Niclas G. Karlsson

Databases and associated tools for glycomics and glycoproteomics

The access to biodatabases for glycomics and glycoproteomics has proven to be essential for current glycobiological research. This chapter presents available databases that are devoted to different aspects of glycobioinformatics. This includes oligosaccharide sequence databases, experimental databases, 3D structure databases (of both glycans and glycorelated proteins) and association of glycans with tissue, disease, and proteins. Specific search protocols are also provided using tools associated with experimental databases for converting primary glycoanalytical data to glycan structural information. In particular, researchers using glycoanalysis methods by U/HPLC (GlycoBase), MS (GlycoWorkbench, UniCarb-DB, GlycoDigest), and NMR (CASPER) will benefit from this chapter. In addition we also include information on how to utilize glycan structural information to query databases that associate glycans with proteins (UniCarbKB) and with interactions with pathogens (SugarBind).

Bisecting Galactose as a Feature of N-Glycans of Wild-type and Mutant Caenorhabditis elegans

The N-glycosylation of the model nematode Caenorhabditis elegans has proven to be highly variable and rather complex; it is an example to contradict the existing impression that “simple” organisms possess also a rather simple glycomic capacity. In previous studies in a number of laboratories, N-glycans with up to four fucose residues have been detected. However, although the linkage of three fucose residues to the N,N′-diacetylchitobiosyl core has been proven by structural and enzymatic analyses, the nature of the fourth fucose has remained uncertain. By constructing a triple mutant with deletions in the three genes responsible for core fucosylation (fut-1, fut-6 and fut-8), we have produced a nematode strain lacking products of these enzymes, but still retaining maximally one fucose residue on its N-glycans. Using mass spectrometry and HPLC in conjunction with chemical and enzymatic treatments as well as NMR, we examined a set of α-mannosidase-resistant N-glycans. Within this glycomic subpool, we can reveal that the core β-mannose can be trisubstituted and so carries not only the ubiquitous α1,3- and α1,6-mannose residues, but also a “bisecting” β-galactose, which is substoichiometrically modified with fucose or methylfucose. In addition, the α1,3-mannose can also be α-galactosylated. Our data, showing the presence of novel N-glycan modifications, will enable more targeted studies to understand the biological functions and interactions of nematode glycans.

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 GalNAcβ1-4GlcNAc-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.

Comparison of analytical methods for profiling N- and O-linked glycans from cultured cell lines : HUPO Human Disease Glycomics/Proteome Initiative multi-institutional study.

The Human Disease Glycomics/Proteome Initiative (HGPI) is an activity in the Human Proteome Organization (HUPO) supported by leading researchers from international institutes and aims at development of disease-related glycomics/glycoproteomics analysis techniques. Since 2004, the initiative has conducted three pilot studies. The first two were N- and O-glycan analyses of purified transferrin and immunoglobulin-G and assessed the most appropriate analytical approach employed at the time. This paper describes the third study, which was conducted to compare different approaches for quantitation of N- and O-linked glycans attached to proteins in crude biological samples. The preliminary analysis on cell pellets resulted in wildly varied glycan profiles, which was probably the consequence of variations in the pre-processing sample preparation methodologies. However, the reproducibility of the data was not improved dramatically in the subsequent analysis on cell lysate fractions prepared in a specified method by one lab. The study demonstrated the difficulty of carrying out a complete analysis of the glycome in crude samples by any single technology and the importance of rigorous optimization of the course of analysis from preprocessing to data interpretation. It suggests that another collaborative study employing the latest technologies in this rapidly evolving field will help to realize the requirements of carrying out the large-scale analysis of glycoproteins in complex cell samples.

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

Abstract 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.

Aeromonas salmonicida Binds Differentially to Mucins Isolated from Skin and Intestinal Regions of Atlantic Salmon in an N-Acetylneuraminic Acid-Dependent Manner

ABSTRACT Aeromonas salmonicida subsp. salmonicida infection, also known as furunculosis disease, is associated with high morbidity and mortality in salmonid aquaculture. The first line of defense the pathogen encounters is the mucus layer, which is predominantly comprised of secreted mucins. Here we isolated and characterized mucins from the skin and intestinal tract of healthy Atlantic salmon and studied how A. salmonicida bound to them. The mucins from the skin, pyloric ceca, and proximal and distal intestine mainly consisted of mucins soluble in chaotropic agents. The mucin density and mucin glycan chain length from the skin were lower than were seen with mucin from the intestinal tract. A. salmonicida bound to the mucins isolated from the intestinal tract to a greater extent than to the skin mucins. The mucins from the intestinal regions had higher levels of sialylation than the skin mucins. Desialylating intestinal mucins decreased A. salmonicida binding, whereas desialylation of skin mucins resulted in complete loss of binding. In line with this, A. salmonicida also bound better to mammalian mucins with high levels of sialylation, and N-acetylneuraminic acid appeared to be the sialic acid whose presence was imperative for binding. Thus, sialylated structures are important for A. salmonicida binding, suggesting a pivotal role for sialylation in mucosal defense. The marked differences in sialylation as well as A. salmonicida binding between the skin and intestinal tract suggest interorgan differences in the host-pathogen interaction and in the mucin defense against A. salmonicida.

