Terminal Monosaccharide Research Articles

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.

Elucidating the conformational dynamics of histo-blood group antigens and their interactions with the rotavirus spike protein through computational lens

In the present study, we investigated the conformational dynamics of histo-blood group antigens (HBGAs) and their interactions with the VP8* domain of four rotavirus genotypes (P[4], P[6], P[19], and P[11]) utilizing all-atom molecular dynamics simulations in explicit water. Our study revealed distinct changes in the dynamic behavior of the same glycan due to linkage variations. We observed that LNFPI HBGA having a terminal β linkage shows two dominant conformations after complexation, whereas only one was obtained for LNFPI with a terminal α linkage. Interestingly, both variants displayed a single dominant structure in the free state. Similarly, LNT and LNnT show a shift in their dihedral linkage profile between their two terminal monosaccharides because of a change in the linkage from β(1–3) to β(1–4). The molecular mechanics generalized Born surface area (MM/GBSA) calculations yielded the highest binding affinity for LNFPI(β)/P[6] (−13.93 kcal/mol) due to the formation of numerous hydrogen bonds between VP8* and HBGAs. LNnT binds more strongly to P[11] (−12.88 kcal/mol) than LNT (−4.41 kcal/mol), suggesting a single change in the glycan linkage might impact its binding profile significantly. We have also identified critical amino acids and monosaccharides (Gal and GlcNAc) that contributed significantly to the protein-ligand binding through the per-residue decomposition of binding free energy. Moreover, we found that the interaction between the same glycan and different protein receptors within the same rotavirus genogroup influenced the micro-level dynamics of the glycan. Overall, our study helps a deeper understanding of the H-type HBGA and rotavirus spike protein interaction. Communicated by Ramaswamy H. Sarma

Hepatic sialic acid synthesis modulates glucose homeostasis in both liver and skeletal muscle

ObjectiveSialic acid is a terminal monosaccharide of glycans in glycoproteins and glycolipids, and its derivation from glucose is regulated by the rate-limiting enzyme UDP-GlcNAc 2-epimerase/ManNAc kinase (GNE). Although the glycans on key endogenous hepatic proteins governing glucose metabolism are sialylated, how sialic acid synthesis and sialylation in the liver influence glucose homeostasis is unknown. Studies were designed to fill this knowledge gap. MethodsTo decrease the production of sialic acid and sialylation in hepatocytes, a hepatocyte-specific GNE knockdown mouse model was generated, and systemic glucose metabolism, hepatic insulin signaling and glucagon signaling were evaluated in vivo or in primary hepatocytes. Peripheral insulin sensitivity was also assessed. Furthermore, the mechanisms by which sialylation in the liver influences hepatic insulin signaling and glucagon signaling and peripheral insulin sensitivity were identified. ResultsLiver GNE deletion in mice caused an impairment of insulin suppression of hepatic glucose production. This was due to a decrease in the sialylation of hepatic insulin receptors (IR) and a decline in IR abundance due to exaggerated degradation through the Eph receptor B4. Hepatic GNE deficiency also caused a blunting of hepatic glucagon receptor (GCGR) function which was related to a decline in its sialylation and affinity for glucagon. An accompanying upregulation of hepatic FGF21 production caused an enhancement of skeletal muscle glucose disposal that led to an overall increase in glucose tolerance and insulin sensitivity. ConclusionThese collective observations reveal that hepatic sialic acid synthesis and sialylation modulate glucose homeostasis in both the liver and skeletal muscle. By interrogating how hepatic sialic acid synthesis influences glucose control mechanisms in the liver, a new metabolic cycle has been identified in which a key constituent of glycans generated from glucose modulates the systemic control of its precursor.

