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Previously, we showed in the human umbilical vein endothelial cells (HUVECs) model that a liposome formulation of melphalan lipophilic prodrug (MlphDG) decorated with selectin ligand tetrasaccharide Sialyl Lewis X (SiaLeX) undergoes specific uptake by activated cells and in an in vivo tumor model causes a severe antivascular effect. Here, we cultured HUVECs in a microfluidic chip and then applied the liposome formulations to study their interactions with the cells in situ under hydrodynamic conditions close to capillary blood flow using confocal fluorescent microscopy. The incorporation of 5 to 10% SiaLeX conjugate in the bilayer of MlphDG liposomes increased their consumption exclusively by activated endotheliocytes. The increase of serum concentration from 20 to 100% in the flow resulted in lower liposome uptake by the cells. To elucidate the possible roles of plasma proteins in the liposome–cell interactions, liposome protein coronas were isolated and analyzed by shotgun proteomics and immunoblotting of selected proteins. Proteomic analysis showed that a gradual increase in SiaLeX content correlated with the overall enrichment of the liposome-associated proteins with several apolipoproteins, including the most positively charged one, ApoC1, and serum amyloid A4, associated with inflammation, on the one hand, and a decrease in the content of bound immunoglobulins, on the other. The article discusses the potential interference of the proteins in the binding of liposomes to selectins of endothelial cells.

A controlled reaction of a mixture of structural isomers of diacylglycerol (DAG) with N,N-disuccinimidyl carbonate and then with b-alanine provided intermediates of 1,2-diacylglycerol (1,2-DAG) which were further modified with carboxy- and amino-terminated oligoethylene glycol spacer arms of different length. As an example of bioactive molecules intended for the incorporation in lipid bilayer of artificial or cell membranes, 1,2-DAGs bearing tetrasaccharide Sialyl Lewis X or triglycine at the terminus of polar spacer were synthesized. The synthetic scheme can be readily scaled-up by the use of DAGs from food industry.

In the near future, the increase in the number of required tests for COVID-19 antibodies is expected to be many hundreds of millions. Obviously, this will be done using a variety of analytical methods and using different antigens, including peptides. In this work, we compare three method variations for detecting specific immunoglobulins directed against peptides of approximately 15-aa of the SARS-CoV-2 spike protein. These linear peptide epitopes were selected using antigenicity algorithms, and were synthesized with an additional terminal cysteine residue for their bioconjugation. In two of the methods, constructs were prepared where the peptide (F, function) is attached to a negatively charged hydrophilic spacer (S) linked to a dioleoylphosphatidyl ethanolamine residue (L, lipid) to create a function-spacer-lipid construct (FSL). These FSLs were easily and controllably incorporated into erythrocytes for serologic testing or in a lipid bilayer deposited on a polystyrene microplate for use in an enzyme immunoassays (EIA). The third method, also an EIA, used polyacrylamide conjugated peptides (peptide-PAA) prepared by controlled condensation of the cysteine residue of the peptide with the maleimide-derived PAA polymer which were immobilized on polystyrene microplates by physisorption of the polymer. In this work, we describe the synthesis of the PAA and FSL peptide bioconjugates, design of test systems, and comparison of the bioassays results, and discuss potential reasons for higher performance of the FSL conjugates, particularly in the erythrocyte-based serologic assay.

The transmission of Plasmodium spp. sporozoites to the mammalian host is the first step in the initiation of the mosquito-borne disease known as malaria. The exact route of transmission from the bloodstream to the liver is still not clearly elucidated, and identification of the host glycan structures bound by the sporozoites may inform as to which host cells are involved. Here, we provide a comprehensive analysis of the glycan structures that sporozoites from the human pathogen, P. falciparum, and the rodent pathogen, P. yoelii, recognize and bind. Glycan array analysis was used to profile the glycans bound by the sporozoites, and the binding affinities of these sporozoite-glycan interactions were then determined by surface plasmon resonance. Data showed that the different Plasmodium spp. bind different classes of glycans. P. falciparum was observed to bind to glycans with terminal N-acetylgalactosamine (GalNAc) or Galactose (Gal) linked to a GalNAc, and the highest-affinity observed was with the GalNAc monosaccharide (12.5 nM). P. yoelii bound glycosaminoglycans, mannosyl glycans, Gal linked to N-acetylglucosamine structures, and the αGal epitope. The highest-affinity interaction for P. yoelii was with the αGal epitope (31.4 nM). This is the first study to identify the key host glycan structures recognized by human and rodent Plasmodium spp. sporozoites. An understanding of how Plasmodium sporozoites interact with the specific glycan structures identified here may provide further insight into this infectious disease that could help direct the design of an effective therapeutic.

