Glycobiology and Extracellular Matrices
N-glycosylation modulates enzymatic activity of Trypanosoma congolense trans-sialidase
Trypanosomes cause the devastating disease trypanosomiasis, in which the action of trans-sialidase (TS) enzymes harbored on their surface is a key virulence factor. TS enzymes are N-glycosylated, but the biological functions of their glycans have remained elusive. In this study, we investigated the influence of N-glycans on the enzymatic activity and structural stability of TconTS1, a recombinant TS from the African parasite Trypanosoma congolense. We expressed the enzyme in Chinese hamster ovary Lec1 cells, which produce high-mannose type N-glycans similar to the TS N-glycosylation pattern in vivo.Human brain sialoglycan ligand for CD33, a microglial inhibitory Siglec implicated in Alzheimer’s disease
Alzheimer’s disease (AD) is characterized by accumulation of misfolded proteins. Genetic studies implicate microglia, brain-resident phagocytic immune cells, in AD pathogenesis. As positive effectors, microglia clear toxic proteins, whereas as negative effectors, they release proinflammatory mediators. An imbalance of these functions contributes to AD progression. Polymorphisms of human CD33, an inhibitory microglial receptor, are linked to AD susceptibility; higher CD33 expression correlates with increased AD risk.Sialoglycan-binding patterns of bacterial AB5 toxin B subunits correlate with host range and toxicity, indicating evolution independent of A subunits
Many pathogenic bacteria secrete AB5 toxins that can be virulence factors. Cytotoxic A subunits are delivered to the cytosol following B subunit binding to specific host cell surface glycans. Some B subunits are not associated with A subunits, for example, YpeB of Yersinia pestis, the etiologic agent of plague. Plague cannot be eradicated because of Y. pestis' adaptability to numerous hosts. We previously showed selective binding of other B5 pentamers to a sialoglycan microarray, with sialic acid (Sia) preferences corresponding to those prominently expressed by various hosts, for example, N-acetylneuraminic acid (Neu5Ac; prominent in humans) or N-glycolylneuraminic acid (Neu5Gc; prominent in ruminant mammals and rodents).The sialidase NEU1 directly interacts with the juxtamembranous segment of the cytoplasmic domain of mucin-1 to inhibit downstream PI3K-Akt signaling
The extracellular domain (ED) of the membrane-spanning sialoglycoprotein, mucin-1 (MUC1), is an in vivo substrate for the lysosomal sialidase, neuraminidase-1 (NEU1). Engagement of the MUC1-ED by its cognate ligand, Pseudomonas aeruginosa-expressed flagellin, increases NEU1-MUC1 association and NEU1-mediated MUC1-ED desialylation to unmask cryptic binding sites for its ligand. However, the mechanism(s) through which intracellular NEU1 might physically interact with its surface-expressed MUC1-ED substrate are unclear.Identification and functional characterization of a Siglec-7 counter-receptor on K562 cells
Sialic acid (Sia)-binding immunoglobulin-like lectin 7 (Siglec-7) is an inhibitory receptor primarily expressed on natural killer (NK) cells and monocytes. Siglec-7 is known to negatively regulate the innate immune system through Sia binding to distinguish self and nonself; however, a counter-receptor bearing its natural ligand remains largely unclear. Here, we identified a counter-receptor of Siglec-7 using K562 hematopoietic carcinoma cells presenting cell surface ligands for Siglec-7. We affinity-purified the ligands using Fc-ligated recombinant Siglec-7 and diSia-dextran polymer, a strong inhibitor for Siglec-7.The promiscuous binding pocket of SLC35A1 ensures redundant transport of CDP-ribitol to the Golgi
The glycoprotein α-dystroglycan helps to link the intracellular cytoskeleton to the extracellular matrix. A unique glycan structure attached to this protein is required for its interaction with extracellular matrix proteins such as laminin. Up to now, this is the only mammalian glycan known to contain ribitol phosphate groups. Enzymes in the Golgi apparatus use CDP-ribitol to incorporate ribitol phosphate into the glycan chain of α-dystroglycan. Since CDP-ribitol is synthesized in the cytoplasm, we hypothesized that an unknown transporter must be required for its import into the Golgi apparatus.Glycosyltransferase ST6Gal-I promotes the epithelial to mesenchymal transition in pancreatic cancer cells
