer's spent yeasts with different repitching numbers were studied. Glucans were the main component of S. cerevisiae whereas mannoproteins were abundant in S. pastorianus. The major changes were noticed on glucans of both species, ß1,3-glucans decrease more pronounced in S. cerevisiae. The increase of α1,4-Glc, related with osmotolerance, was higher in S. cerevisiae while ß1,4-Glc, related with cell-wall strength, had a small increase. In addition, these structural details showed different binding profiles to immune receptors, important to develop tailored bioactive applications.

Mapping Molecular Recognition of ß1,3-1,4-Glucans by a Surface Glycan-Binding Protein from the Human Gut Symbiont Bacteroides ovatus.

A multigene polysaccharide utilization locus (PUL) encoding enzymes and surface carbohydrate (glycan)-binding proteins (SGBPs) was recently identified in prominent members of Bacteroidetes in the human gut and characterized in Bacteroides ovatus. This PUL-encoded system specifically targets mixed-linkage ß1,3-1,4-glucans, a group of diet-derived carbohydrates that promote a healthy microbiota and have potential as prebiotics. The BoSGBPMLG-A protein encoded by the BACOVA_2743 gene is a SusD-like protein that plays a key role in the PUL's specificity and functionality. Here, we perform a detailed analysis of the molecular determinants underlying carbohydrate binding by BoSGBPMLG-A, combining carbohydrate microarray technology with quantitative affinity studies and a high-resolution X-ray crystallography structure of the complex of BoSGBPMLG-A with a ß1,3-1,4-nonasaccharide. We demonstrate its unique binding specificity toward ß1,3-1,4-gluco-oligosaccharides, with increasing binding affinities up to the octasaccharide and dependency on the number and position of ß1,3 linkages. The interaction is defined by a 41-Å-long extended binding site that accommodates the oligosaccharide in a mode distinct from that of previously described bacterial ß1,3-1,4-glucan-binding proteins. In addition to the shape complementarity mediated by CH-π interactions, a complex hydrogen bonding network complemented by a high number of key ordered water molecules establishes additional specific interactions with the oligosaccharide. These support the twisted conformation of the ß-glucan backbone imposed by the ß1,3 linkages and explain the dependency on the oligosaccharide chain length. We propose that the specificity of the PUL conferred by BoSGBPMLG-A to import long ß1,3-1,4-glucan oligosaccharides to the bacterial periplasm allows Bacteroidetes to outcompete bacteria that lack this PUL for utilization of ß1,3-1,4-glucans. IMPORTANCE With the knowledge of bacterial gene systems encoding proteins that target dietary carbohydrates as a source of nutrients and their importance for human health, major efforts are being made to understand carbohydrate recognition by various commensal bacteria. Here, we describe an integrative strategy that combines carbohydrate microarray technology with structural studies to further elucidate the molecular determinants of carbohydrate recognition by BoSGBPMLG-A, a key protein expressed at the surface of Bacteroides ovatus for utilization of mixed-linkage ß1,3-1,4-glucans. We have mapped at high resolution interactions that occur at the binding site of BoSGBPMLG-A and provide evidence for the role of key water-mediated interactions for fine specificity and affinity. Understanding at the molecular level how commensal bacteria, such as prominent members of Bacteroidetes, can differentially utilize dietary carbohydrates with potential prebiotic activities will shed light on possible ways to modulate the microbiome to promote human health.

Cracking the Breast Cancer Glyco-Code through Glycan-Lectin Interactions: Targeting Immunosuppressive Macrophages.

The immune microenvironment of breast cancer (BC) is composed by high macrophage infiltrates, correlated with the most aggressive subtypes. Tumour-associated macrophages (TAM) within the BC microenvironment are key regulators of immune suppression and BC progression. Nevertheless, several key questions regarding TAM polarisation by BC are still not fully understood. Recently, the modulation of the immune microenvironment has been described via the recognition of abnormal glycosylation patterns at BC cell surface. These patterns rise as a resource to identify potential targets on TAM in the BC context, leading to the development of novel immunotherapies. Herein, we will summarize recent studies describing advances in identifying altered glycan structures in BC cells. We will focus on BC-specific glycosylation patterns known to modulate the phenotype and function of macrophages recruited to the tumour site, such as structures with sialylated or N-acetylgalactosamine epitopes. Moreover, the lectins present at the surface of macrophages reported to bind to such antigens, inducing tumour-prone TAM phenotypes, will also be highlighted. Finally, we will discuss and give our view on the potential and current challenges of targeting these glycan-lectin interactions to reshape the immunosuppressive landscape of BC.

Siglec-15 recognition of sialoglycans on tumor cell lines can occur independently of sialyl Tn antigen expression.

Siglec-15 is a conserved sialic acid-binding Ig-like lectin expressed on osteoclast progenitors, which plays an important role in osteoclast development and function. It is also expressed by tumor-associated macrophages and by some tumors, where it is thought to contribute to the immunosuppressive microenvironment. It was shown previously that engagement of macrophage-expressed Siglec-15 with tumor cells expressing its ligand, sialyl Tn (sTn), triggered production of TGF-ß. In the present study, we have further investigated the interaction between Siglec-15 and sTn on tumor cells and its functional consequences. Based on binding assays with lung and breast cancer cell lines and glycan-modified cells, we failed to see evidence for recognition of sTn by Siglec-15. However, using a microarray of diverse, structurally defined glycans, we show that Siglec-15 binds with higher avidity to sialylated glycans other than sTn or related antigen sequences. In addition, we were unable to demonstrate enhanced TGF-ß secretion following co-culture of Siglec-15-expressing monocytic cell lines with tumor cells expressing sTn or following Siglec-15 cross-linking with monoclonal antibodies. However, we did observe activation of the SYK/MAPK signaling pathway following antibody cross-linking of Siglec-15 that may modulate the functional activity of macrophages.