Interferons (IFNs) mediate innate antiviral activity against many types of viruses, including influenza viruses. In light of their potential use as anti-influenza agents, we examined whether resistance to these host antiviral proteins can develop. We generated IFN-β-resistant variants of the A/California/04/09 (H1N1) virus by serial passage in a human airway epithelial cell line, Calu-3, under IFN-β selective pressure. The combination of specific mutations (i.e., L373I in PB1, K154E1, D222G1, I56V2, and V122I2 in HA, and M269I in NA) correlated with decreased ability of the virus to induce expression of IFN (IFNB1, IFNL1, and IFNL2/3) and IFN-stimulated genes (IFIT1, IFIT3, OAS1, IRF7, and MX1) by target respiratory epithelial cells. In addition, the IFN-induced mutations were associated with decreased HA binding affinity to α2,6 sialyl receptors, reduced NA enzyme catalytic activity, and decreased polymerase transcription activity. Our findings demonstrate that the mutations in the influenza HA, NA, and PB1 proteins induced by IFN-b selective pressure significantly increase viral ability to productively infect and replicate in host cells. Keywords: influenza A virus; interferon-β; lung epithelial cells; interferon response.

In this report, we describe the fluorescent labeling of bacterial polysaccharides (Escherichia coli O86:B7, Escherichia coli O19ab, Pseudomonas aeruginosa O10a10b, and Shigella flexneri 2b) at the “natural” amino group of their phosphoethanolamine moiety. Two protocols for labeling are compared: 1) on a scale of a few mg of the polysaccharide, with a dialysis procedure for purification from excessive reagents; and 2) on a scale of 0.1 mg of the polysaccharide, with a simple precipitation procedure instead of dialysis. The microscale version is sufficient for comfortable cytofluorometric analysis. The resulting probes were found to specifically bind to human dendritic cells in a dose-dependent manner. The used limited set of polysaccharides did not allow us even to get close to understanding which dendritic cell-associated lectins and which cognate polysaccharide epitopes are involved in recognition, but the proposed microscale protocol allows to generate a library of fluorescent probes for further mapping of the polysaccharide specificity of the dendritic cells.

Background The Coronavirus disease 2019 (COVID‐19) pandemic is having a major global impact, and the resultant response in the development of new diagnostics is unprecedented. The detection of antibodies against severe acute respiratory syndrome coronavirus 2 (SARS‐CoV‐2) has a role in managing the pandemic. We evaluated the feasibility of using SARS‐CoV‐2 peptide Kode Technology‐modified red cells (C19‐kodecytes) to develop an assay compatible with existing routine serologic platforms. Study Design and Methods A panel of eight unique red cells modified using Kode Technology function‐spacer‐lipid constructs and bearing short SARS‐CoV‐2 peptides was developed (C19‐kodecyte assay). Kodecytes were tested against undiluted expected antibody‐negative and ‐positive plasma samples in manual tube and three column agglutination technology (CAT) platforms. Parallel analysis with the same peptides in solid phase by enzyme immunoassays was performed. Evaluation samples included >120 expected negative blood donor samples and >140 COVID‐19 convalescent plasma samples, with independent serologic analysis from two centers. Results Specificity (negative reaction rate against expected negative samples) in three different CAT platforms against novel C19‐kodecytes was >91%, which correlated with published literature. Sensitivity (positive reaction rate against expected positive convalescent, PCR‐confirmed samples) ranged from 82% to 97% compared to 77% with the Abbott Architect SARS‐CoV‐2 IgG assay. Manual tube serology was less sensitive than CAT. Enzyme immunoassay results with some Kode Technology constructs also had high sensitivity. Conclusions C19‐kodecytes are viable for use as serologic reagent red cells for the detection of SARS‐CoV‐2 antibody with routine blood antibody screening equipment.

Many viruses, beside binding to their main cell target, interact with other molecules that promote virus adhesion to the cell; often, these additional targets are glycans. The main receptor for SARS-CoV-2 is a peptide motif in the ACE2 protein. We studied interaction of the recombinant SARS-CoV-2 spike (S) protein with an array of glycoconjugates, including various sialylated, sulfated, and other glycans, and found that the S protein binds some (but not all) glycans of the lactosamine family. We suggest that parallel influenza infection will promote SARS-CoV-2 adhesion to the respiratory epithelial cells due to the unmasking of lactosamine chains by the influenza virus neuraminidase. Electronic supplementary material Supplementary material is available in the online version of this article at 10.1134/S0006297921030019 and on the journal website (http://protein.bio.msu.ru/biokhimiya).

Polyacrylamide conjugates of glycans have long been widely used in many research areas of glycobiology, mainly for immobilizing glycans in solid-phase assays and as multivalent inhibitors. Pending biotin tag allows immobilizing Glyc-PAA quantitatively on any surface, and acts as a tracer for detection of carbohydrate-binding proteins. However, the scope of already realized capabilities of these probes is immeasurably richer than those listed above. This review is not so much about routine as about less common, but not less significant applications. Also, the data on the glycopolymers themselves, their molecular weight, size and polymer chain flexibility are presented, as well as the methods of synthesis, clusterisation and entropy factor in their interaction with proteins.

