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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.

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).

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.

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.

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.

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.

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

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.

Cells are highly decorated with glycans (carbohydrates) and this complex and dominating coating on cells, which also carries the glycan blood group antigens, is critically important to immunology a...

Sperm storage in the female reproductive tract after mating and before ovulation is a reproductive strategy used by many species. When insemination and ovulation are poorly synchronized, the formation and maintenance of a functional sperm reservoir improves the possibility of fertilization. In mammals, the oviduct regulates sperm functions, such as Ca2+ influx and processes associated with sperm maturation, collectively known as capacitation. A fraction of the stored sperm is released by unknown mechanisms and moves to the site of fertilization. There is an empirical association between the hormonal milieu in the oviduct and sperm detachment; therefore, we tested directly the ability of progesterone to induce sperm release from oviduct cell aggregates. Sperm were allowed to bind to oviduct cells or an immobilized oviduct glycan and then challenged with progesterone, which stimulated the release of 48% of sperm from oviduct cells or 68% of sperm from an immobilized oviduct glycan. The effect of progesterone on sperm release was specific; pregnenolone and 17α-OH-progesterone did not affect sperm release. Ca2+ influx into sperm is associated with capacitation and development of hyperactivated motility. Progesterone increased sperm intracellular Ca2+, which was abrogated by blocking the sperm–specific Ca2+ channel CatSper with NNC 055-0396. NNC 055-0396 also blocked the progesterone-induced sperm release from oviduct cells or immobilized glycan. An inhibitor of the non-genomic progesterone receptor that activates CatSper similarly blocked sperm release. This is the first report indicating that release of sperm from the sperm reservoir is induced by progesterone action through CatSper channels.

Protein–carbohydrate interactions are significant in a wide range of biological processes, disruption of which has been implicated in many different diseases. The capability of glycan-binding proteins (GBPs) to specifically bind to the corresponding glycans allows GBPs to be utilized in glycan biomarker detection or conversely to serve as targets for therapeutic intervention. However, understanding the structural origins of GBP specificity has proven to be challenging due to their typically low binding affinities (mM) and their potential to display broad or complex specificities. Here we perform molecular dynamics (MD) simulations and post-MD energy analyses with the Poisson–Boltzmann and generalized Born solvent models (MM-PB/GBSA) of the Erythrina cristagalli lectin (ECL) with its known ligands, and with new cocrystal structures reported herein. While each MM-PB/GBSA parametrization resulted in different estimates of the desolvation free energy, general trends emerged that permit us to define GBP binding preferences in terms of ligand substructure specificity. Additionally, we have further decomposed the theoretical interaction energies into contributions made between chemically relevant functional groups. Based on these contributions, the functional groups in each ligand can be assembled into a pharmacophore comprised of groups that are either critical for binding, or enhance binding, or are noninteracting. It is revealed that the pharmacophore for ECL consists of the galactopyranose (Gal) ring atoms along with C6 and the O3 and O4 hydroxyl groups. This approach provides a convenient method for identifying and quantifying the glycan pharmacophore and provides a novel method for interpreting glycan specificity that is independent of residue-level glycan nomenclature. A pharmacophore approach to defining specificity is readily transferable to molecular design software and, therefore, may be particularly useful in designing therapeutics (glycomimetics) that target GBPs.

Innate immunity natural Abs (NAbs) execute a number of functions, including protection and surveillance. Despite active research, the stimuli that induce the formation of NAbs are still described only hypothetically. Here, we compared repertoires of anti-glycan Abs in the peripheral blood of mice that received per os various bacteria. The repertoires of Abs of mice primed in this way were compared using a microarray that included about 350 glycans, as well as 150 bacterial polysaccharides. Sterile mice did not possess anti-glycan Abs. Oral inoculation of a single strain or combination of two to four strains of bacteria, as well as putting the animals on short-term nutrition with non-sterile food, did not contribute significantly to the formation of Abs, whereas a single gavage of digested food of non-sterile mice induced the formation of a repertoire close to the natural ones. Interestingly, the priming with polysaccharide Ags (in a composition of the bacterial cell envelope), that is, dominant Ags of bacteria, led to the induction of Abs against typical glycans of mammalian glycoproteins and glycolipids (e.g. Abs of the ABH blood group system) that do not have a structural similarity to the polysaccharides. The results support the importance of early contact with a naïve immune system with microorganisms of the environment to form a normal NAbs repertoire.

We describe a rapid one-step method to biotinylate virtually any biological or non-biological surface. Contacting a solution of biotin-spacer-lipid constructs with a surface will form a coating within seconds on non-biological surfaces or within minutes on most biological membranes including membrane viruses. The resultant biotinylated surface can then be used to interact with avidinylated conjugates, beads, vesicles, surfaces or cells.

