Surface Glycans of Microvesicles Derived from Endothelial Cells, as Probed Using Plant Lectins.

Glycans of MVs are proposed to be candidates for mediating targeting specificity or at least promoting it. In contrast to exosomes, glycomic studies of MVs are largely absent. We studied the glycoprofile of endothelial cell-derived MVs using 21 plant lectins, and the results show the dominance of oligolactosamines and their α2-6-sialylated forms as N-glycans and low levels of α2-3-sialylated glycans. The low levels of α2-3-sialosides could not be explained by the action of extracellular glycosidases. Additionally, the level of some Man-containing glycans was also decreased in MVs. Spatial masking as the causative relationship between these low level glycans (as glycosphingolipids) by integral proteins or proteoglycans (thus, their lack of interaction with lectins) seems unlikely. The results suggest that integral proteins do not pass randomly into MVs, but instead only some types, differing in terms of their specific glycosylation, are integrated into MVs.

Chemical Resolution of an Epitope Recognized by Blood Group Antibodies Capable of Binding Both A and B Red Blood Cells.

The high specificity of human antibodies to blood group A and B antigens is impressive, especially when considering the structural difference between these antigens (tetrasaccharides) is a NHAc versus a hydroxyl group on the terminal monosaccharide residue. It is well established that in addition to anti-A and anti-B there is a third antibody, anti-A,B capable of recognizing both A and B antigens. To analyze this AB specificity, we synthesized a tetrasaccharide, where the NHAc of the A antigen was replaced with an NH2. This NH2 group was then used to attach the glycan to an affinity resin, creating an AB epitope (ABep) adsorbent where the critical site for recognition by A and B antibodies was not accessible, while the rest of the (conformationally compact) tetrasaccharide remained accessible. Anti-ABep antibodies were then isolated from blood group O donors and found to have expected A,B specificity against immobilized and red cell bound synthetic antigens, including ABep, and were able to agglutinate both A and B red cells. The amount of these anti-ABep (anti-A,B) antibodies found in the blood of group O donors was comparable to levels of anti-A and anti-B found in group B and A individuals. Using STD-NMR the location for the AB epitope on the tetrasaccharide was found.

Galectin-9 as a Potential Modulator of Lymphocyte Adhesion to Endothelium via Binding to Blood Group H Glycan.

The recruitment of leukocytes from blood is one of the most important cellular processes in response to tissue damage and inflammation. This multi-step process includes rolling leukocytes and their adhesion to endothelial cells (EC), culminating in crossing the EC barrier to reach the inflamed tissue. Galectin-8 and galectin-9 expressed on the immune system cells are part of this process and can induce cell adhesion via binding to oligolactosamine glycans. Similarly, these galectins have an order of magnitude higher affinity towards glycans of the ABH blood group system, widely represented on ECs. However, the roles of gal-8 and gal-9 as mediators of adhesion to endothelial ABH antigens are practically unknown. In this work, we investigated whether H antigen-gal-9-mediated adhesion occurred between Jurkat cells (of lymphocytic origin and known to have gal-9) and EA.hy 926 cells (immortalized endothelial cells and known to have blood group H antigen). Baseline experiments showed that Jurkat cells adhered to EA.hy 926 cells; however when these EA.hy 926 cells were defucosylated (despite the unmasking of lactosamine chains), adherence was abolished. Restoration of fucosylation by insertion of synthetic glycolipids in the form of H (type 2) trisaccharide Fucα1-2Galß1-4GlcNAc restored adhesion. The degree of lymphocyte adhesion to native and the "H-restored" (glycolipid-loaded) EA.hy 926 cells was comparable. If this gal-9/H (type 2) interaction is similar to processes that occur in vivo, this suggests that only the short (trisaccharide) H glycan on ECs is required.

The International Bathymetric Chart of the Southern Ocean Version 2.

