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Chronic obstructive pulmonary disease (COPD) kills millions of people annually and patients suffering from exacerbations of this disorder display high morbidity and mortality. The clinical course of COPD is associated with dysbiosis and infections, but the underlying mechanisms are poorly understood. Glycosylation of proteins play roles in regulating interactions between microbes and immune cells, and knowledge on airway glycans therefore contribute to the understanding of infections. Furthermore, glycans have biomarker potential for identifying smokers with enhanced risk for developing COPD as well as COPD subgroups. Here, we characterized the N-glycosylation in the lower airways of healthy never-smokers (HNS, n = 5) and long-term smokers (LTS) with (LTS+, n = 4) and without COPD (LTS-, n = 8). Using mass spectrometry, we identified 57 highly confident N-glycan structures whereof 38 oligomannose, complex, and paucimannose type glycans were common to BAL samples from HNS, LTS- and LTS+ groups. Hybrid type N-glycans were identified only in the LTS+ group. Qualitatively and quantitatively, HNS had lower inter-individual variation between samples compared to LTS- or LTS+. Cluster analysis of BAL N-glycosylation distinguished LTS from HNS. Correlation analysis with clinical parameters revealed that complex N-glycans were associated with health and absence of smoking whereas oligomannose N-glycans were associated with smoking and disease. The N-glycan profile from monocyte-derived macrophages differed from the BAL N-glycan profiles. In conclusion, long-term smokers display substantial alterations of N-glycosylation in the bronchoalveolar space, and the hybrid N-glycans identified only in long-term smokers with COPD deserve to be further studied as potential biomarkers., (© The Author(s) 2023. Published by Oxford University Press. All rights reserved. For permissions, please e-mail: journals.permissions@oup.com.)

MUC5AC has been indicated to be a marker for mucinous ovarian cancer (OC). We investigated the use of in situ proximity ligation assay (PLA) for blood group ABH expressing MUC5AC to differentiate between serous and mucinous OC, to validate preceding observations that also MUC5AC ABH expression is increased in mucinous OC. We developed PLA for anti-A, B, and H/anti-MUC5AC and a PLA using a combined lectin Ulex europaeus agglutinin I (UEA I)/anti-MUC5AC assay. The PLAs were verified with mass spectrometry, where mucinous OC secretor positive patients' cysts fluids containing ABH O-linked oligosaccharides also showed positive OC tissue PLA staining. A nonsecretor mucinous OC cyst fluid was negative for ABH and displayed negative PLA staining of the matched tissue. Using the UEA I/MUC5AC PLA, we screened a tissue micro array of 410 ovarian tissue samples from patients with various stages of mucinous or serous OC, 32 samples with metastasis to the ovaries and 34 controls. The PLA allowed differentiating mucinous tumors with a sensitivity of 84% and a specificity of 97% both against serous cancer but also compared to tissues from controls. This sensitivity is close to the expected incidence of secretor individuals in a population. The recorded sensitivity was also found to be higher compared to mucinous type cancer with metastasis to the ovaries, where only 32% were positive. We conclude that UEA 1/MUC5AC PLA allows glycospecific differentiation between serous and mucinous OC in patients with positive secretor status and will not identify secretor negative individuals with mucinous OC., (© The Author(s) 2021. Published by Oxford University Press.)

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., (© The Author 2017. Published by Oxford University Press. All rights reserved. For permissions, please e-mail: journals.permissions@oup.com.)

The minimum information required for a glycomics experiment (MIRAGE) project was established in 2011 to provide guidelines to aid in data reporting from all types of experiments in glycomics research including mass spectrometry (MS), liquid chromatography, glycan arrays, data handling and sample preparation. MIRAGE is a concerted effort of the wider glycomics community that considers the adaptation of reporting guidelines as an important step towards critical evaluation and dissemination of datasets as well as broadening of experimental techniques worldwide. The MIRAGE Commission published reporting guidelines for MS data and here we outline guidelines for sample preparation. The sample preparation guidelines include all aspects of sample generation, purification and modification from biological and/or synthetic carbohydrate material. The application of MIRAGE sample preparation guidelines will lead to improved recording of experimental protocols and reporting of understandable and reproducible glycomics datasets., (© The Author 2016. Published by Oxford University Press. All rights reserved. For permissions, please e-mail: journals.permissions@oup.com.)