Background. Serologic assays detecting antibodies against the SARS-CoV-2 spike or nucleocapsid proteins have been developed to advance our understanding of the prognosis and clinical course of the COVID-19 disease. We recently developed a red cell agglutination-based assay to detect severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) antibodies. This assay uses peptide fragments of the SARS-CoV-2 spike protein to label red cells (C19-kodecytes). We performed a clinical evaluation of this C19-kodecyte assay in COVID-19 convalescent plasma (CCP) donors previously assessed with 2 commercial immunoassays and a virus neutralizing assay. Methods. Red cells were coated with peptide fragments of the SARS-CoV-2 spike protein. We tested plasma samples from 140 CCP donors. The results were compared with those of a virus neutralizing assay and 2 commercial chemiluminescent antibody tests: anti-SARS-CoV-2 Total (IgG, IgM and IgA) assay and anti-SARS-CoV-2 IgG assay (Ortho). Inter-rater agreement between the different assays was measured using Cohen's kappa. Specificity was tested with 150 plasma samples, collected in 2008, more than a decade before the COVID-19 outbreak and with 125 plasma samples, collected in 2020 from consecutive healthy volunteer donors, who tested negative for the Ortho Total assay. Testing was performed using the column agglutination technique commonly employed for blood typing. Results. The area under the ROC curve (AUC) for the C19-kodecyte assay reached 0.95 (95% CI: 0.93 - 0.97) with sensitivity of 92.8% (95% CI: 86.9% - 96.3%) and specificity of 96.3% (95% CI: 93.2% - 98.1%). In almost all of the 40 CCP donors with longitudinal data, the antibody concentration decreased during the follow-up, which ranged from 7 to 44 weeks. In the 140 CCP donors, we compared the C19-kodecyte score to the antibody concentrations from the 2 FDA authorized assays (Ortho Total and Ortho IgG) and the titer in the neutralizing assay. There was a positive relationship between the results of all 4 assays. The Spearman's correlation of our assay was 0.20 with the neutralizing assay (0.49 with IgG, and 0.41 with Total assays). Conclusions. Sensitivity and specificity of the C19-kodecyte assay were within the minimum performance range required by the FDA for EUA authorization of serology tests. The limited correlation in assay reaction strengths suggested that the assays may be influenced by different antibody specificities. Unlike the other 84 FDA authorized serologic tests for SARS-CoV-2, this C19-kodecyte assay is a simple and rapid test that can be easily established in any blood typing laboratory worldwide using its routine setup for column agglutination or tube technique. The technique could vastly improve assay capacity, particularly in resource limited hospital blood banks. Disclosures Bovin: Kode Biotech: Current Employment, Current holder of stock options in a privately-held company. Henry: Kode Biotech: Current Employment, Current holder of stock options in a privately-held company.

Modification of the cell surface with synthetic glycolipids opens up a wide range of possibilities for studying the function of glycolipids. Synthetic glycolipids called Function-Spacer-Lipids (FSL; where F is a glycan or label, S is a spacer, and L is dioleoylphosphatidyl ethanolamine) easily and controllably modify the membrane of a living cells. This current study investigates the dynamics and mechanism of the FSL insertion and release/loss. FSL insert into the cell membrane (~1 million molecules per cell) within tens of minutes, almost regardless of the nature of the cells (including the thickness of their glycocalyx) and the size of the FSL glycan. FSLs do not accumulate uniformly, but instead form patches >300 nm in size either entrapped in the glycocalyx, or integrated in the plane of the plasma membrane, but always outside the cell rafts. The natural release (loss) of FSL from the modified cell was two orders of magnitude slower than attachment/insertion and occurred mainly in the form of released microvesicles with a size of 140 ± 5 nm. The accumulation of FSL as patches in the cell membrane is similar to the coalescence of natural glycosphingolipids and supports (along with their long residence time in the membrane) the use of FSL as probes for the study of glycosphingolipid-protein interactions.