Glycosylation of lactosamine acceptors with Kdn thioglycoside donors in the presence of NIS/TfOH as a promoter affords products with both α- and β-ketosidic linkage (2–6' or 2–3') between the Kdn and Gal residues. After deprotection, the synthesized trisaccharides and glycans containing Neu5Ac were printed to a chip and their comparative interaction with human serum antibodies was explored.

The concept of biomedical significance of the functional pairing between tissue lectins and their glycoconjugate counterreceptors has reached the mainstream of research on the flow of biological information. A major challenge now is to identify the principles of structure-activity relationships that underlie specificity of recognition and the ensuing post-binding processes. Toward this end, we focus on a distinct feature on the side of the lectin, i.e. its architecture to present the carbohydrate recognition domain (CRD). Working with a multifunctional human lectin, i.e. galectin-3, as model, its CRD is used in protein engineering to build variants with different modular assembly. Hereby, it becomes possible to compare activity features of the natural design, i.e. CRD attached to an N-terminal tail, with those of homo- and heterodimers and the tail-free protein. Thermodynamics of binding disaccharides proved full activity of all proteins at very similar affinity. The following glycan array testing revealed maintained preferential contact formation with N-acetyllactosamine oligomers and histo-blood group ABH epitopes irrespective of variant design. The study of carbohydrate-inhibitable binding of the test panel disclosed up to qualitative cell-type-dependent differences in sections of fixed murine epididymis and especially jejunum. By probing topological aspects of binding, the susceptibility to inhibition by a tetravalent glycocluster was markedly different for the wild-type vs the homodimeric variant proteins. The results teach the salient lesson that protein design matters: the type of CRD presentation can have a profound bearing on whether basically suited oligosaccharides, which for example tested positively in an array, will become binding partners in situ. When lectin-glycoconjugate aggregates (lattices) are formed, their structural organization will depend on this parameter. Further testing (ga)lectin variants will thus be instrumental (i) to define the full range of impact of altering protein assembly and (ii) to explain why certain types of design have been favored during the course of evolution, besides opening biomedical perspectives for potential applications of the novel galectin forms. Open Access funding provided by Projekt DEAL. We gratefully acknowledge inspiring discussions with Drs. B. Friday, A. Leddoz and A. W. L. Nose, valuable input during the review process and generous fnancial support by an NIH Grant (No. CA242351; to M.C.). peer-reviewed

Strong discrepancies in published data on the levels and epitope specificities of antibodies against the xenogenic N-glycolyl forms of sialoglycans (Hanganutziu-Deicher Neu5Gcɑ2-3Galβ1-4Glc and related antigens) in healthy donors prompted us to carry out a systematic study in this area using the printed glycan array and other methods. This article summarizes and discusses our published and previously unpublished data, as well as publicly available data from the Consortium for Functional Glycomics. As a result, we conclude that (1) the level of antibodies referred to as anti-Neu5Gc in healthy individuals is low; (2) there are antibodies that seem to interact with Neu5Gc-containing epitopes, but in fact they recognize internal fragments of Neu5Gc-containing glycans (without sialic acids), which served as antigens in the assays used and; (3) a population capable of interacting specifically with Neu5Gc (it does not bind the corresponding NAc analogs) does exist, but it binds the monosaccharide Neu5Gc better than the entire glycans containing it. In other words, in healthy donors, there are populations of antibodies capable of binding the Neu5Gc monosaccharide or the inner core -Galβ1-4Glc, but very few true anti-Neu5Gcɑ2-3Galβ1-4Glc antibodies, i.e., antibodies capable of specifically recognizing the entire trisaccharide.

A possible mechanism of the immune tolerance in pregnancy is production of blocking antibodies which reside in placenta and protect foetal allogeneic cells from the mother's immune system. Their epitope specificity, as well as the nature of the biomolecules masked by them, is unknown. For better understanding of this phenomenon, we attempted to characterize the specificity of antibodies isolated from placentas of women with healthy pregnancy and pre-eclampsia (PE). It was found that: (1) the repertoire of placental antibodies is significantly less variable and qualitatively different from the peripheral blood; (2) with PE, the repertoire of placental antibodies is narrower than in healthy pregnancy; (3) some antibodies are found almost exclusively in the placenta, and some - only in the placenta of healthy women.

