Oxidative Release of O-Glycans under Neutral Conditions for Analysis of Glycoconjugates Having Base-Sensitive Substituents

Protein O-glycosylation is one of the most diverse post-translational modifications. A critical step in the analysis of O-glycomes is the release of glycans from glycoconjugates. Current release methods rely mainly on β-elimination, which can result in peeling reactions and loss of base-sensitive functionalities leading to misidentification of glycans. To address this challenge, well-defined synthetic glycopeptides were used to establish a robust workflow for the oxidative release of O-glycans suitable for glycomics. Treatment of O-glycopeptides with neutralized hypochlorite resulted in the selective formation of lactic/glycolic acid glycosides, thereby retaining unique information of the parent amino acid (serine/threonine) that is lost by β-elimination. It locks the glycan in a closed ring configuration, thereby preventing peeling, and furthermore, the carboxylate of the anomeric tag promotes ionization in negative ion mode mass spectrometry, thereby increasing signal intensities. Labile modifications such as sialic acids, sulfates, and acetyl esters are maintained during the release procedure. The promise of the approach was demonstrated by the analysis of O-glycans from bovine submaxillary mucin, which identified mono- and di- O-acetylated sialoglycans as well as previously undetected tri- O-acetylated and sulfated glycans. The use of well-defined glycopeptide standards made it also possible to identify reaction intermediates, which in turn allowed us to postulate a reaction mechanism for oxidative O-glycan release under neutral conditions

Contemporary human H3N2 influenza A viruses require a low threshold of suitable glycan receptors for efficient infection

Recent human H3N2 influenza A viruses 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, which are commonly employed to propagate influenza A virus, resulting in low or no viral propagation. Here, we examined whether the overexpression of the glycosyltransferases β-1,3-N-acetylglucosaminyltransferase and β-1,4-galactosyltransferase 1, which are responsible for the elongation of poly-N-acetyllactosamines (LacNAcs), would result in improved A/H3N2 propagation. Stable overexpression of β-1,3-N-acetylglucosaminyltransferase and β-1,4-galactosyltransferase 1 in Madin-Darby Canine Kidney and "humanized" Madin-Darby Canine Kidney 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 β-1,3-N-acetylglucosaminyltransferase and/or β-1,4-galactosyltransferase 1 knock-in cells demonstrated increased binding of viral hemagglutinins and the presence of a larger number of LacNAc repeating units, especially on "humanized" Madin-Darby Canine Kidney-β-1,3-N-acetylglucosaminyltransferase 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 influenza A viruses 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.</p

Contemporary human H3N2 influenza a viruses require a low threshold of suitable glycan receptors for efficient infection

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

Contemporary human H3N2 influenza A viruses require a low threshold of suitable glycan receptors for efficient infection

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 cells commonly employed to propagate these viruses (MDCK and hCK), 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

A Yersinia Effector with Enhanced Inhibitory Activity on the NF-κB Pathway Activates the NLRP3/ASC/Caspase-1 Inflammasome in Macrophages

A type III secretion system (T3SS) in pathogenic Yersinia species functions to translocate Yop effectors, which modulate cytokine production and regulate cell death in macrophages. Distinct pathways of T3SS-dependent cell death and caspase-1 activation occur in Yersinia-infected macrophages. One pathway of cell death and caspase-1 activation in macrophages requires the effector YopJ. YopJ is an acetyltransferase that inactivates MAPK kinases and IKKβ to cause TLR4-dependent apoptosis in naïve macrophages. A YopJ isoform in Y. pestis KIM (YopJKIM) has two amino acid substitutions, F177L and K206E, not present in YopJ proteins of Y. pseudotuberculosis and Y. pestis CO92. As compared to other YopJ isoforms, YopJKIM causes increased apoptosis, caspase-1 activation, and secretion of IL-1β in Yersinia-infected macrophages. The molecular basis for increased apoptosis and activation of caspase-1 by YopJKIM in Yersinia-infected macrophages was studied. Site directed mutagenesis showed that the F177L and K206E substitutions in YopJKIM were important for enhanced apoptosis, caspase-1 activation, and IL-1β secretion. As compared to YopJCO92, YopJKIM displayed an enhanced capacity to inhibit phosphorylation of IκB-α in macrophages and to bind IKKβ in vitro. YopJKIM also showed a moderately increased ability to inhibit phosphorylation of MAPKs. Increased caspase-1 cleavage and IL-1β secretion occurred in IKKβ-deficient macrophages infected with Y. pestis expressing YopJCO92, confirming that the NF-κB pathway can negatively regulate inflammasome activation. K+ efflux, NLRP3 and ASC were important for secretion of IL-1β in response to Y. pestis KIM infection as shown using macrophages lacking inflammasome components or by the addition of exogenous KCl. These data show that caspase-1 is activated in naïve macrophages in response to infection with a pathogen that inhibits IKKβ and MAPK kinases and induces TLR4-dependent apoptosis. This pro-inflammatory form of apoptosis may represent an early innate immune response to highly virulent pathogens such as Y. pestis KIM that have evolved an enhanced ability to inhibit host signaling pathways

Endocrinologic, neurologic, and visual morbidity after treatment for craniopharyngioma

Craniopharyngiomas are locally aggressive tumors which typically are focused in the sellar and suprasellar region near a number of critical neural and vascular structures mediating endocrinologic, behavioral, and visual functions. The present study aims to summarize and compare the published literature regarding morbidity resulting from treatment of craniopharyngioma. We performed a comprehensive search of the published English language literature to identify studies publishing outcome data of patients undergoing surgery for craniopharyngioma. Comparisons of the rates of endocrine, vascular, neurological, and visual complications were performed using Pearson’s chi-squared test, and covariates of interest were fitted into a multivariate logistic regression model. In our data set, 540 patients underwent surgical resection of their tumor. 138 patients received biopsy alone followed by some form of radiotherapy. Mean overall follow-up for all patients in these studies was 54 ± 1.8 months. The overall rate of new endocrinopathy for all patients undergoing surgical resection of their mass was 37% (95% CI = 33–41). Patients receiving GTR had over 2.5 times the rate of developing at least one endocrinopathy compared to patients receiving STR alone or STR + XRT (52 vs. 19 vs. 20%, χ2P < 0.00001). On multivariate analysis, GTR conferred a significant increase in the risk of endocrinopathy compared to STR + XRT (OR = 3.45, 95% CI = 2.05–5.81, P < 0.00001), after controlling for study size and the presence of significant hypothalamic involvement. There was a statistical trend towards worse visual outcomes in patients receiving XRT after STR compared to GTR or STR alone (GTR = 3.5% vs. STR 2.1% vs. STR + XRT 6.4%, P = 0.11). Given the difficulty in obtaining class 1 data regarding the treatment of this tumor, this study can serve as an estimate of expected outcomes for these patients, and guide decision making until these data are available

Contemporary human H3N2 influenza A viruses require a low threshold of suitable glycan receptors for efficient infection

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 cells commonly employed to propagate these viruses (MDCK and hCK), 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