A recent study has demonstrated that porcine spermatozoa recognize with high affinity carbohydrate structures containing Lewis X motifs. Sperm adhesion to Lewis X is proposed to mediate sperm binding to the oviduct epithelium to form a reservoir. The objective of this study was to identify Lewis X-binding proteins from porcine spermatozoa as candidate receptors for oviduct glycans. To identify low-abundance proteins typically masked by proteins originating from seminal fluid, Lewis X candidate receptors were enriched from cauda epididymal boar spermatozoa. Plasma membrane preparations from cauda epididymal spermatozoa were subjected to RP-HPLC and glycan blotting assays to isolate and detect proteins that bind Lewis X. Following bottom-up LC-MS/MS analysis, among the two bands that bound sulfated Lewis X, ADAM5, which spermatozoa, was confidently identified. ADAM family members have been established as contributors to sperm entry into the oviduct. A second sulfated Lewis X-binding protein identified was the peripheral membrane protein lactadherin (also known as P47, SED1 and MFG-E8 in different species). The interaction between Lewis X and lactadherin was functionally important because competitive inhibition by soluble recombinant lactadherin reduced sperm binding to the oviduct epithelium. Furthermore, far-western blotting demonstrated that purified lactadherin could bind oviduct cells. In summary, these findings reveal that, in addition to the previously reported glycan affinity of accessory gland proteins that adhere to spermatozoa, multiple proteins intrinsic to spermatozoa have affinity for a specific oviduct glycan. Further, in addition to binding to the zona pellucida, lactadherin is now implicated in binding to oviduct glycans to promote formation of the sperm reservoir.

International audience; Burkholderia pseudomallei and Chromobacterium violaceum are bacteria of tropical and subtropical soil and water that occasionally cause fatal infections in humans and animals. Microbial lectins mediate the adhesion of organisms to host cells, which is the first phase in the development of infection. Here we report the discovery of two novel lectins from the above-mentioned bacteria-BP39L and CV39L. The crystal structures revealed that the lectins possess a seven-bladed -propeller fold. Functional studies conducted on a series of oligo-and polysaccharides confirmed the preference of BP39L for mannosylated saccharides and CV39L for rather more complex polysaccharides with a monosaccharide preference for β-L-fucose. The presented data indicate that the proteins belong to a currently unknown family of lectins.

Discoveries on involvement of glycan–protein recognition in many (patho)physiological processes are directing attention to exploring the significance of a fundamental structural aspect of sugar receptors beyond glycan specificity, i.e., occurrence of distinct types of modular architecture. In order to trace clues for defining design–functionality relationships in human lectins, a lectin's structural unit has been used as source material for engineering custom-made variants of the wild-type protein. Their availability facilitates comparative analysis toward the stated aim. With adhesion/growth-regulatory human galectin-1 as example, the strategy of evaluating how changes of its design (here, from the homodimer of non-covalently associated domains to (i) linker-connected di- and tetramers and (ii) a galectin-3-like protein) affect activity is illustrated by using three assay systems of increasing degree of glycan complexity. Whereas calorimetry with two cognate disaccharides and array testing with 647 (glyco)compounds disclosed no major changes, galectin histochemical staining profiles of tissue sections that present natural glycome complexity revealed differences between wild-type and linker-connected homo-oligomers as well as between the galectin-3-like variant and wild-type galectin-3 for cell-type positivity, level of intensity at the same site and susceptibility for inhibition by a bivalent glycocompound. These results underscore the strength of the documented approach. Moreover, they give direction to proceed to (i) extending its application to other members of this lectin family, especially galectin-3 and (ii) then analyzing impact of architectural alterations on cell surface lattice formation and ensuing biosignaling systematically, considering the variants’ potential for translational medicine.

Immune system failure in primary antibody deficiencies (PADs) has been linked to recurrent infections, autoimmunity, and cancer, yet clinical judgment is often based on the reactivity to a restricted panel of antigens. Previously, we demonstrated that the human repertoire of carbohydrate-specific immunoglobulin G (IgG) exhibits modular organization related to glycan epitope structure. The current study compares the glycan-specific IgG repertoires between different PAD entities. Distinct repertoire profiles with extensive qualitative glycan-recognition defects were observed, which are characterized by the common loss of Galα and GalNAc reactivity and disease-specific recognition of microbial antigens, self-antigens, and tumor-associated carbohydrate antigens. Antibody repertoire analysis may provide a useful tool to elucidate the degree and the clinical implications of immune system failure in individual patients.

Background Endogenous lectins are multifunctional effectors in cell physiology. Adding the sixth member of the galectin family in chicken, a model organism for systematic profiling of these adhesion/growth-regulatory proteins, is a step toward comprehensive network monitoring. Methods Database mining and computational data processing are applied for gene detection, chromosomal location and sequence alignments. Cloning, recombinant production and fusion-protein technology gain access to the protein, mass spectrometry and gel electrophoresis/filtration provide analytical data. Haemagglutination, glycan microarray and cell assays assess binding capacity, and crystallography of a shortened variant (also analyzed by ultracentrifugation and small angle X-ray scattering) determines its structure. Results The gene for the galectin-related protein (GRP) is present exclusively in vertebrates with high-level sequence conservation and similar chromosomal positioning. The chicken protein is monomeric and has lost the canonical galectin property of binding lactose. The crystal structure of the variant without the 36-amino-acid extension at the start provides explanations for this lack of binding. Conclusions Chicken GRP is special within this family of six proteins by being unable to bind lactose. The documented high degree of sequence conservation among vertebrate orthologues confers the status of a model for delineating an assumedly shared functionality to this GRP. General significance Biochemical characterization of a product of a gene under strong positive selection is a prerequisite for functional characterization. It is also essential for network monitoring by adding a new member to this lectin family.

