Glycobiology最新文献

Glycosylphosphatidylinositols (GPIs) are glycolipids found ubiquitously in eukaryotes. They consist of a glycan and an inositol phospholipid, and act as membrane anchors of many cell-surface proteins by covalently linking to their C-termini. GPIs also exist as unlinked, free glycolipids on the cell surface. In human cells, at least 160 proteins with various functions are GPI-anchored proteins. Because the attachment of GPI is required for the cell-surface expression of GPI-anchored proteins, a thorough knowledge of the molecular basis of mammalian GPI-anchored protein biosynthesis is important for understanding the basic biochemistry and biology of GPI-anchored proteins and their medical significance. In this paper, I review our previous knowledge of the biosynthesis of mammalian GPI-anchored proteins and then examine new findings made since 2020.

Broadly neutralizing antibodies (bNAbs) isolated from HIV-1 infected donors are vaccine paradigms. These bNAbs recognize envelope glycoprotein trimers that carry 75-90 oligomannose and complex-type glycans. Although bNAbs and their precursors must navigate past glycans, they usually also make some glycan contacts. Glycan-modified vaccines may therefore be useful to initiate and guide bNAb development. Here, we describe two ways to modify Env glycans for possible vaccine use: 1) using a cocktail of glycosidases (termed "NGAF3" (Neuraminidase, β-Galactosidase, N-Acetylglucosaminidase, endoglycosidase F3 (endo F3)) to deplete complex glycans to try to minimize bNAb-glycan clashes and 2) co-expressing β-1,4-galactosyltransferase 1 (B4G) and β-galactoside α-2,6 sialyltransferase 1 (ST6) during Env biosynthesis, creating bNAb-preferred glycan structures. Mass spectrometry revealed that NGAF3 removed glycan heads at 3/7 sites occupied by complex glycans. B4G overexpression resulted in hybrid glycan development whenever complex glycans were closely spaced. The glycan at position 611 in of Env's gp41 transmembrane subunit was uniquely isolated from the effects of both endo F3 and B4G. B4G and ST6 co-expression increased hybrid and sialylated glycan abundance, reducing glycan complexity. In rabbit vaccinations, B4G + ST6 virus-like particles (VLPs) induced less frequent, weaker titer NAbs, implying that ST6-mediated increased Env charge dampens vaccine antibodies. In some cases, vaccine sera preferentially neutralized B4G + ST6-modified pseudovirus. HIV-1+ donor plasma NAbs were generally more effective against B4G + ST6 modified pseudovirus, suggesting a preference for less complex and/or α-2,6 sialylated Env trimers. Collectively, our data suggest that B4G and ST6 Env modifications are best suited for intermediate or late vaccine shots.

Escherichia coli serotype O117 (ECO117) are pathogenic bacteria that produce Shiga toxin. Repeating units of the O antigen of ECO117 have the pentasaccharide structure [4-D-GalNAcβ1-3-L-Rhaα1-4-D-Glcα1-4-D-Galβ1-3-D-GalNAcα1-]n. The related non-pathogenic serotype (ECO107) contains a GlcNAc residue instead of Glc in the repeating unit, and the biosynthetic enzymes involved are almost identical. We assembled these repeating units based on GalNAcα-diphosphate-phenylundecyl (GalNAcα-PP-PhU), an analog of the natural intermediate GalNAc-diphosphate-undecaprenyl. We previously characterized α1,4-Glc-transferase WclY from ECO117 that transfers the Glc residue to Galβ1-3GalNAcα-PP-PhU and showed that Arg194Cys mutants of WclY are active α1,4-GlcNAc-transferases. In this work, the reaction products of WclY were used as acceptor substrates for the final enzymes in pathway, L-Rha-transferase WclX, and GalNAc-transferase WclW, demonstrating a complete synthesis of the ECO117 and O107 repeating units. WclX transfers L-Rha with high specificity for the WclY enzyme product as the acceptor and for TDP-L-Rha as the donor substrate. A number of highly conserved sequence motifs were identified (DDGSxD, DxDD, and YR). Mutational analysis revealed several Asp residues are essential for the catalysis of L-Rha transfer, while mutations of Asp44 and R212 substantially reduced the activity of WclX. WclW is a GT2 enzyme specific for UDP-GalNAc but with broad specificity for the acceptor substrate. Using L-Rhaα-p-nitrophenyl as an acceptor for WclW, the reaction product was analyzed by NMR demonstrating that GalNAc was transferred in a β1-3 linkage to L-Rha. The in vitro synthesis of the repeating units allows the production of vaccine candidates and identifies potential targets for inhibition of O antigen biosynthesis.