The binding of Shiga-like toxin 1 (Stx1) and Shiga-like toxin 2 (Stx2) to a mucin-like fusion protein, P-selectin glycoprotein ligand-1/mouse IgG2b (PSGL-1/mIgG2b), carrying multiple copies of the blood group P1 determinant on O-glycans was investigated with western blot and the biosensor Biacore. Chinese hamster ovary K-1 (CHO-K1) cells were stably transfected with linearized plasmids encoding the PSGL-1/mIgG2b fusion protein, the pigeon α1,4-galactosyltransferase (α4Gal-T) and the core 2 β1,6-N-acetylglucosaminyltransferase (C2GnT-I). Western blot analyses of purified PSGL-1/mIgG2b and liquid chromatography-mass spectrometry (LC-MS) of released O-glycans confirmed the presence of the P1 determinant. Western blot analysis indicated strong binding of Stx1, but not Stx2, to PSGL-1/mIgG2b. In a Biacore assay, Stx1 and Stx2 were immobilized on a dextran chip and the binding of purified PSGL-1/mIgG2b and a P(k)-albumin neoglycoprotein was analyzed. Stx1 and Stx2 bound with high avidity to both PSGL-1/mIgG2b and P(k)-albumin, while the Stx1 binding was the strongest. In summary, we have shown that the pigeon α4Gal-T can be aberrantly expressed in CHO cells together with the core 2 enzyme to generate multiple, O-linked P1 determinants on a simultaneously expressed mucin-type fusion protein. P1-decorated PSGL-1/mIgG2b bound with high avidity to both Stx1 and Stx2, and as such constitutes a potential therapeutic inhibitor of these toxins.

The O-glycans of a recombinant mucin-type protein expressed in insect cell lines derived from Trichoplusia ni (Hi-5) and Spodoptera frugiperda (Sf9) were characterized. The P-selectin glycoprotein ligand-1/mouse IgG2b (PSGL-1/mIgG2b) fusion protein carrying 106 potential O-glycosylation sites and 6 potential N-glycosylation sites was expressed and purified from the Hi-5 and Sf9 cell culture medium using affinity chromatography and gel filtration. Liquid chromatography mass spectrometry (LC-MS) of O-glycans released from PSGL-1/mIgG2b revealed a large repertoire of structurally diverse glycans, which is in contrast to previous reports of only simple glycans. O-Glycans containing hexuronic acid (HexA, here glucuronic acid and galacturonic acid) were found to be prevalent. Also sulfate (Hi-5 and Sf9) and phosphocholine (PC; Sf9) O-glycan substitutions were detected. Western blotting confirmed the presence of O-linked PC on PSGL-1/mIG2b produced in Sf9 cells. To our knowledge, this is the first structural characterization of PC-substituted O-glycans in any species. The MS analyses revealed that Sf9 oligosaccharides consisted of short oligosaccharides (<6 residues) low in hexose (Hex) and with terminating N-acetylhexosamine (HexNAc) units, whereas Hi-5 produced a family of large O-glycans with (HexNAc-HexA-Hex) repeats and sulfate substitution on terminal residues. In both cell lines, the core N-acetylgalactosamine was preferentially non-branched, but small amounts of O-glycan cores with single fucose or hexose branches were found.

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.

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 GalNAcβ1-4GlcNAcβ1- 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 GalNAcβ1-4GlcNAcβ1- 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.

The O-linked glycosylation of the main acidic high-molecular-weight glycoprotein from ascites fluid from patients with ovarian cancer were analyzed. The O-linked oligosaccharides were shown to consist of mainly highly sialylated core 1 and 2 structures with a smaller amount of sulfated core 2 structures. These structures were shown to be able to be further extended into small keratan sulfate (KS)-type oligosaccharides with up to four N-acetyllactosamine units. Proteomic studies of the acidic fraction of ascites fluid from patients with ovarian cancer showed that this fraction was enriched in proteoglycans. Among them, lumican, agrin, versican and dystroglycans were potential candidates, with threonine- and serine-rich domains that could carry a significant amount of O-linked glycosylation, including also the O-linked KS. Glycomic analysis using liquid chromatography (LC)-tandem mass spectrometry (MS/MS) also showed that the disialic acid NeuAc-NeuAc- was frequently found as the terminating structure on the O-linked core 1 and 2 oligosaccharides from one ascites sample. Also, a small amount of the epidermal growth factor (EGF)-associated O-linked fucose structure Gal-GlcNAc-Fucitol was detected with and without sialic acid in the LC-MS/MS analysis. Candidate proteins containing O-linked fucose were suggested to be proteoglycan-type molecules containing the O-linked fucose EGF consensus domain.

