Exo-enzymatic addition of diazirine-modified sialic acid to cell surfaces enables photocrosslinking of glycoproteins

Glycan binding often mediates extracellular macromolecular recognition events. Accurate characterization of these binding interactions can be difficult because of dissociation and scrambling that occur during purification and analysis steps. Use of photocrosslinking methods has been pursued to covalently capture glycan-dependent interactions in situ however use of metabolic glycan engineering methods to incorporate photocrosslinking sugar analogs is limited to certain cell types. Here we report an exo-enzymatic labeling method to add a diazirine-modified sialic acid (SiaDAz) to cell surface glycoconjugates. The method involves chemoenzymatic synthesis of diazirine-modified CMP-sialic acid (CMP-SiaDAz), followed by sialyltransferase-catalyzed addition of SiaDAz to desialylated cell surfaces. Cell surface SiaDAz-ylation is compatible with multiple cell types and is facilitated by endogenous extracellular sialyltransferase activity present in Daudi B cells. This method for extracellular addition of α2-6-linked SiaDAz enables UV-induced crosslinking of CD22, demonstrating the utility for covalent capture of glycan-mediated binding interactions.

An essential, kinetoplastid-specific GDP-Fuc: β-D-Gal α-1,2-fucosyltransferase is located in the mitochondrion of Trypanosoma brucei

Fucose is a common component of eukaryotic cell-surface glycoconjugates, generally added by Golgi-resident fucosyltransferases. Whereas fucosylated glycoconjugates are rare in kinetoplastids, the biosynthesis of the nucleotide sugar GDP-Fuc has been shown to be essential in Trypanosoma brucei. Here we show that the single identifiable T. brucei fucosyltransferase (TbFUT1) is a GDP-Fuc: β-D-galactose α-1,2-fucosyltransferase with an apparent preference for a Galβ1,3GlcNAcβ1-O-R acceptor motif. Conditional null mutants of TbFUT1 demonstrated that it is essential for both the mammalian-infective bloodstream form and the insect vector-dwelling procyclic form. Unexpectedly, TbFUT1 was localized in the mitochondrion of T. brucei and found to be required for mitochondrial function in bloodstream form trypanosomes. Finally, the TbFUT1 gene was able to complement a Leishmania major mutant lacking the homologous fucosyltransferase gene (Guo et al., 2021). Together these results suggest that kinetoplastids possess an unusual, conserved and essential mitochondrial fucosyltransferase activity that may have therapeutic potential across trypanosomatids.

Multiple evolutionary origins reflect the importance of sialic acid transporters in the colonisation potential of bacterial pathogens and commensals

Located at the tip of cell surface glycoconjugates, sialic acids are at the forefront of host-microbe interactions and, being easily liberated by sialidase enzymes, are used as metabolites by numerous bacteria, particularly by pathogens and commensals living on or near diverse mucosal surfaces. These bacteria rely on specific transporters for the acquisition of host-derived sialic acids. Here, we present the first comprehensive genomic and phylogenetic analysis of bacterial sialic acid transporters, leading to the identification of multiple new families and subfamilies. Our phylogenetic analysis suggests that sialic acid-specific transport has evolved independently at least 8 times during the evolution of bacteria, from within 4 of the major families/superfamilies of bacterial transporters, and we propose a robust classification scheme to bring together a myriad of different nomenclatures that exist to date. The new transporters discovered occur in diverse bacteria including Spirochaetes, Bacteroidetes, Planctomycetes, and Verrucomicrobia, many of which are species that have not been previously recognised to have sialometabolic capacities. Two subfamilies of transporters stand out in being fused to the sialic acid mutarotase enzyme, NanM, and these transporter fusions are enriched in bacteria present in gut microbial communities. We also provide evidence for a possible function of a sialic acid transporter component in chemotaxis that is independent of transport. Our analysis supports the increasing experimental evidence that competition for host-derived sialic acid is a key phenotype for successful colonisation of complex mucosal microbiomes, such that a strong evolutionary selection has occurred for the emergence of sialic acid specificity within existing transporter architectures.