Chilensosides E, F, and G—New Tetrasulfated Triterpene Glycosides from the Sea Cucumber Paracaudina chilensis (Caudinidae, Molpadida): Structures, Activity, and Biogenesis

Three new tetrasulfated triterpene glycosides, chilensosides E (1), F (2), and G (3), have been isolated from the Far-Eastern sea cucumber Paracaudina chilensis (Caudinidae, Molpadida). The structures were established based on extensive analysis of 1D and 2D NMR spectra and confirmed by HR-ESI-MS data. The compounds differ in their carbohydrate chains, namely in the number of monosaccharide residues (five or six) and in the positions of sulfate groups. Chilensosides E (1) and F (2) are tetrasulfated pentaosides with the position of one of the sulfate groups at C-3 Glc3, and chilensoside G (3) is a tetrasulfated hexaoside. The biogenetic analysis of the glycosides of P. chilensis has revealed that the structures form a network due to the attachment of sulfate groups to almost all possible positions. The upper semi-chain is sulfated earlier in the biosynthetic process than the lower one. Noticeably, the presence of a sulfate group at C-3 Glc3-a terminal monosaccharide residue in the bottom semi-chain of compounds 1 and 2-excludes the possibility of this sugar chain's further elongation. Presumably, the processes of glycosylation and sulfation are concurrent biosynthetic stages. They can be shifted in time in relation to each other, which is a characteristic feature of the mosaic type of biosynthesis. The hemolytic action of compounds 1-3 against human erythrocytes and cytotoxic activities against five human cancer cell lines were tested. The compounds showed moderate hemolytic activity but were inactive against cancer cells, probably because of their structural peculiarities, such as the combination of positions of four sulfate groups.

The structure of a Bacteroides thetaiotaomicron carbohydrate-binding module provides new insight into the recognition of complex pectic polysaccharides by the human microbiome

TheBacteroides thetaiotaomicronhas developed a consortium of enzymes capable of overcoming steric constraints and degrading, in a sequential manner, the complex rhamnogalacturonan II (RG-II) polysaccharide. BT0996 protein acts in the initial stages of the RG-II depolymerisation, where its two catalytic modules remove the terminal monosaccharides from RG-II side chains A and B. BT0996 is modular and has three putative carbohydrate-binding modules (CBMs) for which the roles in the RG-II degradation are unknown. Here, we present the characterisation of the module at the C-terminal domain, which we designated BT0996-C. The high-resolution structure obtained by X-ray crystallography reveals that the protein displays a typical β-sandwich fold with structural similarity to CBMs assigned to families 6 and 35. The distinctive features are: 1) the presence of several charged residues at the BT0996-C surface creating a large, broad positive lysine-rich patch that encompasses the putative binding site; and 2) the absence of the highly conserved binding-site signatures observed in CBMs from families 6 and 35, such as region A tryptophan and region C asparagine. These findings hint at a binding mode of BT0996-C not yet observed in its homologues. In line with this, carbohydrate microarrays and microscale thermophoresis show the ability of BT0996-C to bind α1-4-linked polygalacturonic acid, and that electrostatic interactions are essential for the recognition of the anionic polysaccharide. The results support the hypothesis that BT0996-C may have evolved to potentiate the action of BT0996 catalytic modules on the complex structure of RG-II by binding to the polygalacturonic acid backbone sequence.

Efficient TurboID-based proximity labelling method for identifying terminal sialic acid glycosylation in living cells.

TurboID, a proximity labelling method based on mutant biotin ligase, is an efficient new technique for recognizing protein-protein interactions and has been successfully applied to living cells. Sialic acid is typically the terminal monosaccharide attached to many glycoproteins and plays many important roles in many biological processes. However, the lack of enrichment methods for terminal sialic acid glycosylation in vivo hinders the identification and analysis of this glycosylation. Here, we introduce TurboID to identify terminal sialic acid glycosylation in living cells. SpCBM, the carbohydrate-binding domain of sialidase from Streptococcus pneumoniae, is fused with TurboID and overexpressed in HeLa cells. After streptavidin-based purification and detection by mass spectrometry, 31 terminal sialic acid N-glycosylated sites and 1359 putative terminal sialic acid glycosylated proteins are identified, many of which are located in the cytoplasm and nucleus.

