Reinvestigation into the role of lipopolysaccharide Glycosyltransferases in protein glycosylation.

Protein glycosylation has been considered as a fundamental phenomenon shared by all domains of life. In , glycosylation of flagellins A and B with pseudaminic acid have been rigorously confirmed and shown to be essential for flagella assembly and bacterial colonization. In addition to flagellins, several other proteins including RecA, AlpA/B, and BabA/B in have also been reported to be glycosylated and to be dependent on the lipopolysaccharide (LPS) biosynthetic pathway.

Glycomics of cervicovaginal fluid from women at risk of preterm birth reveals immuno-regulatory epitopes that are hallmarks of cancer and viral glycosylation.

During pregnancy the immune system needs to maintain immune tolerance of the foetus while also responding to infection, which can cause premature activation of the inflammatory pathways leading to the onset of labour and preterm birth. The vaginal microbiome is an important modifier of preterm birth risk, with Lactobacillus dominance during pregnancy associated with term delivery while high microbial diversity is associated with an increased risk of preterm birth. Glycans on glycoproteins along the lower female reproductive tract are fundamental to microbiota-host interactions and the mediation of inflammatory responses.

Decreased fucosylation impacts epithelial integrity and increases risk for COPD.

COPD causes significant morbidity and mortality worldwide. Epithelial damage is fundamental to disease pathogenesis, although the mechanisms driving disease remain undefined. Published evidence from a COPD cohort (SPIROMICS) and confirmed in a second cohort (COPDgene) demonstrate a polymorphism in is a trans-pQTL for E-cadherin, which is critical in COPD pathogenesis.

Glyco-engineered MDCK cells display preferred receptors of H3N2 influenza absent in eggs used for vaccines.

Evolution of human H3N2 influenza viruses driven by immune selection has narrowed the receptor specificity of the hemagglutinin (HA) to a restricted subset of human-type (Neu5Acα2-6 Gal) glycan receptors that have extended poly-LacNAc (Galβ1-4GlcNAc) repeats. This altered specificity has presented challenges for hemagglutination assays, growth in laboratory hosts, and vaccine production in eggs. To assess the impact of extended glycan receptors on virus binding, infection, and growth, we have engineered N-glycan extended (NExt) cell lines by overexpressing β3-Ν-acetylglucosaminyltransferase 2 in MDCK, SIAT, and hCK cell lines.

Development of a novel glycoengineering platform for the rapid production of conjugate vaccines.

Conjugate vaccines produced either by chemical or biologically conjugation have been demonstrated to be safe and efficacious in protection against several deadly bacterial diseases. However, conjugate vaccine assembly and production have several shortcomings which hinders their wider availability. Here, we developed a tool, Mobile-element Assisted Glycoconjugation by Insertion on Chromosome, MAGIC, a novel biotechnological platform that overcomes the limitations of the current conjugate vaccine design method(s).

Roles of N-linked glycosylation and glycan-binding proteins in placentation: trophoblast infiltration, immunomodulation, angiogenesis, and pathophysiology.

Protein N-linked glycosylation is a structurally diverse post-translational modification that stores biological information in a larger order of magnitude than other post-translational modifications such as phosphorylation, ubiquitination and acetylation. This gives N-glycosylated proteins a diverse range of properties and allows glyco-codes (glycan-related information) to be deciphered by glycan-binding proteins (GBPs). The intervillous space of the placenta is richly populated with membrane-bound and secreted glycoproteins.

The pleiotropic role of galectin-3 in melanoma progression: Unraveling the enigma.

Melanoma is a highly aggressive skin cancer with poor outcomes associated with distant metastasis. Intrinsic properties of melanoma cells alongside the crosstalk between melanoma cells and surrounding microenvironment determine the tumor behavior. Galectin-3 (Gal-3), a ß-galactoside-binding lectin, has emerged as a major effector in cancer progression, including melanoma behavior.

Perturbation of placental protein glycosylation by endoplasmic reticulum stress promotes maladaptation of maternal hepatic glucose metabolism.

Placental hormones orchestrate maternal metabolic adaptations to support pregnancy. We hypothesized that placental ER stress, which characterizes early-onset pre-eclampsia (ePE), compromises glycosylation, reducing hormone bioactivity and these maladaptations predispose the mother to metabolic disease in later life. We demonstrate ER stress reduces the complexity and sialylation of trophoblast protein N-glycosylation, while aberrant glycosylation of vascular endothelial growth factor reduced its bioactivity.

A combinatorial DNA assembly approach to biosynthesis of N-linked glycans in E. coli.

Glycoengineering of recombinant glycans and glycoconjugates is a rapidly evolving field. However, the production and exploitation of glycans has lagged behind that of proteins and nucleic acids. Biosynthetic glycoconjugate production requires the coordinated cooperation of three key components within a bacterial cell: a substrate protein, a coupling oligosaccharyltransferase, and a glycan biosynthesis locus.

N-glycosylation of cervicovaginal fluid reflects microbial community, immune activity, and pregnancy status.

