The Flexibility of Oligosaccharides Unveiled Through Residual Dipolar Coupling Analysis

The intrinsic flexibility of glycans complicates the study of their structures and dynamics, which are often important for their biological function. NMR has provided insights into the conformational, dynamic and recognition features of glycans, but suffers from severe chemical shift degeneracy. We employed labelled glycans to explore the conformational behaviour of a β(1-6)-Glc hexasaccharide model through residual dipolar couplings (RDCs). RDC delivered information on the relative orientation of specific residues along the glycan chain and provided experimental clues for the existence of certain geometries. The use of two different aligning media demonstrated the adaptability of flexible oligosaccharide structures to different environments.

Purified EDEM3 or EDEM1 alone produces determinant oligosaccharide structures from M8B in mammalian glycoprotein ERAD

Sequential mannose trimming of N-glycan, from M9 to M8B and then to oligosaccharides exposing the a1,6-linked mannosyl residue (M7A, M6 and M5), facilitates endoplasmic reticulum-associated degradation of misfolded glycoproteins (gpERAD). We previously showed that EDEM2 stably disulfide-bonded to the thioredoxin domain-containing protein TXNDC11 is responsible for the first step (George et al., 2020). Here, we show that EDEM3 and EDEM1 are responsible for the second step. Incubation of pyridylamine-labeled M8B with purified EDEM3 alone produced M7 (M7A and M7C), M6 and M5. EDEM1 showed a similar tendency, although much lower amounts of M6 and M5 were produced. Thus, EDEM3 is a major a1,2-mannosidase for the second step from M8B. Both EDEM3 and EDEM1 trimmed M8B from a glycoprotein efficiently. Our confirmation of the Golgi localization of MAN1B indicates that no other a1,2-mannosidase is required for gpERAD. Accordingly, we have established the entire route of oligosaccharide processing and the enzymes responsible.

The possible association of clusterin fucosylation changes with male fertility disorders

In the seminal plasma (n = 118) and serum (n = 90) clusterin (CLU) the fucosylation and the expression of selected fucosyltransferases (FUTs) were analyzed. Samples from infertile men were divided into groups based on the results of the standard semen analysis: normozoospermic (N), teratozoospermic (T), asthenoteratozoospermic (AT) and oligoasthenoteratozoospermic (OAT). The CLU fucosylation was analyzed using lectin-ELISAs with biotinylated lectins specific to α1,3-, α1,2-linked antennary fucose, and α1,6-linked core fucose (LTA, UEA, and LCA, respectively). The concentrations of FUT3 and FUT4, reflecting the expression of Le oligosaccharide structures, were measured using ELISA tests. The differences in serum CLU and FUT4 concentrations, and in the expression of core fucose and antennary fucose α1,2-linked in CLU glycans between the N group and other groups examined suggest that the disturbances in sperm count, motility, and morphology are not the only cause of male infertility. Lack of similarities between levels of examined parameters in blood serum and seminal plasma may suggest the differences in mechanisms leading to glycoproteins glycosylation. It confirmed the observed differences in concentrations of seminal plasma CLU, FUT3, and FUT4 between the OAT group and N, T, AT groups, indicating that decreased sperm count may be related to these parameters expression. The serum CLU concentrations and expression of core fucose and fucose α1,2-linked in CLU, seem to be good markers differentiating normozoospermic men from those with abnormal sperm parameters, which was not observed for seminal plasma.

Deciphering the structural attributes of protein–heparan sulfate interactions using chemo-enzymatic approaches and NMR spectroscopy

Heparan sulfates (HS) is a polysaccharide found at the cell surface, where it mediates interactions with hundreds of proteins and regulates major pathophysiological processes. HS is highly heterogeneous and structurally complex and examples that define their structure–activity relationships remain limited. Here, in order to characterize a protein–HS interface and define the corresponding saccharide-binding domain, we present a chemo-enzymatic approach that generates 13C-labeled HS-based oligosaccharide structures. Nuclear magnetic resonance (NMR) spectroscopy, which efficiently discriminates between important or redundant chemical groups in the oligosaccharides, is employed to characterize these molecules alone and in interaction with proteins. Using chemokines as model system, docking based on NMR data on both proteins and oligosaccharides enable the identification of the structural determinant involved in the complex. This study shows that both the position of the sulfo groups along the chain and their mode of presentation, rather than their overall number, are key determinant and further points out the usefulness of these 13C-labeled oligosaccharides in obtaining detailed structural information on HS–protein complexes.

Characterization of O-glycan binding lectin from the red alga Hydropuntia eucheumatoides

The red alga, Hydropuntia eucheumatoides is one of the algal genera from which agar is commercially extracted, and is the main source of agar in the world. The lectin HEL from the red alga H. eucheumatoides was isolated by a combination of aqueous ethanol extraction, ethanol precipitation, ion exchange and filtration chromatography. Lectin gave a single band with molecular mass of 17,000 Da in both non-reducing and reducing SDS-PAGE conditions, therefore lectin exists in monomeric form. The hemagglutination activities of HEL were stable over a wide range of pH from 3 to 10, temperature up 60 oC and not affected by either the presence of EDTA or addition of divalent cations, indicating that lectin requires no metal for biological activity. The hemagglutination activities of HEL were not inhibited by monosaccharides and glycoproteins, D-glucose, D-mannose, D-galactose, D-xylose, N-acety-D-mannosamine, transferin, fetuin and yeast mannan, but strongly inhibited by monosaccharides containing acetamido groups at equatorial C2 position, such as N-acetyl-galactosamine, N-acetyl-glucosamine, N-acetyl-neuraminic acid and glycoprotein porcine stomach mucin bearing O-glycans. Thus, lectin is specific for O-glycans and may recognize the sequences GalNAcαSer/Thr, GalNAc(α1-3)[Fuc(α1-2)]Gal(β1-4)GlcNAc(β1-3)GalNAc- and GluNAc(α1-4)Gal- under interacting with the acetamido groups at equatorial C2 position of the terminal sugar residues in oligosaccharide structures of O-glycans. The red alga H. eucheumatoides could promise to be a source of valuable lectins for application in biochemistry and biomedicine.