Preeclampsia (PE) is a multisystem pathologic state that clinically manifests itself after the 20th week of pregnancy. It is characterized by high maternal and perinatal morbidity and mortality. According to modern concepts, the impairment of trophoblast invasion into maternal spiral arteries, leading to the development of ischemia in placenta, is considered to be the major pathogenetic factor of PE development. Ischemic lesions initiate the development of a systemic inflammatory response (SIR) and endothelial dysfunction, which is the main cause of the multiple organ failure in PE. Some data has appear indicating the importance of a glycans-forming endothelial glycocalyx and extracellular matrix (ECM) for placenta morphogenesis, as well as their role in the regulation of vascular permeability and vascular tone in hypertension disorders and, in particular, PE. Since intact glycocalyx and ECM are considered to be the major factors that maintain the physiological vascular tone and adequate intercellular interactions, their value in PE pathogenesis is underestimated. This review is focused on hyaluronic acid (HA) as the key glycan providing the organization and stabilization of the ECM and glycocalyx, its distribution in tissues in the case of presence or absence of placental pathology, as well as on the regulatory function of hyaluronic acids of various molecular weights in different physiological and pathophysiological processes. The summarized data will provide a better understanding of the PE pathogenesis, with the main focus on glycopathology.

Colorectal cancer (CRC) is the third most common malignancy in industrialized countries. Despite the advances in diagnostics and development of new drugs, the 5-year survival remains only 60-65%. Our approach to early diagnostics of CRC is based on the determination of serological signatures with an array of hemispherical hydrogel cells containing immobilized proteins and oligosaccharides (glycochip). The compounds immobilized on the glycochip include tumor-associated glycans (SiaTn, Tn, TF, Le(C) , Le(Y) , SiaLe(A) , and Manβ1-4GlcNAcβ) and antibodies against human immunoglobulins IgG, IgA, and IgM. The glycochip detects antibodies against tumor-associated glycans in patients' sera. The simultaneous measurement of the levels of immunoglobulins enhances the diagnostic impact of the signatures. In this work, we found previously unreported increase in antibodies against oligosaccharide Manβ1-4GlcNAcβ in patients with CRC. In parallel with these experiments, we determined the levels of oncomarkers carcinoembryonic antigen (CEA), cancer antigen (CA) 19-9, CA 125, CA 15-3, human chorionic gonadotropin (HCG), and alpha-fetoprotein (AFP) using another gel-based biochip with immobilized antibodies (oncochip) developed earlier in our laboratory. In total, 69 samples from healthy donors, 33 from patients with colorectal carcinoma, and 27 from patients with inflammatory bowel diseases were studied. The use of combined signatures of antiglycan antibodies and oncomarkers provides much better predictive value than the conventional measurement of oncomarkers CEA and CA 19-9. Positive predictive value of CRC diagnoses using together glycochip and oncochip reached 95% with the sensitivity and specificity 88% and 98%, respectively. Thus, the combination of antibody profiling with detection of conventional oncomarkers proved to be a promising tool in diagnostics of CRC.

Today pre-eclampsia (PE) is considered as a disease of various theories; still all of them agree that endothelial dysfunction is the leading pathogenic factor. Endothelial dysfunction is a sequence of permanent immune activation, resulting in the change of both the phenotype and the functions of an endothelial cell and of the extracellular layer associated with the cell membrane—endothelial glycocalyx (eGC). Numerous studies demonstrate that eGC mediates and regulates the key functions of endothelial cells including regulation of vascular tone and thromboresistance; and these functions are disrupted during PE. Taking into account that eGC and its components undergo alterations under pathological conditions leading to endothelial activation, it is supposed that eGC plays a certain role in pathogenesis of PE. Envisaging the eGC damage as a key factor of PE, might be a new approach to prevention, treatment, and rehabilitation of patients with PE. This approach could include the development of drugs protecting eGC and promoting regeneration of this structure. Since the issue of PE is far from being solved, any effort in this direction might be valuable.