Modification of vaccine carriers by decoration with glycans can enhance binding to and even targeting of dendritic cells (DCs), thus augmenting vaccine efficacy. To find a specific glycan-“vector” it is necessary to know glycan-binding profile of DCs. This task is not trivial; the small number of circulating blood DCs available for isolation hinders screening and therefore advancement of the profiling. It would be more convenient to employ long-term cell cultures or even primary DCs from murine blood. We therefore examined whether THP-1 (human monocyte cell line) and DC2.4 (immature murine DC-like cell line) could serve as a model for human DCs. These cells were probed with a set of glycans previously identified as binding to circulating human CD14low/-CD16+CD83+ DCs. In addition, we tested a subpopulation of murine CD14low/-CD80+СD11c+CD16+ cells reported as relating to the human CD14low/-CD16+CD83+ cells. Manα1–3(Manα1–6)Manβ1–4GlcNAcβ1–4GlcNAcβ bound to both the cell lines and the murine CD14low/-CD80+СD11c+CD16+ cells. Primary cells, but not the cell cultures, were capable of binding GalNAcα1–3Galβ (Adi), the most potent ligand for binding to human circulating DCs. In conclusion, not one of the studied cell lines proved an adequate model for DCs processes involving lectin binding. Although the glycan-binding profile of BYRB-Rb (8.17)1Iem mouse DCs could prove useful for assessing human DCs, important glycan interactions were missing, a situation which was aggravated when employing cells from the BALB/c strain. Accordingly, one must treat results from murine work with caution when seeking vaccine targeting of human DCs, and certainly should avoid cell lines such as THP-1 and DC2.4 cells.

Previous studies have shown that in the blood of healthy donors (1) there are no natural antibodies against sialylated glycoproteins, which contain Neu5Ac (N-acetylneuraminic acid) as the most widespread form of human sialic acid, and (2) there is a moderate level of antibodies capable of binding unnatural oligosaccharides, where Neu5Ac is beta-linked to a typical mammalian glycan core. In the present study, we investigated antibodies against Neu5Ac in more detail and verified the presence of Kdn (2-keto-3-deoxy-D-glycero-D-galacto-nonulosonic acid) as a possible cause behind their appearance in humans, taking into account the expected cross-reactivity to Kdn glycans, which are found in bacterial glycoconjugates in both the - and -forms. We observed the binding of peripheral blood immunoglobulins to sialyllactosamines (where sialyl is Kdn or neuraminic acid) in only a very limited number of donors, while the binding to monosaccharide Kdn occurred in all samples, regardless of the configuration of the glycosidic bond of the Kdn moiety. In some individuals, the binding level of some of the immunoglobulins was high. This means that bacterial Kdn glycoconjugates are very unlikely to induce antibodies to Neu5Ac glycans in humans. To determine the reason for the presence of these antibodies, we focused on noninfectious pathologies, as well as on a normal state in which a significant change in the immune system occurs: namely, pregnancy. As a result, we found that 2/3 of pregnant women have IgM in the blood against Neu5Ac2-3Gal1-4GlcNAc. Moreover, IgG class antibodies against Neu5Ac2-3Gal1-4GlcNAc and Neu5Ac2-6Gal1-4GlcNAc were also detected in eluates from the placenta. Presumably, these antibodies block fetal antigens.

Objective Gastric cancer (GC) is traditionally considered a difficult disease to diagnose and treat. The search for new markers for GC is an extremely urgent purpose. Previously has been shown, that serum anti-glycan antibodies (AGAT) are very large reservoir of markers which can be reliably detected using an instrument called glycoarray (PGA). A “;signature”; approach, i.e. searching of combinations of diagnostically significant markers – AGAT detected by PGA, is used in this study. Methodology The cohort of the serum of apparently healthy donors from the National Medical Research Center of Oncology (NMRC) (n = 55, 69%/31% - m/f) and previously untreated patients with an established diagnosis of GC I-IV stages from the NMRC (n = 146, 52%/48% - m/f) were collected. To study serum AGATs glycoarray containing 300 different glycans was used. To search for a diagnostic signature, the mathematical apparatus “;Immunoruler”; [Int. J. Bioinformatics Res. Appl., 7, 402-426 (2011)] was applied. Results Using glycoarray IgG and IgM profiles of donors and GC patients were obtained and data quality control has been performed. The mathematical apparatus Immunoruler was applied to the resulting database and a signature was obtained. It includes antibodies to 11 glycans: 7 IgM (directed to KDNb6’LN-C3, b3’SLN, LN-C8, Aa4A, TF, 3’SiaLeC and Tn3Su) and 4 IgG (GN6Su, TF, para-Fs and bGU). The quality of the developed diagnostic approach was assessed: the AUC value was 0.87, and the accuracy was 0.81. Conclusion Thus, the use of glycoarray technology in combination with a mathematical signature search apparatus has made it possible to find a reliable combination of molecular markers for the diagnosis of gastric cancer. Since the tumor can dramatically change as it progresses, the AGAT profile can also change. This opens up the possibility for a differentiated diagnosis of GC depending on the stage of the disease and, first of all, to develop early diagnosis of this disease.

