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Carbohydrate binding modules (CBMs) are protein domains that typically reside near catalytic domains, increasing substrate-protein proximity by constraining the conformational space of carbohydrates. Due to the flexibility and variability of glycans, the molecular details of how these protein regions recognize their target molecules are not always fully understood. Computational methods, including molecular docking and molecular dynamics simulations, have been employed to investigate lectin-carbohydrate interactions. In this study, we introduce a novel approach that integrates multiple computational techniques to identify the critical amino acids involved in the interaction between a CBM located at the tip of bacteriophage J-1's tail and its carbohydrate counterparts. Our results highlight three amino acids that play a significant role in binding, a finding we confirmed through in vitro experiments. By presenting this approach, we offer an intriguing alternative for pinpointing amino acids that contribute to protein-sugar interactions, leading to a more thorough comprehension of the molecular determinants of protein-carbohydrate interactions. [ABSTRACT FROM AUTHOR]

The Middle East Respiratory Syndrome Coronavirus (MERS-CoV) causes severe and fatal acute respiratory disease in humans. High fatality rates and continued infectiousness remain a pressing concern for global health preparedness. Antibodies targeted at the receptor-binding domain (RBD) are major countermeasures against human viral infection. Here, we report four potent nanobodies against MERS-CoV, which are isolated from alpaca, and especially the potency of Nb14 is highest in the pseudotyped virus assay. Structural studies show that Nb14 framework regions (FRs) are mainly involved in interactions targeting a novel epitope, which is entirely distinct from all previously reported antibodies, and disrupt the protein-carbohydrate interaction between residue W535 of RBD and hDPP4 N229-linked carbohydrate moiety (hDPP4-N229-glycan). Different from Nb14, Nb9 targets the cryptic face of RBD, which is distinctive from the hDPP4 binding site and the Nb14 epitope, and it induces the β5-β6 loop to inflect towards a shallow groove of the RBD and dampens the accommodation of a short helix of hDPP4. The particularly striking epitopes endow the two Nbs administrate synergistically in the pseudotyped MERS-CoV assays. These results not only character unprecedented epitopes for antibody recognition but also provide promising agents for prophylaxis and therapy of MERS-CoV infection. Author summary: MERS-CoV is one of the most prevalent zoonotic virus that has spread through 27 countries and infected more than 2,605 people since its first outbreak in Saudi Arabia in 2012. The high fatality rate and its persistent wide spread infectiousness in animal reservoirs have generated tremendous global public health concern. However, no licensed therapeutic agents or vaccines against MERS-CoV are currently available. To address the risk of MERS-CoV and its variants re-emergence, we developed four highly effective Nbs from alpaca and solved two crystal complexes of Nb9 and Nb14 with RBD. Remarkably, the neutralizing activity of Nb14 represents the highest of MERS-CoV antibodies reported to date. The novel epitopes of Nb14 and Nb9 locate outside the RBD and hDPP4 interface, and the unique different epitopes and mechanisms of Nb14 and Nb9 provide a better neutralizing for synergistically against MERS-CoV infection. Our findings provide insights into the cryptic epitopes on RBD and the development of antibodies against MERS-CoV infection. [ABSTRACT FROM AUTHOR]

Lectins are a group of proteins that are widespread in all kingdoms of living nature, but plants are the undisputed "champions" in terms of the abundance and diversity of lectins. The fundamental property of reversibly binding to specific carbohydrates makes lectins important participants in the "glycocode" system, which has a special functional significance for plants with their incredible carbohydrate diversity. The structural diversity of lectins underlies their numerous functions, including signaling associated with growth and development as well as plant responses to biotic and abiotic stimuli. The review presents a retrospective of the development of plant lectinology and last data about the classification of plant lectins, their localization, and known and potential functions. [ABSTRACT FROM AUTHOR]

Protein-carbohydrate interactions are involved in several cellular and biological functions. Integrating structure and function of carbohydrate-binding proteins with disease-causing mutations help to understand the molecular basis of diseases. Although databases are available for protein-carbohydrate complexes based on structure, binding affinity and function, no specific database for mutations in human carbohydrate-binding proteins is reported in the literature. We have developed a novel database, CarbDisMut, a comprehensive integrated resource for disease-causing mutations with sequence and structural features. It has 1.17 million disease-associated mutations and 38,636 neutral mutations from 7,187 human carbohydrate-binding proteins. The database is freely available at https://web.iitm.ac.in/bioinfo2/carbdismut. The web-site is implemented using HTML, PHP and JavaScript and supports recent versions of all major browsers, such as Firefox, Chrome and Opera. [ABSTRACT FROM AUTHOR]

Mucus is a defining feature of many gastropod phenotypes. Its material properties are a product of selection acting on the relationship between mucus composition, structure and performance; however, to date, there are very few studies addressing this. Here, we introduce attenuated total reflection Fourier transform infrared (FTIR) spectroscopy as an accessible analytical technique to record, identify and classify gastropod locomotive mucus, as deposited, with practically no intervention. Advancing the field beyond previous spectroscopic studies, we also probe mucus response to a temperature increase, determining its propensity to remain hydrated (i.e. resist denaturation) and serving as the basis for building cladograms based on spectral similarities. Specifically, the relative content of proteins, their secondary structures, protein-carbohydrate interactions, and thermal stability were investigated and shown to have significant variation between species. Collating and comparing over 648 spectra across 12 species and 7 superfamilies, we report that it is even possible to classify gastropods based solely on their secreted pedal mucus. This was further compared to previously published phylogenetic data and taxonomies enabling a 100% hit rate of genus assignment, implying that mucus FTIR may also be a powerful tool for field-based biodiversity studies. [ABSTRACT FROM AUTHOR]

Protein-carbohydrate interactions are essential in maintaining immune homeostasis and orchestrating inflammatory and regulatory immune processes. This review elucidates the immune interactions of macrophage galactose-type lectin (MGL, CD301) and Tn carbohydrate antigen. MGL is a C-type lectin receptor (CLR) primarily expressed by myeloid cells such as macrophages and immature dendritic cells. MGL recognizes terminal O-linked N-acetylgalactosamine (GalNAc) residue on the surface proteins, also known as Tn antigen (Tn). Tn is a truncated form of the elongated cell surface O-glycan. The hypoglycosylation leading to Tn may occur when the enzyme responsible for O-glycan elongation—T-synthase—or its associated chaperone—Cosmc—becomes functionally inhibited. As reviewed here, Tn expression is observed in many different neoplastic and non-neoplastic diseases, and the recognition of Tn by MGL plays an important role in regulating effector T cells, immune suppression, and the recognition of pathogens. [ABSTRACT FROM AUTHOR]