Immunological Compatibility of Novel Enzyme-Converted Universal Blood Donor Cells

Introduction: In transfusion, unintentional mismatching of blood groups remains one of the most common causes of serious and sometimes fatal adverse reactions. Scarcity of certain blood types is another major transfusion challenge. The ABO blood group specificity is determined by the terminal monosaccharides, N-acetylgalactosamine on A and galactose on B RBCs. To create a universal blood supply, cell surface engineering has been studied as a method to minimize the immunological rejection of donor cells. Enzymatic removal of immunodominant sugars would uncover universally acceptable H-antigen (O group RBC). However, enzymatic conversion of A-type RBCs was lagging due to a lack of very active and specific glycosidases and the complex nature of the A blood group antigen. In this research, the performance of two novel enzymes that act together extremely efficiently to convert A-type RBCs to universal donor O RBCs, with high specificity for A antigen, is evaluated. Optimal conversion conditions are explored, as well as the immunological compatibility of the converted RBCs. Materials & Methods: Whole blood samples are collected from consenting A donors (with approval from the University of British Columbia clinical research ethics board), washed and diluted to 10 % hematocrit in PBS buffer (pH7.4). The washed RBCs were treated with the newly developed glycosidases for 1 h at 37 oC. The efficiency of the newly developed glycosidases and the characterization of enzyme converted O (ECO)-RBCs were evaluated by gel-column based micro typing system (MTS) cards against A antigen and flow cytometry using anti A and H antibodies. Safety and compatibility of ECO-RBCs were assessed by blood group serology studies. In brief, ECO-RBCs were incubated in Rh-matched sera collected from diverse donors and any serum reactivity against ECO-RBCs was assessed using anti IgG MTS cards and flow cytometry using anti C3d (complement protein) antibody. To further confirm the cross-matching of ECO-RBCs, the monocyte monolayer assay (MMA), a clinically used method, was performed using monocytes collected from B and O blood donors to assess the phagocytic activity against ECO-RBCs pre-incubated with serum samples. Phagocytic activity against ECO-RBCs was calculated using the monocyte index (MI). Results: Anti A MTS cards and flow cytometry analysis of antigen A and H expression, after enzymatic treatment, confirmed successful conversion of A blood group to universal O group. Our studies showed no significant hemolysis and no complement protein (C3d) deposition on ECO-RBCs pre-incubated with diverse sera, demonstrating the compatibility. Serology studies showed that ECO-RBCs are compatible with most sera (~50 donors) studied under transfusion conditions, though serum reactivity was shown by some O and B sera. A MI value of ≤ 6 % was obtained for all ECO-RBCs indicating minimal risk of an acute hemolytic reaction associated with ECO-RBC transfusion. Conclusions: We have developed novel glycosidases that efficiently remove immunodominant A antigen from RBC surfaces with high specificity to create universal blood donor cells at neutral pH and at enzyme concentrations that are much lower than those previously required. Based on preliminary results, ECO-RBCs have the potential to increase donor blood supply and thereby greatly advance the field of transfusion.

OR24-6 Liver-Specific Inhibition of Sialic Acid Biosynthesis Improves Systemic Glucose Homeostasis in Mice

Abstract Glycosylation is an important posttranslational modification that modulates protein expression and function. Sialic acid is the most abundant terminal monosaccharide of N-linked glycosylation in mammalian cells, and its biosynthesis is controlled by the rate-limited enzyme UDP-GlcNAc 2-epimerase/ManNac kinase (GNE). There is a positive association between the abundance of terminally sialylated plasma glycoproteins and the risk of type 2 diabetes (T2DM). The majority of glycosylated plasma proteins are liver-derived, and key hepatic receptors involved in glucose metabolism, such as the insulin receptor (IR) and glucagon receptor (GCGR), are terminally sialylated. Whether and how protein sialylation in the liver influences glucose homeostasis is unknown. To address this question we generated floxed GNE mice and at 4 weeks of age injected males with either AAV8 encoding a liver-specific targeted Cre recombinase or GFP, to yield mice lacking or normally expressing GNE in liver (GNELKO and GNELWT, respectively). Two weeks post AAV8 injection, GNELKO showed a >98% loss of GNE protein selectively in liver and drastic reductions in the sialylation of liver and plasma proteins detected by glycan-specific lectin-based analyses. Whereas body weights/body composition and plasma lipids were unaltered, GNELKO had lower fasting serum glucose and greater glucose tolerance and insulin sensitivity compared to GNELWT; serum insulin or glucose-stimulated insulin secretion were unchanged. In GNELKO mice hyperinsulinemic-euglycemic clamps displayed a loss of insulin suppression of hepatic glucose production, hepatic IRa and IRb subunit expression and Akt and GSK phosphorylation were decreased, and liver glycogen content was lowered >85%. However, there was also evidence of markedly impaired GCGR function in GNELKO mice, with serum glucagon levels raised 18-fold and attenuation of hepatic cAMP production, CREB phosphorylation and gluconeogenesis in response to glucagon. Regarding extrahepatic mechanisms, GNELKO mice displayed enhanced peripheral glucose disposal, and this was associated with increased IRb, Akt and GSK phosphorylation in skeletal muscle, and with serum FGF21 levels elevated by 406%. In additional studies, when invoked following 12 weeks of high fat diet feeding, liver GNE silencing caused dramatic improvements in glucose tolerance and insulin sensitivity. To evaluate the relevance of these findings to humans, liver GNE transcript levels were queried in a publicly available cohort, and they were higher in T2DM patients compared to non-diabetic controls in two independent cohorts (n= 54 and n=9 for Cohort I and n=4 and n=4 for Cohort II, respectively). Collectively these observations reveal that protein sialylation in the liver has dramatic impact on both insulin and glucagon action in the liver, and on peripheral glucose disposal. Further interrogation of the modulation of glucose control mechanisms by protein sialylation may lead to novel approaches to combatting glucose intolerance in T2DM and other conditions. Presentation: Monday, June 13, 2022 12:15 p.m. - 12:30 p.m.

