Carbohydrate Receptors Research Articles

Determination of Binding Constants and Gas Phase Stabilities of Artificial Carbohydrate Receptor Complexes Using Electrospray Mass Spectrometry.

In recent years, binding studies to determine complex stabilities and selectivities of artificial carbohydrate receptors with glycosides have been mainly performed using 1H NMR, isothermal titration calorimetry (ITC), and other spectroscopic titration techniques. Native electrospray ionization (ESI) mass spectrometry is used only to verify the complex stoichiometries, although determination of dissociation constants is also possible. Herein, the binding of a 1,3,5-substituted 2,4,6-triethylbenzene-based receptor (CHR) to four alkyl-β-d-glucosides with varying alkyl side chain lengths (methyl (MGP), hexyl (HGP), octyl (OGP), and dodecyl (DGP)-β-d-glucosides), which was analyzed by ESI Fourier transform ion cyclotron resonance mass spectrometry (ESI-FT-ICR-MS) under optimized spray conditions in both ion modes, is reported. The complexes of the receptor with different sugars could be detected in 1:1 and 2:1 stoichiometries. Dissociation constants calculated for the 1:1 complexes showed a stability trend depending on the length of the alkyl side chain of the sugar: CHR:DGP > CHR:OGP > CHR:HGP > CHR:MGP. Gas phase stabilities determined by CID-MS confirm this relative trend in binding affinities. These findings substantiate the validity and applicability of ESI-MS as a method for investigating noncovalent complex stabilities and thus support research in the field of molecular recognition of carbohydrates by artificial receptors.

Boronolipids

AbstractThe interactions between boron and polyols significantly impact the functioning of bioactive metabolites, algae, plant growth, and quorum determination in certain bacterial species. This wide array of boron‐inclusive compounds aligns with the expectation as boron ranks among the top ten most plentiful elements in seawater. Moreover, carbohydrates, largely comprising polyols, represent the most abundant class of biomass on earth. Lately, the complexes formed between boron and sugars have garnered interest. They have been employed to construct synthetic carbohydrate receptors. These complexes are typified by covalent interactions, enabling their conversion into aqueous solutions. The physical and chemical traits of boron and its established compounds suggest the feasibility of generating boron‐based lipids and glycolipids. Building upon the chemical and mathematical models that demonstrate the formation of protomembranes and biological membranes from complex lipids using low molecular weight polyols as their framework, it appears that the abiotic synthesis of boronolipids follows a parallel route. This review offers a succinct overview of naturally occurring boron compounds and delves into the possible existence of boronolipids within the makeup of protomembranes and biological membranes.

Harnessing the Power of Sugar-Based Nanoparticles: A Drug-Free Approach to Enhance Immune Checkpoint Inhibition against Glioblastoma and Pancreatic Cancer.

Cancer cells have a high demand for sugars and express diverse carbohydrate receptors, offering opportunities to improve delivery with multivalent glycopolymer materials. However, effectively delivering glycopolymers to tumors while inhibiting cancer cell activity, altering cellular metabolism, and reversing tumor-associated macrophage (TAM) polarization to overcome immunosuppression remains a challenging area of research due to the lack of reagents capable of simultaneously achieving these objectives. Here, the glycopolymer-like condensed nanoparticle (∼60 nm) was developed by a one-pot carbonization reaction with a single precursor, promoting multivalent interactions for the galactose-related receptors of the M2 macrophage (TAM) and thereby regulating the STAT3/NF-κB pathways. The subsequently induced M2-to-M1 transition was increased with the condensed level of glycopolymer-like nanoparticles. We found that the activation of the glycopolymer-like condensed galactose (CG) nanoparticles influenced monocarboxylate transporter 4 (MCT-4) function, which caused inhibited lactate efflux (similar to inhibitor effects) from cancer cells. Upon internalization via galactose-related endocytosis, CG NPs induced cellular reactive oxygen species (ROS), leading to dual functionalities of cancer cell death and M2-to-M1 macrophage polarization, thereby reducing the tumor's acidic microenvironment and immunosuppression. Blocking the nanoparticle-MCT-4 interaction with antibodies reduced their toxicity in glioblastoma (GBM) and affected macrophage polarization. In orthotopic GBM and pancreatic cancer models, the nanoparticles remodeled the tumor microenvironment from "cold" to "hot", enhancing the efficacy of anti-PD-L1/anti-PD-1 therapy by promoting macrophage polarization and activating cytotoxic T lymphocytes (CTLs) and dendritic cells (DCs). These findings suggest that glycopolymer-like nanoparticles hold promise as a galactose-elicited adjuvant for precise immunotherapy, particularly in targeting hard-to-treat cancers.

