Protein Paucimannosylation Is an Enriched N-Glycosylation Signature of Human Cancers.

While aberrant protein glycosylation is a recognized characteristic of human cancers, advances in glycoanalytics continue to discover new associations between glycoproteins and tumorigenesis. This glycomics-centric study investigates a possible link between protein paucimannosylation, an under-studied class of human N-glycosylation [Man1-3 GlcNAc2 Fuc0-1 ], and cancer. The paucimannosidic glycans (PMGs) of 34 cancer cell lines and 133 tissue samples spanning 11 cancer types and matching non-cancerous specimens are profiled from 467 published and unpublished PGC-LC-MS/MS N-glycome datasets collected over a decade. PMGs, particularly Man2-3 GlcNAc2 Fuc1 , are prominent features of 29 cancer cell lines, but the PMG level varies dramatically across and within the cancer types (1.0-50.2%). Analyses of paired (tumor/non-tumor) and stage-stratified tissues demonstrate that PMGs are significantly enriched in tumor tissues from several cancer types including liver cancer (p = 0.0033) and colorectal cancer (p = 0.0017) and is elevated as a result of prostate cancer and chronic lymphocytic leukaemia progression (p < 0.05). Surface expression of paucimannosidic epitopes is demonstrated on human glioblastoma cells using immunofluorescence while biosynthetic involvement of N-acetyl-ß-hexosaminidase is indicated by quantitative proteomics. This intriguing association between protein paucimannosylation and human cancers warrants further exploration to detail the biosynthesis, cellular location(s), protein carriers, and functions of paucimannosylation in tumorigenesis and metastasis.

Helicobacter suis infection alters glycosylation and decreases the pathogen growth inhibiting effect and binding avidity of gastric mucins.

Helicobacter suis is the most prevalent non-Helicobacter pylori Helicobacter species in the human stomach and is associated with chronic gastritis, peptic ulcer disease, and gastric mucosa-associated lymphoid tissue (MALT) lymphoma. H. suis colonizes the gastric mucosa of 60-95% of pigs at slaughter age, and is associated with chronic gastritis, decreased weight gain, and ulcers. Here, we show that experimental H. suis infection changes the mucin composition and glycosylation, decreasing the amount of H. suis-binding glycan structures in the pig gastric mucus niche. Similarly, the H. suis-binding ability of mucins from H. pylori-infected humans is lower than that of noninfected individuals. Furthermore, the H. suis growth-inhibiting effect of mucins from both noninfected humans and pigs is replaced by a growth-enhancing effect by mucins from infected individuals/pigs. Thus, Helicobacter spp. infections impair the mucus barrier by decreasing the H. suis-binding ability of the mucins and by decreasing the antiprolific activity that mucins can have on H. suis. Inhibition of these mucus-based defenses creates a more stable and inhabitable niche for H. suis. This is likely of importance for long-term colonization and outcome of infection, and reversing these impairments may have therapeutic benefits.

Plasma N-glycans in colorectal cancer risk.

Aberrant glycosylation has been associated with a number of diseases including cancer. Our aim was to elucidate changes in whole plasma N-glycosylation between colorectal cancer (CRC) cases and controls in one of the largest cohorts of its kind. A set of 633 CRC patients and 478 age and gender matched controls was analysed. Additionally, patients were stratified into four CRC stages. Moreover, N-glycan analysis was carried out in plasma of 40 patients collected prior to the initial diagnosis of CRC. Statistically significant differences were observed in the plasma N-glycome at all stages of CRC, this included a highly significant decrease in relation to the core fucosylated bi-antennary glycans F(6)A2G2 and F(6)A2G2S(6)1 (P < 0.0009). Stage 1 showed a unique biomarker signature compared to stages 2, 3 and 4. There were indications that at risk groups could be identified from the glycome (retrospective AUC = 0.77 and prospective AUC = 0.65). N-glycome biomarkers related to the pathogenic progress of the disease would be a considerable asset in a clinical setting and it could enable novel therapeutics to be developed to target the disease in patients at risk of progression.

Helicobacter suis binding to carbohydrates on human and porcine gastric mucins and glycolipids occurs via two modes.