The isolation from organisms and readily available glycoproteins has become an increasingly convenient source of N‐glycans for multiple applications including glycan microarrays, as reference standards in glycan analysis or as reagents that improve bioavailability of protein and peptide therapeutics through conjugation. A problematic step in the isolation process on a preparative scale can be the attachment of a linker for the improved purification, separation, immobilization and quantification of the glycan structures. Addressing this issue, we firstly aimed for the development of an UV active linker for a fast and reliable attachment to anomeric glycosylamines via urea bond formation. Secondly, we validated the new linker on glycan arrays in a comparative study with a collection of N‐glycans which were screened against various lectins. In total, we coupled four structurally varied N‐glycans to four different linkers, immobilized all constructs on a microarray and compared their binding affinities to four plant and fungal lectins of widely described specificity. Our study shows that the urea type linker showed an overall superior performance for lectin binding and once more, highlights the often neglected influence of the choice of linker on lectin recognition. [ABSTRACT FROM AUTHOR]

Glycovesicles mimic synthetic cell membrane surfaces and aid to delineate intricate, weak carbohydrate–protein interactions. In this report, the dependence of the hydrodynamic diameters in relation to the molar fractions of carbohydrate moieties in the mixed polydiacetylene (PDA) glycovesicles is evaluated. The glycovesicles are constituted with diacetylene monomers of varying molar fractions of carbohydrate moieties and the hydrodynamic diameters are assessed without and with polymerization of the vesicles. A strong dependence of the hydrodynamic diameter of glycovesicles is seen as a function of the molar fractions and the nature of the sugar moiety being either mono- or disaccharide. A monotonous increase in the hydrodynamic diameters of the glycovesicles occurs with the increase in mole fractions of the sugar monomer lipids. Upon polymerization, the hydrodynamic diameters reduce for the vesicles with lower mole fractions of sugar monomer, whereas the reverse occurs for glycovesicles possessing higher mole fractions. Disaccharide glycovesicles possess higher hydrodynamic diameters than monosaccharide-containing vesicles. Ligand–lectin interactions were probed with lactose disaccharide-containing glycovesicles with tetrameric peanut agglutinin lectin, from which an increase in the hydrodynamic diameters is observed, as the mole fraction of sugar monomer is increased in the PDA-glycovesicles. [ABSTRACT FROM AUTHOR]

Protein–carbohydrate interactions happen to be a crucial facet of biology, discharging a myriad of functions. Microarrays have become a premier choice to discern the selectivity, sensitivity and breadth of these interactions in a high-throughput manner. The precise recognition of target glycan ligands among the plethora of others is central for any glycan-targeting probe being tested by microarray analyses. Ever since the introduction of the microarray as an elemental tool for high-throughput glycoprofiling, numerous distinct array platforms possessing different customizations and assemblies have been developed. Accompanying these customizations are various factors ushering variances across array platforms. In this primer, we investigate the influence of various extrinsic factors, namely printing parameters, incubation procedures, analyses and array storage conditions on the protein–carbohydrate interactions and evaluate these factors for the optimal performance of microarray glycomics analysis. We hereby propose a 4D approach (Design–Dispense–Detect–Deduce) to minimize the effect of these extrinsic factors on glycomics microarray analyses and thereby streamline cross-platform analyses and comparisons. This work will aid in optimizing microarray analyses for glycomics, minimize cross-platform disparities and bolster the further development of this technology. [ABSTRACT FROM AUTHOR]

Gastropods, such as snails and slugs, can excrete mucus to aid in movement and adhesion. However, very few studies have examined the physical relationship between mucus composition and function. Here, we explore the role of mucus polymers (specifically their proteins) and their influence on the material properties of locomotive mucus. Using a range of spectroscopic, thermal, and rheological analytical tools, we characterised locomotive mucus from six gastropod species across four families. We report that all mucus tested consisted of 97%–99% water, and the remaining 1%–3% solid content contained a range of proteins (41–377 kDa, 18 of which are previously undocumented), which we propose contribute to its weak gel behaviour (1.58–36.33 Pa•s at 1 rad/s). Our results indicate that mucus properties are also grouped at the family level, suggesting that niche-specific adaptation occurs in these materials. We expect our study to offer a broader approach to how a correlation between properties is crucial for understanding the stability and functionality of snail mucus.

Nuclear Magnetic Resonance (NMR) has been widely employed to assess diverse features of glycan- protein molecular recognition events. Different types of qualitative and quantitative information at different degrees of resolution and complexity can be extracted from the proper application of the available NMR-techniques. In fact, affinity, structural, kinetic, conformational, and dynamic characteristics of the binding process are available. Nevertheless, except in particular cases, the affinity of lectin-sugar interactions is weak, mostly at the low mM range. This feature is overcome in biological processes by using multivalency, thus augmenting the strength of the binding. However, the application of NMR methods to monitor multivalent lectin-glycan interactions is intrinsically challenging. It is well known that when large macromolecular complexes are formed, the NMR signals disappear from the NMR spectrum, due to the existence of fast transverse relaxation, related to the large size and exchange features. Indeed, at the heart of the molecular recognition event, the associated free-bound chemical exchange process for both partners takes place in a particular timescale. Thus, these factors have to be considered and overcome. In this review article, we have distinguished, in a subjective manner, the existence of multivalent presentations in the glycan or in the lectin. From the glycan perspective, we have also considered whether multiple epitopes of a given ligand are presented in the same linear chain of a saccharide (i.e., poly-LacNAc oligosaccharides) or decorating different arms of a multiantennae scaffold, either natural (as in multiantennae N-glycans) or synthetic (of dendrimer or polymer nature). From the lectin perspective, the presence of an individual binding site at every monomer of a multimeric lectin may also have key consequences for the binding event at different levels of complexity. [ABSTRACT FROM AUTHOR]

Cell surface protein–carbohydrate interactions are essential for tissue-specific recognition and endocytosis of viruses, some bacteria and their toxins, and many glycoproteins. Often protein–carbohydrate interactions are multivalent – multiple copies of glycans bind simultaneously to multimeric receptors. Multivalency enhances both affinity and binding specificity, and is of interest for targeted delivery of drugs to specific cell types. The first such example of carbohydrate-mediated drug delivery to reach the clinic is Givosiran, a small interfering ribonucleic acid (siRNA) that is conjugated to a trivalent N-acetylgalactosamine (GalNAc) ligand. This ligand enables efficient uptake of the nucleic acid by the asialoglycoprotein receptor (ASGP-R) on hepatocytes. Synthetic multivalent ligands for ASGP-R were among the first 'cluster glycosides' developed at the birth of multivalent glycoscience around 40 years ago. In this review we trace the history of 'GalNAc targeting' from early academic studies to current pharmaceuticals and consider what other opportunities could follow the success of this delivery technology. [ABSTRACT FROM AUTHOR]