Sandwich-Like Complexation of Carbohydrates by Hydrogen Bonding and CH-π Interactions

Introduction: The design of carbohydrate-binding agents (artificial carbohydrate receptors) that enable selective and effective biomimetic recognition via noncovalent interactions is aimed either at a better understanding of natural recognition phenomena or at various potential applications in medicine and other fields. Although very interesting artificial receptors have been developed, the exact prediction of the receptor selectivity remains a challenge. Results and Methods: A molecular architecture based on a 1,3,5-substituted 2,4,6-triethylbenzene backbone bearing two aminopyridine- or aminopyrimidine-based recognition units and a purine moiety, which acts as both a hydrogen bonding site and a bridging component for the incorporation of additional substituents, has proved to be very useful for the development of effective carbohydrate-binding agents. This type of compounds has the ability to bind suitable carbohydrates through combined noncovalent interactions, where CH···π interactions can be formed on both faces of the carbohydrate substrate. The successful syntheses of the target compounds can be realized by the use of microwave irradiation and sealed tubes. The performed binding studies included 1H NMR spectroscopic titrations and measurements by isothermal titration calorimetry. Conclusion: The new compounds were developed as artificial receptors especially for carbohydrates with an all-equatorial substitution pattern and have the ability to predictably form strong 1:1 complexes with a suitable substrate. The use of the purine moiety in the construction of carbohydrate receptors with a 1,3,5-substituted 2,4,6-triethylbenzene backbone has proved to be a very promising approach. The possibilities for structural variation of this molecular architecture are manifold. As a result, a wide range of compounds can be synthesized to perform extensive studies on the relationships between structure and binding efficiency. Graphical Abstract Short Text: New compounds were developed as artificial receptors especially for carbohydrates with an all-equatorial substitution pattern and have the ability to predictably form 1:1 complexes with a suitable substrate. Key Topics: Artificial carbohydrate receptors; Combination of hydrogen bonding and CH۔۔۔π interactions; Molecular recognition

Endogenous IFN-γ facilitates Pneumocystis infection and downregulates carbohydrate receptors in CD4+ Tcell-depleted mice.

IFN-γ plays a critical role in host defense against intracellular pathogens. IFN-γ is produced in the bronchoalveolar lavage fluid of mice infected with Pneumocystis, but the role of IFN-γ in host defense against Pneumocystis remains controversial. It has been previously reported that although exogenous IFN-γ has beneficial effects on eradication of Pneumocystis, endogenous IFN-γ has a negative impact on innate immunity in immunocompromised hosts. Surprisingly, CD4+ T cell-depleted IFN-γ deficient (GKO) mice exhibit resistance to Pneumocystis. Alveolar macrophages (AM) from GKO mice exhibit higher expression of macrophage mannose receptor (MMR) and Dectin-1. Concomitantly, they exhibited greater ability to phagocytize Pneumocystis, and this activity was suppressed by inhibitors of these receptors. Incubation with IFN-γ resulted in a reduction in both the expression of these receptors on AM and their Pneumocystis-phagocytic activity. These results indicate that endogenous IFN-γ facilitates Pneumocystis to escape from host innate immunity by attenuating the phagocytic activity of AM via downregulation of MMR and Dectin-1.

Re-Print: New Perspectives in Immunotherapy: The importance of Dendritic Cells in Allergen-Specific Immunotherapy

Allergen-specific immunotherapy is the only treatment capable of altering the natural course of the allergic disease. Clinical trials show that immunotherapy is safe and effective for many patients. However, it still faces problems related to efficacy, safety, long treatment duration and low patient adherence. Contrary to what was imagined, the shift in immunity from Th2 to Th1 is not the key to successful treatment. Recent advances in the knowledge of Treg and Breg cells and tolerance mechanisms have been essential to explain the immunological changes resulting from immunotherapy. In this context, there has been intense research into the development of adjuvants with the aim of increasing safety, optimizing treatment regimens and improving patient adherence. Allergens were modified (glycoconjugates) with carbohydrates derived from Saccharomyces Cerevisae to increase their uptake and presentation through carbohydrate receptors present in Dendritic Cells, benefiting from the ability to act in inducing tolerance to initiate immune responses. Dendritic cells are the most professional antigen presenters of the APC system. They represent the bridge between innate and adaptive immunity and are capable of initiating and maintaining immune responses. In light of new evidence, they constitute a key therapeutic target to obtain an adequate response to allergen-specific immunotherapy with the potential to contribute to innovation in the field of immunotherapy.