ST6Gal-I, an enzyme upregulated in numerous malignancies, adds α2-6-linked sialic acids to select membrane receptors, thereby modulating receptor signaling and cell phenotype. In this study, we investigated ST6Gal-I’s role in epithelial to mesenchymal transition (EMT) using the Suit2 pancreatic cancer cell line, which has low endogenous ST6Gal-I and limited metastatic potential, along with two metastatic Suit2-derived subclones, S2-013 and S2-LM7AA, which have upregulated ST6Gal-I. RNA-Seq results suggested that the metastatic subclones had greater activation of EMT-related gene networks than parental Suit2 cells, and forced overexpression of ST6Gal-I in the Suit2 line was sufficient to activate EMT pathways.Adaptation of influenza viruses to human airway receptors
Through annual epidemics and global pandemics, influenza A viruses (IAVs) remain a significant threat to human health as the leading cause of severe respiratory disease. Within the last century, four global pandemics have resulted from the introduction of novel IAVs into humans, with components of each originating from avian viruses. IAVs infect many avian species wherein they maintain a diverse natural reservoir, posing a risk to humans through the occasional emergence of novel strains with enhanced zoonotic potential.Decrease of core 2 O-glycans on synovial lubricin in osteoarthritis reduces galectin-3 mediated crosslinking
The synovial fluid glycoprotein lubricin (also known as proteoglycan 4) is a mucin-type O-linked glycosylated biological lubricant implicated to be involved in osteoarthritis (OA) development. Lubricin's ability to reduce friction is related to its glycosylation consisting of sialylated and unsialylated Tn-antigens and core 1 and core 2 structures. The glycans on lubricin have also been suggested to be involved in crosslinking and stabilization of the lubricating superficial layer of cartilage by mediating interaction between lubricin and galectin-3.Modulation of glycosyltransferase ST6Gal-I in gastric cancer-derived organoids disrupts homeostatic epithelial cell turnover
Programmed cell death promotes homeostatic cell turnover in the epithelium but is dysregulated in cancer. The glycosyltransferase ST6Gal-I is known to block homeostatic apoptosis through α2,6-linked sialylation of the death receptor TNFR1 in many cell types. However, its role has not been investigated in gastric epithelial cells or gastric tumorigenesis. We determined that human gastric antral epithelium rarely expressed ST6Gal-I, but the number of ST6Gal-I–expressing epithelial cells increased significantly with advancing premalignancy leading to cancer.α2,3 linkage of sialic acid to a GPI anchor and an unpredicted GPI attachment site in human prion protein
Prion diseases are transmissible, lethal neurodegenerative disorders caused by accumulation of the aggregated scrapie form of the prion protein (PrPSc) after conversion of the cellular prion protein (PrPC). The glycosylphosphatidylinositol (GPI) anchor of PrPC is involved in prion disease pathogenesis, and especially sialic acid in a GPI side chain reportedly affects PrPC conversion. Thus, it is important to define the location and structure of the GPI anchor in human PrPC. Moreover, the sialic acid linkage type in the GPI side chain has not been determined for any GPI-anchored protein.Influence of sulfonated and diet-derived human milk oligosaccharides on the infant microbiome and immune markers
Human milk oligosaccharides (HMOs) promote the development of the neonatal intestinal, immune, and nervous systems and has recently received considerable attention. Here we investigated how the maternal diet affects HMO biosynthesis and how any diet-induced HMO alterations influence the infant gut microbiome and immunity. Using capillary electrophoresis and MS-based analyses, we extracted and measured HMOs from breast milk samples and then correlated their levels with results from validated 24-h diet recall surveys and breast milk fatty acids.N-Glycosylation regulates ligand-dependent activation and signaling of vascular endothelial growth factor receptor 2 (VEGFR2)