Salivary agglutinin plays a vital biological role modulating the protective effect in the oral cavity by interacting with a broad range of oral pathogens. Here, we describe the first characterization of the O-linked oligosaccharides of salivary agglutinin identified by negative ion liquid chromatography-mass spectrometry. The dominating structures were neutral or monosialylated core 1 (Galbeta1-3GalNAcalpha1-Ser/Thr) and core 2 (Galbeta1-3(GlcNAcbeta1-6)GalNAcalpha1-Ser/Thr) structures extended by fucosylated oligo-N-acetyllactosamine units. Oligosaccharides detected as [M-H](-) or [M-2H](2)(-) ions ranged from the disaccharide Galbeta1-3GalNAcol up to structures of almost 4000 Da, corresponding to core 1/2 structures with five N-acetyllactosamine units and 11 fucoses. Fucose was found either as terminal or internal blood group H structures in type 1 (Galbeta1-3GlcNAcbeta1-R), type 2 (Galbeta1-4GlcNAcbeta1-R) and type 3 (Galbeta1-3GalNAcalpha1-Ser/Thr) units, where the chains also could be fucosylated on GlcNAc yielding repeated Lewis a/b or Lewis x/y structures. Sialylation was located either at the non-reducing end of the N-acetyllactosamine chains as sialyl-Lewis x or as sialyl-T (NeuAcalpha2-3Galbeta1-3GalNAcalpha1-Ser/Thr) type structures with or without further extension of the C-6 branch of GalNAc with neutral fucosylated N-acetyllactosamine chains. The data indicated that sialylation, fucosylation and type 1 N-acetyllactosamine termination are important regulatory elements for controlling the oligosaccharide chain length. Furthermore, it was shown that these regulatory oligosaccharide elements could be utilized by the pathogen Helicobacter pylori to colonize the oral cavity, reside in dental plaque and serve as a reservoir for reinfection after successful clearance of H. pylori gastric infection.

Isolation of salivary MUC7 with gel electrophoresis allowed analysis by LC-MS and LC-MS(2) of released O-linked oligosaccharides and a thorough description of the glycosylation of this molecule, where high-molecular-weight oligosaccharides up to the size of 2790 Da and with up to three sialic acid residues were identified. A common theme of these novel high abundant oligosaccharides on MUC7 showed that the C-3 branch of the oligosaccharides consisted of branched I-antigen type structural epitopes (GlcNAc beta 1-3(GlcNAc beta 1-6)Gal beta 1-), where the branch point was initiated on core 1 and core 2 galactose residues, and the branches were terminated by sialyl type 2 and sialyl Lewis x epitopes. Six sulfated sialylated oligosaccharides of low intensity were also identified, with the sulfate mainly on N-acetyl glucosamine residues located close to the reducing termini. One of these oligosaccharides was identified as a candidate for the high-affinity L-selectin ligand 6'-sulfo sialyl Lewis x. Neutral oligosaccharides and blood group antigens were found to be less abundant on MUC7 and the glycosylation appeared to be more preserved between individuals as compared to salivary MUC5B. This was illustrated by comparing the LC-MS spectra of MUC7 and MUC5B glycans from secretors (23 individuals) and nonsecretors (6 individuals). The data show that MUC7 provides a multivalent scaffold for sialylation, meeting the requirement for high-avidity binding via its glycosylation and mediator of the interaction between immune cells such as salivary neutrophils and oral bacteria.

Cystic fibrosis (CF) is characterized by chronic lung infection and inflammation, with periods of acute exacerbation causing severe and irreversible lung tissue damage. We used protein and glycosylation analysis of high-molecular mass proteins in saline-induced sputum from CF adults with and without an acute exacerbation, CF children with stable disease and preserved lung function, and healthy non-CF adult and child controls to identify potential biomarkers of lung condition. While the main high-molecular mass proteins in the sputum from all subjects were the mucins MUC5B and MUC5AC, these appeared degraded in CF adults with an exacerbation. The glycosylation of these mucins also showed reduced sulfation, increased sialylation, and reduced fucosylation in CF adults compared with controls. Despite improvements in pulmonary function after hospitalization, these differences remained. Two CF children showed glycoprotein profiles similar to those of CF adults with exacerbations and also presented with pulmonary flares shortly after sampling, while the remaining CF children had profiles indistinguishable from those of healthy non-CF controls. Sputum mucin glycosylation and degradation are therefore not inherently different in CF, and may also be useful predictive biomarkers of lung condition.

