GlycoDigest: a tool for the targeted use of exoglycosidase digestions in glycan structure determination

Summary: Sequencing oligosaccharides by exoglycosidases, either sequentially or in an array format, is a powerful tool to unambiguously determine the structure of complex N- and O-link glycans. Here, we introduce GlycoDigest, a tool that simulates exoglycosidase digestion, based on controlled rules acquired from expert knowledge and experimental evidence available in GlycoBase. The tool allows the targeted design of glycosidase enzyme mixtures by allowing researchers to model the action of exoglycosidases, thereby validating and improving the efficiency and accuracy of glycan analysis. Availability and implementation: http://www.glycodigest.org. Contact: [email protected] or [email protected]

Anti-D monoclonal antibodies from 23 human and rodent cell lines display diverse IgG Fc-glycosylation profiles that determine their clinical efficacy.

Anti-D immunoglobulin (Anti-D Ig) prophylaxis prevents haemolytic disease of the fetus and newborn. Monoclonal IgG anti-Ds (mAb-Ds) would enable unlimited supplies but have differed in efficacy in FcγRIIIa-mediated ADCC assays and clinical trials. Structural variations of the oligosaccharide chains of mAb-Ds are hypothesised to be responsible. Quantitative data on 12 Fc-glycosylation features of 23 mAb-Ds (12 clones, 5 produced from multiple cell lines) and one blood donor-derived anti-D Ig were obtained by HPLC and mass spectrometry using 3 methods. Glycosylation of mAb-Ds from human B-lymphoblastoid cell lines (B) was similar to anti-D Ig although fucosylation varied, affecting ADCC activity. In vivo, two B mAb-Ds with 77-81% fucosylation cleared red cells and prevented D-immunisation but less effectively than anti-D Ig. High fucosylation (>89%) of mouse-human heterohybridoma (HH) and Chinese hamster ovary (CHO) mAb-Ds blocked ADCC and clearance. Rat YB2/0 mAb-Ds with 60%) together with lower fucosylation (<60%) as safe features of mAb-Ds for mediating rapid red cell clearance at low doses, to enable effective, inexpensive prophylaxis

Differential Modulation of Angiogenesis by Erythropoiesis-Stimulating Agents in a Mouse Model of Ischaemic Retinopathy

BACKGROUND: Erythropoiesis stimulating agents (ESAs) are widely used to treat anaemia but concerns exist about their potential to promote pathological angiogenesis in some clinical scenarios. In the current study we have assessed the angiogenic potential of three ESAs; epoetin delta, darbepoetin alfa and epoetin beta using in vitro and in vivo models. METHODOLOGY/PRINCIPAL FINDINGS: The epoetins induced angiogenesis in human microvascular endothelial cells at high doses, although darbepoetin alfa was pro-angiogenic at low-doses (1-20 IU/ml). ESA-induced angiogenesis was VEGF-mediated. In a mouse model of ischaemia-induced retinopathy, all ESAs induced generation of reticulocytes but only epoetin beta exacerbated pathological (pre-retinal) neovascularisation in comparison to controls (p<0.05). Only epoetin delta induced a significant revascularisation response which enhanced normality of the vasculature (p<0.05). This was associated with mobilisation of haematopoietic stem cells and their localisation to the retinal vasculature. Darbepoetin alfa also increased the number of active microglia in the ischaemic retina relative to other ESAs (p<0.05). Darbepoetin alfa induced retinal TNFalpha and VEGF mRNA expression which were up to 4 fold higher than with epoetin delta (p<0.001). CONCLUSIONS: This study has implications for treatment of patients as there are clear differences in the angiogenic potential of the different ESAs

Genomics Meets Glycomics—The First GWAS Study of Human N-Glycome Identifies HNF1α as a Master Regulator of Plasma Protein Fucosylation

Over half of all proteins are glycosylated, and alterations in glycosylation have been observed in numerous physiological and pathological processes. Attached glycans significantly affect protein function; but, contrary to polypeptides, they are not directly encoded by genes, and the complex processes that regulate their assembly are poorly understood. A novel approach combining genome-wide association and high-throughput glycomics analysis of 2,705 individuals in three population cohorts showed that common variants in the Hepatocyte Nuclear Factor 1α (HNF1α) and fucosyltransferase genes FUT6 and FUT8 influence N-glycan levels in human plasma. We show that HNF1α and its downstream target HNF4α regulate the expression of key fucosyltransferase and fucose biosynthesis genes. Moreover, we show that HNF1α is both necessary and sufficient to drive the expression of these genes in hepatic cells. These results reveal a new role for HNF1α as a master transcriptional regulator of multiple stages in the fucosylation process. This mechanism has implications for the regulation of immunity, embryonic development, and protein folding, as well as for our understanding of the molecular mechanisms underlying cancer, coronary heart disease, and metabolic and inflammatory disorders

Fragmentation and ion mobility properties of negative ions from N-linked carbohydrates : part 7. reduced glycans

