Distinguishing N-acetylneuraminic acid linkage isomers on glycopeptides by ion mobility-mass spectrometry

Differentiating the structure of isobaric glycopeptides represents a major challenge for mass spectrometry-based characterisation techniques. Here we show that the regiochemistry of the most common N-acetylneuraminic acid linkages of N-glycans can be identified in a site-specific manner from individual glycopeptides using ion mobility-mass spectrometry analysis of diagnostic fragment ions

Exploring the glycans of <i>Euglena gracilis</i>

Euglena gracilis is an alga of great biotechnological interest and extensive metabolic capacity, able to make high levels of bioactive compounds, such as polyunsaturated fatty acids, vitamins and β-glucan. Previous work has shown that Euglena expresses a wide range of carbohydrate-active enzymes, suggesting an unexpectedly high capacity for the synthesis of complex carbohydrates for a single-celled organism. Here, we present an analysis of some of the carbohydrates synthesised by Euglena gracilis. Analysis of the sugar nucleotide pool showed that there are the substrates necessary for synthesis of complex polysaccharides, including the unusual sugar galactofuranose. Lectin- and antibody-based profiling of whole cells and extracted carbohydrates revealed a complex galactan, xylan and aminosugar based surface. Protein N-glycan profiling, however, indicated that just simple high mannose-type glycans are present and that they are partially modified with putative aminoethylphosphonate moieties. Together, these data indicate that Euglena possesses a complex glycan surface, unrelated to plant cell walls, while its protein glycosylation is simple. Taken together, these findings suggest that Euglena gracilis may lend itself to the production of pharmaceutical glycoproteins

Cross Reactive Material 197 glycoconjugate vaccines contain privileged conjugation sites

Production of glycoconjugate vaccines involves the chemical conjugation of glycans to an immunogenic carrier protein such as Cross-Reactive-Material-197 (CRM197). Instead of using glycans from natural sources recent vaccine development has been focusing on the use of synthetically defined minimal epitopes. While the glycan is structurally defined, the attachment sites on the protein are not. Fully characterized conjugates and batch-to-batch comparisons are the key to eventually create completely defined conjugates. A variety of glycoconjugates consisting of CRM197 and synthetic oligosaccharide epitopes was characterised using mass spectrometry techniques. The primary structure was assessed by combining intact protein MALDI-TOF-MS, LC-MALDI-TOF-MS middle-down and LC-ESI-MS bottom-up approaches. The middle-down approach on CNBr cleaved glycopeptides provided almost complete sequence coverage, facilitating rapid batch-to-batch comparisons, resolving glycan loading and identification of side products. Regions close to the N- and C-termini were most efficiently conjugated.Full Tex

Glycomic analysis of gastric carcinoma cells discloses glycans as modulators of RON receptor tyrosine kinase activation in cancer

