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Sialic acid is a diverse group of monosaccharides often found on the termini of N- and O-linked glycans as well as being components of glycoconjugates. Hypersialylation has been associated with the progression of chronic inflammation-mediated diseases such as cardiovascular disease and cancer. Given its role in infection and disease-related processes, sialic acid is a promising target for therapeutic approaches that utilize carbohydrate-binding molecules. In this study, we screened for sialic acid-recognizing variable lymphocyte receptors (VLRBs) or ccombodies from inshore hagfish (Eptatretus burgeri) using a synthetic Neu5Ac-glycoconjugate as an antigen in immunoassay. Resulting ccombodies, 2D8, 5G11, 4A1, and 5F8 were further characterized in terms of their binding activity and specificity. A competitive ELISA using free haptens showed strong inhibition using either N-acetylneuraminic acid (Neu5Ac) and N-glycolylneuraminic acid (Neu5Gc). The half-maximal inhibitory concentrations (IC50) for Neu5Ac ranged from 7.02 to 17.06 mM, with candidates 4A1 and 5G11 requiring the least and highest amounts, respectively. IC50 values for Neu5Gc ranged from 8.12 to 13.91 mM, for 4A1 and 5G11, respectively. Candidate ccombodies also detected naturally occurring sialic acid from known sialoglycoproteins using a dot blot assay. Neu5Gc-5G11 and Neu5Ac-2D8 yielded the strongest and weakest docking interactions with affinity values of −5.9 kcal/mol and −4.9 kcal/mol, respectively. Hydrogen bonding and hydrophobic interactions were predicted to be the predominant noncovalent forces observed between the ccombodies and sialic acid. This study demonstrates that glycan-binding VLRBs from hagfish hold promise in augmenting the glycobiologists’ toolkit in investigating the roles of glycans in human and animal health and disease.
Sialic acid is a diverse group of monosaccharides often found on the termini of N- and O-linked glycans as well as being components of glycoconjugates. Hypersialylation has been associated with the progression of chronic inflammation-mediated diseases such as cardiovascular disease and cancer. Given its role in infection and disease-related processes, sialic acid is a promising target for therapeutic approaches that utilize carbohydrate-binding molecules. In this study, we screened for sialic acid-recognizing variable lymphocyte receptors (VLRBs) or ccombodies from inshore hagfish (Eptatretus burgeri) using a synthetic Neu5Ac-glycoconjugate as an antigen in immunoassay. Resulting ccombodies, 2D8, 5G11, 4A1, and 5F8 were further characterized in terms of their binding activity and specificity. A competitive ELISA using free haptens showed strong inhibition using either N-acetylneuraminic acid (Neu5Ac) and N-glycolylneuraminic acid (Neu5Gc). The half-maximal inhibitory concentrations (IC50) for Neu5Ac ranged from 7.02 to 17.06 mM, with candidates 4A1 and 5G11 requiring the least and highest amounts, respectively. IC50 values for Neu5Gc ranged from 8.12 to 13.91 mM, for 4A1 and 5G11, respectively. Candidate ccombodies also detected naturally occurring sialic acid from known sialoglycoproteins using a dot blot assay. Neu5Gc-5G11 and Neu5Ac-2D8 yielded the strongest and weakest docking interactions with affinity values of −5.9 kcal/mol and −4.9 kcal/mol, respectively. Hydrogen bonding and hydrophobic interactions were predicted to be the predominant noncovalent forces observed between the ccombodies and sialic acid. This study demonstrates that glycan-binding VLRBs from hagfish hold promise in augmenting the glycobiologists’ toolkit in investigating the roles of glycans in human and animal health and disease.
Antihypertensive therapy is an essential part of management of patients with preeclampsia (PE). Methyldopa (Dopegyt®) and nifedipine (Cordaflex®) are basic medications of therapy since they stabilize blood pressure without affecting the fetus. Their effect on the endothelium of placental vessels has not yet been studied. In this study, we analyzed the effect of antihypertensive therapy on the expression of fucosylated glycans in fetal capillaries of placental terminal villi in patients with early-onset PE (EOPE) and late-onset PE (LOPE), and determined correlation between their expression and mother’s hemodynamic parameters, fetoplacental system, factors reflecting inflammatory response, and destructive processes in the endothelial glycocalyx (eGC). A total of 76 women were enrolled in the study: the comparison group consisted of 15 women with healthy pregnancy, and the main group comprised 61 women with early-onset and late-onset PE, who received one-component or two-component antihypertensive therapy. Hemodynamic status was assessed by daily blood pressure monitoring, dopplerometry of maternal placental and fetoplacental blood flows, and the levels of IL-18, IL-6, TNFα, galectin-3, endocan-1, syndecan-1, and hyaluronan in the blood of the mother. Expression of fucosylated glycans was assessed by staining placental sections with AAL, UEA-I, LTL lectins, and anti-LeY MAbs. It was found that (i) expression patterns of fucosylated glycans in eGC capillaries of placental terminal villi in EOPE and LOPE are characterized by predominant expression of structures with a type 2 core and have a similar pattern of quantitative changes, which seems to be due to the impact of one-component and two-component antihypertensive therapy on their expression; (ii) correlation patterns indicate interrelated changes in the molecular composition of eGC fucoglycans and indicators reflecting changes in maternal hemodynamics, fetoplacental hemodynamics, and humoral factors associated with eGC damage. The presented study is the first to demonstrate the features of placental eGC in women with PE treated with antihypertensive therapy. This study also considers placental fucoglycans as a functional part of the eGC, which affects hemodynamics in the mother–placenta–fetus system.
Regular monitoring of patients with a history of hepatitis C virus (HCV) infection is critical for the detection and management of hepatocellular carcinoma (HCC). Mac-2 binding protein glycosylation isomer (M2BPGi) has been used to monitor fibrosis progression and predict HCC. However, HCC prediction based on M2BPGi has not been optimized. Here, we identified HCC risk-related glycan signatures of M2BP using a newly developed automated bead array with multiplexed lectins. Among 955 patients with HCV who achieved sustained virological response following direct-acting antiviral treatment, we compared M2BP glycosylation from sera of 42 patients diagnosed with HCC during follow-up and 43 without HCC (control) by the lectin microarray. At the HCC observation point, we found significant differences in 17 lectins. Using an automated bead array with 12 of 17 lectins, a principal component analysis (PCA) biplot differentiated HCC from control, along the PC1 axis, explaining 75.2% of variance. Based on PC1, we generated a scoring formula for an HCC-related glycosylation signature on M2BP (M2BPgs-HCC), showing good diagnostic performance for HCC (p = 2.92 × 10−8, AUC = 0.829). This automated multilectin bead array improved the ability of M2BP to detect HCC, providing a candidate test for HCC surveillance in combination with other HCC markers.
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