In Vitro Anticancer and Cancer-Preventive Activity of New Triterpene Glycosides from the Far Eastern Starfish Solaster pacificus.

Sea stars or starfish (class Asteroidea) and holothurians or sea cucumbers (class Holothuroidea), belonging to the phylum Echinodermata (echinoderms), are characterized by different sets of glycosidic metabolites: the steroid type in starfish and the triterpene type in holothurians. However, herein we report the isolation of eight new triterpene glycosides, pacificusosides D–K (1–3, 5–9) along with the known cucumarioside D (4), from the alcoholic extract of the Far Eastern starfish Solaster pacificus. The isolated new compounds are closely related to the metabolites of sea cucumbers, and their structures of 1–3 and 5–9 were determined by extensive NMR and ESIMS techniques. Compounds 2, 5, and 8 have a new type of tetrasaccharide chain with a terminal non-methylated monosaccharide unit. Compounds 3, 6, and 9 contain another new type of tetrasaccharide chain, having 6-O-SO3-Glc as one of the sugar units. The cytotoxic activity of 1–9 against non-cancerous mouse epidermal cells JB6 Cl41 and human melanoma cell lines SK-MEL-2, SK-MEL-28, and RPMI-7951 was determined by MTS assay. Compounds 1, 3, 4, 6, and 9 showed potent cytotoxicity against these cell lines, but the cancer selectivity (SI > 9) was observed only against the SK-MEL-2 cell line. Compounds 1, 3, 4, 6, and 9 at the non-toxic concentration of 0.1 μM significantly inhibited neoplastic cell transformation of JB6 Cl41 cells induced by chemical carcinogens (EGF, TPA) or ionizing radiation (X-rays and UVB). Moreover, compounds 1 and 4 at the non-toxic concentration of 0.1 µM possessed the highest inhibiting activity on colony formation among the investigated compounds and decreased the colonies number of SK-MEL-2 cells by 64% and 70%, respectively. Thus, triterpene glycosides 1 and 4 can be considered as prospective cancer-preventive and anticancer-compound leaders.

The S-layer homology domains of Paenibacillus alvei surface protein SpaA bind to cell wall polysaccharide through the terminal monosaccharide residue