Fluorine (19F) incorporation into glycan‐binding proteins (lectins) has been achieved and exploited to monitor the binding to carbohydrate ligands by nuclear magnetic resonance (NMR) spectroscopy. Galectins are a family of lectins that bind carbohydrates, generally with weak affinities, through a combination of intermolecular interactions including a key CH‐π stacking involving a conserved tryptophan residue. Herein, Galectin‐3 (Gal3) and Galectin‐8 (Gal8) with one and two carbohydrate recognition domains (CRDs), respectively, were selected. Gal3 contains one Trp, whereas Gal8 contains three, one at each binding site and a third one not involved in sugar binding; these were substituted by the corresponding F‐Trp analogues. The presence of fluorine did not significantly modify the affinity for glycan binding, which was in slow exchange on the 19F NMR chemical‐shift timescale, even for weak ligands, and allowed binding events taking place at two different binding sites within the same lectin to be individualized. [ABSTRACT FROM AUTHOR]

Carbohydrate-protein interactions play fundamental roles in numerous aspects of biological activities, and the search for new carbohydrate (CHO)-binding proteins (CBPs) has long been a research focus. In this study, through the analysis of CBP structures, we identified significant enrichment of aromatic residues in CHO-binding regions. We further summarized the structural features of these aromatic rings within the CHO-stacking region, namely "exposing" and "proximity" features, and developed a screening algorithm that can identify CHO-stacking Trp (tryptophan) residues based on these two features. Our Trp screening algorithm can achieve high accuracy in both CBP (specificity score 0.93) and CBS (Carbohydrate binding site, precision score 0.77) prediction using experimentally determined protein structures. We also applied our screening algorithm on AlphaGO pan-species predicted models and observed significant enrichment of carbohydrate-related functions in predicted CBP candidates across different species. Moreover, through carbohydrate arrays, we experimentally verified the CHO-binding ability of four candidate proteins, which further confirms the robustness of the algorithm. This study provides another perspective on proteome-wide CBP and CBS prediction. Our results not only help to reveal the structural mechanism of CHO-binding, but also provide a pan-species CBP dataset for future CHO-protein interaction exploration. [ABSTRACT FROM AUTHOR]

Lectins are ubiquitous proteins that selectively bind to carbohydrates, serving as vital models for understanding protein-carbohydrate interactions. While extensively distributed across various life forms, plant lectins, especially from the Leguminosae family, have garnered significant attention. However, limited research exists on lectins from the Caesalpinioideae subfamily, suggesting a source of untapped biotechnological potential. This underscores the imperative for further exploration, particularly in isolating lectins from the Bauhinia genus, which remains relatively understudied, despite harboring lectins with diverse characteristics and promising biotechnological activities. In this study, a novel lectin extracted from Bauhinia catingae Harms seeds (BCL) was isolated in three chromatographic steps. BCL exhibited affinity for galactose and derivatives, akin to other Bauhinia lectins, with SDS-PAGE confirming its molecular weight around 30 kDa. Notably, BCL demonstrated stability across temperature and pH ranges and lacked metalloprotein characteristics. Electrospray ionization mass spectrometry revealed a partial sequence covering 81 % of the total protein sequence with nearly 80 % identity to Bauhinia forficata. Structural analysis suggested a β -sheet-rich secondary structure similar to that of other lectins. Further structural elucidation of BCL is essential to unveil its full potential and applications. [ABSTRACT FROM AUTHOR]

[Display omitted] • Rg NanH-GH33 is an intramolecular trans -sialidase expressed by the human gut symbiont Ruminococcus gnavus , to utilise intestinal sialylated mucin glycan epitopes. • The use of multifrequency STD NMR in combination with computational techniques (docking and full matrix relaxation calculations with CORCEMA-ST) provided the first 3D structures of the complexes of Rg NanH-GH33 with the sialylated trisaccharides 3′SL and 6′SL ligands (3′SL being the enzymatic substrate). • The sialic acid moiety is confirmed to be the core recognition element for the interaction in the enzymatic cleft. The main difference observed between the two oligosaccharides resides in the binding orientation of the lactose moiety, with the lactose of 6′SL being oriented at a less favourable angle, preventing the CH-π interaction with Trp698, known to provide selectivity for α2-3 sialoglycans. • This is reflected in a faster kinetics of binding and overall less favourable interactions for 6′SL in comparison to 3′SL, as also observed by variable temperature and competition STD NMR experiments. • Multifrequency-STD NMR is shown to be instrumental for the structural elucidation of an elusive Michaelis-Menten complex for this intramolecular trans -sialidase crucial to R. gnavus ' adaptation to the mucin environment. Rg NanH is an intramolecular trans -sialidase expressed by the human gut symbiont Ruminococcus gnavus , to utilise intestinal sialylated mucin glycan epitopes. Its catalytic domain, belonging to glycoside hydrolase GH33 family, cleaves off terminal sialic acid residues from mucins, releasing 2,7-anhydro-Neu5Ac which is then used as metabolic substrate by R. gnavus to proliferate in the mucosal environment. Rg NanH is one of the three intramolecular trans -sialidases (IT-sialidases) characterised to date, and the first from a gut commensal organism. Here, saturation transfer difference NMR (STD NMR) in combination with computational techniques (molecular docking and CORCEMA-ST) were used to elucidate the specificity, kinetics and relative affinity of Rg NanH for sialoglycans and 2,7-anhydro-Neu5Ac. We propose the first 3D model for the Michaelis complex of an IT-sialidase. This confirms the sialic acid to be the main recognition element for the interaction in the enzymatic cleft and highlights the crucial role of Trp698 to make CH-π stacking with the galactose residue of the substrate 3′-sialyllactose. The same contact is shown not to be possible for 6′-sialyllactose, due to geometrical constrains of the α-2,6 linkage. Indeed 6′-sialyllactose is not a substrate, even though it is shown to bind to Rg NanH by STD NMR. These findings corroborate the role of Trp698 for the α-2,3 specificity of IT-sialidases. In this structural study, the use of Differential Epitope Mapping STD NMR (DEEP-STD NMR) approach allowed the validation of the proposed 3D models in solution. These structural approaches are shown to be instrumental in shedding light on the molecular mechanisms underpinning enzymatic reactions in the absence of enzyme-substrate X-ray structures. [ABSTRACT FROM AUTHOR]

This study utilized whey protein concentrate (WPC) as the primary protein source, employing the Maillard reaction to link it with three distinct carbohydrates: lactose, maltodextrin, and gum Arabic. Mixtures were prepared with a 1:4 protein-to-carbohydrate ratio, and conjugation was performed using dry heating at a temperature of 60 °C, 50% relative humidity for 8 h processing time. Under the same conditions, native WPC was heated as a control group. The formation of conjugates was tracked by monitoring absorbance changes, browning index (BI), and reduction in free amino groups (FAG). All conjugates exhibited significant loss of FAG, correlating with an increase in BI. Shifts in protein fingerprint bands observed via Fourier Transform Infrared Spectroscopy suggested potential protein-carbohydrate interactions, while X-ray diffraction showed increased sample crystallinity post-conjugation. Solubility assessments across pH levels (3.0–8.0) indicated a 10–13% rise in solubility for conjugated samples compared to mixtures, broadening their potential applications. Moreover, the emulsion stability index demonstrated improved stability after conjugation. Overall, the Maillard reaction-mediated conjugation enhanced the solubility and stability of emulsions. [ABSTRACT FROM AUTHOR]