Background Terminal a2-3 and a2-6 sialylation of glycans precludes further chain elongation, leading to the biosynthesis of cancer relevant epitopes such as sialyl-Lewis X (SLe X ). SLe X overexpression is associated with tumor aggressive phenotype and patients' poor prognosis. Methods MKN45 gastric carcinoma cells transfected with the sialyltransferase ST3GAL4 were established as a model overexpressing sialylated terminal glycans. We have evaluated at the structural level the glycome and the sialoproteome of this gastric cancer cell line applying liquid chromatography and mass spectrometry. We further validated an identified target expression by proximity ligation assay in gastric tumors. Results Our results showed that ST3GAL4 overexpression leads to several glycosylation alterations, including reduced O-glycan extension and decreased bisected and increased branched N-glycans. A shift from a2-6 towards a2-3 linked sialylated N-glycans was also observed. Sialoproteomic analysis further identified 47 proteins with significantly increased sialylated N-glycans. These included integrins, insulin receptor, carcinoembryonic antigens and RON receptor tyrosine kinase, which are proteins known to be key players in malignancy. Further analysis of RON confirmed its modification with SLe X and the concomitant activation. SLe X and RON co-expression was validated in gastric tumors. Conclusion The overexpression of ST3GAL4 interferes with the overall glycophenotype of cancer cells affecting a multitude of key proteins involved in malignancy. Aberrant glycosylation of the RON receptor was shown as an alternative mechanism of oncogenic activation. General significance This study provides novel targets and points to an integrative tumor glycomic/proteomic-profiling for gastric cancer patients' stratification. This article is part of a Special Issue entitled "Glycans in personalised medicine" Guest Editor: Professor Gordan Lauc.We acknowledge the support from the European Union, Seventh Framework Programme, Gastric Glyco Explorer initial training network: grant number 316929. IPATIMUP integrates the i3S Research Unit, which is partially supported by FCT, the Portuguese Foundation for Science and Technology. This work is funded by FEDER funds through the Operational Programme for Competitiveness Factors-COMPETE (FCOMP-01-0124-FEDER028188) and National Funds through the FCT-Foundation for Science and Technology, under the projects: PEst-C/SAU/LA0003/2013, PTDC/BBB-EBI/0786/2012, and PTDC/BBB-EBI/0567/2014 (to CAR). This work was also supported by"Glycoproteomics" project grant number PCIG09-GA-2011-293847(to DK) and the Danish Natural Science Research Council and a generous grant from the VILLUM Foundation to the VILLUM Center for Bioanalytical Sciences at the University of Southern Denmark (to MRL). Grants were received from FCT, POPH (Programa Operacional Potencial Humano) and FSE (Fundo Social Europeu): SFRH/BPD/75871/2011 to AM; SFRH/BPD/111048/2015 to JAF; SFRH/BPD/96510/2013 to CG. The UPLC instrument was obtained with a grant from the Ingabritt and Arne Lundbergs Research Foundation (to NK). C.J. was supported by the Knut and Alice Wallenberg Foundation. The mass spectrometer (LTQ) was obtained by a grant from the Swedish Research Council (342-2004-4434) (to NK)

Better Than You Think—Appropriate Use of Implantable Cardioverter-Defibrillators at a Single Academic Center: A Retrospective Review

Background: Implantable cardioverter-defibrillators (ICDs) can be life-saving devices, although they are expensive and may cause complications. In 2013, several professional societies published joint appropriate use criteria (AUC) assessing indications for ICD implantation. Data evaluating the clinical application of AUC are limited. Previous registry-based studies estimated that 22.5% of primary prevention ICD implantations were “non-evidence-based” implantations. On the basis of AUC, we aimed to determine the prevalence of “rarely appropriate” ICD implantation at our institution for comparison with previous estimates. Methods: We reviewed 286 patients who underwent ICD implantation between 2013 and 2016. Appropriateness of each ICD implantation was assessed by independent review and rated on the basis of AUC. Results: Of 286 ICD implantations, two independent reviewers found that 89.5% and 89.2%, respectively, were appropriate, 5.6% and 7.3% may be appropriate, and 1.8% and 2.1% were rarely appropriate. No AUC indication was found for 3.5% and 3.4% of ICD implantations, respectively. Secondary prevention ICD implantations were more likely rarely appropriate (2.6% vs. 1.2% and 3.6% vs. 1.1%) or unrated (6.0% vs. 1.2% and 2.7% vs. 0.6%). The reviewers found 3.5% and 3.4% of ICD implantations, respectively, were non-evidence-based implantations. The difference in rates between reviewers was not statistically significant. Conclusion: Compared with prior reports, our prevalence of rarely appropriate ICD implantation was very low. The high appropriate use rate could be explained by the fact that AUC are based on current clinical practice. The AUC could benefit from additional secondary prevention indications. Most importantly, clinical judgement and individualized care should determine which patients receive ICDs irrespective of guidelines or criteria. </p

The minimum information required for a glycomics experiment (MIRAGE) project: improving the standards for reporting glycan microarray-based data

MIRAGE (Minimum Information Required for A Glycomics Experiment) is an initiative that was created by experts in the fields of glycobiology, glycoanalytics and glycoinformatics to produce guidelines for reporting results from the diverse types of experiments and analyses used in structural and functional studies of glycans in the scientific literature. As a sequel to the guidelines for sample preparation (Struwe et al. 2016, Glycobiology, 26:907–910) and mass spectrometry  data (Kolarich et al. 2013, Mol. Cell Proteomics, 12:991–995), here we present the first version of guidelines intended to improve the standards for reporting data from glycan microarray analyses. For each of eight areas in the workflow of a glycan microarray experiment, we provide guidelines for the minimal information that should be provided in reporting results. We hope that the MIRAGE glycan microarray guidelines proposed here will gain broad acceptance by the community, and will facilitate interpretation and reproducibility of the glycan microarray results with implications in comparison of data from different laboratories and eventual deposition of glycan microarray data in international databases

