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.

Identification of O-glycan Structures from Chicken Intestinal Mucins Provides Insight into Campylobactor jejuni Pathogenicity.

The Gram-negative bacteria Campylobactor jejuni is the primary bacteria responsible for food poisoning in industrialized countries, and acute diarrheal illness is a leading cause of mortality among children in developing countries. C. jejuni are commensal in chickens. They are particularly abundant in the caecal crypts, and poultry products are commonly infected as a result of cross-contamination during processing. The interactions between C. jejuni and chicken intestinal tissues as well as the pathogenic molecular mechanisms of colonization in humans are unknown, but identifying these factors could provide potential targets to reduce the incidence of campylobacteriosis. Recently, purified chicken intestinal mucin was shown to attenuate adherence and invasion of C. jejuni in the human colorectal adenocarcinoma cell line HCT-8 in vitro, and this effect was attributed to mucin O-glycosylation. Mucins from different regions of the chicken intestine inhibited C. jejuni binding and internalization differentially, with large intestine>small intestine>caecum. Here, we use LC-MS to perform a detailed structural analysis of O-glycans released from mucins purified from chicken large intestine, small intestine, and caecum. The O-glycans identified were abundantly sulfated compared with the human intestines, and sulfate moieties were present throughout the chicken intestinal tract. Interestingly, alpha 1-2 linked fucose residues, which have a high binding affinity to C. jejuni, were identified in the small and large intestines. Additionally, N-glycolylneuraminic/N-acetylneuraminic acid containing structures present as Sd(a)-like epitopes were identified in large intestine samples but not small intestine or caecum. O-glycan structural characterization of chicken intestinal mucins provides insights into adherence and invasion properties of C. jejuni, and may offer prospective candidate molecules aimed at reducing the incidence of infection.

Presence of terminal N-acetylgalactosamineß1-4N-acetylglucosamine residues on O-linked oligosaccharides from gastric MUC5AC: involvement in Helicobacter pylori colonization?

Isolation of MUC5AC mucins from the gastric mucosa from two secretor individuals (one from normal mucosa from a patient with gastric cancer and one from a control) showed different abilities to bind and induce the proliferation of the Helicobacter pylori strain J99. Analysis of the released O-linked oligosaccharides by LC-MS from these individuals showed a very heterogeneous mixture of species from the cancer patient containing both neutral and sialylated structures, whereas the normal sample showed dominating neutral blood group H terminating structures as well as neutral structures containing the di-N-acetyllactosamine (lacdiNAc) unit GalNAcß1-4GlcNAcß1- on the C-6 branch of the reducing end GalNAc. The linkage configuration of these epitopes were determined using C-4-specific fragmentation for the GalNAcß1-4GlcNAcß1- glycosidic linkage, comparison of the MS(3) fragmentation with standards for linkage configuration and N-acetylhexosamine type as well as exoglycosidase treatment. It was also shown that the lacdiNAc epitope is present in both human and porcine gastric mucins, indicating that this is an epitope preserved between species. We hypothesize that the termination on gastric MUC5AC with lacdiNAc is in competition with complex glycosylation such as the Le(b) and H type 1 as well as complex sialylated structures. These are epitopes known to bind the H. pylori BabA and SabA adhesins.

Sulfate migration in oligosaccharides induced by negative ion mode ion trap collision-induced dissociation.

Migration of sulfate groups between hydroxyl groups was identified after collision-induced dissociation (CID) of sulfated oligosaccharides in an ion trap mass spectrometer in negative ion mode. Analysis of various sulfated oligosaccharides showed that this was a common phenomenon and was particularly prominent in sulfated oligosaccharides also containing sialic acid. It was also shown that the level of migration was increased when the sulfate was positioned on the flexible areas of the oligosaccharides not involved in the pyranose ring, such as the extra-cyclic C-6 carbon of hexoses or N-acetylhexosamines, or on reduced oligosaccharide. This suggested that migration is dependent on the spatial availability of the sulfate in the ion trap during collision. It is proposed that the migration is initiated when the negatively charged -SO3 (-) residue attached to the oligosaccharide precursor becomes protonated by a CID-induced proton transfer. This is supported by the CID fragmentation of precursor ions depleted of acidic protons such as doubly charged [M - 2H](2-) ions or the sodiated [M + Na - 2H](-) ions of oligosaccharides containing one sulfate and one sialic acid in the same molecule. Compared to the CID fragmentation of their monocharged [M - H](-) ions, no migration was observed in CID of proton depleted precursors. Alternative fragmentation parameters to suppress migration of sulfated oligosaccharides also showed that it was not present when sulfated oligosaccharides were fragmented by HCD (High-Energy C-trap Dissociation) in an Orbitrap mass spectrometer.

Mass spectrometric analysis of O-linked oligosaccharides from various recombinant expression systems.

Analysis of O-linked glycosylation is one of the main challenges during structural validation of recombinant glycoproteins. With methods available for N-linked glycosylation in regard to oligosaccharide analysis as well as glycopeptide mapping, there are still challenges for O-linked glycan analysis. Here, we present mass spectrometric methodology for O-linked oligosaccharides released by reductive ß-elimination. Using LC-MS and LC-MS(2) with graphitized carbon columns, oligosaccharides are analyzed without derivatization. This approach provides a high-throughput method for screening during clonal selection, as well as product structure verification, without impairing sequencing ability. The protocols are exemplified by analysis of glycoproteins from mammalian cell cultures (CHO cells) as well as insect cells and yeast. The data shows that the method can be successfully applied to both neutral and acidic O-linked oligosaccharides, where sialic acid, hexuronic acid, and sulfate are common substituents. Further characterization of O-glycans can be achieved using permethylation. Permethylation of O-linked oligosaccharides followed by direct infusion into the mass spectrometer provide information about oligosaccharide composition, and subsequent MS (n) experiments can be carried out to elucidate oligosaccharide structure including linkage information and sequence.

Structural Identification of O-Linked Oligosaccharides Using Exoglycosidases and MSn Together with UniCarb-DB Fragment Spectra Comparison.

The availability of specific exoglycosidases alongside a spectral library of O-linked oligosaccharide collision induced dissociation (CID) MS fragments, UniCarb-DB, provides a pathway to make the elucidation of O-linked oligosaccharides more efficient. Here, we advise an approach of exoglycosidase-digestion of O-linked oligosaccharide mixtures, for structures that do not provide confirmative spectra. The combination of specific exoglycosidase digestion and MS2 matching of the exoglycosidase products with structures from UniCarb-DB, allowed the assignment of unknown structures. This approach was illustrated by treating sialylated core 2 O-linked oligosaccharides, released from the human synovial glycoprotein (lubricin), with a α2-3 specific sialidase. This methodology demonstrated the exclusive 3 linked nature of the sialylation of core 2 oligosaccharides on lubricin. When specific exoglycosidases were not available, MS3 spectral matching using standards was used. This allowed the unusual 4-linked terminal GlcNAc epitope in a porcine stomach to be identified in the GlcNAc1-4Galb1-3(GlcNAcb1-6)GalNAcol structure, indicating the antibacterial epitope GlcNAca1-4. In total, 13 structures were identified using exoglycosidase and MSn, alongside UniCarb-DB fragment spectra comparison. UniCarb-DB could also be used to identify the specificity of unknown exoglycosidases in human saliva. Endogenous salivary exoglycosidase activity on mucin oligosaccharides could be monitored by comparing the generated tandem MS spectra with those present in UniCarb-DB, showing that oral exoglycosidases were dominated by sialidases with a higher activity towards 3-linked sialic acid rather than 6-linked sialic acid.