Meningococcal glycoconjugate vaccines sourced from capsular polysaccharides (CPSs) of pathogenic Neisseria meningitidis strains are well-established measures to prevent meningococcal disease. However, the exact structural factors responsible for antibody recognition are not known. CPSs of Neisseria meningitidis serogroups Y and W differ by a single stereochemical center, yet they evoke specific immune responses. Herein, we developed specific monoclonal antibodies (mAbs) targeting serogroups C, Y, and W and evaluated their ability to kill bacteria. We then used these mAbs to dissect structural elements responsible for carbohydrate-protein interactions. First, Men oligosaccharides were screened against the mAbs using ELISA to select putative lengths representing the minimal antigenic determinant. Next, molecular interaction features between the mAbs and serogroup-specific sugar fragments were elucidated using STD-NMR. Moreover, X-ray diffraction data with the anti-MenW CPS mAb enabled the elucidation of the sugar-antibody binding mode. Our findings revealed common traits in the epitopes of all three sialylated serogroups. The minimal binding epitopes typically comprise five to six repeating units. Moreover, the O -acetylation of the neuraminic acid moieties was fundamental for mAb binding. These insights hold promise for the rational design of optimized meningococcal oligosaccharides, opening new avenues for novel production methods, including chemical or enzymatic approaches. [Display omitted] [ABSTRACT FROM AUTHOR]

Real-time detection of glycosylation on label-free cancer cell surfaces is of significance for the diagnosis and treatment of cancer. In this work, we have successfully developed a novel dynamic reversible sensor based on pH-sensitive phenylboronic esters to determine in real-time the binding kinetics of protein–carbohydrate interactions on suspension cancer cell surfaces using a quartz crystal microbalance (QCM) technique. [ABSTRACT FROM AUTHOR]

Cell surface carbohydrates mediate a wide range of carbohydrate–protein interactions key to healthy and disease mechanisms. Many of such interactions are multivalent in nature and in order to study these processes at a molecular level, many glycan-presenting platforms have been developed over the years. Among those, carbon nanoforms such as graphene and their derivatives, carbon nanotubes, carbon dots and fullerenes, have become very attractive as biocompatible platforms that can mimic the multivalent presentation of biologically relevant glycosides. The most recent examples of carbon-based nanoplatforms and their applications developed over the last few years to study carbohydrate-mediate interactions in the context of cancer, bacterial and viral infections, among others, are highlighted in this review. [ABSTRACT FROM AUTHOR]

Mannose/glucose-binding lectin from Canavalia ensiformis seeds (Concanavalin A - ConA) has several biological applications, such as mitogenic and antitumor activity. However, most of the mechanisms involved in the in vivo toxicity of ConA are not well known. In this study, the Drosophila melanogaster model was used to assess the toxicity and genotoxicity of different concentrations of native ConA (4.4, 17.5 and 70 μg/mL) in inhibited and denatured forms of ConA. The data show that native ConA affected: the survival, in the order of 30.6 %, and the locomotor performance of the flies; reduced cell viability to levels below 50 % (4.4 and 17.5 μg/mL); reduced nitric oxide levels; caused lipid peroxidation and increased protein and non-protein thiol content. In the Comet assay, native ConA (17.5 e 70 μg/mL) caused DNA damage higher than 50 %. In contrast, treatments with inhibited and denatured ConA did not affect oxidative stress markers and did not cause DNA damage. We believe that protein-carbohydrate interactions between ConA and carbohydrates of the plasma membrane are probably the major events involved in these activities, suggesting that native ConA activates mechanisms that induce oxidative stress and consequently DNA damage. • The Lectin ConA induces ROS production. • ConA causes oxidative stress in Drosophila melanogaster. • The toxic effect of ConA results in damage to DNA and membranes. • The toxic effect of ConA is related to its CRD. [ABSTRACT FROM AUTHOR]

Dystroglycan (DG), which constitutes a part of the dystrophin–glycoprotein complex, connects the extracellular matrix to the cytoskeleton. The matriglycans presented by the extracellular α-DG serve as a contact point with extracellular matrix proteins (ECM) containing laminin G-like domains, providing cellular stability. However, it remains unknown whether core M1 (GlcNAcβ1-2Man) structures can serve as ligands among the various O-Mannosylated glycans. Therefore, based on the presence of N-acetylLactosamine (LacNAc) in this glycan following the core extension, the binding interactions with adhesion/growth-regulatory galectins were explored. To elucidate this process, the interaction between galectin (Gal)-1, -3, -4 and -9 with α-DG fragment 372TRGAIIQTPTLGPIQPTRV390 core M1-based glycopeptide library were profiled, using glycan microarray and nuclear magnetic resonance studies. The binding of galectins was revealed irrespective of its modular architecture, adding galectins to the list of possible binding partners of α-DG core M1 glycoconjugates by cis-binding (via peptide- and carbohydrate-protein interactions), which can be abrogated by α2,3-sialylation of the LacNAc units. The LacNAc-terminated α-DG glycopeptide interact simultaneously with both the S- and F-faces of Gal-1, thereby inducing oligomerization. Furthermore, Gal-1 can trans-bridge α-DG core M1 structures and laminins, which proposed a possible mechanism by which Gal-1 ameliorates muscular dystrophies; however, this proposal warrants further investigation. [ABSTRACT FROM AUTHOR]

The study of multivalent carbohydrate–protein interactions remains highly complicated and sometimes rendered impossible due to aggregation problems. Biolayer interferometry is emerging as a tool to monitor such complex interactions. In this study, various glycoclusters and dendrimers were prepared and evaluated as ligands for lectins produced by pathogenic bacteria Pseudomonas aeruginosa (LecA and Lec B) and Burkholderia ambifaria (BambL). Reliable kinetic and thermodynamic parameters could be measured, and immobilization of either lectin or ligands resulted in high quality data. The methods gave results in full agreement with previous isothermal titration calorimetry experiments, and presented strong advantages because they require less quantity and purity for the biomolecules. [ABSTRACT FROM AUTHOR]