Mass spectrometry (MS) of glycoproteins is an emerging field in proteomics, poised to meet the technical demand for elucidation of the structural complexity and functions of the oligosaccharide components of molecules. Considering the divergence of the mass spectrometric methods employed for oligosaccharide analysis in recent publications, it is necessary to establish technical standards and demonstrate capabilities. In the present study of the Human Proteome Organisation (HUPO) Human Disease Glycomics/Proteome Initiative (HGPI), the same samples of transferrin and immunoglobulin-G were analyzed for N-linked oligosaccharides and their relative abundances in 20 laboratories, and the chromatographic and mass spectrometric analysis results were evaluated. In general, matrix-assisted laser desorption/ionization (MALDI) time-of-flight MS of permethylated oligosaccharide mixtures carried out in six laboratories yielded good quantitation, and the results can be correlated to those of chromatography of reductive amination derivatives. For underivatized oligosaccharide alditols, graphitized carbon-liquid chromatography (LC)/electrospray ionization (ESI) MS detecting deprotonated molecules in the negative ion mode provided acceptable quantitation. The variance of the results among these three methods was small. Detailed analyses of tryptic glycopeptides employing either nano LC/ESI MS/MS or MALDI MS demonstrated excellent capability to determine site-specific or subclass-specific glycan profiles in these samples. Taking into account the variety of MS technologies and options for distinct protocols used in this study, the results of this multi-institutional study indicate that MS-based analysis appears as the efficient method for identification and quantitation of oligosaccharides in glycomic studies and endorse the power of MS for glycopeptide characterization with high sensitivity in proteomic programs.

This study aimed to characterize human salivary glycoforms and the natural glycosylation variation of the major ABO blood group bearing high molecular weight glycoprotein fraction MG1, which mainly consists of MUC5B mucin. Reduced and alkylated mucins from individuals of blood group A, B, and O were purified by sodium dodecyl sulfate-agarose/polyacrylamide composite gel electrophoresis (SDS-AgPAGE), blotted to polyvinylidene fluoride (PVDF) membranes, and visualized with alcian blue. O-linked oligosaccharides were released from MUC5B glycoform bands by reductive beta-elimination and analyzed by liquid chromatography (LC) electrospray ion trap mass spectrometry (MS). Slow electrophoretically migrating MUC5B components (sm) were found to be dominated by neutral oligosaccharides, and fast-migrating (fm) components were dominated by sulfated oligosaccharides. ABO blood group-specific sequences were found on all glycoforms, and novel oligosaccharides containing blood group A and B type sequences were sequenced. This is the first molecular description of the influence of the blood group ABO system on salivary MUC5B oligosaccharides. Expanding these results from the three A, B, and O individuals into larger population (29 individuals), we found oligosaccharide sequences corresponding to the blood group of the donor on MUC5B from 23 individuals. The remaining six individuals were characterized by a high degree of sialylation. These individuals were assigned as nonsecretors, whereas blood group-expressing individuals were assigned as secretors. Western blot assays with antibodies confirmed increased expression of Sialyl Lewis a (Si-Le(a)) in the nonsecretors. Our results highlight that salivary MUC5B consists of glycoforms with distinct glycosylation that vary extensively between individuals and that some of this variation is owing to blood group and secretor status.

Addition of the weak base ammonium chloride (NH4Cl) or the proton pump inhibitor bafilomycin A1 to cultured HeLa and LS 174T cells effectively neutralized the pH gradient of the secretory pathway. This resulted in relocalization of the three studied glycosyltransferases, N-acetylgalactosaminyltransferase 2, beta1,2 N-acetylglucosaminyltransferase I, and beta1,4 galactosyltransferase 1, normally localized to the Golgi stack, the medial/trans-Golgi and the trans-Golgi/TGN, respectively. Indirect immunofluorescence microscopy, immunoelectron microscopy, and subcellular fractionation of the tagged or native glycosyltransferases showed that NH4Cl caused a relocalization of the enzymes mainly to vesicles of endosomal type, whereas bafilomycin A1 gave mainly cell surface staining. The general morphology of the endoplasmic reticulum and Golgi apparatus was retained as judged from immunofluorescence and electron microscopy studies. When the O-glycans on the guanidinium chloride insoluble gel-forming mucins from the LS 174T cells were analyzed by gas chromatography-mass spectrometry after neutralization of the secretory pathway pH by NH4Cl over 10 days shorter O-glycans were observed. However, no decrease in the number of oligosaccharide chains was indicated. Together, the results suggest that pH is a contributing factor for proper steady-state distribution of glycosyltransferases over the Golgi apparatus and that altered pH may cause alterations in glycosylation possibly due to a relocalization of glycosyltransferases.