The hemagglutinin (HA) of A/H3N2 pandemic influenza viruses (IAVs) of 1968 differed from its inferred avian precursor by eight amino acid substitutions. To determine their phenotypic effects, we studied recombinant variants of A/Hong Kong/1/1968 virus containing either human-type or avian-type amino acids in the corresponding positions of HA. The precursor HA displayed receptor binding profile and high conformational stability typical for duck IAVs. Substitutions Q226L and G228S, in addition to their known effects on receptor specificity and replication, marginally decreased HA stability. Substitutions R62I, D63N, D81N and N193S reduced HA binding avidity. Substitutions R62I, D81N and A144G promoted viral replication in human airway epithelial cultures. Analysis of HA sequences revealed that substitutions D63N and D81N accompanied by the addition of N-glycans represent common markers of avian H3 HA adaptation to mammals. Our results advance understanding of genotypic and phenotypic changes in IAV HA required for avian-to-human adaptation and pandemic emergence., Author summary Pandemic influenza A viruses (IAVs) originate from animal IAVs and always contain the envelope protein hemagglutinin (HA) of the animal parent. Molecular changes essential for the animal-to-human adaptation of the HA are not well defined. To address this problem, we studied phenotypic effects of eight amino acid substitutions that accompanied evolution of the HA of the 1968 pandemic IAV from its inferred avian precursor. We also analysed patterns of evolution of these HA positions of IAVs in birds and mammals. Our results predict that the 1968 pandemic IAV acquired its HA from a wild duck virus. We showed that two substitutions were primarily responsible for increased HA binding to human-type receptors and, in addition, marginally decreased stability of the HA, whereas three other substitutions reduced binding avidity of the HA. We demonstrated that substitutions accompanied by the addition of N-glycan at either position 63 or 81 represent a previously unrecognized common marker of avian-to-mammalian adaptation of IAVs. Our findings highlight the importance of the studies on previous pandemic IAVs for the influenza risk assessment and pandemic preparedness.

Since the discovery of carbohydrate nature of the antigens related to ABO blood group system in the middle of 20th century, an interest to synthetic approaches to these oligosaccharides is very high. ABH glycans are required for clinical applications, namely, immunoadsorption and blood typing, as well as for scientific investigations in the areas of hematology, immunology and glycobiology. In this article, the chemical syntheses of various ABH glycans and related structures are reviewed, and examples of their use for clinical purposes are collected.

A fraction of sperm deposited at mating or insemination reaches the oviduct isthmus, where sperm are retained and thereby form a reservoir. This reservoir delays capacitation, prevents polyspermy, selects a fertile population of sperm, and, foremost, increases sperm lifespan. The molecular interactions underlying the formation of a sperm reservoir are becoming clearer in mammals. Sperm lectins bind to oviductal glycans to form the reservoir. Herein, we found that the highest percentage of bovine sperm bound to the 3′-O-sulfated form of Lewis A (suLe(A)) trisaccharide and sialylated Lewis A and that fluoresceinated versions of each localized to receptors on the anterior head of the sperm. Following capacitation, binding to suLe(A) decreased significantly, a potential explanation for sperm release from the reservoir. MS and immunohistochemistry analyses indicated that suLe(A) motifs were present predominantly on O-linked glycans initiated by GalNAc residues, but no sialylated Lewis A was detected. To determine whether sperm binding to isolated suLe(A) in vitro could mimic in vivo sperm binding to oviduct cells and increase sperm longevity, we immobilized suLe(A) and incubated it with sperm. Using free-swimming sperm and sperm bound to immobilized laminin as controls, we observed that over 96 h, the viability of free-swimming sperm decreased to 10%, and that of sperm bound to immobilized laminin decreased to about 50%, whereas viability of sperm bound to immobilized suLe(A) was highest throughout the incubation and 60% at 96 h. These results indicate that bovine sperm binding to oviduct suLe(A) retains sperm for reservoir formation and extends sperm lifespan.

Pseudomonas aeruginosa is an opportunistic pathogen that causes nosocomial infections most commonly in immunocompromised, cystic fibrosis (CF) and burns patients. The pilin and Pseudomonas lectins 1 (PA-IL) and 2 (PA-IIL) are known glycan-binding proteins of P. aeruginosa that are involved in adherence to host cells, particularly CF host airways. Recently, new P. aeruginosa surface proteins were identified by reverse vaccinology and tested in vivo as potential vaccine antigens. Three of these, namely PSE17-1, PSE41-5 and PSE54, were screened for glycan binding using glycan arrays displaying glycan structures representative of those found on human cells. Surface plasmon resonance was used to confirm the lectin activity of these proteins, and determined affinities with several host glycans to be in the nanomolar range. PSE17-1 binds hyaluronic acid and sialyl Lewis A and X. PSE41-5 binds terminal β-linked galactose structures, Lewis and ABO blood group antigens. PSE54 binds to ABO blood group antigens and some terminal β-linked galactose. All three proteins are novel lectins of P. aeruginosa with potential roles in infection of host cells.