GlyTouCan: an accessible glycan structure repository

Rapid and continued growth in the generation of glycomic data has revealed the need for enhanced development of basic infrastructure for presenting and interpreting these datasets in a manner that engages the broader biomedical research community. Early in their growth, the genomic and proteomic fields implemented mechanisms for assigning unique gene and protein identifiers that were essential for organizing data presentation and for enhancing bioinformatic approaches to extracting knowledge. Similar unique identifiers are currently absent from glycomic data. In order to facilitate continued growth and expanded accessibility of glycomic data, the authors strongly encourage the glycomics community to coordinate the submission of their glycan structures to the GlyTouCan Repository and to make use of GlyTouCan identifiers in their communications and publications. The authors also deeply encourage journals to recommend a submission workflow in which submitted publications utilize GlyTouCan identifiers as a standard reference for explicitly describing glycan structures cited in manuscripts.

Mucin-type fusion proteins with blood group A or B determinants on defined O-glycan core chains produced in glycoengineered Chinese hamster ovary cells and their use as immunoaffinity matrices

Assays for quantification, and methods for removal, of anti-A and anti-B antibodies are the key for the success of ABO incompatible organ transplantation programs. In order to produce tools that can be used as substrates in tests for anti-A/anti-B quantification and specificity determination or as affinity matrices in extracorporeal immunoadsorption (IA) columns, we engineered Chinese hamster ovary (CHO) cells secreting mucin-type fusion proteins carrying blood group A or B determinants on defined O-glycan core saccharide chains. Besides the P-selectin glycoprotein ligand-1/mouse immunoglobulin G(2b) (PSGL-1/mIgG(2b)) cDNA, CHO cells were transfected with plasmids encoding core 2 (β1,6GlcNAc-T1) or core 3 (β1,3GlcNAc-T6 and β1,3Gal-T5) enzymes together with α1,2Fuc-T1 or α1,2Fuc-T2 and the A or B gene-encoded α1,3GalNAcT or α1,3Gal-T, respectively. Selected clones with the correct glycophenotype were expanded and cultured in shaker flasks and Wave bioreactors. Western blotting was used to characterize purified fusion protein and liquid chromatography-mass spectrometry was used to characterize the released O-glycans. Clones producing PSGL-1/mIgG(2b) carrying O-glycans with A and B determinants on type 1 (Galβ3GlcNAc), type 2 (Galβ4GlcNAc) and type 3 (Galβ3GalNAcα) outer core saccharide chains were established. The conversion of CHO cells from exclusive inner core 1 (Galβ3GalNAc) to core 3 (GlcNAcβ3GalNAc) O-glycan producers was almost complete, whereas conversion to inner core 2 (GlcNAcβ6GalNAc) O-glycans was incomplete as was the α2-fucosylation of the core 1 chain. Sialylation may prevent these biosynthetic steps. The clinical utility of the blood group A and B substituted mucin-type fusion proteins as substrates in enzyme-linked immunosorbent assay or as affinity matrices in IA columns is explored.

Negative Ion CID Fragmentation of O-linked Oligosaccharide Aldoses—Charge Induced and Charge Remote Fragmentation

Collision induced dissociation (CID) fragmentation was compared between reducing and reduced sulfated, sialylated, and neutral O-linked oligosaccharides. It was found that fragmentation of the [M – H]– ions of aldoses with acidic residues gave unique Z-fragmentation of the reducing end GalNAc containing the acidic C-6 branch, where the entire C-3 branch was lost. This fragmentation pathway, which is not seen in the alditols, showed that the process involved charge remote fragmentation catalyzed by a reducing end acidic anomeric proton. With structures containing sialic acid on both the C-3 and C-6 branch, the [M – H]– ions were dominated by the loss of sialic acid. This fragmentation pathway was also pronounced in the [M – 2H]2– ions revealing both the C-6 Z-fragment plus its complementary C-3 C-fragment in addition to glycosidic and cross ring fragmentation. This generation of the Z/C-fragment pairs from GalNAc showed that the charges were not participating in their generation. Fragmentation of neutral aldoses showed pronounced Z-fragmentation believed to be generated by proton migration from the C-6 branch to the negatively charged GalNAc residue followed by charge remote fragmentation similar to the acidic oligosaccharides. In addition, A-type fragments generated by charge induced fragmentation of neutral oligosaccharides were observed when the charge migrated from C-1 of the GalNAc to the GlcNAc residue followed by rearrangement to accommodate the 0,2A-fragmentation. LC-MS also showed that O-linked aldoses existed as interchangeable α/β pyranose anomers, in addition to a third isomer (25% of the total free aldose) believed to be the furanose form.

Atlantic Salmon Carries a Range of Novel O-Glycan Structures Differentially Localized on Skin and Intestinal Mucins

Aquaculture is a growing industry, increasing the need for understanding host-pathogen interactions in fish. The skin and mucosal surfaces, covered by a mucus layer composed of mucins, is the first point of contact between fish and pathogens. Highly O-glycosylated mucins have been shown to be an important part of the defense against pathogens, and pathogens bind to host surfaces using lectin-like adhesins. However, knowledge of piscine O-glycosylation is very limited. We characterized mucin O-glycosylation of five freshwater acclimated Atlantic salmon, using mass spectrometry. Of the 109 O-glycans found, most were sialylated and differed in distribution among skin, pyloric ceca, and proximal and distal intestine. Skin O-glycans were shorter (2-6 residues) and less diverse (33 structures) than intestinal O-glycans (2-13 residues, 93 structures). Skin mucins carried O-glycan cores 1, 2, 3, and 5 and three types of sialic acids (Neu5Ac, Neu5Gc, and Kdn) and had sialyl-Tn as the predominant structure. Intestinal mucins carried only cores 1, 2, and 5, Neu5Ac was the only sialic acid present, and sialylated core 5 was the most dominant structure. This structural characterization can be used for identifying structures of putative importance in host-pathogen interactions for further testing in biological assays and disease intervention therapies.