The Southern Ocean surrounding Antarctica is a region that is key to a range of climatic and oceanographic processes with worldwide effects, and is characterised by high biological productivity and biodiversity. Since 2013, the International Bathymetric Chart of the Southern Ocean (IBCSO) has represented the most comprehensive compilation of bathymetry for the Southern Ocean south of 60°S. Recently, the IBCSO Project has combined its efforts with the Nippon Foundation - GEBCO Seabed 2030 Project supporting the goal of mapping the world's oceans by 2030. New datasets initiated a second version of IBCSO (IBCSO v2). This version extends to 50°S (covering approximately 2.4 times the area of seafloor of the previous version) including the gateways of the Antarctic Circumpolar Current and the Antarctic circumpolar frontal systems. Due to increased (multibeam) data coverage, IBCSO v2 significantly improves the overall representation of the Southern Ocean seafloor and resolves many submarine landforms in more detail. This makes IBCSO v2 the most authoritative seafloor map of the area south of 50°S.

SARS-CoV-2 Peptide Bioconjugates Designed for Antibody Diagnostics.

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.

Localization of synthetic glycolipids in the cell and the dynamics of their insertion/loss.

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.

Recombinant SARS-CoV-2 S Protein Binds to Glycans of the Lactosamine Family in vitro.

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.

COVID-19 antibody screening with SARS-CoV-2 red cell kodecytes using routine serologic diagnostic platforms.

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.

Structure of Supramers Formed by the Amphiphile Biotin-CMG-DOPE.

Invited for this month's cover is the group of Prof. Nicolai Bovin from the Russian Academy of Sciences. The cover picture shows how a biotin residue initially hidden in a monolayer formed on the surface of a material by biot-CMG-DOPE (see top left) is pulled out of the layer by the streptavidin molecule (Str) that has come close to it (see below). This can be considered as a model of certain events (in particular, cis protein-ligand interactions) occurring on the surface of a living cell when it is necessary to hide the ligand from undesirable interactions, but leave the possibility of its recognition by a high-affinity protein. The picture is inspired by the legendary Yellow Submarine cartoon. Read the full text of their Full Paper at https://doi.org/10.1002/open.201900276.

Structure of Supramers Formed by the Amphiphile Biotin-CMG-DOPE.

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 <10 % of biotin residues on the highly water dispersible globules and only 1 % of biotin residues in layer coatings are in an linear conformation and exposing biotin into the aqueous medium. This explains why in biotin-CMG-DOPE apolar biotin residues both in water dispersible globules and coatings on solid surfaces are still capable of interacting with streptavidin.

Glycan-binding profile of DC-like cells.

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)Manß1-4GlcNAcß1-4GlcNAcß 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 GalNAcα1-3Galß (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.

Blood Group O→A Transformation by Chemical Ligation of Erythrocytes.

Agglutination of red blood cells (RBCs) remains the only practical method for routine use for ABH typing in clinical practice. However, exact mechanistic details of agglutination are not yet thoroughly studied. In this research, RBCs of blood group O were converted to blood group A through two approaches: by chemical ligation of the cells' glycocalyx with synthetic blood group A tetrasaccharide, and by insertion of synthetic glycolipid carrying the same A antigen into the cells' membranes. The O→A ligated RBCs and natural A RBCs showed comparable agglutination characteristics with antibodies. As expected, RBCs with inserted glycolipid showed lower agglutination scores. This approach could help cell biologists in site-specific and cell-friendly modification of glycocalyx by other ligands.

Glycan recognition by human blood mononuclear cells with an emphasis on dendritic cells.

Dendritic cells (DCs) play crucial roles in innate and adaptive immune response, for which reason targeting antigen to these cells is an important strategy for improvement of vaccine development. To this end, we explored recognition of DCs lectins by glycans. For selection of the glycan "vector", a library of 229 fluorescent glycoprobes was employed to assess interaction with the CD14low/-CD16+CD83+ blood mononuclear cell population containing the DCs known for their importance in antigen presentation to T-lymphocytes. It was found that: 1) the glycan-binding profiles of this CD14low/-CD16+CD83+ subpopulation were similar but not identical to DCs of monocyte origin (moDCs); 2) the highest percentage of probe-positive cells in this CD14 low/-CD16+CD83+ subpopulation was observed for GalNAcα1-2Galß (Adi), (Neu5Acα)3 and three mannose-reach glycans; 3) subpopulation of CD14low/-CD16+ cells preferentially bound 4'-O-Su-LacdiNAc. Considering the published data on specificity of DCs binding, the glycans showing particular selectivity for the CD14 low/-CD16+CD83+ cells are likely interacting with macrophage galactose binding lectin (MGL), siglec-7 and dectin-2. In contrast, DC-SIGN is not apparently involved, even in case of mannose-rich glycans. Taking into consideration potential in vivo competition between glycan "vectors" and glycans within glycocalyx, attempting to target vaccine to DCs glycan-binding receptors should focus on Adi and (Neu5Acα)3 as the most promising vectors.