Trans-sialylation: a strategy used to incorporate sialic acid into oligosaccharides

Sialic acid, as component of cell surface glycoconjugates, plays a crucial role in recognition events. Efficient synthetic methods are necessary for the supply of sialosides in enough quantities for biochemical...

Unraveling the sugar code: the role of microbial extracellular glycans in plant–microbe interactions

To defend against microbial invaders but also to establish symbiotic programs, plants need to detect the presence of microbes through the perception of molecular signatures characteristic of a whole class of microbes. Among these molecular signatures, extracellular glycans represent a structurally complex and diverse group of biomolecules that has a pivotal role in the molecular dialog between plants and microbes. Secreted glycans and glycoconjugates such as symbiotic lipochitooligosaccharides or immunosuppressive cyclic β-glucans act as microbial messengers that prepare the ground for host colonization. On the other hand, microbial cell surface glycans are important indicators of microbial presence. They are conserved structures normally exposed and thus accessible for plant hydrolytic enzymes and cell surface receptor proteins. While the immunogenic potential of bacterial cell surface glycoconjugates such as lipopolysaccharides and peptidoglycan has been intensively studied in the past years, perception of cell surface glycans from filamentous microbes such as fungi or oomycetes is still largely unexplored. To date, only few studies have focused on the role of fungal-derived cell surface glycans other than chitin, highlighting a knowledge gap that needs to be addressed. The objective of this review is to give an overview on the biological functions and perception of microbial extracellular glycans, primarily focusing on their recognition and their contribution to plant–microbe interactions.

Topsy-turvy binding of negatively charged homogalacturonan oligosaccharides to galectin-3

Galectin-3 is crucial to many physiological and pathological processes. The generally accepted dogma is that galectins function extracellularly by binding specifically to β(1→4)-galactoside epitopes on cell surface glycoconjugates. Here, we used crystallography and NMR spectroscopy to demonstrate that negatively charged homogalacturonans (HG, linear polysaccharides of α(1→4)-linked-D-galacturonate (GalA)) bind to the galectin-3 carbohydrate recognition domain. The HG carboxylates at the C6 positions in GalA rings mandate that this saccharide bind galectin-3 in an unconventional, “topsy-turvy” orientation that is flipped by about 180o relative to that of the canonical β-galactoside lactose. In this binding mode, the reducing end GalA β-anomer of HGs takes the position of the nonreducing end galactose residue in lactose. This novel orientation maintains interactions with the conserved tryptophan and seven of the most crucial lactose-binding residues, albeit with different H-bonding interactions. Nevertheless, the HG molecular orientation and new interactions have essentially the same thermodynamic binding parameters as lactose. Overall, our study provides structural details for a new type of galectin–sugar interaction that broadens glycospace for ligand binding to Gal-3 and suggests how the lectin may recognize other negatively charged polysaccharides like glycoaminoglycans (e.g. heparan sulfate) on the cell surface. This discovery impacts on our understanding of galectin-mediated biological function.

TLR2-stimulating contaminants in glycoconjugate fractions prepared from Bacteroides fragilis

Bacteroides fragilis is a member of the normal intestinal flora and is involved in host immunostimulation via TLR2. On the bacterial cell surface, glycoconjugates, such as LPS and capsular polysaccharide A (PSA), have been reported to participate in host immunostimulation via TLR2. Previously, we identified a TLR2-stimulating lipoprotein in B. fragilis cells. In this study, we demonstrated that TLR2-stimulating principal molecules in glycoconjugate fractions prepared from B. fragilis are contaminating proteinous molecules, which may also be lipoproteins. The glycoconjugate fractions were prepared by phenol–hot water extraction of B. fragilis wild type and PSA-deficient strains, followed by hydrophobic interaction chromatography. TLR2-stimilating activities of the fractions were not affected by PSA deficiency. By in-gel TLR2-stimulation assay, molecules in high-molecular-mass area, where capsular polysaccharides were migrated, were found not to stimulate TLR2, but those in the range of 15–40 kDa were active. Further, proteinase K could digest the latter molecules and the TLR2-stimulating activities were migrated to the area of below 15 kDa. These results support that proteinous molecules, which are estimated to be lipoproteins, are responsible for almost all TLR2-stimulating activity in the glycoconjugate fractions prepared from B. fragilis.