Self-assembling (glyco)protein surface layers (S-layers) are ubiquitous prokaryotic cell-surface structures involved in structural maintenance, nutrient diffusion, host adhesion, virulence, and other processes, which makes them appealing targets for therapeutics and biotechnological applications as biosensors or drug delivery systems. However, unlocking this potential requires expanding our understanding of S-layer properties, especially the details of surface-attachment. S-layers of Gram-positive bacteria often are attached through the interaction of S-layer homology (SLH) domain trimers with peptidoglycan-linked secondary cell wall polymers (SCWPs). Cocrystal structures of the SLH domain trimer from the Paenibacillus alvei S-layer protein SpaA (SpaASLH) with synthetic, terminal SCWP disaccharide and trisaccharide analogs, together with isothermal titration calorimetry binding analyses, reveal that while SpaASLH accommodates longer biologically relevant SCWP ligands within both its primary (G2) and secondary (G1) binding sites, the terminal pyruvylated ManNAc moiety serves as the nearly exclusive SCWP anchoring point. Binding is accompanied by displacement of a flexible loop adjacent to the receptor site that enhances the complementarity between protein and ligand, including electrostatic complementarity with the terminal pyruvate moiety. Remarkably, binding of the pyruvylated monosaccharide SCWP fragment alone is sufficient to cause rearrangement of the receptor-binding sites in a manner necessary to accommodate longer SCWP fragments. The observation of multiple conformations in longer oligosaccharides bound to the protein, together with the demonstrated functionality of two of the three SCWP receptor-binding sites, reveals how the SpaASLH-SCWP interaction has evolved to accommodate longer SCWP ligands and alleviate the strain inherent to bacterial S-layer adhesion during growth and division.

NamZ1 and NamZ2 from the Oral Pathogen Tannerella forsythia Are Peptidoglycan Processing Exo-β-N-Acetylmuramidases with Distinct Substrate Specificities.

The Gram-negative periodontal pathogen Tannerella forsythia is inherently auxotrophic for N-acetylmuramic acid (MurNAc), which is an essential carbohydrate constituent of the peptidoglycan (PGN) of the bacterial cell wall. Thus, to build up its cell wall, T. forsythia strictly depends on the salvage of exogenous MurNAc or sources of MurNAc, such as polymeric or fragmentary PGN, derived from cohabiting bacteria within the oral microbiome. In our effort to elucidate how T. forsythia satisfies its demand for MurNAc, we recognized that the organism possesses three putative orthologs of the exo-β-N-acetylmuramidase BsNamZ from Bacillus subtilis, which cleaves nonreducing end, terminal MurNAc entities from the artificial substrate pNP-MurNAc and the naturally-occurring disaccharide substrate MurNAc-N-acetylglucosamine (MurNAc-GlcNAc). TfNamZ1 and TfNamZ2 were successfully purified as soluble, pure recombinant His6-fusions and characterized as exo-lytic β-N-acetylmuramidases with distinct substrate specificities. The activity of TfNamZ1 was considerably lower compared to TfNamZ2 and BsNamZ, in the cleavage of MurNAc-GlcNAc. When peptide-free PGN glycans were used as substrates, we revealed striking differences in the specificity and mode of action of these enzymes, as analyzed by mass spectrometry. TfNamZ1, but not TfNamZ2 or BsNamZ, released GlcNAc-MurNAc disaccharides from these glycans. In addition, glucosamine (GlcN)-MurNAc disaccharides were generated when partially N-deacetylated PGN glycans from B. subtilis 168 were applied. This characterizes TfNamZ1 as a unique disaccharide-forming exo-lytic β-N-acetylmuramidase (exo-disaccharidase), and, TfNamZ2 and BsNamZ as sole MurNAc monosaccharide-lytic exo-β-N-acetylmuramidases. IMPORTANCE Two exo-N-acetylmuramidases from T. forsythia belonging to glycosidase family GH171 (www.cazy.org) were shown to differ in their activities, thus revealing a functional diversity within this family: NamZ1 releases disaccharides (GlcNAc-MurNAc/GlcN-MurNAc) from the nonreducing ends of PGN glycans, whereas NamZ2 releases terminal MurNAc monosaccharides. This work provides a better understanding of how T. forsythia may acquire the essential growth factor MurNAc by the salvage of PGN from cohabiting bacteria in the oral microbiome, which may pave avenues for the development of anti-periodontal drugs. On a broad scale, our study indicates that the utilization of PGN as a nutrient source, involving exo-lytic N-acetylmuramidases with different modes of action, appears to be a general feature of bacteria, particularly among the phylum Bacteroidetes.