Helicobacter suis colonizes the stomach of most pigs and is the most prevalent non-Helicobacter pylori Helicobacter species found in the human stomach. In the human host, H. suis contributes to the development of chronic gastritis, peptic ulcer disease and MALT lymphoma, whereas in pigs it is associated with gastritis, decreased growth and ulcers. Here, we demonstrate that the level of H. pylori and H. suis binding to human and pig gastric mucins varies between individuals with species dependent specificity. The binding optimum of H. pylori is at neutral pH whereas that of H. suis has an acidic pH optimum, and the mucins that H. pylori bind to are different than those that H. suis bind to. Mass spectrometric analysis of mucin O-glycans from the porcine mucin showed that individual variation in binding is reflected by a difference in glycosylation; of 109 oligosaccharide structures identified, only 14 were present in all examined samples. H. suis binding to mucins correlated with glycans containing sulfate, sialic acid and terminal galactose. Among the glycolipids present in pig stomach, binding to lactotetraosylceramide (Galß3GlcNAcß3Galß4Glcß1Cer) was identified, and adhesion to Galß3GlcNAcß3Galß4Glc at both acidic and neutral pH was confirmed using other glycoconjugates. Together with that H. suis bound to DNA (used as a proxy for acidic charge), we conclude that H. suis has two binding modes: one to glycans terminating with Galß3GlcNAc, and one to negatively charged structures. Identification of the glycan structures H. suis interacts with can contribute to development of therapeutic strategies alternative to antibiotics.

Sample handling of gastric tissue and O-glycan alterations in paired gastric cancer and non-tumorigenic tissues.

Sample collection, handling and storage are the most critical steps for ensuring the highest preservation of specimens. Pre-analytical variability can influence the results as protein signatures alter rapidly after tissue excision or during long-term storage. Hence, we evaluated current state-of-the-art biobank preservation methods from a glycomics perspective and analyzed O-glycan alterations occurring in the gastric cancer tissues. Paired tumor and adjacent normal tissue samples were obtained from six patients undergoing gastric cancer surgery. Collected samples (n = 24) were either snap-frozen or heat stabilized and then homogenized. Glycans were released from extracted glycoproteins and analyzed by LC-MS/MS. In total, the relative abundance of 83 O-glycans and 17 derived structural features were used for comparison. There was no statistically significant difference found in variables between snap frozen and heat-stabilized samples, which indicated the two preservation methods were comparable. The data also showed significant changes between normal and cancerous tissue. In addition to a shift from high sialylation in the cancer area towards blood group ABO in the normal area, we also detected that the LacdiNAc epitope (N,N'-diacetyllactosamine) was significantly decreased in cancer samples. The O-glycan alterations that are presented here may provide predictive power for the detection and prognosis of gastric cancer.

Structural Diversity of Human Gastric Mucin Glycans.

The mucin O-glycosylation of 10 individuals with and without gastric disease was examined in depth in order to generate a structural map of human gastric glycosylation. In the stomach, these mucins and their O-glycosylation protect the epithelial surface from the acidic gastric juice and provide the first point of interaction for pathogens such as Helicobacter pylori, reported to cause gastritis, gastric and duodenal ulcers and gastric cancer. The rational of the present study was to map the O-glycosylation that the pathogen may come in contact with. An enormous diversity in glycosylation was found, which varied both between individuals and within mucins from a single individual: mucin glycan chain length ranged from 2-13 residues, each individual carried 34-103 O-glycan structures and in total over 258 structures were identified. The majority of gastric O-glycans were neutral and fucosylated. Blood group I antigens, as well as terminal α1,4-GlcNAc-like and GalNAcß1-4GlcNAc-like (LacdiNAc-like), were common modifications of human gastric O-glycans. Furthemore, each individual carried 1-14 glycan structures that were unique for that individual. The diversity and alterations in gastric O-glycosylation broaden our understanding of the human gastric O-glycome and its implications for gastric cancer research and emphasize that the high individual variation makes it difficult to identify gastric cancer specific structures. However, despite the low number of individuals, we could verify a higher level of sialylation and sulfation on gastric O-glycans from cancerous tissue than from healthy stomachs.

Structural diversity of human gastric mucin glycans.

The mucin O-glycosylation of 10 individuals with and without gastric disease was examined in depth in order to generate a structural map of human gastric glycosylation. In the stomach, these mucins and their O-glycosylation protect the epithelial surface from the acidic gastric juice and provide the first point of interaction for pathogens such as Helicobacter pylori, reported to cause gastritis, gastric and duodenal ulcers and gastric cancer. The rational of the present study was to map the O-glycosylation that the pathogen may come in contact with. An enormous diversity in glycosylation was found, which varied both between individuals and within mucins from a single individual: mucin glycan chain length ranged from 2-13 residues, each individual carried 34-103 O-glycan structures and in total over 258 structures were identified. The majority of gastric O-glycans were neutral and fucosylated. Blood group I antigens, as well as terminal α1,4-GlcNAc-like and GalNAcß1-4GlcNAc-like (LacdiNAc-like), were common modifications of human gastric O-glycans. Furthemore, each individual carried 1-14 glycan structures that were unique for that individual. The diversity and alterations in gastric O-glycosylation broaden our understanding of the human gastric O-glycome and its implications for gastric cancer research and emphasize that the high individual variation makes it difficult to identify gastric cancer specific structures. However, despite the low number of individuals, we could verify a higher level of sialylation and sulfation on gastric O-glycans from cancerous tissue than from healthy stomachs.