Discovery of Orally Bioavailable FmlH Lectin Antagonists as Treatment for Urinary Tract Infections.

FmlH, a bacterial adhesin of uropathogenic Escherichia coli (UPEC), has been shown to provide a fitness advantage in colonizing the bladder during chronic urinary tract infections (UTIs). Previously reported ortho-biphenyl glycosides based on βGal and βGalNAc have excellent binding affinity to FmlH and potently block binding to its natural carbohydrate receptor, but they lack oral bioavailability. In this paper, we outline studies where we have optimized compounds for improved pharmacokinetics, leading to the discovery of novel analogues with good oral bioavailability. We synthesized galactosides with the anomeric O-linker replaced with more stable S- and C-linked linkers. We also investigated modifications to the GalNAc sugar and modifications to the biphenyl aglycone. We identified GalNAc 69 with an IC50 of 0.19 μM against FmlH and 53% oral bioavailability in mice. We also obtained a FimlH-bound X-ray structure of lead compound 69 (AM4085) which has potential as a new antivirulence therapeutic for UTIs.

A protocol to isolate, identify, and verify glucose- or carbohydrate-binding receptors.

Sensing, transport, and utilization of glucose is pivotal to the maintenance of energy homeostasis in animals. Although transporters involved in mobilizing glucose across different cellular compartments are fairly well known, the receptors that bind glucose to mediate its effects independently of glucose metabolism remain largely unrecognized. Establishing precise and reproducible methods to identify glucose receptors in the brain or other peripheral organs will pave the way for comprehending the role of glucose signaling pathways in maintaining, regulating, and reprogramming cellular metabolic needs. Identification of such potential glucose receptors will also likely lead to development of effective therapeutics for treatment of diabetes and related metabolic disorders. Commercially available biotin or radiolabeled glucose conjugates have low molecular weight; therefore, they do not provide enough sensitivity and density to isolate glucose receptors. Here, we describe a protocol to isolate, identify, and verify glucose-binding receptor/s using high molecular weight glucose (or other carbohydrate) conjugates. We have produced 30 kDa glucose- (or other carbohydrate-) biotin-polyacrylamide (PAA) conjugates with mole fractions of 80:5:15% respectively. These conjugates are used with biotin-streptavidin biochemistry, In-cell ELISA, and surface plasmon resonance (SPR) methods to isolate, identify, and verify glucose- or carbohydrate-binding receptors. We first demonstrate how streptavidin-coated magnetic beads are employed to immobilize glucose-biotin-PAA conjugates. Then, these beads are used to enrich and isolate glucose-binding proteins from tissue homogenates or from single-cell suspensions. The enriched or isolated proteins are subjected to mass spectrometry/proteomics to reveal the identity of top candidate proteins as potential glucose receptors. We then describe how the In-cell ELISA method is used to verify the interaction of glucose with its potential receptor through stable expression of the receptor in-vitro. We further demonstrate how a highly sensitive SPR method can be used to measure the binding kinetics of glucose with its receptor. In summary, we describe a protocol to isolate, identify, and verify glucose- or carbohydrate-binding receptors using magnetic beads, In-cell ELISA, and SPR. This protocol will form the future basis of studying glucose or carbohydrate receptor signaling pathways in health and in disease.

From small changes to big gains: pyridinium-based tetralactam macrocycle for enhanced sugar recognition in water.

The complex distribution of functional groups in carbohydrates, coupled with their strong solvation in water, makes them challenging targets for synthetic receptors. Despite extensive research into various molecular frameworks, most synthetic carbohydrate receptors have exhibited low affinities, and their interactions with sugars in aqueous environments remain poorly understood. In this work, we present a simple pyridinium-based hydrogen-bonding receptor derived from a subtle structural modification of a well-known tetralactam macrocycle. This small structural change resulted in a dramatic enhancement of glucose binding affinity, increasing from 56 M-1 to 3001 M-1. Remarkably, the performance of our synthetic lectin surpasses that of the natural lectin, concanavalin A, by over fivefold. X-ray crystallography of the macrocycle-glucose complex reveals a distinctive hydrogen bonding pattern, which allows for a larger surface overlap between the receptor and glucose, contributing to the enhanced affinity. Furthermore, this receptor possesses allosteric binding sites, which involve chloride binding and trigger receptor aggregation. This unique allosteric process reveals the critical role of structural flexibility in this hydrogen-bonding receptor for the effective recognition of sugars. We also demonstrate the potential of this synthetic lectin as a highly sensitive glucose sensor in aqueous solutions.

