N-linked protein glycosylation is a post-translational modification that exists in all domains of life. It involves two consecutive steps: (1) biosynthesis of a lipid-linked oligosaccharide (LLO), and (2) glycan transfer from the LLO to asparagine residues in secretory proteins, which is catalyzed by the integral membrane enzyme oligosaccharyltransferase (OST). In the last decade, structural and functional studies of the N-glycosylation machinery have increased our mechanistic understanding of the pathway. The structures of bacterial and eukaryotic glycosyltransferases involved in LLO elongation provided insight into the mechanism of LLO biosynthesis, whereas structures of OST enzymes revealed the molecular basis of sequon recognition and catalysis. In this review, we will discuss approaches used and insight obtained from these studies with a special emphasis on the design and preparation of substrate analogs.

Non-alcoholic fatty liver disease (NAFLD) is caused by an imbalance in lipid metabolism and immune response to pose a risk factor for liver fibrosis. Recent evidence indicates that M2 macrophages secrete transforming growth factor (TGF)-β1, which contributes to liver fibrosis. Galectin-12 (Gal12) has been demonstrated to regulate lipid metabolism and macrophage polarization. The purpose of this study is to investigate the role of Gal12 in the development of NAFLD and fibrosis. Liver tissue from wild-type C57BL/6 mice fed with a high-fat diet containing cholesterol and cholic acid diet (HFCCD) for 4-12 weeks was used to examine Gal12 expression and its correlation with NAFLD. Furthermore, the effects of Gal12 on M2 macrophages during the progression of NAFLD was investigated by studying Kupffer cells from Gal12 knockout mice and doxycycline-inducible Gal12-/-THP-1 cells. Ablation of Gal12 promoted M2 polarization of Kupffer cells, as indicated by higher levels of M2 markers, such as arginase I and chitinase 3-like protein 3. Furthermore, the activation of signal transducer and activator of transcription 6 was significantly higher in Gal12-/- macrophages activated by IL-4, which was correlated with higher levels of TGF-β1. Moreover, Gal12-/- macrophage-conditioned medium promoted hepatic stellate cells (HSCs) myofibroblast differentiation, which was indicated by higher α-SMA expression levels compared to those treated with LacZ control medium. Finally, we demonstrated that Gal12 knockdown negatively regulated the suppressor of cytokine signaling 3 levels. These findings suggested that Gal12 balances M1/M2 polarization of Kupffer cells to prevent NAFLD progression.

Glycans are essential to all scales of biology, with their intricate structures being crucial for their biological functions. The structural complexity of glycans is communicated through simplified and unified visual representations according to the Symbol Nomenclature for Glycans (SNFG) guidelines adopted by the community. Here, we introduce GlycoDraw, a Python-native implementation for high-throughput generation of high-quality, SNFG-compliant glycan figures with flexible display options. GlycoDraw is released as part of our glycan analysis ecosystem, glycowork, facilitating integration into existing workflows by enabling fully automated annotation of glycan-related figures and thus assisting the analysis of e.g. differential abundance data or glycomics mass spectra.

Glycans play a pivotal role in biology. However, due to the low-affinity of glycan-protein interactions, many interaction pairs remain unknown. Two important glycoproteins involved in B cell biology are the B cell receptor (BCR) and its secreted counterpart, antibodies (Abs). It has been indicated that glycans expressed by these B cell-specific molecules can modulate immune activation via glycan-binding proteins (GBPs). In several autoimmune diseases, an increased prevalence of variable domain glycosylation of IgG autoantibodies has been observed. Especially, the hallmarking autoantibodies in rheumatoid arthritis (RA), anti-citrullinated protein antibodies (ACPA), carry a substantial amount of variable domain glycans (VDGs). The VDGs expressed by these autoantibodies are N-linked, complex-type, α2-6 sialylated and BCRs carrying VDGs have been hypothesized to promote selection of autoreactive B cells via interactions with GBPs. Here, we use the ACPA response as a prototype to study potential in solution and in situ BCR VDG interactors. We employed SiaDAz, a UV-activatable sialic acid analogue carrying a diazirine moiety that can form covalent bonds with proximal GPBs. We show, using oligosaccharide engineering that SiaDAz can be readily incorporated into VDGs of both Abs and BCRs. Our data show that Ab VDGs are able to interact with inhibitory receptor, CD22. Interestingly, although we did not detect this interaction on the cell surface, we captured CD79 β glycan-BCR interactions. These results show the utility of combining photoaffinity labeling and oligosaccharide engineering for identifying Ab and BCR interactions and indicate that VDGs appear not to be lectin cis ligands in our tested conditions.