Featured Application: The dextranase variant (r–TmDEX49A–ΔSP–ΔN30) can be used for dextran removal from sugar during the production process. In the sugar industry, dextran generates difficulties in the manufacturing process. Using crude dextranase (EC 3.2.1.11) to eliminate dextran in sugar is an effective practice. In this study, a synthetic dextranase-encoding gene of the filamentous fungus Talaromyces minioluteus, lacking its putative native signal peptide (1–20 amino acids) and the next 30 amino acids (r–TmDEX49A–ΔSP–ΔN30), was fused to the Saccharomyces cerevisiae prepro α–factor (MFα–2) signal sequence and expressed in Komagataella phaffii under the constitutive GAP promoter. K. phaffii DEX49A–ΔSP–ΔN30, constitutively producing and secreting the truncated dextranase, was obtained. The specific activity of the truncated variant resulted in being nearly the same in relation to the full-length mature enzyme (900–1000 U·mg−1 of protein). At shaker scale (100 mL) in a YPG medium, the enzymatic activity was 273 U·mL−1. The highest production level was achieved in a fed-batch culture (30 h) at 5 L fermenter scale using the FM21–PTM1 culture medium. The enzymatic activity in the culture supernatant reached 1614 U·mL−1, and the productivity was 53,800 U·L−1·h−1 (53.8 mg·L−1·h−1), the highest reported thus far for a DEX49A variant. Dextran decreased r–TmDEX49A–ΔSP–ΔN30 mobility in affinity gel electrophoresis, providing evidence of carbohydrate–protein interactions. K. phaffii DEX49A–ΔSP–ΔN30 shows great potential as a methanol-free, commercial dextranase production system. [ABSTRACT FROM AUTHOR]

Azobenzene photoswitches are valuable tools for controlling properties of molecular systems with light. We have been investigating azobenzene glycoconjugates to probe carbohydrate‐protein interactions and to design glycoazobenzene macrocycles with chiroptical and physicochemical properties modulated by light irradiation. To date, direct conjugation of glycosides to azobenzenes was performed by reactions providing target compounds in limited yields. We therefore sought a more effective and reliable coupling method. In this paper, we report on a straightforward thioarylation of azobenzene derivatives with glycosyl thiols as well as other thiols, thereby increasing the scope of azobenzene conjugation. Even challenging unsymmetrical conjugates can be achieved in good yields via sequential or one‐pot procedures. Importantly, red‐shifted azoswitches, which are addressed with visible light, were easily functionalized. Additionally, by oxidation of the sulfide bridge to the respective sulfones, both the photochromic and the thermal relaxation properties of the core azobenzene can be tuned. Utilizing this option, we realized orthogonal three‐state photoswitching in mixtures containing two distinct azobenzene thioglycosides. [ABSTRACT FROM AUTHOR]

The study examined the impact of glycation with five-carbon and six-carbon monosaccharides on the structure and physicochemical properties of coconut protein isolate (CPI). Compared to native CPI, CPI/monosaccharide complexes exhibited higher absorbance ratios (A 294 /A 420) and glycation degrees. Structural analysis revealed an increase in disordered structures (β-turn and random coil), a blue shift in fluorescence emission, and elevated UV absorbance, indicating protein unfolding and exposure of hydrophobic groups. Glycation significantly enhanced CPI's solubility, emulsifying capacity, oil holding capacity, and thermal stability. Molecular docking simulations emphasized the importance of hydrogen bonding in complex formation. These findings provide insights into protein-carbohydrate interactions, offering potential for diverse industrial applications and guiding future research. [Display omitted] • CPI/monosaccharide complexes were first obtained by wet-heating glycation. • Five-carbon sugars showed higher glycosylation extent on CPI than six-carbon. • The foaming ability of CPI reached up to 166.25% after galactose glycation. • Molecular docking revealed interaction mechanism between CPI and monosaccharides. [ABSTRACT FROM AUTHOR]

[Display omitted] • High temperature and pH are required to overcome the complex leaves cell wall structure. • Protein content of concentrates is influenced by precipitation pH. • Carbohydrates solubilized in the extraction step prevent protein precipitation. • Protein-carbohydrate interactions could improve functional properties. Alternative protein sources have been required to meet the significant plant protein demand. Agro-industrial by-products such as leaves have considerable potential as a source of macromolecules once they are mostly discarded as waste. The current study evaluated dried cassava leaves as a protein source. First, alkaline extraction parameters (solid–liquid ratio, pH, and temperature) were optimized and the run that result in the highest protein yield were acidified at pH 2.5 or 4. The influence of carbohydrate solubilized on protein precipitation was also evaluated by removing it via alcoholic extraction prior to precipitation. The experimental design showed that high pH and temperature conditions associated with a low solid–liquid ratio led to increased protein yields. The presence of carbohydrates in the supernatant significantly influenced protein precipitation. The protein concentrate had around 17.51% protein when it was obtained from a supernatant with carbohydrates, while protein content increased to 26.88% when it was obtained from carbohydrate-free supernatant. The precipitation pH also influenced protein content, whereas protein content significantly decreased when pH increased from 2.5 to 4. The natural interaction between carbohydrates and proteins from cassava leaves positively influenced the emulsion stability index and the foaming capacity and stability. Thus, the presented results bring insights into challenges in extracting and precipitation proteins from agro-industrial by-products. [ABSTRACT FROM AUTHOR]

Sialic acid‐binding immunoglobulin‐type lectins (Siglecs) are a family of immunoglobulin‐type lectins that mediate protein‐carbohydrate interactions via sialic acids attached to glycoproteins or glycolipids. Most of the CD33‐related Siglecs (CD33rSiglecs), a major subfamily of rapidly evolving Siglecs, contain a cytoplasmic signaling domain consisting of the immunoreceptor tyrosine‐based inhibitory motif (ITIM) and immunoreceptor tyrosine‐based switch motif (ITSM) and mediate suppressive signals for lymphoid and myeloid cells. While most CD33rSiglecs are expressed by innate immune cells, such as monocytes and neutrophils, to date, the expression of Siglecs in human T cells has not been well appreciated. In this study, we found that Siglec‐5, a member of the CD33rSiglecs, is expressed by most activated T cells upon antigen receptor stimulation. Functionally, Siglec‐5 suppresses T cell activation. In support of these findings, we found that Siglec‐5 overexpression abrogates antigen receptor induced activation of NFAT and AP‐1. Furthermore, we show that GBS β‐protein, a known bacterial ligand of Siglec‐5, reduces the production of cytokines and cytolytic molecules by activated primary T cells in a Siglec‐5 dependent manner. Our data also show that some cancer cell lines express a putative Siglec‐5 ligand(s), and that the presence of soluble Siglec‐5 enhances tumor‐cell specific T cell activation, suggesting that some tumor cells inhibit T cell activation via Siglec‐5. Together, our data demonstrate that Siglec‐5 is a previously unrecognized inhibitory T cell immune checkpoint molecule and suggest that blockade of Siglec‐5 could serve as a new strategy to enhance anti‐tumor T cell functions. [ABSTRACT FROM AUTHOR]