Interactions between DC-SIGN and the envelope protein from Dengue and Zika viruses: a structural perspective based on molecular dynamics and MM/GBSA analyses

Zika virus (ZIKV) and dengue virus (DENV) share a lot of similarities being both phylogenetically closely related, share the same insect vector passage for reaching the host, affinity for the same carbohydrate receptor domains (CRDs), indicating feasible competition between them on the natural field. Here, we prospected interactions of both envelope proteins with a DC-SIGN, a transmembrane c-type lectine receptor with the most implicated CRD with the Flavivirus infection presents on dendritic cells involved in viruses replication processes into the host, and among rares CRD receptors susceptible to interacting with a broad of subtypes of DENV. Protein–protein docking procedures produced structures for molecular dynamics experiments, suggesting the most energetically favorable complex. The difference found in the deltaG results prompted the experimentation with molecular dynamics. To investigate further specific residues involved with such interactions we produced a decomposition analysis using molecular dynamics of the docked proteins evaluated afterward with the Generalized Born Surface Area method. Solvent-accessible surface area (SASA) analysis for both showed very similar but with a slight reduction for ZIKV_E, which agreed with residues SASA analysis highlighting regions more exposed in the ZIVK protein than in DENV. Despite residues PHE313 is reponsible for most of the interactions with the envelope of these arboviruses, ZIKV interacted with this residue in DC-SIGN with lower energies and using more interactions with not expexted residues GLU241 and ARG386. Taken together these results suggest better competitive interaction of ZIKV with the DC-SIGN receptor, particularly in the CRD portion.

Human Coronavirus Cell Receptors Provide Challenging Therapeutic Targets.

Coronaviruses interact with protein or carbohydrate receptors through their spike proteins to infect cells. Even if the known protein receptors for these viruses have no evolutionary relationships, they do share ontological commonalities that the virus might leverage to exacerbate the pathophysiology. ANPEP/CD13, DPP IV/CD26, and ACE2 are the three protein receptors that are known to be exploited by several human coronaviruses. These receptors are moonlighting enzymes involved in several physiological processes such as digestion, metabolism, and blood pressure regulation; moreover, the three proteins are expressed in kidney, intestine, endothelium, and other tissues/cell types. Here, we spot the commonalities between the three enzymes, the physiological functions of the enzymes are outlined, and how blocking either enzyme results in systemic deregulations and multi-organ failures via viral infection or therapeutic interventions is addressed. It can be difficult to pinpoint any coronavirus as the target when creating a medication to fight them, due to the multiple processes that receptors are linked to and their extensive expression.

Cycloalkyl Groups as Building Blocks of Artificial Carbohydrate Receptors: Studies with Macrocycles Bearing Flexible Side-Arms.

The cyclopentyl group was expected to act as a building block for artificial carbohydrate receptors and to participate in van der Waals contacts with the carbohydrate substrate in a similar way as observed for the pyrrolidine ring of proline in the crystal structures of protein-carbohydrate complexes. Systematic binding studies with a series of 1,3,5-trisubstituted 2,4,6-triethylbenzenes bearing various cycloalkyl groups as recognition units provided indications of the involvement of these groups in the complexation process and showed the influence of the ring size on the receptor efficiency. Representatives of compounds that exhibit a macrocyclic backbone and flexible side arms were now chosen as further model systems to investigate whether the previously observed effects represent a general trend. Binding studies with these macrocycles towards β-D-glucopyranoside, an all-equatorial substituted carbohydrate substrate, included 1H NMR spectroscopic titrations and microcalorimetric investigations. The performed studies confirmed the previously observed tendency and showed that the compound bearing cyclohexyl groups displays the best binding properties.