Glycomic and sialoproteomic data of gastric carcinoma cells overexpressing ST3GAL4.

Gastric carcinoma MKN45 cells stably transfected with the full-length ST3GAL4 gene were characterised by glycomic and sialoproteomic analysis. Complementary strategies were applied to assess the glycomic alterations induced by ST3GAL4 overexpression. The N- and O-glycome data were generated in two parallel structural analyzes, based on PGC-ESI-MS/MS. Data on glycan structure identification and relative abundance in ST3GAL4 overexpressing cells and respective mock control are presented. The sialoproteomic analysis based on titanium-dioxide enrichment of sialopeptides with subsequent LC-MS/MS identification was performed. This analysis identified 47 proteins with significantly increased sialylation. The data in this article is associated with the research article published in Biochim Biophys Acta "Glycomic analysis of gastric carcinoma cells discloses glycans as modulators of RON receptor tyrosine kinase activation in cancer" [1].

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

BACKGROUND: Terminal α2-3 and α2-6 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 α2-6 towards α2-3 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.

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.

Loci associated with N-glycosylation of human immunoglobulin G show pleiotropy with autoimmune diseases and haematological cancers.

Glycosylation of immunoglobulin G (IgG) influences IgG effector function by modulating binding to Fc receptors. To identify genetic loci associated with IgG glycosylation, we quantitated N-linked IgG glycans using two approaches. After isolating IgG from human plasma, we performed 77 quantitative measurements of N-glycosylation using ultra-performance liquid chromatography (UPLC) in 2,247 individuals from four European discovery populations. In parallel, we measured IgG N-glycans using MALDI-TOF mass spectrometry (MS) in a replication cohort of 1,848 Europeans. Meta-analysis of genome-wide association study (GWAS) results identified 9 genome-wide significant loci (P<2.27 × 10(-9)) in the discovery analysis and two of the same loci (B4GALT1 and MGAT3) in the replication cohort. Four loci contained genes encoding glycosyltransferases (ST6GAL1, B4GALT1, FUT8, and MGAT3), while the remaining 5 contained genes that have not been previously implicated in protein glycosylation (IKZF1, IL6ST-ANKRD55, ABCF2-SMARCD3, SUV420H1, and SMARCB1-DERL3). However, most of them have been strongly associated with autoimmune and inflammatory conditions (e.g., systemic lupus erythematosus, rheumatoid arthritis, ulcerative colitis, Crohn's disease, diabetes type 1, multiple sclerosis, Graves' disease, celiac disease, nodular sclerosis) and/or haematological cancers (acute lymphoblastic leukaemia, Hodgkin lymphoma, and multiple myeloma). Follow-up functional experiments in haplodeficient Ikzf1 knock-out mice showed the same general pattern of changes in IgG glycosylation as identified in the meta-analysis. As IKZF1 was associated with multiple IgG N-glycan traits, we explored biomarker potential of affected N-glycans in 101 cases with SLE and 183 matched controls and demonstrated substantial discriminative power in a ROC-curve analysis (area under the curve = 0.842). Our study shows that it is possible to identify new loci that control glycosylation of a single plasma protein using GWAS. The results may also provide an explanation for the reported pleiotropy and antagonistic effects of loci involved in autoimmune diseases and haematological cancer.

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.

Glycans as cancer biomarkers.

BACKGROUND: Non-invasive biomarkers, such as those from serum, are ideal for disease prognosis, staging and monitoring. In the past decade, our understanding of the importance of glycosylation changes with disease has evolved. SCOPE OF REVIEW: We describe potential biomarkers derived from serum glycoproteins for liver, pancreatic, prostate, ovarian, breast, lung and stomach cancers. Methods for glycan analysis have progressed and newly developed high-throughput platform technologies have enabled the analysis of large cohorts of samples in an efficient manner. We also describe this evolution and trends to follow in the future. MAJOR CONCLUSIONS: Many convincing examples of aberrant glycans associated with cancer have come about from glycosylation analyses. Most studies have been carried out to identify changes in serum glycan profiles or through the isolation and identification of glycoproteins that contain these irregular glycan structures. In a majority of cancers the fucosylation and sialylation expression are found to be significantly modified. Therefore, these aberrations in glycan structures can be utilized as targets to improve existing cancer biomarkers. GENERAL SIGNIFICANCE: The ability to distinguish differences in the glycosylation of proteins between cancer and control patients emphasizes glycobiology as a promising field for potential biomarker identification. Furthermore, the high-throughput and reproducible nature of the chromatography platform have highlighted extensive applications in biomarker discovery and allowed integration of glycomics with other -omics fields, such as proteomics and genomics, making systems glycobiology a reality. This article is part of a Special Issue entitled Glycoproteomics.