Deciphering how enzymes interact, modify, and recognize carbohydrates has long been a topic of interest in academic, pharmaceutical, and industrial research. Carbohydrate-binding modules (CBMs) are noncatalytic globular protein domains attached to carbohydrate-active enzymes that strengthen enzyme affinity to substrates and increase enzymatic efficiency via targeting and proximity effects. CBMs are considered auspicious for various biotechnological purposes in textile, food, and feed industries, representing valuable tools in basic science research and biomedicine. Here, we present the first crystallographic structure of a CBM8 family member (CBM8), DdCBM8, from the slime mold Dictyostelium discoideum, which was identified attached to an endo-β-1,4-glucanase (glycoside hydrolase family 9). We show that the planar carbohydrate-binding site of DdCBM8, composed of aromatic residues, is similar to type A CBMs that are specific for crystalline (multichain) polysaccharides. Accordingly, pull-down assays indicated that DdCBM8 was able to bind insoluble forms of cellulose. However, affinity gel electrophoresis demonstrated that DdCBM8 also bound to soluble (single chain) polysaccharides, especially glucomannan, similar to type B CBMs, although it had no apparent affinity for oligosaccharides. Therefore, the structural characteristics and broad specificity of DdCBM8 represent exceptions to the canonical CBM classification. In addition, mutational analysis identified specific amino acid residues involved in ligand recognition, which are conserved throughout the CBM8 family. This advancement in the structural and functional characterization of CBMs contributes to our understanding of carbohydrate-active enzymes and protein-carbohydrate interactions, pushing forward protein engineering strategies and enhancing the potential biotechnological applications of glycoside hydrolase accessory modules. [ABSTRACT FROM AUTHOR]

In the yeast Saccharomyces cerevisiae and other ascomycetes, the maintenance of cell wall integrity is governed by a family of plasma-membrane spanning sensors that include the Wsc-type proteins. These cell wall proteins apparently sense stress-induced mechanical forces at the cell surface and target the cell wall integrity (CWI) signaling pathway, but the structural base for their sensor function is yet unknown. Here, we solved a high-resolution crystal structure of the extracellular cysteine-rich domain (CRD) of yeast Wsc1, which shows the characteristic PAN/Apple domain fold with two of the four Wsc1 disulfide bridges being conserved in other PAN domain cores. Given the general function of PAN domains in mediating protein–protein and protein–carbohydrate interactions, this finding underpins the importance of Wsc domains in conferring sensing and localization functions. Our Wsc1 CRD structure reveals an unusually high number of surface-exposed aromatic residues that are conserved in other fungal CRDs, and can be arranged into three solvent-exposed clusters. Mutational analysis demonstrates that two of the aromatic clusters are required for conferring S. cerevisiae Wsc1-dependent resistance to the glucan synthase inhibitor caspofungin, and the chitin-binding agents Congo red and Calcofluor white. These findings suggest an essential role of surface-exposed aromatic clusters in fungal Wsc-type sensors that might include an involvement in stress-induced sensor-clustering required to elicit appropriate cellular responses via the downstream CWI pathway. [ABSTRACT FROM AUTHOR]

Carbohydrate microarrays, featuring high-throughput and rapid detection, provide a powerful tool for getting better insights into carbohydrate-mediated interactions. In this study, poly(2-methylacrylic acid) (pMAA)-modified substrates have been developed for fabricating carbohydrate microarrays. The abundant carboxyl groups of the pMAA polymer provide large amounts of reaction sites for subsequent carbohydrate immobilization. After the activation of the carboxyl groups, amino-modified carbohydrates were covalently and site-specifically immobilized on the pMAA-based substrates. As a typical example, the specific interaction between 4-aminophenyl α- D -mannopyranoside (α-Man) and fluorescein isothiocyanate labelled concanavalin A (ConA-FITC) was investigated using a pMAA-based carbohydrate microarray. It exhibited a low limit of detection of both spotted α-Man (10 μM) and ConA-FITC in solution (9.26 nM) without any further signal amplification. The practical application of the pMAA-based microarrays was validated by investigating the specific interactions of polysaccharides and SARS-CoV-2 spike protein, and fabricating microarrays containing other biomolecules (e.g., glycoprotein and DNA). The proposed pMAA-based microarrays provide a promising tool in bioanalytical and biomedical studies. [ABSTRACT FROM AUTHOR]

Lactose-binding lectin from Vatairea macrocarpa seeds (VML) has attracted great attention due to its interesting biological activities, such as pro-inflammatory effects and macrophage activation. This study evaluated the cytotoxicity and genotoxicity/antigenotoxicity of VML in human lymphocytes using the CometChip assay, and angiogenic activity by the chick embryo chorioallantoic membrane (CAM) assay. In genotoxicity, lymphocytes were treated with different concentrations of VML (0.5, 2 and 8 μM). In antigenotoxicity, lymphocytes were treated with the same concentrations of VML concomitant doxorubicin (90 μM DXR). To evaluate angiogenesis, all CAM were treated with different concentrations of VML (0.5, 2 and 8 μM) alone or co-treated with lactose (0.1 M). Furthermore, the levels of vascular endothelial growth factor (VEGF) and tumor necrosis factor-alpha (TNF-α) in CAM were assessed by immunohistochemistry. The results showed that VML was cytotoxic to lymphocytes, genotoxic at the highest concentration (8 μM) and antigenotoxic at low concentrations (0.5, and 2 μM). Regarding the CAM assay and immunohistochemistry, VML was angiogenic and significantly increased VEGF and TNF-α levels. In contrast, co-treatment with lactose significantly reduced the angiogenic effect and VEGF levels. We propose that protein-carbohydrate interactions between VML and glycans in the cell membrane are probably the major events involved in these activities. It seems likely that VML elicits a pro-inflammatory response through VEGF and TNF-α expression, resulting in increased vascularization at the site of inflammation. Therefore, our results show novel information on the effects of VML on DNA, as well as provide data regarded the neovascularization process involving this lectin. [Display omitted] • Cytotoxic, genotoxic/antigenotoxic effects of VML were assessed by CometChip assay. • Angiogenesis of VML was assessed alone or co-treated with lactose by CAM assay. • VML was cytotoxic, genotoxic at the highest concentration, and induced angiogenesis. • VML was antigenotoxic at low concentrations, and co-treatment reduced angiogenesis. • Protein-carbohydrate interactions are probably involved in these activities. [ABSTRACT FROM AUTHOR]

Lactose-functionalized supramolecular nanoprodrugs were fabricated in water without help of any carriers or adjuvants via supramolecular self-assembly technology for targeted and fluorescence imaging-guided chemo-photodynamic therapy. [Display omitted] Most carrier-based nano drug delivery systems (nano-DDSs) are subjected to complex preparation or purification processes, metabolic instability and potential systemic toxicity. To overcome these issues, it is urgent to develop a multifunctional carrier-free nano-DDS that can be fabricated by a simple approach for enhanced anticancer efficacy. In this work, the carrier-free supramolecular nanoprodrug (CF SNPD) based on lactose (Lac) functionalized dimeric camptothecin (CPT) was developed, in which Lac and CPT were conjugated by the aromatized thioacetal (ATA, a reactive oxygen species (ROS)-responsive bond). The obtained Lac-ATA-CPT 2 prodrug and the photosensitizer Chlorin e6 (Ce6) formed CF SNPD (denoted as Ce6@Lac-ATA-CPT 2 NPs) in water by supramolecular self-assembly. The design of dimeric CPT endowed Ce6@Lac-ATA-CPT 2 NPs with ultrahigh drug-loading capacity (up to 94%) and excellent stability. The Lac-functionalized CF SNPD displayed active specific targetability to HepG2 cells resulting from the carbohydrate-protein interactions. Furthermore, the fluorescence signal of Ce6 facilitated the precise tracking and localization of Ce6@Lac-ATA-CPT 2 NPs within the cell. Meanwhile, the ROS generated by Ce6 not only cleaved ATA linker to trigger on-demand CPT release, but also exhibited a killing effect on tumor cells, enabling synergistic therapy via CPT-mediated chemotherapy (CT) and Ce6-induced photodynamic therapy (PDT). Therefore, the multifunctional CF SNPD may be one of the promising therapeutic options for liver cancer. [ABSTRACT FROM AUTHOR]