The tumor microenvironment and proinflammatory signals significantly alter glycosylation of cell-surface proteins on endothelial cells. By altering the N-glycosylation machinery in the endoplasmic reticulum and Golgi, proinflammatory cytokines promote the modification of endothelial glycoproteins such as vascular endothelial growth factor receptor 2 (VEGFR2) with sialic acid–capped N-glycans. VEGFR2 is a highly N-glycosylated receptor tyrosine kinase involved in pro-angiogenic signaling in physiological and pathological contexts, including cancer.The female reproductive tract contains multiple innate sialic acid-binding immunoglobulin-like lectins (Siglecs) that facilitate sperm survival
A sperm that fertilizes an egg has successfully survived multiple checkpoints within the female reproductive tract, termed pre-fertilization events. The leukocytic response is a pre-fertilization event in which sperm trigger an immune response that promotes homing of circulating leukocytes to the uterine lumen to destroy most sperm. Various glycoconjugates decorate the sperm surface, including sialic acids, which are abundant at the sperm surface where they cap most glycan chains and regulate sperm migration through cervical mucus, formation of the sperm oviductal reservoir, and sperm capacitation.Glycosylation of the viral attachment protein of avian coronavirus is essential for host cell and receptor binding
Avian coronaviruses, including infectious bronchitis virus (IBV), are important respiratory pathogens of poultry. The heavily glycosylated IBV spike protein is responsible for binding to host tissues. Glycosylation sites in the spike protein are highly conserved across viral genotypes, suggesting an important role for this modification in the virus life cycle. Here, we analyzed the N-glycosylation of the receptor-binding domain (RBD) of IBV strain M41 spike protein and assessed the role of this modification in host receptor binding.Combined sialic acid and histone deacetylase (HDAC) inhibitor treatment up-regulates the neuroblastoma antigen GD2
Neuroblastoma cells highly express the disialoganglioside GD2, a tumor-associated carbohydrate antigen, which is only sparsely expressed on healthy tissue. GD2 is a primary target for the development of immunotherapy for neuroblastoma. Immunotherapy with monoclonal anti-GD2 antibodies has proven safety and efficacy in clinical trials and is included in the standard treatment for children with high-risk neuroblastoma. Strategies to modulate GD2 expression in neuroblastoma could further improve anti-GD2–targeted immunotherapy.A complex between phosphatidylinositol 4-kinase IIα and integrin α3β1 is required for N-glycan sialylation in cancer cells
Aberrant N-glycan sialylation of glycoproteins is closely associated with malignant phenotypes of cancer cells and metastatic potential, which includes cell adhesion, migration, and growth. Recently, phosphatidylinositol 4-kinase IIα (PI4KIIα), which is localized to the trans-Golgi network, was identified as a regulator of Golgi phosphoprotein 3 (GOLPH3) and of vesicle transport in the Golgi apparatus. GOLPH3 is a target of PI4KIIα and helps anchor sialyltransferases and thereby regulates sialylation of cell surface receptors.Neuraminidase 1–mediated desialylation of the mucin 1 ectodomain releases a decoy receptor that protects against Pseudomonas aeruginosa lung infection
Pseudomonas aeruginosa (Pa) expresses an adhesin, flagellin, that engages the mucin 1 (MUC1) ectodomain (ED) expressed on airway epithelia, increasing association of MUC1-ED with neuraminidase 1 (NEU1) and MUC1-ED desialylation. The MUC1-ED desialylation unmasks both cryptic binding sites for Pa and a protease recognition site, permitting its proteolytic release as a hyperadhesive decoy receptor for Pa. We found here that intranasal administration of Pa strain K (PAK) to BALB/c mice increases MUC1-ED shedding into the bronchoalveolar compartment.Identification of a Kdn biosynthesis pathway in the haptophyte Prymnesium parvum suggests widespread sialic acid biosynthesis among microalgae
Sialic acids are a family of more than 50 structurally distinct acidic sugars on the surface of all vertebrate cells where they terminate glycan chains and are exposed to many interactions with the surrounding environment. In particular, sialic acids play important roles in cell–cell and host–pathogen interactions. The sialic acids or related nonulosonic acids have been observed in Deuterostome lineages, Eubacteria, and Archaea but are notably absent from plants. However, the structurally related C8 acidic sugar 3-deoxy-d-manno-2-octulosonic acid (Kdo) is present in Gram-negative bacteria and plants as a component of bacterial lipopolysaccharide and pectic rhamnogalacturonan II in the plant cell wall.Sialidase down-regulation reduces non-HDL cholesterol, inhibits leukocyte transmigration, and attenuates atherosclerosis in ApoE knockout mice