Human cervicovaginal fluid (CVF) is a complex, functionally important and glycan rich biological fluid, fundamental in mediating physiological events associated with reproductive health. Using a comprehensive glycomic strategy we reveal an extremely rich and complex N-glycome in CVF of pregnant and non-pregnant women, abundant in paucimannose and high mannose glycans, complex glycans with 2-4 N-Acetyllactosamine (LacNAc) antennae, and Poly-LacNAc glycans decorated with fucosylation and sialylation. N-glycosylation profiles were observed to differ in relation to pregnancy status, microbial composition, immune activation, and pregnancy outcome.

Hypoxia Controls the Glycome Signature and Galectin-8-Ligand Axis to Promote Protumorigenic Properties of Metastatic Melanoma.

The prognosis for patients with metastatic melanoma (MM) involving distant organs is grim, and treatment resistance is potentiated by tumor-initiating cells (TICs) that thrive under hypoxia. MM cells, including TICs, express a unique glycome featuring i-linear poly-N-acetyllactosamines through the loss of I-branching enzyme, β1,6 N-acetylglucosaminyltransferase 2. Whether hypoxia instructs MM TIC development by modulating the glycome signature remains unknown.

The Tip of O-Polysaccharide Is a Potent Epitope in Response to Brucellosis Infection and Enables Short Synthetic Antigens to Be Superior Diagnostic Reagents.

Brucellosis is a global disease and the world's most prevalent zoonosis. All cases in livestock and most cases in humans are caused by members of the genus that possess a surface O-polysaccharide (OPS) comprised of a rare monosaccharide 4-deoxy-4-formamido-D-mannopyranose assembled with α1,2 and α1,3 linkages. The OPS of the bacterium is the basis for serodiagnostic tests for brucellosis.

Activation of regulatory T cells triggers specific changes in glycosylation associated with Siglec-1-dependent inflammatory responses.

: Siglec-1 is a macrophage lectin-like receptor that mediates sialic acid-dependent cellular interactions. Its upregulation on macrophages in autoimmune disease was shown previously to promote inflammation through suppressing the expansion of regulatory T cells (Tregs). Here we investigate the molecular basis for Siglec-1 binding to Tregs using -induced cells as a model system.

Strategies to control therapeutic antibody glycosylation during bioprocessing: Synthesis and separation.

Glycosylation can be a critical quality attribute in biologic manufacturing. In particular, it has implications on the half-life, immunogenicity, and pharmacokinetics of therapeutic monoclonal antibodies (mAbs), and must be closely monitored throughout drug development and manufacturing. To address this, advances have been made primarily in upstream processing, including mammalian cell line engineering, to yield more predictably glycosylated mAbs and the addition of media supplements during fermentation to manipulate the metabolic pathways involved in glycosylation.

Proteome-wide prediction of bacterial carbohydrate-binding proteins as a tool for understanding commensal and pathogen colonisation of the vaginal microbiome.

Bacteria use carbohydrate-binding proteins (CBPs), such as lectins and carbohydrate-binding modules (CBMs), to anchor to specific sugars on host surfaces. CBPs in the gut microbiome are well studied, but their roles in the vagina microbiome and involvement in sexually transmitted infections, cervical cancer and preterm birth are largely unknown. We established a classification system for lectins and designed Hidden Markov Model (HMM) profiles for data mining of bacterial genomes, resulting in identification of >100,000 predicted bacterial lectins available at unilectin.

Modified recombinant human IgG1-Fc is superior to natural intravenous immunoglobulin at inhibiting immune-mediated demyelination.

Intravenous immunoglobulin (IVIG) is an established treatment for numerous autoimmune conditions. Although Fc fragments derived from IVIG have shown efficacy in controlling immune thrombocytopenia in children, the mechanisms of action are unclear and controversial. The aim of this study was to dissect IVIG effector mechanisms using further adapted Fc fragments on demyelination in an ex vivo model of the central nervous system-immune interface.

Loss of α2-6 sialylation promotes the transformation of synovial fibroblasts into a pro-inflammatory phenotype in arthritis.

In healthy joints, synovial fibroblasts (SFs) provide the microenvironment required to mediate homeostasis, but these cells adopt a pathological function in rheumatoid arthritis (RA). Carbohydrates (glycans) on cell surfaces are fundamental regulators of the interactions between stromal and immune cells, but little is known about the role of the SF glycome in joint inflammation. Here we study stromal guided pathophysiology by mapping SFs glycosylation pathways.

Red blood cell mannoses as phagocytic ligands mediating both sickle cell anaemia and malaria resistance.

In both sickle cell disease and malaria, red blood cells (RBCs) are phagocytosed in the spleen, but receptor-ligand pairs mediating uptake have not been identified. Here, we report that patches of high mannose N-glycans (ManGlcNAc), expressed on diseased or oxidized RBC surfaces, bind the mannose receptor (CD206) on phagocytes to mediate clearance. We find that extravascular hemolysis in sickle cell disease correlates with high mannose glycan levels on RBCs.

Efficient inhibition of O-glycan biosynthesis using the hexosamine analog AcGalNTGc.

There is a critical need to develop small-molecule inhibitors of mucin-type O-linked glycosylation. The best-known reagent currently is benzyl-GalNAc, but it is effective only at millimolar concentrations. This article demonstrates that AcGalNTGc, a peracetylated C-2 sulfhydryl-substituted GalNAc, fulfills this unmet need.