Comparative studies on the structure, biological activity and molecular mechanisms of polysaccharides from Craterellus cornucopioide (CC-M) and Dictyophora indusiata (Vent.ex Pers) Fisch (DI-Z)

In this study, two new polysaccharides were extracted from the fruiting bodies of Craterellus cornucopioide and Dictyophora indusiata (Vent.ex Pers) Fisch, respectively. The results showed that CC-M was composed of Xyl: Glc: Gal in a ratio of 2:5:4. The CC-M takes 1,6-glucose and 1,6-galactose as skeletons, extends a branched chain from galactose 2-O to connect 1,3,4-xylose, and connects β-4-glucose terminal monosaccharide to xylose. DI-Z was also composed of Xyl: Glc: Gal in the ratio of 2:6:5, which takes 1,6-glucose and 1,6-galactose as skeletons, extends a branched chain from galactose 2-O to connect 1,4-xylose and β-4-glucose as terminal group. The results of immunoactivity showed that CC-M and DI-Z have the proliferation activity of B cells, T cells and RAW264.7 cells in vitro, and the effect of CC-M on the proliferation of immune cells was higher than that of DI-Z. In addition, CC-M could significantly promote the secretion of TNF - α, IL-6, IL-1 β, IL-10 and CD163 and enhance the phagocytic ability of RAW264.7 cells. The results of RNA-sequencing showed that CC-M exerted its proliferative activity and immunoregulatory activity on RAW264.7 cells through the interaction of MAPK signaling pathway, NOD like receptor signaling pathway, PI3K-Akt signaling pathway and NF-κB signaling pathway.

Comparative studies on the structure, biological activity and molecular mechanisms of polysaccharides from Boletus aereus (BA-T) and Pleurotus cornucopiae (PC-1)

In this study, two new polysaccharides were extracted from the fruiting bodies of Boletus aereus and Pleurotus cornucopiae, respectively. The results of structure identification showed that BA-T was composed of xylose residues, glucose residues and galactose residues, and the ratio is 3:6:6. The BA-T takes 1,6-glucose and 1,6-galactose as skeletons, extends a branched chain from galactose 2-O to connect 1,4-xylose, and connects β-4-glucose terminal monosaccharide to xylose. PC-1 was composed of of xylose, glucose and galactose in the ratio of 2:8:2 and takes 1,6-glucose as skeletons, extends a branched chain from galactose 4-O to connect (1→4,,6)-α-D-Glcp and (1→2,6)-α-D-galactose, and →4)-β-D-Glcp and →1)-α-D-Xylp as terminal group. The results of immunoactivity showed that BA-T and PC-1 have the proliferation activity of B cells, T cells and RAW264.7 cells in vitro, and the effect of BA-T on the proliferation of T cells was the greatest. The results of RNA-sequencing showed that the protein synthesis and metabolism of T cells are more vigorous under the stimulation of BA-T, and oxidative phosphorylation is the main energy source in the process of T cell proliferation.

Synthesis of a cyclic tetramer of 3-amino-3-deoxyallose with axially oriented amino groups

A linear tetramer of β-(1 → 6)-linked 3-azido-3-deoxy-d-allose containing glycosyl donor and glycosyl acceptor functions in the terminal monosaccharide units was prepared starting from 3-azido-3-deoxy-1,2:5,6-di-O-isopropylidene-α-d-allofuranose. Cyclization of the linear tetramer under glycosylation conditions afforded the corresponding cyclic tetrasaccharide in 77% yield; its deprotection and reduction of the azido groups resulted in the formation of the cyclic tetramer of 3-amino-3-deoxy-d-allose with axial amino groups, a potential scaffold for the synthesis of tetravalent functional clusters.

The Role of Fucose-Containing Glycan Motifs Across Taxonomic Kingdoms.

The extraordinary diversity of glycans leads to large differences in the glycomes of different kingdoms of life. Yet, while most monosaccharides are solely found in certain taxonomic groups, there is a small set of monosaccharides with widespread distribution across nearly all domains of life. These general monosaccharides are particularly relevant for glycan motifs, as they can readily be used by commensals and pathogens to mimic host glycans or hijack existing glycan recognition systems. Among these, the monosaccharide fucose is especially interesting, as it frequently presents itself as a terminal monosaccharide, primed for interaction with proteins. Here, we analyze fucose-containing glycan motifs across all taxonomic kingdoms. Using a hereby presented large species-specific glycan dataset and a plethora of methods for glycan-focused bioinformatics and machine learning, we identify characteristic as well as shared fucose-containing glycan motifs for various taxonomic groups, demonstrating clear differences in fucose usage. Even within domains, fucose is used differentially based on an organism’s physiology and habitat. We particularly highlight differences in fucose-containing motifs between vertebrates and invertebrates. With the example of pathogenic and non-pathogenic Escherichia coli strains, we also demonstrate the importance of fucose-containing motifs in molecular mimicry and thereby pathogenic potential. We envision that this study will shed light on an important class of glycan motifs, with potential new insights into the role of fucosylated glycans in symbiosis, pathogenicity, and immunity.

