Classes Of Glycans Research Articles

(Sialyl)Lewis Antigen Expression on Glycosphingolipids, N-, and O-Glycans in Colorectal Cancer Cell Lines is Linked to a Colon-Like Differentiation Program

Alterations in the glycomic profile are a hallmark of cancer, including colorectal cancer (CRC). While, the glycosylation of glycoproteins and glycolipids has been widely studied for CRC cell lines and tissues, a comprehensive overview of CRC glycomics is still lacking due to the usage of different samples and analytical methods. In this study, we compared glycosylation features of N-, O-glycans, and glycosphingolipid glycans for a set of 22 CRC cell lines, all measured by porous graphitized carbon nano-liquid chromatography-tandem mass spectrometry. An overall, high abundance of (sialyl)Lewis antigens for colon-like cell lines was found, while undifferentiated cell lines showed high expression of H blood group antigens and α2-3/6 sialylation. Moreover, significant associations of glycosylation features were found between the three classes of glycans, such as (sialyl)Lewis and H blood group antigens. Integration of the datasets with transcriptomics data revealed positive correlations between (sialyl)Lewis antigens, the corresponding glycosyltransferase FUT3 and transcription factors CDX1, ETS, HNF1/4A, MECOM, and MYB. This indicates a possible role of these transcription factors in the upregulation of (sialyl)Lewis antigens, particularly on glycosphingolipid glycans, via FUT3/4 expression in colon-like cell lines. In conclusion, our study provides insights into the possible regulation of glycans in CRC and can serve as a guide for the development of diagnostic and therapeutic biomarkers.

Particle-attached bacteria act as gatekeepers in the decomposition of complex phytoplankton polysaccharides

BackgroundMarine microalgae (phytoplankton) mediate almost half of the worldwide photosynthetic carbon dioxide fixation and therefore play a pivotal role in global carbon cycling, most prominently during massive phytoplankton blooms. Phytoplankton biomass consists of considerable proportions of polysaccharides, substantial parts of which are rapidly remineralized by heterotrophic bacteria. We analyzed the diversity, activity, and functional potential of such polysaccharide-degrading bacteria in different size fractions during a diverse spring phytoplankton bloom at Helgoland Roads (southern North Sea) at high temporal resolution using microscopic, physicochemical, biodiversity, metagenome, and metaproteome analyses.ResultsProminent active 0.2–3 µm free-living clades comprised Aurantivirga, “Formosa”, Cd. Prosiliicoccus, NS4, NS5, Amylibacter, Planktomarina, SAR11 Ia, SAR92, and SAR86, whereas BD1-7, Stappiaceae, Nitrincolaceae, Methylophagaceae, Sulfitobacter, NS9, Polaribacter, Lentimonas, CL500-3, Algibacter, and Glaciecola dominated 3–10 µm and > 10 µm particles. Particle-attached bacteria were more diverse and exhibited more dynamic adaptive shifts over time in terms of taxonomic composition and repertoires of encoded polysaccharide-targeting enzymes. In total, 305 species-level metagenome-assembled genomes were obtained, including 152 particle-attached bacteria, 100 of which were novel for the sampling site with 76 representing new species. Compared to free-living bacteria, they featured on average larger metagenome-assembled genomes with higher proportions of polysaccharide utilization loci. The latter were predicted to target a broader spectrum of polysaccharide substrates, ranging from readily soluble, simple structured storage polysaccharides (e.g., laminarin, α-glucans) to less soluble, complex structural, or secreted polysaccharides (e.g., xylans, cellulose, pectins). In particular, the potential to target poorly soluble or complex polysaccharides was more widespread among abundant and active particle-attached bacteria.ConclusionsParticle-attached bacteria represented only 1% of all bloom-associated bacteria, yet our data suggest that many abundant active clades played a pivotal gatekeeping role in the solubilization and subsequent degradation of numerous important classes of algal glycans. The high diversity of polysaccharide niches among the most active particle-attached clades therefore is a determining factor for the proportion of algal polysaccharides that can be rapidly remineralized during generally short-lived phytoplankton bloom events.9xVFsdNaK4F9f5nWnV_ZPhVideo

SSSMuG: Same Sample Sequential Multi-Glycomics.