N-glycosylation is a ubiquitous posttranslational modification that may influence folding, subcellular localization, secretion, solubility and oligomerization of proteins. In this study, we examined the effects of N-glycans on the activity of human Gb3/CD77 synthase, which catalyzes the synthesis of glycosphingolipids with terminal Galα1→4Gal (Gb3 and the P1 antigen) and Galα1→4GalNAc disaccharides (the NOR antigen). The human Gb3/CD77 synthase contains two occupied N-glycosylation sites at positions N121 and N203. Intriguingly, we found that while the N-glycan at N203 is essential for activity and correct subcellular localization, the N-glycan at N121 is dispensable and its absence did not reduce, but, surprisingly, even increased the activity of the enzyme. The fully N-glycosylated human Gb3/CD77 synthase and its glycoform missing the N121 glycan correctly localized in the Golgi, whereas a glycoform without the N203 site partially mislocalized in the endoplasmic reticulum. A double mutein missing both N-glycans was inactive and accumulated in the endoplasmic reticulum. Our results suggest that the decreased specific activity of human Gb3/CD77 synthase glycovariants results from their improper subcellular localization and, to a smaller degree, a decrease in enzyme solubility. Taken together, our findings show that the two N-glycans of human Gb3/CD77 synthase have opposing effects on its properties, revealing a dual nature of N-glycosylation and potentially a novel regulatory mechanism controlling the biological activity of proteins.

In this report, we describe the fluorescent labeling of bacterial polysaccharides (Escherichia coli O86:B7, Escherichia coli O19ab, Pseudomonas aeruginosa O10a10b, and Shigella flexneri 2b) at the "natural" amino group of their phosphoethanolamine moiety. Two protocols for labeling are compared: 1) on a scale of a few mg of the polysaccharide, with a dialysis procedure for purification from excessive reagents; and 2) on a scale of 0.1 mg of the polysaccharide, with a simple precipitation procedure instead of dialysis. The microscale version is sufficient for comfortable cytofluorometric analysis. The resulting probes were found to specifically bind to human dendritic cells in a dose-dependent manner. The used limited set of polysaccharides did not allow us even to get close to understanding which dendritic cell-associated lectins and which cognate polysaccharide epitopes are involved in recognition, but the proposed microscale protocol allows to generate a library of fluorescent probes for further mapping of the polysaccharide specificity of the dendritic cells.

Summary The small 3-O-sulfated galactose head group of sulfatides, an abundant glycosphingolipid class, poses the (sphinx-like) riddle on involvement of glycan bridging by tissue lectins (sugar code). First, synthesis of head group derivatives for functionalization of amphiphilic dendrimers is performed. Aggregation of resulting (biomimetic) vesicles, alone or in combination with lactose, demonstrates bridging by a tissue lectin (galectin-4). Physiologically, this can stabilize glycolipid-rich microdomains (rafts) and associate sulfatide-rich regions with specific glycoproteins. Further testing documents importance of heterobivalency and linker length. Structurally, sulfatide recognition by galectin-8 is shown to involve sphingosine's OH group as substitute for the 3′-hydroxyl of glucose of lactose. These discoveries underscore functionality of this small determinant on biomembranes intracellularly and on the cell surface. Moreover, they provide a role model to examine counterreceptor capacity of more complex glycans of glycosphingolipids and to start their bottom-up glycotope surface programming., Graphical abstract, Highlights • Nanoparticle programming detects sulfatide-(N)-glycan bridging by galectins-4 and -8 • Protein design (linker/domain type) is a switch for aggregation activity • Sphingosine's OH group is involved in contact building with a galectin, Supramolecular Chemistry; Biochemistry; Biophysics

BACKGROUND The α1,3-galactosyltransferase gene-knockout (GalT KO) mice are able to produce natural anti-αGal antibodies apparently without any specific immunization. GalT KO mice are commonly used as a model immunological system for studying anti-αGal responses to Gal-positive xenografts in human. In this study, we compared the specificity of mouse and human αGal antibodies to realize the adequacy of the murine model. METHODS Using hapten-specific affinity chromatography antibodies against Galα1-3Galβ1-4GlcNAcβ epitope were isolated from both human and GalT KO mice blood sera. Specificity of isolated antibodies was determined using a printed glycan array (PGA) containing 400 mammalian glycans and 200 bacterial polysaccharides. RESULTS The quantity of isolated specific anti-Galα antibodies corresponds to a content of