One critical step of metastasis formation is the extravasation of circulating tumor cells from the bloodstream. This process requires the dynamic interaction of cell adhesion molecules (CAMs) like E-selectin on endothelial cells (ECs) with carbohydrate ligands on tumor cells (TCs). To characterize these glycans in a comprehensible approach, the rolling, tethering and firm adhesion of nine human TC lines on human umbilical vein ECs was analyzed using laminar flow adhesion assays. The TC lines were grouped into three subsets by their canonical E-selectin ligand status (sialyl-Lewis A and X (sLeA/X) +/+, -/+, -/-) and their adhesiveness was compared after enzymatic, pharmacologic, chemical treatment or antibody blockade of the TCs or ECs, respectively. TCs were also screened regarding their glycosyltransferase expression profile. We found that although E-selectin and terminal α2,3-sialic acid largely determined firm adhesion, adhesive events did not exclusively depend on the presence of sLeA and/or sLeX. Two of the three sLeA/X-/- TCs additionally or fully depended on VCAM-1 for firm adhesion. The significance of O-GalNAc- and N-glycans for adhesion varied remarkably among the TCs. The sLeA/X+/+ subset showed glycoprotein-independent adhesion suggesting a role for glycolipids as well. All sLeA/X-/- TCs lacked FUT3 and FUT7 expression as opposed to sLeA/X+/+ or -/+ cell lines. Summarized, the glycans on TCs mediating endothelial adhesion are not as much restricted to sLeA/X as previously assumed. The present study specifically suggests α2,3-linked sialic acid, O-GalNAc glycans, glycosphingolipids, and FUT3/FUT7 products as promising targets for future studies.

Recent human H3N2 influenza A viruses (IAV) have evolved to employ elongated glycans terminating in α2,6-linked sialic acid as their receptors. These glycans are displayed in low abundancies by (humanized) Madin-Darby Canine Kidney cells (MDCK and hCK) which are commonly employed to propagate IAV, resulting in low or no viral propagation. Here, we examined whether the overexpression of the glycosyltransferases B3GNT2 and B4GALT1, which are responsible for the elongation of poly-N-acetyllactosamines (LacNAc), would result in improved A/H3N2 propagation. Stable overexpression of B3GNT2 and B4GALT1 in MDCK and hCK cells was achieved by lentiviral integration and subsequent antibiotic selection and confirmed by qPCR and protein mass spectrometry experiments. Flow cytometry and glycan mass spectrometry experiments using the B3GNT2 and/or B4GALT1 knock-in cells demonstrated increased binding of viral hemagglutinins and the presence of a larger number of LacNAc repeating units, especially on hCK-B3GNT2 cells. An increase in the number of glycan receptors did, however, not result in a greater infection efficiency of recent human H3N2 viruses. Based on these results, we propose that H3N2 IAVs require a low number of suitable glycan receptors to infect cells and that an increase in the glycan receptor display above this threshold does not result in improved infection efficiency.

Keratan sulfate glycosaminoglycan is composed of repeating N-acetyllactosamine (LacNAc) disaccharide units consisting of galactose (Gal) and N-acetylglucosamine (GlcNAc), both often 6-O-sulfated. Sulfate contents of keratan sulfate are heterogeneous depending upon the origins. In this study, keratan sulfate is classified as either highly sulfated (in which both GlcNAc and Gal residues are 6-O-sulfated) or low-sulfated (in which only GlcNAc residues are 6-O-sulfated). It is reported that highly sulfated keratan sulfate detected by the 5D4 monoclonal antibody is preferentially expressed in normal epithelial cells lining the female genital tract and in their neoplastic counterparts; however, expression of low-sulfated keratan sulfate in either has not been characterized. In the present study, we generated the 294-1B1 monoclonal antibody, which selectively recognizes low-sulfated keratan sulfate, and performed precise glycan analysis of sulfated glycans expressed on human serous ovarian carcinoma OVCAR-3 cells. We found that OVCAR-3 cells do not express highly sulfated keratan sulfate but rather express low-sulfated form, which was heterogeneous in 294-1B1 reactivity. Comparison of mass spectrometry spectra of sulfated glycans in 294-1B1-positive versus -negative OVCAR-3 cells indicated that the 294-1B1 epitope is likely at least two, and possibly three or more, tandem GlcNAc-6-O-sulfated LacNAc units. Then, using the 294-1B1 antibody, we performed quantitative immunohistochemical analysis of 40 specimens from patients with ovarian cancer, consisting of 10 each of serous, endometrioid, clear cell and mucinous carcinomas, and found that among them low-sulfated keratan sulfate was widely expressed in all but mucinous ovarian carcinoma.

Chondroitin sulfate (CS) has been widely administered orally to improve knee osteoarthritis (OA). CS also has various biological properties, such as anti-inflammatory, immunomodulatory, anti-oxidative, and anti-tumour activity. However, CS absorption in the digestive system and bioavailability remain controversial owing to its large molecular weight (MW). In this study, we aimed to evaluate the absorption of CS oligosaccharides (CSOS), depolymerized CS with low MW, in oral administration to humans. Four types of CS with varying MW [CS tetrasaccharide (MW. 980), CSOS-1 (MW. 1500), CSOS-2 (MW. 2800), and HMWCS (MW. 70,000)] were orally administered and quantified in plasma and urine. Exogenous CS (Exo-CS) in these samples was quantified using a high-performance liquid chromatography system equipped with a fluorescence detector. Quantitative changes of administered CS tetrasaccharide showed similar patterns in plasma and urine, therefore it was presumed that the amount of Exo-CS excreted in urine reflects its quantitative profile in blood. Considering urinary Exo-CS as a parameter of intestinal CS absorption, urinary contents of orally administered CS with varying MW were compared. Consequently, the amount of urinary Exo-CS in 24 hours after administration was higher in the CSOS group than that in the HMWCS group. Additionally, in the MW distribution, urinary Exo-CS after CSOS administration showed a lower content of CSOS with a higher MW than that observed before administration. In summary, our results demonstrated for the first time that lower MW of CS is more efficiently absorbed through the digestive tract in human, and the improvement of its bioavailability is expected.