Rationale: Negative ion collision-induced dissociation (CID) spectra of released N-glycans provide very informative structural information relating to branching patterns and location of residues such as fucose. For some structural studies, particularly those involving chromatography, glycans are often reduced to avoid production of multiple peaks from α- and β-anomers. We examined the effect of reduction on the production of diagnostic fragment ions and on the ion mobility properties of N-glycans. Methods: Released N-glycans from the glycoproteins bovine fetuin, ribonuclease B, chicken ovalbumin, and porcine thyroglobulin were reduced with sodium cyanoborohydride and both negative ion CID spectra and ion mobility properties of their phosphate adducts were examined with a Waters Synapt G2Si travelling-wave ion mobility mass spectrometer with electrospray sample introduction. Estimated collisional cross sections were measured with dextran as the calibrant. Results: Fragment ions were similar to those from the unreduced glycans with the exception that the prominent ², ⁴A cleavage ion from the reducing terminus was replaced by a prominent [M-H₃PO₄]⁻ ion. Other ions arising from the chitobiose core were of lower relative abundance than those from the unreduced glycans. Estimated collisional cross sections were similar to those of the unreduced compounds but with symmetrical arrival time distribution (ATD) profiles, unlike those of the unreduced glycans whose peaks often contained prominent asymmetry. This observation showed that this asymmetry was due to anomer separation. Conclusions: Reduction of the reducing terminal GlcNAc residue resulted in fewer diagnostic ions from the chitobiose core but fragmentation of the remainder of the molecules generally paralleled that of the unreduced glycans. Thus, most structural information, with the exception of the linkage position of fucose on the core GlcNAc, was available. ATD peaks were symmetrical with the result that cross sections were more appropriate for data-base searching than those from the non-reduced compounds where asymmetry produced lower precision in the measurement.8 page(s

The Glycosylation profile of metastatic melanoma lymph node tumours

Metastasis accounts for the majority of mortality associated with melanoma, as limited treatment options exist for advanced stages of the disease. Alterations in cell surface glycosylation, in particular, increases in highly branched sialylated tetra-antennary N-glycan structures contribute to the invasive and metastatic potential of melanoma cells. Despite a growing understanding of the role of N-linked oligosaccharides in melanoma biology, there has been little progress in using glycans as a screening tool for the early diagnosis of metastasis and predictor of patient prognosis. Here, we demonstrate a targeted method combining PGC-LC-MS with exoglycosidase digestion for the complete structural characterisation and relative quantitation of N-glycans released from metastatic melanoma lymph node tumours. Released glycans were treated with a full array of exoglycosidase enzymes to assign monosaccharide linkage and confirm terminal epitopes from pools of good and poor prognosis patient samples. The global membrane N-glycosylation profile of tumour tissue from individual patient samples have been compared. Structures were quantitated before and after selected exoglycosidase combinations to investigate differences in structural features including the degree of branching, sialylation and fucosylation. Over 80 glycan structures were identified, including high mannose, pauci mannose, hybrid and complex type glycans. This study contributes to our understanding of glycosylation alterations in melanoma metastasis towards using specific glycosylation changes as prognostic markers and the identification of specific targets for therapeutic intervention.2 page(s

Recent advances in glycoinformatic platforms for glycomics and glycoproteomics

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Cell surface protein glycosylation in cancer

Glycosylation of proteins is one of the most important PTMs, with more than half of all human proteins estimated to be glycosylated. It is widely known that aberrant glycosylation has been implicated in many different diseases due to changes associated with biological function and protein folding. In cancer, there is increasing evidence pertaining to the role of glycosylation in tumour formation and metastasis. Alterations in cell surface glycosylation, particularly terminal motifs, can promote invasive behaviour of tumour cells that ultimately lead to the progression of cancer. While a majority of studies have investigated protein glycosylation changes in cancer cell lines and tumour tissue for individual cancers, the review presented here represents a comprehensive, in-depth overview of literature on the structural changes of glycosylation and their associated synthetic enzymes in five different cancer types originating from the breast, colon, liver, skin and ovary. More importantly, this review focuses on key similarities and differences between these cancers that reflect the importance of structural changes of cell surface N- and O-glycans, such as sialylation, fucosylation, degree of branching and the expression of specific glycosyltransferases for each cancer. It is envisioned that the understanding of these biologically relevant glycan alterations on cellular proteins will facilitate the discovery of novel glycan-based biomarkers which could potentially serve as diagnostic and prognostic indicators of cancer.22 page(s

Comprehensive analysis of the N-glycan biosynthetic pathway using bioinformatics to generate UniCorn : a theoretical N-glycan structure database

Glycan structures attached to proteins are comprised of diverse monosaccharide sequences and linkages that are produced from precursor nucleotide-sugars by a series of glycosyltransferases. Databases of these structures are an essential resource for the interpretation of analytical data and the development of bioinformatics tools. However, with no template to predict what structures are possible the human glycan structure databases are incomplete and rely heavily on the curation of published, experimentally determined, glycan structure data. In this work, a library of 45 human glycosyltransferases was used to generate a theoretical database of N-glycan structures comprised of 15 or less monosaccharide residues. Enzyme specificities were sourced from major online databases including Kyoto Encyclopedia of Genes and Genomes (KEGG) Glycan, Consortium for Functional Glycomics (CFG), Carbohydrate-Active enZymes (CAZy), GlycoGene DataBase (GGDB) and BRENDA. Based on the known activities, more than 1.1 million theoretical structures and 4.7 million synthetic reactions were generated and stored in our database called UniCorn. Furthermore, we analyzed the differences between the predicted glycan structures in UniCorn and those contained in UniCarbKB (www.unicarbkb.org), a database which stores experimentally described glycan structures reported in the literature, and demonstrate that UniCorn can be used to aid in the assignment of ambiguous structures whilst also serving as a discovery database.8 page(s