The Minimum Information Required for a Glycomics Experiment (MIRAGE) project: improving the standards for reporting glycan microarray-based data

MIRAGE (Minimum Information Required for A Glycomics Experiment) is an initiative that was created by experts in the fields of glycobiology, glycoanalytics, and glycoinformatics to produce guidelines for reporting results from the diverse types of experiments and analyses used in structural and functional studies of glycans in the scientific literature. As a sequel to the guidelines for sample preparation (Struwe et al. 2016, Glycobiology, 26, 907-910) and mass spectrometry (MS) data (Kolarich et al. 2013, Mol. Cell Proteomics. 12, 991-995), here we present the first version of guidelines intended to improve the standards for reporting data from glycan microarray analyses. For each of eight areas in the workflow of a glycan microarray experiment, we provide guidelines for the minimal information that should be provided in reporting results. We hope that the MIRAGE glycan microarray guidelines proposed here will gain broad acceptance by the community, and will facilitate interpretation and reproducibility of the glycan microarray results with implications in comparison of data from different laboratories and eventual deposition of glycan microarray data in international databases

Production of α-L-iduronidase in maize for the potential treatment of a human lysosomal storage disease

Lysosomal storage diseases are a class of over 70 rare genetic diseases that are amenable to enzyme replacement therapy. Towards developing a plant-based enzyme replacement therapeutic for the lysosomal storage disease mucopolysaccharidosis I, here we expressed α-L-iduronidase in the endosperm of maize seeds by a previously uncharacterized mRNA-targeting-based mechanism. Immunolocalization, cellular fractionation and in situ RT-PCR demonstrate that the α-L-iduronidase protein and mRNA are targeted to endoplasmic reticulum (ER)-derived protein bodies and to protein body-ER regions, respectively, using regulatory (5′-and 3′-UTR) and signal-peptide coding sequences from the γ-zein gene. The maize α-L-iduronidase exhibits high activity, contains high-mannose N-glycans and is amenable to in vitro phosphorylation. This mRNA-based strategy is of widespread importance as plant N-glycan maturation is controlled and the therapeutic protein is generated in a native form. For our target enzyme, the N-glycan structures are appropriate for downstream processing, a prerequisite for its potential as a therapeutic protein.9 page(s

Linkage Specific Fucosylation of Alpha-1-Antitrypsin in Liver Cirrhosis and Cancer Patients: Implications for a Biomarker of Hepatocellular Carcinoma

We previously reported increased levels of protein-linked fucosylation with the development of liver cancer and identified many of the proteins containing the altered glycan structures. One such protein is alpha-1-antitrypsin (A1AT). To advance these studies, we performed N-linked glycan analysis on the five major isoforms of A1AT and completed a comprehensive study of the glycosylation of A1AT found in healthy controls, patients with hepatitis C- (HCV) induced liver cirrhosis, and in patients infected with HCV with a diagnosis of hepatocellular carcinoma (HCC).Patients with liver cirrhosis and liver cancer had increased levels of triantennary glycan-containing outer arm (alpha-1,3) fucosylation. Increases in core (alpha-1,6) fucosylation were observed only on A1AT from patients with cancer. We performed a lectin fluorophore-linked immunosorbent assay using Aleuria Aurantia lectin (AAL), specific for core and outer arm fucosylation in over 400 patients with liver disease. AAL-reactive A1AT was able to detect HCC with a sensitivity of 70% and a specificity of 86%, which was greater than that observed with the current marker of HCC, alpha-fetoprotein. Glycosylation analysis of the false positives was performed; results indicated that these patients had increases in outer arm fucosylation but not in core fucosylation, suggesting that core fucosylation is cancer specific.This report details the stepwise change in the glycosylation of A1AT with the progression from liver cirrhosis to cancer and identifies core fucosylation on A1AT as an HCC specific modification