A Synthetic Glycan Array Containing Cryptococcus Neoformans Glucuronoxylomannan Capsular Polysaccharide Fragments Allows the Mapping of Protective Epitopes

A block synthetic strategy to Cryptococcus neoformans glucuronoxylomannan (GXM) capsular polysaccharide part structures has been developed based on di-, tri-, tetra-, penta- and hexasaccharide thioglycoside building blocks. The approach permitted the synthesis of a library of spacer-containing serotype A and D related GXM oligosaccharide structures, ranging from di- to octadecasaccharides. Ten deprotected GXM compounds (mono- to decasaccharide) were printed onto microarray plates and screened with seventeen mouse monoclonal antibodies (mAbs) to GXM. For the first time a GXM oligosaccharide structure (a serotype A decasaccharide), capable of being recognized by neutralizing forms of these GXMspecific mAbs, has been identified, offering insight into the binding epitopes of a range of protective monoclonal antibodies and furthering our efforts to develop semi-synthetic conjugate vaccine candidates against C. neoformans.<br>

A Synthetic Glycan Array Containing Cryptococcus Neoformans Glucuronoxylomannan Capsular Polysaccharide Fragments Allows the Mapping of Protective Epitopes

A block synthetic strategy to Cryptococcus neoformans glucuronoxylomannan (GXM) capsular polysaccharide part structures has been developed based on di-, tri-, tetra-, penta- and hexasaccharide thioglycoside building blocks. The approach permitted the synthesis of a library of spacer-containing serotype A and D related GXM oligosaccharide structures, ranging from di- to octadecasaccharides. Ten deprotected GXM compounds (mono- to decasaccharide) were printed onto microarray plates and screened with seventeen mouse monoclonal antibodies (mAbs) to GXM. For the first time a GXM oligosaccharide structure (a serotype A decasaccharide), capable of being recognized by neutralizing forms of these GXMspecific mAbs, has been identified, offering insight into the binding epitopes of a range of protective monoclonal antibodies and furthering our efforts to develop semi-synthetic conjugate vaccine candidates against C. neoformans.<br>

Glycosylation at an evolutionary nexus: the brittle star Ophiactis savignyi expresses both vertebrate and invertebrate N-glycomic features

Echinoderms are among the most primitive deuterostomes and have been used as model organisms to understand chordate biology because of their close evolutionary relationship to this phylogenetic group. However, there are almost no data available regarding the N-glycomic capacity of echinoderms, which are otherwise known to produce a diverse set of species-specific glycoconjugates, including ones heavily modified by fucose, sulfate, and sialic acid residues. To increase the knowledge of diversity of carbohydrate structures within this phylum, here we conducted an in-depth analysis of N-glycans from a brittle star (Ophiactis savignyi) as an example member of the class Ophiuroidea. To this end, we performed a multi-step N-glycan analysis by HPLC and various exoglyosidase and chemical treatments in combination with MALDI-TOF MS and MS/MS. Using this approach, we found a wealth of hybrid and complex oligosaccharide structures reminiscent of those in higher vertebrates as well as some classical invertebrate glycan structures. 70% of these N-glycans were anionic, carrying either sialic acid, sulfate, or phosphate residues. In terms of glycophylogeny, our data position the brittle star between invertebrates and vertebrates and confirm the high diversity of N-glycosylation in lower organisms.

Induction of specific adaptive immune responses by immunization with newly designed artificial glycosphingolipids

We previously found that artificial glycosphingolipids (artGSLs) containing very-long-chain fatty acids behave as strong immunogens in mice and promote the production of antibodies recognizing the oligosaccharide portion of artGSLs as the epitope. Here, we report that the oligosaccharide structure of artGSLs influences these immunogenic properties. We evaluated the antibody-inducing activity of artGSLs with different oligosaccharide structures in mice and found strong IgG-inducing activity only with an artGSL containing a core-fucosylated tetraoligosaccharide (Manβ1,4GlcNAcβ1,4[Fucα1,6]GlcNAc). To characterize the immunogenic properties of this artGSL, we analyzed various derivatives and found that the non-reducing terminal mannose structure was critical for the antibody-inducing activity. These artGSLs also exhibited IgG-inducing activity dependent on co-administration of lipid A adjuvant, but no cytokine-inducing activity similar to α-galactosylceramide was detected. Furthermore, repetitive immunization with the artGSL promoted the production of antibodies against a core-fucosylated α-fetoprotein isoform (AFP-L3) known as a hepatocellular carcinoma–specific antigen. These results indicate that the newly designed artGSLs specifically induce adaptive immune responses and promote antibody production by B cells, which can be utilized to develop anti-glycoconjugate antibodies and cancer vaccines targeting tumor-associated carbohydrate antigens.