Jodie L. Abrahams

HPLC-based analysis of serum N-glycans on a 96-well plate platform with dedicated database software

We present a robust, fully automatable technology platform that includes computer software for the detailed analysis of low femtomoles of N-linked sugars released from glycoproteins. Features include (i) sample immobilization in 96-well plates, glycan release, and fluorescent labeling; (ii) quantitative HPLC analysis, including monosaccharide sequence, linkage, and arm-specific information for charged and neutral glycans; (iii) automatic structural assignment of peaks from HPLC profiles via web-based software that accesses our database (GlycoBase) of more than 350 N-glycan structures, including 117 present in the human serum glycome; and (iv) software (autoGU) that progressively analyzes data from exoglycosidase digestions to produce a refined list of final structures. The N-glycans from a plate of 96 samples can be released and purified in 2 or 3 days and profiled in 2 days. This strategy can be used for (i) identification and screening of disease biomarkers and (ii) monitoring the production of therapeutic glycoproteins, allowing optimization of production conditions. This technology is also suitable for preparing released glycans for other analytical techniques. Here we demonstrate its application to rheumatoid arthritis using 5 microl of patient serum.

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.

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.

Method for milk oligosaccharide profiling by 2-aminobenzamide labeling and hydrophilic interaction chromatography

Although the properties of milk oligosaccharides have been of scientific interest for many years, their structural diversity presents a challenging analytical task. In the quest for a simple and robust technology to characterize the different oligosaccharides present in milk, we developed an analytical scheme based on their fluorescent labeling, pre-fractionation by weak anionic exchange chromatography and separation by hydrophilic interaction liquid chromatography (HILIC)-high performance liquid chromatography (HPLC). HILIC relies on the hydrophilic potential of the molecule, which accounts for differences in properties such as molecular volume, lipophilic surface area, charge, composition, structure, linkage and oligosaccharide branching. The robustness of the methodology has been demonstrated using bovine colostrum oligosaccharides as a case study. Structural assignments for 37 free glycans, including 20 sialylated species, were obtained by a combination of HILIC-HPLC, exoglycosidase digestion and offline negative-ion mode mass spectrometry (MS)/MS. Parameters obtained for each glycan, including linkages, enzymatic digestion products and glucose unit values, will be added to GlycoBase, a public access database (http://glycobase.nibrt.ie/glycobase.html). This approach provides a basis for the analysis of free milk oligosaccharides in a fast and sensitive manner and could be adapted for an automated technology platform amenable to diverse environments. Indeed, our approach, in conjunction with bacterial-binding assays, can provide a better understanding of the structural elements required for biological activity of free milk oligosaccharides and could serve as a scientific basis for the selection of such bioactives from various food sources.

Erythropoietin produced in a human cell line (Dynepo) has significant differences in glycosylation compared with erythropoietins produced in CHO cell lines.

Recombinant human erythropoietin has been used to treat anemia associated with chronic renal disease. This paper provides a comprehensive comparative analysis of Dynepo and three other commercial erythropoiesis stimulating agents, Eprex, NeoRecormon and Aranesp. We found significant differences in the type, levels and amount of O-acetylation of sialic acids. Sialic acids and O-acetylation present provide protection from clearance from circulation. Aranesp had up to six O-acetyl groups attached to the sialic acids. Eprex and NeoRecormon had only minor amounts of O-acetylation while Dynepo had none. Dynepo had no Neu5Gc, which is potentially immunogenic for humans. Dynepo contained the least amount of disialylated and Aranesp the highest amount of tetrasialylated glycans. NeoRecormon and Eprex contained more trisialylated, but less tetrasialylated glycans than Dynepo and Aranesp. Dynepo had the highest amount of tetraantennary glycans and the lowest amounts of triantennary glycans with a β1-6-GlcNAc linkage. All the samples contained poly-N-acetyl-lactosamine repeats with Dynepo having the least. The major N-acetyl-lactosamine extensions in Dynepo and Aranesp were on biantennary glycans, whereas in NeoRecomon and Eprex they were on triantennary glycans. The sLe(x) epitope was only detected in Dynepo.

Structural Feature Ions for Distinguishing N- and O-Linked Glycan Isomers by LC-ESI-IT MS/MS

AbstractGlycomics is the comprehensive study of glycan expression in an organism, cell, or tissue that relies on effective analytical technologies to understand glycan structure–function relationships. Owing to the macro- and micro-heterogeneity of oligosaccharides, detailed structure characterization has required an orthogonal approach, such as a combination of specific exoglycosidase digestions, LC-MS/MS, and the development of bioinformatic resources to comprehensively profile a complex biological sample. Liquid chromatography-electrospray ionization-mass spectrometry (LC-ESI-MS/MS) has emerged as a key tool in the structural analysis of oligosaccharides because of its high sensitivity, resolution, and robustness. Here, we present a strategy that uses LC-ESI-MS/MS to characterize over 200 N- and O-glycans from human saliva glycoproteins, complemented by sequential exoglycosidase treatment, to further verify the annotated glycan structures. Fragment-specific substructure diagnostic ions were collated from an extensive screen of the literature available on the detailed structural characterization of oligosaccharides and, together with other specific glycan structure feature ions derived from cross-ring and glycosidic-linkage fragmentation, were used to characterize the glycans and differentiate isomers. The availability of such annotated mass spectrometric fragmentation spectral libraries of glycan structures, together with such substructure diagnostic ions, will be key inputs for the future development of the automated elucidation of oligosaccharide structures from MS/MS data. Figureᅟ

Initiation of the alternative pathway of murine complement by immune complexes is dependent on N-glycans in IgG antibodies.