Gram scale synthesis of A (type 2) and B (type 2) blood group tetrasaccharides through 1,6-anhydro-N-acetyl-ß-D-glucosamine.

Gram scale synthesis of A (type 2) and B (type 2) tetrasaccharides in the form of 3-aminopropyl glycosides is proposed starting from 3-O-benzoyl-1,6-anhydro-N-acetylglucosamine. Its galactosylation followed by re-protection gave lactosamine derivative with single free 2'-OH group in total yield 75%. Standard fucosylation and next run of re-protection in total yield 88% gave a trisaccharide Fuc-Gal-anhydroGlcNAc with single free 3'-OH group. Its standard α-galactosylation gave protected B (type 2) tetrasaccharide. For synthesis of correspondent A tetrasaccharide seven different 2-azido-2-deoxygalactosyl (GalN3) donors were tested: 6-O-acetyl-3,4-O-isopropylidene-GalN3 thioglycoside was shown to provide the best yield (89%) and stereoselectivity (α/ß = 24:1). Further 1,6-anhydro cycle opening, spacer-arming and complete deprotection resulted in the target 3-aminopropyl glycosides of A (type 2) and B (type 2) tetrasaccharides, yields 87 and 85% correspondingly.

Function-spacer-lipid constructs of Lewis and chimeric Lewis/ABH glycans. Synthesis and use in serological studies.

Seven lipophilic constructs containing Lewis (Lea, Leb, Ley) or chimeric Lewis/ABH (ALeb, BLeb, ALey, BLey) glycans were obtained starting from corresponding oligosaccharides in form of 3-aminopropyl glycosides. ALeb and BLeb pentasaccharides were synthesized via [3 + 1] blockwise approach. The constructs (neoglycolipids, or FSLs) were inserted in erythrocyte membrane, and obtained "kodecytes" were used to map the immunochemical specificity of historical and contemporary monoclonal and polyclonal blood group system Lewis reagents.

Biofunctionalizing nanofibers with carbohydrate blood group antigens.

A rapid and simple method of biofunctionalising nylon, cellulose acetate, and polyvinyl butyral electrospun nanofibers with blood group glycans was achieved by preparing function-spacer-lipid constructs and simply contacting them to fibers with a piezo inkjet printer. A series of water dispersible amphipathic glycan-spacer constructs were synthesized representing a range ABO and related blood group antigens. After immediate contact of the amphipathic glycan-spacer constructs with nanofiber surfaces they self-assembled and were detectable by enzyme immunoassays with high sensitivity and specificity.

Block synthesis of A (type 2) and B (type 2) tetrasaccharides related to the human ABO blood group system.

Herein we report the synthesis of 3-aminopropyl glycosides of A (type 2) and B (type 2) tetrasaccharides via [3 + 1] block scheme. Peracetylated trichloroacetimidates of A and B trisaccharides were used as glycosyl donors. The well-known low reactivity of 4-OH group of N-acetyl-d-glucosamine forced us to test four glucosamine derivatives (3-Bz-1,6-anhydro-GlcNAc and 3-trifluoroacetamidopropyl ß-glycosides of 3-Ac-6-Bn-GlcNAc, 3-Ac-6-Bn-GlcN3, and 3-Ac-6-Bn-GlcNAc2) to select the best glycosyl acceptor for the synthesis of type 2 tetrasaccharides. The desired tetrasacchrides were not isolated, when 3-trifluoroacetamidopropyl glycosyde of 3-Ac-6-Bn-GlcNAcß was glycosylated. Glycosylation of 3-Bz-1,6-anhydro-GlcNAc derivative resulted in α-glycoside as a major product. High stereospecificity was achieved only in the synthesis of B (type 2) tetrasaccharide, when 3-trifluoroacetamidopropyl 3-Ac-6-Bn-GlcNAc2ß was applied as the glycosyl acceptor (ß/α 5:1), whereas glycosylation with trichloroacetimidate of A trisaccharide was not stereospecific (ß/α 1.3:1). Glycosylation of 3-trifluoroacetamidopropyl glycoside of 3-Ac-6-Bn-GlcN3ß with trichloroacetimidates of A and B trisaccharides provided the same stereochemical yield (ß/α 1.5:1).