Physiological features of development and options for technology for obtaining pluripotent stem cells

Topical issues related to the technology of isolation and mechanisms of development of pluripotent stem cells and their application in medicine are considered. The isolation, as well as the subsequent use of stem cells, still remains an unsolved problem both from a scientific point of view and especially in practical health care. There are three ways to produce pluripotent stem cells. First, they can be obtained in vitro from cell culture of the inner layer of early eggs. These are embryonic stem cells. Second, they can be obtained from somatic cells, as a result of the introduction of a group of genes that induce pluripotency. These are induced pluripotent stem cells. Finally, they can be obtained by transplanting the nucleus of somatic cells into an enucleated secondary egg. The microenvironment of the egg contributes to the reprogramming of the nucleus to a state close to the zygote. Mouse embryonic stem cells have many embryonic markers on their surface: carbohydrate receptors CD15, alkaline phosphatase, factor 4 like Kruppel, estrogen-bound receptor, transcription factor CP2 like 1, T-box transcription factor and gastrulation homeobox brain 2. Embryonic mouse stem cells differentiate from the internal mass of cells at the stage of preimplantation, epiblast. This is established by comparing gene expression profiles and directly isolating embryonic stem cells from epiblasts of 4.5-day-old fertilized eggs. Embryonic stem cells derived from mouse embryos of later stages of development lose markers of pluripotency. Approximately 3 days after the elimination of the leukemia inhibition factor, the expression of the Oct4 gene leads to the loss of specificity markers by cells of the early embryo. Currently, the reprogramming of pluripotency is an active area of research in which significant technological progress has been made. So, the original gene cocktail consisting of four genes is used: Oct4, Sox2, Klf4 and cMyc. The obtained types of embryonic stem cells of mouse and human, from fertilized blastocysts, induced pluripotent stem cells undoubtedly exist. However, this does not apply to pluripotent stem cells derived from postnatal animals, humans, or from extraembryonic sources such as amniotic fluid or cord blood. Despite the fact that many laboratories are working to obtain stem cells from these objects, unfortunately, there is little reproducibility in this work, and the properties of the resulting cells and even their existence are still the subject of controversy.

A "Nonsolvent Quenching" Strategy for 3D Printing of Polysaccharide Scaffolds with Immunoregulatory Accuracy.

3D printing enables the customized design of implant structures for accurately regulating host responses. However, polysaccharides, as a major biomaterial category with versatile immune activities, are typically "non-printable" due to the collapse of their filaments extruded during printing. This challenge renders their potential as immunomodulatory scaffolds underexploited. Here, inspired by the quench hardening in metal processing, a nonsolvent quenching (NSQ) strategy is innovatively designed for the 3D printing of polysaccharides. Through rapid solvent exchanging, NSQ instantly induces surface hardening to strengthen the polysaccharide filaments upon extrusion, requiring neither chemical modification nor physical blending that alters the material properties. Tested with five polysaccharides with varying physicochemical properties, NSQ prints predesigned structures at organ-relevant scales and a long shelf-life over 3 months. Glucomannan scaffolds, fabricated via NSQ with different grid spacings (1.5 and 2.5cm), induce distinct host responses upon murine subcutaneous implantation-from specific carbohydrate receptor activation to differential immunocytes accumulation and tissue matrix remodeling-as mechanistically validated in wild-type and Tlr2-/- knockout mice. Overall, NSQ as a facile and generic strategy is demonstrated to fabricate polysaccharide scaffolds with improved shape fidelity, thereby potentially unmasking their accurate immunomodulatory activities for future biomaterials design.

Characterization of Phages YuuY, KaiHaiDragon, and OneinaGillian Isolated from Microbacterium foliorum.

Microbacterium foliorum is a Gram-positive bacteria found in organic matter. Three lytic bacteriophages, KaiHaiDragon, OneinaGillian, and YuuY, were isolated from M. foliorum strain NRRL B-24224. Phage YuuY in particular expresses a broad host range as it possesses the ability to infect closely related bacterial species Microbacterium aerolatum at a high plating efficiency. Characterization tests were performed on all three Microbacterium phage to assess morphology, genomic characteristics, pH and thermal stabilities, life cycle, and the type of receptor used for infection. All three phages showed similar pH stability, ranging from pH 5–11, except for KaiHaiDragon, which had a reduced infection effectiveness at a pH of 11. YuuY possessed a significantly higher temperature tolerance compared to the other Microbacterium phages as some phage particles remained viable after incubation temperatures of up to 80 °C. Based on the one-step growth curve assay, all three Microbacterium phages possessed a relatively short latent period of 90 min and an approximately two-fold burst size factor. Moreover, all three phages utilize a carbohydrate receptor to initiate infection. Based on bioinformatics analysis, YuuY, KaiHaiDragon and OneinaGillian were assigned to clusters EA10, EC, and EG, respectively.