Helicobacter pylori lipopolysaccharide structural domains and their recognition by immune proteins revealed with carbohydrate microarrays.

The structural diversity of the lipopolysaccharides (LPSs) from Helicobacter pylori poses a challenge to establish accurate and strain-specific structure-function relationships in interactions with the host. Here, LPS structural domains from five clinical isolates were obtained and compared with the reference strain 26695. This was achieved combining information from structural analysis (GC-MS and ESI-MSn) with binding data after interrogation of a LPS-derived carbohydrate microarray with sequence-specific proteins. All LPSs expressed Lewisx/y and N-acetyllactosamine determinants. Ribans were also detected in LPSs from all clinical isolates, allowing their distinction from the 26695 LPS. There was evidence for 1,3-d-galactans and blood group H-type 2 sequences in two of the clinical isolates, the latter not yet described for H. pylori LPS. Furthermore, carbohydrate microarray analyses showed a strain-associated LPS recognition by the immune lectins DC-SIGN and galectin-3 and revealed distinctive LPS binding patterns by IgG antibodies in the serum from H. pylori-infected patients.

Structural analysis and potential immunostimulatory activity of Nannochloropsis oculata polysaccharides.

The relevance of microalgae biotechnology for producing high-value compounds with biomedical application, such as polysaccharides, has been increasing. Despite this, the knowledge about the composition and structure of microalgae polysaccharides is still scarce. In this work, water-soluble polysaccharides from Nannochloropsis oculata were extracted, fractionated, structurally analysed, and subsequently tested in terms of immunostimulatory activity. A combination of sugar and methylation analysis with interaction data of carbohydrate-binding proteins using carbohydrate microarrays disclosed the complex structural features of the different polysaccharides. These analyses showed that the water-soluble polysaccharides fractions from N. oculata were rich in (ß1→3, ß1→4)-glucans, (α1→3)-, (α1→4)-mannans, and anionic sulphated heterorhamnans. The immunostimulatory assay highlighted that these fractions could also stimulate murine B-lymphocytes. Thus, the N. oculata water-soluble polysaccharides show potential to be further explored for immune-mediated biomedical applications.

Novel monoclonal antibody L2A5 specifically targeting sialyl-Tn and short glycans terminated by alpha-2-6 sialic acids.

Incomplete O-glycosylation is a feature associated with malignancy resulting in the expression of truncated glycans such as the sialyl-Tn (STn) antigen. Despite all the progress in the development of potential anti-cancer antibodies, their application is frequently hindered by low specificities and cross-reactivity. In this study, a novel anti-STn monoclonal antibody named L2A5 was developed by hybridoma technology. Flow cytometry analysis showed that L2A5 specifically binds to sialylated structures on the cell surface of STn-expressing breast and bladder cancer cell lines. Moreover, immunoblotting assays demonstrated reactivity to tumour-associated O-glycosylated proteins, such as MUC1. Tumour recognition was further observed using immunohistochemistry assays, which demonstrated a high sensitivity and specificity of L2A5 mAb towards cancer tissue, using bladder and colorectal cancer tissues. L2A5 staining was exclusively tumoural, with a remarkable reactivity in invasive and metastasis sites, not detectable by other anti-STn mAbs. Additionally, it stained 20% of cases of triple-negative breast cancers, suggesting application in diseases with unmet clinical needs. Finally, the fine specificity was assessed using glycan microarrays, demonstrating a highly specific binding of L2A5 to core STn antigens and additional ability to bind 2-6-linked sialyl core-1 probes. In conclusion, this study describes a novel anti-STn antibody with a unique binding specificity that can be applied for cancer diagnostic and future development of new antibody-based therapeutic applications.

Primers for mating-type diagnosis in Diaporthe and Phomopsis: their use in teleomorph induction in vitro and biological species definition.

Sexual reproduction in ascomycete fungi is governed by the mating-type (MAT) locus. The MAT loci of Diaporthe and its Phomopsis anamorphs differ in only one gene: MAT1-1-1 in mating-type MAT1-1 and MAT1-2-1 in mating-type MAT1-2. In order to diagnose mating-types in Diaporthe and Phomopsis and evaluate their usefulness in teleomorph induction in vitro and biological species delimitation, we designed primers that amplify part of the MAT1-1-1 and MAT1-2-1 genes. MAT phylogenies were generated and compared with ITS and EF1-α phylograms. Species recognised in the EF1-α phylogeny corresponded directly with those determined in the MAT phylogenies. ITS was shown to be highly variable resulting in a large number of phylogenetic species that were discordant with MAT and EF1-α species. Mating experiments were conducted to evaluate the existence of reproductive barriers between some isolates, and their anamorphic morphologies were compared. The primers proved to be useful in the mating-type diagnosis of isolates, selection of compatible mating pairs, and in the assessment of biological species boundaries.