Determination of terminal glycan and total monosaccharide profiles of reelin glycoprotein in SH-SY5Y neuroblastoma cell line by lectin blotting and capillary liquid chromatography electrospray ionization-ion trap tandem mass spectrometry system

Reelin (400 kDa) is an extracellular matrix glycoprotein that is a key regulator of the many significant biological processes including the brain formation, cell aggregation, and dendrite formation. The glycosylation contributes to the nature of the protein through folding, localization and trafficking, solubility, antigenicity, biological activity, and half-life. Although reelin is to be known as a glycoprotein, the knowledge of its glycosylation is very limited. In this study, we aimed to characterize the terminal glycan profile of reelin by lectin blotting and monosaccharide analysis of glycan chains by capillary liquid chromatography electrospray ionization ion trap tandem mass spectrometry (CapLC-ESI-MS/MS) in SH-SY5Y neuroblastoma cell line. According to our results, reelin was detected in different protein fragments (310, 250, and 85 kDa) in addition to full-length form (400 kDa) in the cell line. The reelin glycoprotein was found to carry the β-N-Acetylglucosamine, α-Mannose, β-Galactose, and α-2,3 and α2,6 linked sialic acids by lectin blotting. Nevertheless, these terminal monosaccharides were found in different intensity according to reelin fragments. Besides, we purified a reelin fragment (250 kDa), and we analyzed it for their monosaccharide by CapLC-ESI-MS/MS. We found that reelin contained five types of monosaccharides, which were consisted of N-Acetylgalactosamine, N-Acetylglucosamine, Galactose, Glucose, Mannose and Sialic acid, from high to low abundance respectively. The present results provide a valuable guide for biochemical, genetic, and glycobiology based further experiments about reelin glycosylation in cancer perspective.

Unliganded and CMP-Neu5Ac bound structures of human α-2,6-sialyltransferase ST6Gal I at high resolution

Sialic acid residues found as terminal monosaccharides in various types of glycan chains in cell surface glycoproteins and glycolipids have been identified as important contributors of cell-cell interactions in normal vs. abnormal cellular behavior and are pivotal in diseases such as cancers. In vertebrates, sialic acids are attached to glycan chains by a conserved subset of sialyltransferases with different enzymatic and substrate specificities. ST6Gal I is a sialyltransferase using activated CMP-sialic acids as donor substrates to catalyze the formation of a α2,6-glycosidic bond between the sialic acid residue and the acceptor disaccharide LacNAc. Understanding sialyltransferases at the molecular and structural level shed light into their function. We present here two human ST6Gal I structures, which show for the first time the enzyme in the unliganded state and with the full donor substrate CMP-Neu5Ac bound. Comparison of these structures reveal flexibility of the catalytic loop, since in the unliganded structure Tyr354 adopts a conformation seen also as an alternate conformation in the substrate bound structure. CMP-Neu5Ac is bound with the side chain at C5 of the sugar residue directed outwards at the surface of the protein. Furthermore, the exact binding mode of the sialic acid moiety of the substrate directly involves sialylmotifs L, S and III and positions the sialylmotif VS in the immediate vicinity. We also present a model for the ternary complex of ST6Gal I with both the donor and the acceptor substrates.

Elevated plasma free sialic acid levels in individuals with reduced glomerular filtration rates.