The traditionally used strategy for the synthesis of blood group A and B tetrasaccharides includes 2′-O-fucosylation of lactosamine followed by insertion of an α1-3 linked N-acetylgalactosamine or a galactose moiety. Here, we report the synthesis of 3-aminopropyl glycosides of A (type 2) and B (type 2) tetrasaccharides via an alternative sequence, i.e. α-galactosaminylation (or α-galactosylation) followed by α-fucosylation. This strategy allows us to synthesize fucose-free trisaccharides GalNAcα1-3Galβ1-4GlcNAc and Galα1-3Galβ1-4GlcNAc, which are promising targets for immunotherapy utilising human natural antibodies against the trisaccharides. The key stage in this scheme was the selective chloroacetylation of the 2′-OH group of βGal in the intermediate trisaccharides having the second (3-OH) unprotected group.The protocol is suitable for multigram syntheses and its further scaling up.

In mammals, the oviduct retains sperm, forming a reservoir from which they are released in synchrony with ovulation. However, the mechanisms underlying sperm release are unclear. Herein, we first examined in greater detail the release of sperm from the oviduct reservoir by sex steroids, and secondly, if the ubiquitin–proteasome system (UPS) mediates this release in vitro. Sperm were allowed to bind to oviductal cells or immobilized oviduct glycans, either bi-SiaLN or a suLeX, and channeled with steroids in the presence or absence of proteasome inhibitors. Previously, we have demonstrated progesterone-induced sperm release from oviduct cells and immobilized glycans in a steroid-specific manner. Herein, we found that the release of sperm from an immobilized oviduct glycan, a six-sialylated branched structure, and from immobilized fibronectin was inhibited by the CatSper blocker NNC 055-0396, akin to the previously reported ability of NNC 055-0396 to inhibit sperm release from another oviduct glycan, sulfated Lewis-X trisaccharide. Thus, CatSper may be required for release of sperm from a variety of adhesion systems. One possible mechanism for sperm release is that glycan receptors on sperm are degraded by proteasomes or shed from the sperm surface by proteasomal degradation. Accordingly, the inhibition of proteasomal degradation blocked sperm release from oviduct cell aggregates both immobilized oviduct glycans as well as fibronectin. In summary, progesterone-induced sperm release requires both active CatSper channels and proteasomal degradation, suggesting that hyperactivation and proteolysis are vital parts of the mechanism by which sperm move from the oviduct reservoir to the site of fertilization.

Each year, 5% to 20% of the population of the United States becomes infected with influenza A virus. Combination therapy with two or more antiviral agents has been considered a potential treatment option for influenza virus infection. However, the clinical results derived from combination treatment with two or more antiviral drugs have been variable. We examined the effectiveness of cotreatment with two distinct classes of anti-influenza drugs, i.e., neuraminidase (NA) inhibitor, laninamivir, and interferon lambda 1 (IFN-λ1), against the emergence of drug-resistant virus variants in vitro. We serially passaged pandemic A/California/04/09 [A(H1N1)pdm09] influenza virus in a human lung epithelial cell line (Calu-3) in the presence or absence of increasing concentrations of laninamivir or laninamivir plus IFN-λ1. Surprisingly, laninamivir used in combination with IFN-λ1 promoted the emergence of the E119G NA mutation five passages earlier than laninamivir alone (passage 2 versus passage 7, respectively). Acquisition of this mutation resulted in significantly reduced sensitivity to the NA inhibitors laninamivir (∼284-fold) and zanamivir (∼1,024-fold) and decreased NA enzyme catalytic activity (∼5-fold) compared to the parental virus. Moreover, the E119G NA mutation emerged together with concomitant hemagglutinin (HA) mutations (T197A and D222G), which were selected more rapidly by combination treatment with laninamivir plus IFN-λ1 (passages 2 and 3, respectively) than by laninamivir alone (passage 10). Our results show that treatment with laninamivir alone or in combination with IFN-λ1 can lead to the emergence of drug-resistant influenza virus variants. The addition of IFN-λ1 in combination with laninamivir may promote acquisition of drug resistance more rapidly than treatment with laninamivir alone.

ChemistryOpen 9(6), 641 - 648 (2020). doi:10.1002/open.201900276, The synthetic function‐spacer‐lipid (FSL) amphiphile biotin‐CMG‐DOPE is widely used for delicate ligation of living cells with biotin residues under physiological conditions. Since this molecule has an “apolar‐polar‐hydrophobic” gemini structure, the supramolecular organization is expected to differ significantly from the classical micelle. Its organization is investigated with experimental methods and molecular dynamics simulations (MDS). Although the linear length of a single biotin‐CMG‐DOPE molecule is 9.5 nm, the size of the dominant supramer globule is only 14.6 nm. Investigations found that while the DOPE tails form a hydrophobic core, the polar CMG spacer folds back upon itself and predominantly places the biotin reside inside the globule or planar layer. MDS demonstrates that, Published by Wiley-VCH-Verl., Weinheim