In-Depth Characterization of a Re-Engineered Cholera Toxin Manufacturing Process Using Growth-Decoupled Production in Escherichia coli.

Non-toxic derivatives of the cholera toxin are extensively used in neuroscience, as neuronal tracers to reveal the location of cells in the central nervous system. They are, also, being developed as vaccine components and drug-delivery vehicles. Production of cholera-toxin derivatives is often non-reproducible; the quality and quantity require extensive fine-tuning to produce them in lab-scale settings. In our studies, we seek a resolution to this problem, by expanding the molecular toolbox of the Escherichia coli expression system with suitable production, purification, and offline analytics, to critically assess the quality of a probe or drug delivery, based on a non-toxic derivative of the cholera toxin. We present a re-engineered Cholera Toxin Complex (rCTC), wherein its toxic A1 domain was replaced with Maltose Binding Protein (MBP), as a model for an rCTC-based targeted-delivery vehicle. Here, we were able to improve the rCTC production by 11-fold (168 mg/L vs. 15 mg/L), in comparison to a host/vector combination that has been previously used (BL21(DE3) pTRBAB5-G1S). This 11-fold increase in the rCTC production capability was achieved by (1) substantial vector backbone modifications, (2) using Escherichia coli strains capable of growth-decoupling (V strains), (3) implementing a well-tuned fed-batch production protocol at a 1 L scale, and (4) testing the stability of the purified product. By an in-depth characterization of the production process, we revealed that secretion of rCTC across the E. coli Outer Membrane (OM) is processed by the Type II secretion-system general secretory pathway (gsp-operon) and that cholera toxin B-pentamerization is, likely, the rate-limiting step in complex formation. Upon successful manufacturing, we have validated the biological activity of rCTC, by measuring its binding affinity to its carbohydrate receptor GM1 oligosaccharide (Kd = 40 nM), or binding to Jurkat cells (93 pM) and delivering the cargo (MBP) in a retrograde fashion to the cell.

C3-Symmetric Aromatic Core of Griffithsin Is Essential for Potent Anti-HIV Activity.

Lectins, carbohydrate-binding proteins of nonimmune origin, bind to carbohydrates and glycan shields present on the surfaces of cells and viral spike proteins. Lectins thus hold great promise as therapeutic and diagnostic proteins, exemplified by their potent antiviral activities and the desire to engineer synthetic carbohydrate receptors based on lectin recognition principles. Here, we describe a new carbohydrate-binding architectural motif─namely, a C3-symmetric tyrosine-based aromatic core, present in the therapeutic lectin griffithsin (GRFT). By using structure-based amino acid substitutions, X-ray crystallography, molecular dynamics (MD) simulations, and HIV-1 neutralization assays, we show that this core is critical for potent (pM) antiviral activity and nanomolar binding to the glycan shield largely consisting of high mannose glycans. Crystal structures and MD simulations show that CH-π interactions stabilize the aromatic cluster to maintain the three pseudo-symmetric carbohydrate-binding sites, nonaromatic amino acid substitutions (Tyr to Ala) abrogate antiviral activity, and increasing the aromatic CH-π edge-to-centroid interface via a Tyr to Trp substitution yields a GRFT variant with improved potency and increased residence time of Man-9 observed in MD simulations. NMR titrations of a Tyr-to-Ala variant indicate that disruption of the aromatic prevents the intermolecular crosslinking between two equivalents of Man-9 and one carbohydrate-binding face observed in wild-type GRFT and known to be critical for picomolar potency of this lectin. This C3-symmetric aromatic core defines a new recognition motif for the design of carbohydrate receptors and suggests principles for engineering known lectins to have increased affinity and stability.