Spin noise explores local magnetic fields in a semiconductor.

Rapid development of spin noise spectroscopy of the last decade has led to a number of remarkable achievements in the fields of both magnetic resonance and optical spectroscopy. In this report, we demonstrate a new - magnetometric - potential of the spin noise spectroscopy and use it to study magnetic fields acting upon electron spin-system of an n-GaAs layer in a high-Q microcavity probed by elliptically polarized light. Along with the external magnetic field, applied to the sample, the spin noise spectrum revealed the Overhauser field created by optically oriented nuclei and an additional, previously unobserved, field arising in the presence of circularly polarized light. This "optical field" is directed along the light propagation axis, with its sign determined by sign of the light helicity. We show that this field results from the optical Stark effect in the field of the elliptically polarized light. This conclusion is supported by theoretical estimates.

Glycomapping the fine specificity of monoclonal and polyclonal Lewis antibodies with type-specific Lewis kodecytes and function-spacer-lipid constructs printed on paper.

BACKGROUND: Lewis serologic reagents frequently give inaccurate phenotyping results. Furthermore these serologic reagents are often used in nonserologic assays such as inhibition and immunohistochemistry. In both scenarios knowledge of the fine specificity and cross-reactivity of these reagents will improve the quality of results obtained. STUDY DESIGN AND METHODS: A range of contemporary and historical workshop and developmental Lewis reagents including mouse monoclonal (MoAb) and human and goat polyclonal (PoAb) reagents were evaluated. All were evaluated both against Lewis kodecytes expressing only single Le(a) , Le(b) , ALe(b) , BLe(b) , Le(x) , Le(y) , ALe(y) , or BLe(y) antigens and against the same antigens inkjet printed on a paper-based microplate and analyzed by enzyme immunoassay. Nine clinical samples were also evaluated. A kodecyte antigen dilution sensitivity assay was used to establish the ratio of Le(b) antigen between group A1 /A2 and O RBCs. RESULTS: A continuum of cross-reactivity from Le(x) through to H was observed with MoAbs. All PoAb and few MoAb anti-Le(a) samples and reagents cross-reacted to some degree with Le(b) antigen. Some PoAb and MoAb anti-Le(b) did not cross-react with Le(a) . All polyclonal goat anti-Le(b) reagents showed substantial activity against ALe(b) and BLe(b) , while no MoAb reagent had this activity. A1 RBCs had less than half the Le(b) antigen of A2 /O RBCs. CONCLUSIONS: Substantial cross-reactivity of both MoAbs and PoAbs with related antigens highlights the risks of using serologic reagents in nonserologic assays or against synthetic antigens. The lack of ALe(b) activity in anti-Le(b) MoAbs explains their poor performance against blood group A1 Le(a-b+) phenotypes.

Comparative lectinology: Delineating glycan-specificity profiles of the chicken galectins using neoglycoconjugates in a cell assay.

A major aspect of carbohydrate-dependent galectin functionality is their cross-linking capacity. Using a cell surface as biorelevant platform for galectin binding and a panel of 40 glycans as sensor part of a fluorescent polyacrylamide neoglycopolymer for profiling galectin reactivity, properties of related proteins can be comparatively analyzed. The group of the chicken galectins (CGs) is an especially suited system toward this end due to its relatively small size, compared with mammalian galectins. The experiments reveal particularly strong reactivity toward N-acetyllactosamine repeats for all tested CGs and shared reactivity of CG-1A and CG-2 to histo-blood group ABH determinants. In cross-species comparison, CG-1B's properties closely resembled those of human galectin-1, as was the case for the galectin-2 (but not galectin-3) ortholog pair. Although binding-site architectures are rather similar, reactivity patterns can well differ.