OBJECTIVE Collagen antibody-induced arthritis in mice exhibits a requirement for amplification by the alternative pathway of complement. Although the alternative pathway is activated by spontaneous hydrolysis, it is not known whether this pathway can also be initiated directly by IgG antibodies in immune complexes (ICs). IgG lacking terminal sialic acid and galactose (G0 IgG) can activate the lectin pathway of complement, but it is not known if G0 IgG can also activate the classical or alternative pathway. The purpose of this study was to examine the mechanism of initiation of the alternative pathway of complement by ICs. METHODS We used adherent ICs containing bovine type II collagen (CII) and 4 monoclonal antibodies (mAb) to CII (adCII-IC). C3 activation was measured in the presence of sera from wild-type C57BL/6 mice or from mice deficient in informative complement components. The mAb were used intact or after enzyme digestion to create G0 IgG or to completely remove the N-glycan. RESULTS Both the classical and alternative pathways, but not the lectin pathway, mediated C3 activation induced by the adCII-IC. Mannose inhibited the alternative pathway-mediated C3 activation but had no effect on the classical pathway, and N-glycans in IgG were required by the alternative pathway but not the classical pathway. Both the classical and alternative pathways mediated C3 activation induced by G0 IgG. Mannose-binding lectin bound avidly to G0 IgG, but lectin pathway-mediated C3 activation was only slightly increased by G0 IgG. CONCLUSION The alternative pathway of complement is capable of initiating C3 activation induced by adCII-IC and requires the presence of N-glycans on the IgG. G0 IgG activates both the classical and alternative pathways more strongly than the lectin pathway.

Association of medication with the human plasma N-glycome.

Glycosylation is highly variable depending on many environmental factors. Using our fully quantitative high-throughput normal phase hydrophilic interaction liquid chromatography platform we have identified glycosylation changes associated with medication in the plasma N-glycome from three different population cohorts: ORCADES from the Orkney Islands in Scotland and CROATIA-Vis and CROATIA-Korcula from the Croatian islands of Vis and Korcula. Associations between glycosylation and the use of hormones (oral contraceptives, hormone replacement therapy), nonsteroidal anti-inflammatory drugs (aspirin and other NSAIDs), oral steroids (prednisolone) and steroid inhalers (beclomethasone) were investigated. Significant differences associated with usage of oral contraceptives were found with increased core-fucosylated biantennary glycans. Decreases in core-fucosylated biantennary glycans, core-fucosylated triantennary glycans with outer-arm fucose, and high mannosylated glycans were associated with the use of anti-inflammatory drugs. All of the changes in glycosylation were independent of blood group status. In conclusion, hormones and anti-inflammatory medication were associated with changes in glycosylation, possibly as a result of the modulatory effect of these drugs on the inflammatory response. In general, cancer is associated with inflammation, and many glycoproteins in the plasma are acute phase related to the host response. These preliminary data indicate the importance of correcting the levels of glycans used as biomarkers for the effects of medication.

Glycosylation status of serum in inflammatory arthritis in response to anti-TNF treatment

OBJECTIVE Glycosylation is the most common post-translational modification and is altered in disease. The typical glycosylation change in patients with inflammatory arthritis (IA) is a decrease in galactosylation levels on IgG. The aim of this study is to evaluate the effect of anti-TNF therapy on whole serum glycosylation from IA patients and determine whether these alterations in the glycome change upon treatment of the disease. METHODS Serum samples were collected from 54 IA patients before treatment and at 1 and 12 months after commencing anti-TNF therapy. N-linked glycans from whole serum samples were analysed using a high-throughput hydrophilic interaction liquid chromatography-based method. RESULTS Glycosylation on the serum proteins of IA patients changed significantly with anti-TNF treatment. We observed an increase in galactosylated glycans from IgG, also an increase in core-fucosylated biantennary galactosylated glycans and a decrease in sialylated triantennary glycans with and without outer arm fucose. This increase in galactosylated IgG glycans suggests a reversing of the N-glycome towards normal healthy profiles. These changes are strongly correlated with decreasing CRP, suggesting a link between glycosylation changes and decreases in inflammatory processes. CONCLUSION Glycosylation changes in the serum of IA patients on anti-TNF therapy are strongly associated with a decrease in inflammatory processes and reflect the effect of anti-TNF on the immune system.

Levels of specific glycans significantly distinguish lymph node-positive from lymph node-negative breast cancer patients

One of the most urgent requirements in breast cancer is the development of a blood-based test for early detection and prognosis. Previously published results found a significant difference between specific glycan levels in patients with advanced breast cancer and healthy controls. The aim of this investigation was to address a more clinically relevant problem, i.e., whether the measurement of specific glycans could identify women with aggressive disease at an early stage. In order to reduce potential bias in this study, blood samples from patients were collected, stored and analyzed in a similar manner. Agalactosyl biantennary glycans (FA2) and glycans containing the sialyl Lewis x epitope (A3F1G1 and A2F1G1) were measured using high throughput normal-phase high-performance liquid chromatography in combination with exoglycosidase digestions in sera from 52 patients with early breast cancer (21 with lymph node-negative and 20 with lymph node-positive disease) and 134 women with benign breast disease. The combined levels of the glycans were significantly higher in patients with lymph node metastases compared to women without these metastases. Lymph node status is the single most important determinant of survival in early stage breast cancer. As high levels of these glycans were associated with nodal metastases, their measurement may provide a new non-invasive approach to determining prognosis in women with newly diagnosed breast cancer.