Influenza B viruses cause seasonal epidemics and are a considerable burden to public health. To understand their adaptation capability, we examined the genetic changes that occurred following 15 serial passages of two influenza B viruses, B/Brisbane/60/2008 and B/Victoria/504/2000, in human epithelial cells. Thirteen distinct amino acid mutations were found in the PB1, PA, hemagglutinin (HA), neuraminidase (NA), and M proteins after serial passage in the human lung epithelial cell line, Calu-3, and normal human bronchial epithelial (NHBE) cells. These changes were associated with significantly decreased viral replication levels. Our results demonstrate that adaptation of influenza B viruses for growth in human airway epithelial cells is partially conferred by selection of HA1, NA, and polymerase mutations that regulate receptor specificity, functional compatibility with the HA protein, and polymerase activity, respectively.

Building on our recent discovery of the zinc signature phenomenon present in boar, bull, and human spermatozoa, we have further characterized the role of zinc ions in the spermatozoa&rsquo, s pathway to fertilization. In boar, the zinc signature differed between the three major boar ejaculate fractions, the initial pre-rich, the sperm-rich, and the post-sperm-rich fraction. These differences set in the sperm ejaculatory sequence establish two major sperm cohorts with marked differences in their sperm capacitation progress. On the subcellular level, we show that the capacitation-induced Zn-ion efflux allows for sperm release from oviductal glycans as analyzed with the oviductal epithelium mimicking glycan binding assay. Sperm zinc efflux also activates zinc-containing enzymes and proteases involved in sperm penetration of the zona pellucida, such as the inner acrosomal membrane matrix metalloproteinase 2 (MMP2). Both MMP2 and the 26S proteasome showed severely reduced activity in the presence of zinc ions, through studies using by gel zymography and the fluorogenic substrates, respectively. In the context of the fertilization-induced oocyte zinc spark and the ensuing oocyte-issued polyspermy-blocking zinc shield, the inhibitory effect of zinc on sperm-borne enzymes may contribute to the fast block of polyspermy. Altogether, our findings establish a new paradigm on the role of zinc ions in sperm function and pave the way for the optimization of animal semen analysis, artificial insemination (AI), and human male-factor infertility diagnostics.

ObjectiveTo explore the repertoire of glycan-specific immunoglobulin G (IgG) antibodies in treatment-naive patients with relapsing-remitting multiple sclerosis (RRMS).MethodsA systems-level approach combined with glycan array technologies was used to determine specificities and binding reactivities of glycan-specific IgGs in treatment-naive patients with RRMS compared with patients with noninflammatory and other inflammatory neurologic diseases.ResultsWe identified a unique signature of glycan-binding IgG in MS with high reactivities to the dietary xenoglycan N-glycolylneuraminic acid (Neu5Gc) and the self-glycan N-acetylneuraminic acid (Neu5Ac). Increased reactivities of serum IgG toward Neu5Gc and Neu5Ac were additionally observed in an independent, treatment-naive cohort of patients with RRMS.ConclusionPatients with MS show increased IgG reactivities to structurally related xenogeneic and human neuraminic acids. The discovery of these glycan-specific epitopes as immune targets and potential biomarkers in MS merits further investigation.

International audience; To understand the role of human natural IgM known as antibodies against the carbohydrate epitope Tn, the antibodies were isolated using GalNAcα−Sepharose affinity chromatography, and their specificity was profiled using microarrays (a glycan array printed with oligosaccharides and bacterial polysaccharides, as well as a glycopeptide array), flow cytometry, and inhibition ELISA. The antibodies bound a restricted number of GalNAcα-terminated oligosaccharides better than the parent monosaccharide, e.g., 6-O-Su-GalNAcα and GalNAcα1−3Galβ1−3(4)GlcNAcβ. The binding with several bacterial polysaccharides that have no structural resemblance to the affinity ligand GalNAcα was quite unexpected. Given that GalNAcα is considered the key fragment of the Tn antigen, it is surprising that these antibodies bind weakly GalNAcα−OSer and do not bind a wide variety of GalNAcα−OSer/Thr-containing mucin glycopeptides. At the same time, we have observed specific binding to cells having Tn-positive glycoproteins containing similar glycopeptide motifs in a con-formationally rigid macromolecule. Thus, specific recognition of the Tn antigen apparently requires that the naturally occurring "anti-Tn" IgM recognize a complex epitope comprising the GalNAcα as an essential component and a fairly long amino acid sequence where the amino acids adjacent to GalNAcα do not contact the antibody paratope; i.e., the antibodies recognize a spatial epitope or a molecular pattern rather than a classical continuous sequence. In addition, we have not found any increase in the binding of natural antibodies when GalNAcα residues were clustered. These results may help in further development of anticancer vaccines based on synthetic Tn constructs.