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

Lectins are carbohydrate-binding proteins broadly used in various therapeutic and biomedical applications. The variable affinity of lectins towards variety of carbohydrates has raised attention for the biologist to explore functional aspects of lectins. Lectins express specificity to simple carbohydrates for example mannose, lactose, sialic acid, complex glycan, and glycoproteins. Lectins are classified based on their sugar specificity and are used as a tool to study protein-carbohydrate interactions. Lectins are ubiquitous in nature and identified from all sources such as bacteria, fungi, algae, and animals. Plants are the most abundant source of lectins, and till now, more than three hundred lectins were characterized from plants. These are distributed to various parts of a plant according to their requirements and function. The physiological role of lectins in a plant is still not well understood. The overabundant presence of lectins in plant seeds and storage tissues indicated their role in plant development. Plant lectins shows a broad range of activities like antibacterial, antifungal, insecticidal, anticancerous, antileishmanial, antiviral, and anticoagulants. In this review, we aim to highlight the plant lectins classification and their application in various biological aspects. [ABSTRACT FROM AUTHOR]

Carbohydrate‐binding proteins (lectins) are auspicious targets in drug discovery to combat antimicrobial resistance; however, their non‐carbohydrate drug‐like inhibitors are still unavailable. Here, we present a druggable pocket in a β‐propeller lectin BambL from Burkholderia ambifaria as a potential target for allosteric inhibitors. This site was identified employing 19F NMR fragment screening and a computational pocket prediction algorithm SiteMap. The structure–activity relationship study revealed the most promising fragment with a dissociation constant of 0.3±0.1 mM and a ligand efficiency of 0.3 kcal mol−1 HA−1 that affected the orthosteric site. This effect was substantiated by site‐directed mutagenesis in the orthosteric and secondary pockets. Future drug‐discovery campaigns that aim to develop small molecule inhibitors can benefit from allosteric sites in lectins as a new therapeutic approach against antibiotic‐resistant pathogens. [ABSTRACT FROM AUTHOR]

Protein-carbohydrate interactions are crucial for many cellular processes but can be challenging to biologically characterise. To improve our understanding and ability to model these molecular interactions, we used a carefully curated set of 370 protein-carbohydrate complexes with experimental structural and biophysical data in order to train and validate a new tool, cutoff scanning matrix (CSM)-carbohydrate, using machine learning algorithms to accurately predict their binding affinity and rank docking poses as a scoring function. Information on both protein and carbohydrate complementarity, in terms of shape and chemistry, was captured using graph-based structural signatures. Across both training and independent test sets, we achieved comparable Pearson's correlations of 0.72 under cross-validation [root mean square error (RMSE) of 1.58 Kcal/mol] and 0.67 on the independent test (RMSE of 1.72 Kcal/mol), providing confidence in the generalisability and robustness of the final model. Similar performance was obtained across mono-, di- and oligosaccharides, further highlighting the applicability of this approach to the study of larger complexes. We show CSM-carbohydrate significantly outperformed previous approaches and have implemented our method and make all data freely available through both a user-friendly web interface and application programming interface, to facilitate programmatic access at http://biosig.unimelb.edu.au/csm%5fcarbohydrate/. We believe CSM-carbohydrate will be an invaluable tool for helping assess docking poses and the effects of mutations on protein-carbohydrate affinity, unravelling important aspects that drive binding recognition. [ABSTRACT FROM AUTHOR]

Amino acids are involved in energy homeostasis, just as are carbohydrates and lipids. Therefore, mechanisms controlling protein intake should operate independently and in combination with systems controlling overall energy intake to coordinate appropriate metabolic and behavioral responses. The objective of this study was to quantify the respective roles of dietary protein and carbohydrate levels on energy balance, plasma fibroblast growth factor 21 (FGF21) and insulin growth factor 1 (IGF-1) concentrations, and hypothalamic neurotransmitters (POMC, NPY, AgRP, and CART). In a simplified geometric framework, 7-wk-old male Wistar rats were fed 12 diets containing 3%-30% protein for 3 wk, in which carbohydrates accounted for 30%-75% of the carbohydrate and fat part of the diet. As a result of this study, most of the studied parameters (body composition, energy expenditure, plasma FGF21 and IGF-1 concentrations, and Pomc/Agrp ratio) responded mainly to the protein content and to a lesser extent to the carbohydrate content in the diet. [ABSTRACT FROM AUTHOR]

Tumor-associated glycolipids such as NeuGc GM3 are auspicious molecular targets in antineoplastic therapies and vaccine strategies. 14F7 is a monoclonal IgG1 with high clinical potential in cancer immunotherapy as it displays extraordinary specificity for NeuGc GM3, while it does not recognize the very similar, ubiquitous NeuAc GM3. Here we present the 2.3 Å crystal structure of the 14F7 antigen-binding domain (14F7 scFv) in complex with the NeuGc GM3 trisaccharide. Modeling analysis and previous mutagenesis data suggest that 14F7 may also bind to an alternative NeuGc GM3 conformation, not observed in the crystal structure. The most intriguing finding, however, was that a water molecule centrally placed in the complementarity-determining region directly mediates the specificity of 14F7 to NeuGc GM3. This has profound impact on the complexity of engineering in the binding site and provides an excellent example of the importance in understanding the water structure in antibody–antigen interactions. [ABSTRACT FROM AUTHOR]

Pseudomonas aeruginosa is a widespread opportunistic pathogen that is capable of colonizing various human tissues and is resistant to many antibiotics. LecA is a galactose binding tetrameric lectin involved in adhesion, infection and biofilm formation. This study reports on the binding characteristics of mono- and divalent (chelating) ligands to LecA using different techniques. These techniques include affinity capillary electrophoresis, bio-layer interferometry, native mass spectrometry and a thermal shift assay. Aspects of focus include: affinity, selectivity, binding kinetics and residence time. The affinity of a divalent ligand was determined to be in the low-nanomolar range for all of the used techniques and with a ligand residence time of approximately 7 h, while no strong binding was seen to related lectin tetramers. Each of the used techniques provides a unique and complementary insight into the chelation based binding mode of the divalent ligand to the LecA tetramer. [ABSTRACT FROM AUTHOR]