Lamprey immunity protein enables detection for bladder cancer through recognizing N-hydroxyacetylneuraminic acid (Neu5Gc)-modified as a diagnostic marker and exploration of its production mechanism

Previous studies have demonstrated that Neu5Gc is highly expressed in breast, ovarian, prostate, colon and lung cancers, but not in normal human cells. The presence of Neu5Gc is important for prognosis and is associated with aggressiveness, metastasis, and tumor grade. However, increased Neu5Gc in bladder cancer remains unclear. LIP from lamprey binds the carbohydrate receptor of N-glycolylneuraminic acid (Neu5Gc). The combination of Neu5Gc and LIP suggested that it might be used as a diagnostic tool for the detection of Neu5Gc tumor antigen. Here, the classical animal model of bladder cancer was successfully induced by MNU bladder perfusion. The ELISA results showed that the expression level of Neu5Gc in the urine of normal rats was 94.96 ± 21.01ng/mg, and that of bladder cancer rats was 158.28 ± 34.86 ng/mg. In addition, the results of SNA and LIP immunohistochemistry demonstrated the high expression of Neu5Gc in bladder cancer. After the addition of Neu5Gc to BIU-87 and SV-HUC-1 cells, transcriptomic sequencing and real-time quantitative PCR analysis demonstrated that the gene expression of Neu5Gc synthesis pathway was significantly increased. These data suggest that LIP provides a new tool for the detection of biological samples, especially urine from patients with bladder cancer or suspected cancer, and that revealing the mechanism of abnormal glycosylation can provide theoretical basis for clinical studies.

Binding of synthetic carbohydrate receptors to enveloped virus glycans: Insights from molecular dynamics simulations

Can envelope glycans be targeted to stop viral pandemics? Here we address this question by using molecular dynamics simulations to study the binding between 10 synthetic carbohydrate receptors (SCRs) and the 33 N-glycans most commonly found on the surfaces of enveloped viruses, including Zika virus and SARS-CoV-2. Based on association quotients derived from these simulations, we classified the SCRs as weak binders, promiscuous binders, or selective binders. The SCRs almost exclusively associate at the Man3GlcNAc2 core, which is common to all N-glycans, but the binding affinity between the SCR⋅glycan pair depends on the noncovalent interactions between the heterocycle rings and the glycan antennae. Systematic variations in the glycan and SCR structures reveal relationships that could guide the design of SCRs to attain affinity and selectivity towards a chosen envelope glycan target. With these results, envelope glycans, which are currently considered “undruggable”, could become viable targets for new therapeutic strategies.

The Pore-Forming Subunit C2IIa of the Binary Clostridium botulinum C2 Toxin Reduces the Chemotactic Translocation of Human Polymorphonuclear Leukocytes.

The binary C2 toxin of Clostridium (C.) botulinum consists of two non-linked proteins, the enzyme subunit C2I and the separate binding/transport subunit C2II. To exhibit toxic effects on mammalian cells, proteolytically activated C2II (C2IIa) forms barrel-shaped heptamers that bind to carbohydrate receptors which are present on all mammalian cell types. C2I binds to C2IIa and the toxin complexes are internalized via receptor-mediated endocytosis. In acidified endosomal vesicles, C2IIa heptamers change their conformation and insert as pores into endosomal membranes. These pores serve as translocation-channels for the subsequent transport of C2I from the endosomal lumen into the cytosol. There, C2I mono-ADP-ribosylates G-actin, which results in depolymerization of F-actin and cell rounding. Noteworthy, so far morphological changes in cells were only observed after incubation with the complete C2 toxin, i.e., C2IIa plus C2I, but not with the single subunits. Unexpectedly, we observed that the non-catalytic transport subunit C2IIa (but not C2II) alone induced morphological changes and actin alterations in primary human polymorphonuclear leukocytes (PMNs, alias neutrophils) from healthy donors ex vivo, but not macrophages, epithelial and endothelial cells, as detected by phase contrast microscopy and fluorescent microscopy of the actin cytoskeleton. This suggests a PMN selective mode of action for C2IIa. The cytotoxicity of C2IIa on PMNs was prevented by C2IIa pore blockers and treatment with C2IIa (but not C2II) rapidly induced Ca2+ influx in PMNs, suggesting that pore-formation by C2IIa in cell membranes of PMNs is crucial for this effect. In addition, incubation of primary human PMNs with C2IIa decreased their chemotaxis ex vivo through porous culture inserts and in co-culture with human endothelial cells which is closer to the physiological extravasation process. In conclusion, the results suggest that C2IIa is a PMN-selective inhibitor of chemotaxis. This provides new knowledge for a pathophysiological role of C2 toxin as a modulator of innate immune cells and makes C2IIa an attractive candidate for the development of novel pharmacological strategies to selectively down-modulate the excessive and detrimental PMN recruitment into organs after traumatic injuries.