N-acetylneuraminic acid (Neu5Ac, sialic acid) is a negatively charged monosaccharide, and the predominant form of sialic acid in human cells (1). Sialic acid is typically found as the terminal monosaccharide on glycoconjugates, where it plays a role in various physiologic and pathologic interactions (1). Sialylated glycoconjugates are critical contributors to the polyanionic component of the glomerular glycocalyx, contributing to size and charge selectivity for plasma macromolecules (1,2). Podocyte foot process morphology is maintained by the anionic charged sialic acid residues on glycoconjugates in podocyte membranes (2). Free sialic acid is filtered but not reabsorbed by the human kidney, in a fashion similar to that for creatinine (3), but in contrast to the handling of other monosaccharides such as glucose, mannose, galactose, and fructose that are reabsorbed by tubular cells (4). Circulating sialic acid levels, both unbound and bound to glycoconjugates, have only been sporadically studied in different conditions, including some renal disorders (3,5−9); a possible causative link to reduced eGFRs has been seldom discussed or investigated. We designed this study to establish a correlation between eGFR and plasma free sialic acid across a range of subjects with glomerular disorders. This would not only emphasize this often-overlooked aspect of renal filtering of free sialic acid, but also inform our developmental program for glomerular diseases using the sialic acid precursor, N-acetylmannosamine (ManNAc) (10), which was expected to significantly increase plasma free sialic acid levels in subjects with reduced eGFR. Peripheral blood was obtained from eight subjects enrolled in National Institute of Diabetes and Digestive and Kidney Diseases (NIDDK) natural history study 94-DK-0127 (ClinicalTrials.gov identifier, NT1392), “Pathogenesis of FSGS,” and from eight subjects enrolled in NIDDK study 16-DK-0036 (ClinicalTrials.gov identifier, NCT02639260), “A Phase 1 Multiple Ascending Dose Study to Evaluate the Safety, Tolerability, and Pharmacokinetics of …

Knockout of sialidase and pro-apoptotic genes in Chinese hamster ovary cells enables the production of recombinant human erythropoietin in fed-batch cultures

Sialic acid, a terminal monosaccharide present in N-glycans, plays an important role in determining both the in vivo half-life and the therapeutic efficacy of recombinant glycoproteins. Low sialylation levels of recombinant human erythropoietin (rhEPO) in recombinant Chinese hamster ovary (rCHO) cell cultures are considered a major obstacle to the production of rhEPO in fed-batch mode. This is mainly due to the accumulation of extracellular sialidases released from the cells. To overcome this hurdle, three sialidase genes (Neu1, 2, and 3) were initially knocked-out using the CRISPR/Cas9-mediated large deletion method in the rhEPO-producing rCHO cell line. Unlike wild type cells, sialidase knockout (KO) clones maintained the sialic acid content and proportion of tetra-sialylated rhEPO throughout fed-batch cultures without exhibiting a detrimental effect with respect to cell growth and rhEPO production. Additional KO of two pro-apoptotic genes, BAK and BAX, in sialidase KO clones (5X KO clones) further improved rhEPO production without any detrimental effect on sialylation. On day 10 in fed-batch cultures, the 5X KO clones had 1.4-times higher rhEPO concentration and 3.0-times higher sialic acid content than wild type cells. Furthermore, the proportion of tetra-sialylated rhEPO on day 10 in fed-batch cultures was 42.2–44.3% for 5X KO clones while it was only 2.2% for wild type cells. Taken together, KO of sialidase and pro-apoptotic genes in rCHO cells is a useful tool for producing heavily sialylated glycoproteins such as rhEPO in fed-batch mode.

Glycosylation Profile of the Transferrin Receptor in Gestational Iron Deficiency and Early-Onset Severe Preeclampsia.

Objective To examine the expression of hypoxia-inducible factor-1α (HIF-1α), TfR1, and TfR1-attached terminal monosaccharides in placentas of women with IDAP and severe preeclampsia. Methods TfR1 and HIF-1α were detected by western blot. Immunoadsorption of TfR1 was performed to characterize the terminal monosaccharides by specific lectin binding. Results There was no difference in the expression of TfR1 and HIF-1α between groups. Lectin blot analysis pointed out an overexpression of galactose β1-4 N-acetylglucosamine (Gal-GlcNAc) and mannose in severe preeclampsia. Conclusion The increase in Gal-GlcNAc may be due to the increased presence of antennary structures and the mannose glycans of TfR1 may indicate the presence of misfolded or incomplete proteins. These findings may be associated with the low expression of placental TfR1 in women with preeclampsia.