High-sugar-tethered glycodendrimers are a remarkable tool in glycobiology for the investigation of carbohydrate-protein interaction using its multivalency property. An enthralling double-stage convergent synthetic approach was selected to build a novel class of chlorine-containing glucose-coated dendrimers using an efficient click catalyst 'dinuclear copper(I) thiodiacetate complex.' In this context, cyclen core was developed through a divergent approach, while the glucodendron was developed via a convergent approach independently. Both azide-alkyne partners were coupled through a modular copper azide-alkyne cycloaddition (CuAAC) strategy to afford a high yield of the desired 36-glucose-coated glycodendrimer. The synthesized glycodendrimer has been elucidated by NMR, gel permeation chromatography (GPC), and IR spectral analysis. [ABSTRACT FROM AUTHOR]

Carbohydrate‐protein interactions are involved in a myriad of biological processes. Thus, glycomimetics have arisen as one of the most promising synthetic targets to that end. Within the broad variety of glycomimetics, thiodisaccharides have proven to be excellent tools to study these processes, and even more, some of them unveiled interesting biological activities. This review brings together research made on the introduction of N‐acetylhexosamine residues into thiodisaccharides to date, passing through classic substitution (as SN2, thioglycosylation and ring‐opening reactions) and addition (as thiol‐ene coupling and Michael‐type additions) reactions. Recent and interesting developments regarding addition reactions to vinyl azides, cross‐coupling reactions and novel chemoenzymatic methods are also discussed. [ABSTRACT FROM AUTHOR]

ABSTRACT 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 β1,3-1,4-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 β1,3-1,4-nonasaccharide. We demonstrate its unique binding specificity toward β1,3-1,4-gluco-oligosaccharides, with increasing binding affinities up to the octasaccharide and dependency on the number and position of β1,3 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 β1,3-1,4-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 β-glucan backbone imposed by the β1,3 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 β1,3-1,4-glucan oligosaccharides to the bacterial periplasm allows Bacteroidetes to outcompete bacteria that lack this PUL for utilization of β1,3-1,4-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 β1,3-1,4-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.

The isolated protein-polysaccharide fraction (AAF) from the coelomic fluid of Dendrobaena veneta earthworm shows effective activity against Candida albicans yeast. Fungal cells of the clinical strain after incubation with the active fraction were characterized by disturbed cell division and different morphological forms due to the inability to separate the cells from each other. Staining of the cells with acridine orange revealed a change in the pH of the AAF-treated cells. It was observed that, after the AAF treatment, the mitochondrial DNA migrated towards the nuclear DNA, whereupon both merged into a single nuclear structure, which preceded the apoptotic process. Cells with a large nucleus were imaged with the scanning electron cryomicroscopy (Cryo-SEM) technique, while enlarged mitochondria and the degeneration of cell structures were shown by transmission electron microscopy (TEM). The loss of the correct cell shape and cell wall integrity was visualized by both the TEM and SEM techniques. Mass spectrometry and relative quantitative SWATH MS analysis were used to determine the reaction of the C. albicans proteome to the components of the AAF fraction. AAF was observed to influence the expression of mitochondrial and oxidative stress proteins. The oxidative stress in C. albicans cells caused by the action of AAF was demonstrated by fluorescence microscopy, proteomic methods, and XPS spectroscopy. The secondary structure of AAF proteins was characterized by Raman spectroscopy. Analysis of the elemental composition of AAF confirmed the homogeneity of the preparation. The observed action of AAF, which targets not only the cell wall but also the mitochondria, makes the preparation a potential antifungal drug killing the cells of the C. albicans pathogen through apoptosis. [ABSTRACT FROM AUTHOR]

Protein–carbohydrate interactions play a major role in several cellular and biological processes. Elucidating the factors influencing the binding affinity of protein–carbohydrate complexes and predicting their free energy of binding provide deep insights for understanding the recognition mechanism. In this work, we have collected the experimental binding affinity data for a set of 389 protein–carbohydrate complexes and derived several structure-based features such as contact potentials, interaction energy, number of binding residues and contacts between different types of atoms. Our analysis on the relationship between binding affinity and structural features revealed that the important factors depend on the type of the complex based on number of carbohydrate and protein chains. Specifically, binding site residues, accessible surface area, interactions between various atoms and energy contributions are important to understand the binding affinity. Further, we have developed multiple regression equations for predicting the binding affinity of protein–carbohydrate complexes belonging to six categories of protein–carbohydrate complexes. Our method showed an average correlation and mean absolute error of 0.731 and 1.149 kcal/mol, respectively, between experimental and predicted binding affinities on a jackknife test. We have developed a web server PCA-Pred, Protein–Carbohydrate Affinity Predictor, for predicting the binding affinity of protein–carbohydrate complexes. The web server is freely accessible at https://web.iitm.ac.in/bioinfo2/pcapred/. The web server is implemented using HTML and Python and supports recent versions of major browsers such as Chrome, Firefox, IE10 and Opera. [ABSTRACT FROM AUTHOR]

Carbohydrate-protein interactions can improve the properties and texture of proteins. The objective of this study was to investigate the effects of pH and preheating to 90 °C on the interaction between chitosan (CS)/chitosan oligosaccharide (COS) and whey protein isolate (WPI). Additionally, by simulating in vitro gastric digestion to evaluate the digestive behavior of the CS/COS-WPI complexes when applied to protein-supplemented beverages. The results demonstrated that only CS exhibited greater efficacy in preventing the denaturation and aggregation of WPI caused by heat treatment at pH 4.0. As pH continued to expand to 6.0, changes in the strength of electrostatic interactions and the tertiary structure of proteins influenced the formation of the complex system structure. The in vitro digestion experiments revealed that CS and COS inhibition of WPI digestion in the gastric resulted in an opposite trend with an increasing ratio. We hope that this work will offer promising strategies for developing protein-enrich beverages. [Display omitted] • OPLI technique monitoring of CS/COS-WPI interactions and digestive behavior. • The effects of CS and COS on WPI interactions and in vitro digestion were compared. • CS inhibited the thermal denaturation of WPI at pH 4.0. • CS/COS-WPI complexes exhibited different behaviors during digestion. • Heat treatment affected the interaction and digestive behavior. [ABSTRACT FROM AUTHOR]

Glycodendrimers are an important class of synthetic macromolecules that can be used to mimic many structural and functional features of cell‐surface glycoconjugates. Their carbohydrate moieties perform key important functions in bacterial and viral infections, often regulated by carbohydrate–protein interactions. Several studies have shown that the molecular structure, valency and spatial organisation of carbohydrate epitopes in glycoconjugates are key factors in the specificity and avidity of carbohydrate–protein interactions. Choosing the right glycodendrimers almost always helps to interfere with such interactions and blocks bacterial or viral adhesion and entry into host cells as an effective strategy to inhibit bacterial or viral infections. Herein, the state of the art in the design and synthesis of glycodendrimers employed for the development of anti‐adhesion therapy against bacterial and viral infections is described. [ABSTRACT FROM AUTHOR]