Atherosclerosis is a complex disease that involves alterations in lipoprotein metabolism and inflammation. Protein and lipid glycosylation events, such as sialylation, contribute to the development of atherosclerosis and are regulated by specific glycosidases, including sialidases. To evaluate the effect of the sialidase neuraminidase 1 (NEU1) on atherogenesis, here we generated apolipoprotein E (ApoE)-deficient mice that express hypomorphic levels of NEU1 (Neu1hypoApoe−/−). We found that the hypomorphic NEU1 expression in male Apoe−/− mice reduces serum levels of very-low-density lipoprotein (VLDL) and LDL cholesterol, diminishes infiltration of inflammatory cells into lesions, and decreases aortic sinus atherosclerosis.Autopolysialylation of polysialyltransferases is required for polysialylation and polysialic acid chain elongation on select glycoprotein substrates
Polysialic acid (polySia) is a large glycan polymer that is added to some glycoproteins by two polysialyltransferases (polySTs), ST8Sia-II and ST8Sia-IV. As polySia modulates cell adhesion and signaling, immune cell function, and tumor metastasis, it is of interest to determine how the polySTs recognize their select substrates. We have recently identified residues within the ST8Sia-IV polybasic region (PBR) that are required for neural cell adhesion molecule (NCAM) recognition and subsequent polysialylation.The Alzheimer's disease–protective CD33 splice variant mediates adaptive loss of function via diversion to an intracellular pool
The immunomodulatory receptor Siglec-3/CD33 influences risk for late-onset Alzheimer's disease (LOAD), an apparently human-specific post-reproductive disease. CD33 generates two splice variants: a full-length CD33M transcript produced primarily by the “LOAD-risk” allele and a shorter CD33m isoform lacking the sialic acid-binding domain produced primarily from the “LOAD-protective” allele. An SNP that modulates CD33 splicing to favor CD33m is associated with enhanced microglial activity. Individuals expressing more protective isoform accumulate less brain β-amyloid and have a lower LOAD risk.Extrinsic sialylation is dynamically regulated by systemic triggers in vivo
Recent reports have documented that extracellular sialyltransferases can remodel both cell-surface and secreted glycans by a process other than the canonical cell-autonomous glycosylation that occurs within the intracellular secretory apparatus. Despite association of the abundance of these extracellular sialyltransferases, particularly ST6Gal-1, with disease states such as cancer and a variety of inflammatory conditions, the prevalence of this extrinsic glycosylation pathway in vivo remains unknown.The sialate O-acetylesterase EstA from gut Bacteroidetes species enables sialidase-mediated cross-species foraging of 9-O-acetylated sialoglycans
The gut harbors many symbiotic, commensal, and pathogenic microbes that break down and metabolize host carbohydrates. Sialic acids are prominent outermost carbohydrates on host glycoproteins called mucins and protect underlying glycan chains from enzymatic degradation. Sialidases produced by some members of the colonic microbiota can promote the expansion of several potential pathogens (e.g. Clostridium difficile, Salmonella, and Escherichia coli) that do not produce sialidases. O-Acetyl ester modifications of sialic acids help resist the action of many sialidases and are present at high levels in the mammalian colon.Effects of altered sialic acid biosynthesis on N-linked glycan branching and cell surface interactions
GNE (UDP-GlcNAc 2-epimerase/ManNAc kinase) myopathy is a rare muscle disorder associated with aging and is related to sporadic inclusion body myositis, the most common acquired muscle disease of aging. Although the cause of sporadic inclusion body myositis is unknown, GNE myopathy is associated with mutations in GNE. GNE harbors two enzymatic activities required for biosynthesis of sialic acid in mammalian cells. Mutations to both GNE domains are linked to GNE myopathy. However, correlation between mutation-associated reductions in sialic acid production and disease severity is imperfect.