Tannerella serpentiformis is a health-associated Gram-negative oral anaerobe, while its closest phylogenetic relative is the periodontal pathogen Tannerella forsythia. The pathogen employs glycan mimicry through protein O-glycosylation, displaying a terminal nonulosonic acid aiding in evasion of host immune recognition. Like T. forsythia, T. serpentiformis cells are covered with a 2D-crystalline S-layer composed of two abundant S-layer glycoproteins-TssA and TssB. In this study, we elucidated the structure of the O-linked glycans of T. serpentiformis using 1D and 2D NMR spectroscopy analyzing S-layer glycopeptides and β-eliminated glycans. We found that T. serpentiformis produces two highly fucosylated, branched glycoforms carrying non-carbohydrate modifications, with the structure [2-OMe-Fuc-(α1,2)]-4-OMe-Glc-(β1,3)-[Fuc-(α1,4)]-2-NAc-GlcA-(β1,4)-[3-NH2, 2,4-OMe-Fuc-(α1,3)]-Fuc-(α1,4)-Xyl-(β1,4)-[3-OMe-Fuc-(α1,3)]-GlcA-(α1,2)-[Rha-(α1,4]-Gal, where the 3OMe-Fuc is variable; each glycoform contains a rare 2,4-methoxy, 3-amino-modified fucose. These glycoforms support the hypothesis that nonulosonic acid is a hallmark of pathogenic Tannerella species. A combined glycoproteomics and bioinformatics approach identified multiple sites within TssA (14 sites) and TssB (21 sites) to be O-glycosylated. LC-MS/MS confirmed the presence of the Bacteroidetes O-glycosylation motif (D)(S/T) (L/V/T/A/I) in Tannerella species, including the newly identified candidate "N" for the third position. Alphfold2 models of the S-layer glycoproteins were created revealing an almost uniform spatial distribution of the two glycoforms at the N-terminal two thirds of the proteins supported by glycoproteomics, with glycans facing outward. Glycoproteomics identified 921 unique glycopeptide sequences corresponding to 303 unique UniProt IDs. GO-term enrichment analysis versus the entire T. serpentiformis proteome classified these proteins as mainly membrane and cell periphery-associated glycoproteins, supporting a general protein O-glycosylation system in T. serpentiformis.

Aim: This study examined the roles of the laminin and proteoglycan receptor dystroglycan (DG) in extracellular matrix stabilization and cellular mechanosensory processes conveyed through communication between the extracellular matrix (ECM) and cytoskeleton facilitated by DG. Specific functional attributes of HS-proteoglycans (HSPGs) are conveyed through interactions with DG and provide synaptic specificity through diverse interactions with an extensive range of cell attachment and adaptor proteins which convey synaptic plasticity. HSPG-DG interactions are important in phototransduction and neurotransduction and facilitate retinal bipolar-photoreceptor neuronal signaling in vision. Besides synaptic stabilization, HSPG-DG interactions also stabilize basement membranes and the ECM and have specific roles in the assembly and function of the neuromuscular junction. This provides neuromuscular control of muscle systems that control conscious body movement as well as essential autonomic control of diaphragm, intercostal and abdominal muscles and muscle systems in the face, mouth and pharynx which assist in breathing processes. DG is thus a multifunctional cell regulatory glycoprotein receptor and regulates a diverse range of biological and physiological processes throughout the human body. The unique glycosylation of the αDG domain is responsible for its diverse interactions with ECM components in cell-ECM signaling. Cytoskeletal cell regulatory switches assembled by the βDG domain in its role as a nuclear scaffolding protein respond to such ECM cues to regulate cellular behavior and tissue homeostasis thus DG has fascinating and diverse roles in health and disease.

Human sialic acid-binding immunoglobulin-like lectins (Siglecs) are expressed on subsets of immune cells. Siglec-8 is an immune inhibitory Siglec on eosinophils and mast cells, which are effectors in allergic disorders including eosinophilic esophagitis. Inhibition occurs when Siglec-8 is crosslinked by multivalent Siglec ligands in target tissues. Previously we discovered a high-affinity Siglec-8 sialoglycan ligand on human airways composed of terminally sialylated keratan sulfate chains carried on a single protein, DMBT1. Here we extend that approach to another allergic inflammatory target tissue, human esophagus. Lectin overlay histochemistry revealed that Siglec-8 ligands are expressed predominantly by esophageal submucosal glands, and are densely packed in submucosal ducts leading to the lumen. Expression is tissue-specific; esophageal glands express Siglec-8 ligand whereas nearby gastric glands do not. Extraction and resolution by gel electrophoresis revealed a single predominant human esophageal Siglec-8 ligand migrating at >2 MDa. Purification by size exclusion and affinity chromatography, followed by proteomic mass spectrometry, revealed the protein carrier to be MUC5B. Whereas all human esophageal submucosal cells express MUC5B, only a portion convert it to Siglec-8 ligand by adding terminally sialylated keratan sulfate chains. We refer to this as MUC5B S8L. Material from the esophageal lumen of live subjects revealed MUC5B S8L species ranging from ~1-4 MDa. We conclude that MUC5B in the human esophagus is a protein canvas on which Siglec-8 binding sialylated keratan sulfate chains are post-translationally added. These data expand understanding of Siglec-8 ligands and may help us understand their roles in allergic immune regulation.

Peritoneal metastasis frequently accompanies metastatic and/or recurrent gastric cancer, leading to a poor prognosis owing to a lack of effective treatment. Hence, there is a pressing need to enhance our understanding of the mechanisms and molecules driving peritoneal metastasis. In a previous study, galectin-4 inhibition impeded peritoneal metastasis in a murine model. This study examined the glycan profiles of cell surface proteins and glycosphingolipids (GSLs) in cells with varying tumorigenic potentials to understand the intricate mechanisms underlying galectin-4-mediated regulation, particularly glycosylation. Detailed mass spectrometry analysis showed that galectin-4 knockout cells exhibit increased expression of lacto-series GSLs with β1,3-linked galactose while showing no significant alterations in neolacto-series GSLs. We conducted real-time polymerase chain reaction (PCR) analysis to identify candidate glycosyltransferases that synthesize increased levels of GSLs. Subsequently, we introduced the candidate B3GALT5 gene and selected the clones with high expression levels. B3GALT5 gene-expressing clones showed GSL glycan profiles like those of knockout cells and significantly reduced tumorigenic ability in mouse models. These clones exhibited diminished proliferative capacity and showed reduced expression of galectin-4 and activated AKT. Moreover, co-localization of galectin-4 with flotillin-2 (a raft marker) decreased in B3GALT5-expressing cells, implicating GSLs in galectin-4 localization to lipid rafts. D-threo-1-phenyl-2-decanoylamino-3-morpholino-1-propanol (a GSL synthase inhibitor) also affected galectin-4 localization in rafts, suggesting the involvement of GSL microdomains. We discovered that B3GALT5 plays a crucial role in regulating peritoneal metastasis of malignant gastric cancer cells by suppressing cell proliferation and modulating lipid rafts and galectin-4 via mechanisms that are yet to be elucidated.

Lipooligosaccharides are the most abundant cell surface glycoconjugates on the outer membrane of Gram-negative bacteria. They play important roles in host-microbe interactions. Certain Gram-negative pathogenic bacteria cap their lipooligosaccharides with the sialic acid, N-acetylneuraminic acid (Neu5Ac), to mimic host glycans that among others protects these bacteria from recognition by the hosts immune system. This process of molecular mimicry is not fully understood and remains under investigated. To explore the functional role of sialic acid-capped lipooligosaccharides at the molecular level, it is important to have tools readily available for the detection and manipulation of both Neu5Ac on glycoconjugates and the involved sialyltransferases, preferably in live bacteria. We and others have shown that the native sialyltransferases of some Gram-negative bacteria can incorporate extracellular unnatural sialic acid nucleotides onto their lipooligosaccharides. We here report on the expanded use of native bacterial sialyltransferases to incorporate neuraminic acids analogs with a reporter group into the lipooligosaccharides of a variety of Gram-negative bacteria. We show that this approach offers a quick strategy to screen bacteria for the expression of functional sialyltransferases and the ability to use exogenous CMP-Neu5Ac to decorate their glycoconjugates. For selected bacteria we also show this strategy complements two other glycoengineering techniques, Metabolic Oligosaccharide Engineering and Selective Exo-Enzymatic Labeling, and that together they provide tools to modify, label, detect and visualize sialylation of bacterial lipooligosaccharides.