The mammalian glycome is structurally complex and diverse, composed of many glycan classes such as N- and O-linked glycans, glycosaminoglycans (GAGs), glycosphingolipids (GSLs), and other distinct glycan features such as polysialic acids (PolySia), sulfation, and proteoglycan attachment stubs. Various methods are used to analyze these different components of the glycome, but they require prefractionated/partitioned samples to target each glycan class individually. To address this need for a knowledge of the relationship between the different glycan components of a biological system, we developed a sequential release workflow for analysis of multiple conjugated glycan classes (PolySia, GAGs, GSL glycans, N-glycans, and O-glycans) from the same tissue lysate, termed SSSMuG─Same Sample Sequential Multi-Glycomics. With this sequential glycan release approach, five glycan classes were characterized (or four glycan classes plus proteomics) using enzymatic or chemical release from a single sample immobilized on a polyvinylidene difluoride membrane. The various released glycan classes were then analyzed by HPLC and MS techniques using commonly available analytical setups. Compared to single glycan class release approaches, SSSMuG was able to identify more glycans and more proteins with higher-intensity analytical peaks and provide a better comparative normalization of the different glycan classes of the complex glycome. To this end, the SSSMuG technology workflow will be a foundation for a paradigm shift in the field, transforming glycoanalytics and facilitating the push toward multiglycomics and systems glycobiology.

Glycan Node Analysis Detects Varying Glycosaminoglycan Levels in Melanoma-Derived Extracellular Vesicles.

Extracellular vesicles (EVs) play important roles in (patho)physiological processes by mediating cell communication. Although EVs contain glycans and glycosaminoglycans (GAGs), these biomolecules have been overlooked due to technical challenges in comprehensive glycome analysis coupled with EV isolation. Conventional mass spectrometry (MS)-based methods are restricted to the assessment of N-linked glycans. Therefore, methods to comprehensively analyze all glyco-polymer classes on EVs are urgently needed. In this study, tangential flow filtration-based EV isolation was coupled with glycan node analysis (GNA) as an innovative and robust approach to characterize most major glyco-polymer features of EVs. GNA is a molecularly bottom-up gas chromatography-MS technique that provides unique information that is unobtainable with conventional methods. The results indicate that GNA can identify EV-associated glyco-polymers that would remain undetected with conventional MS methods. Specifically, predictions based on GNA identified a GAG (hyaluronan) with varying abundance on EVs from two different melanoma cell lines. Enzyme-linked immunosorbent assays and enzymatic stripping protocols confirmed the differential abundance of EV-associated hyaluronan. These results lay the framework to explore GNA as a tool to assess major glycan classes on EVs, unveiling the EV glycocode and its biological functions.

Fucose Migration Pathways Identified Using Infrared Spectroscopy**

AbstractFucose is a ubiquitous monosaccharide associated to major classes of glycans. A main obstacle to the sequencing of fucosylated glycans is the migration of fucose, which leads to misinterpretations in mass spectrometry analysis. Here, using ion vibrational spectroscopy, we resolve the structure of fucosylated fragments of Lewis and blood group H antigen trisaccharides and we unveil the position and linkage of the fucose after migration. Our findings demonstrate that the structure of fragment ions resulting from fucose migration can be characterized. Additionally, we report a new type of fucose migration, which does not feature any change of mass and therefore had not been previously reported: it consists of a local migration where the fucose changes its position remaining on the initial residue. Our approach allows the characterization of glycans, an essential step to interpret glycomics data, as well as to understand underlying processes at play in mass spectrometry.

Fucose Migration Pathways Identified Using Infrared Spectroscopy.

Fucose is a ubiquitous monosaccharide associated to major classes of glycans. A main obstacle to the sequencing of fucosylated glycans is the migration of fucose, which leads to misinterpretations in mass spectrometry analysis. Here, using ion vibrational spectroscopy, we resolve the structure of fucosylated fragments of Lewis and blood group H antigen trisaccharides and we unveil the position and linkage of the fucose after migration. Our findings demonstrate that the structure of fragment ions resulting from fucose migration can be characterized. Additionally, we report a new type of fucose migration, which does not feature any change of mass and therefore had not been previously reported: it consists of a local migration where the fucose changes its position remaining on the initial residue. Our approach allows the characterization of glycans, an essential step to interpret glycomics data, as well as to understand underlying processes at play in mass spectrometry.

Assessing the use of host-guest chemistry in conjunction with cyclic ion mobility separations for the linkage-specific characterization of human milk oligosaccharides

Human milk oligosaccharides (HMOs) are a class of glycans that are highly abundant in human milk and contribute to the healthy growth of an infant's immune system. While new advancements in analytical methodologies have been made in glycomics, the high degree of isomeric heterogeneity and lack of authentic standards have made the high-resolution separation and accurate characterization of linkage positioning of all HMO species very challenging. Herein, we present an evaluation of the use of host-guest chemistry in conjunction with cyclic ion mobility spectrometry-mass spectrometry (cIMS-MS)-based separations for the identification of linkage positioning in three pairs of di-, tetra-, and hexasaccharide HMO isomers that only differ in the positioning of one glycosidic linkage (β1,3 versus β1,4). Suitable hosts, such as α/β cyclodextrins, cucurbit[n]urils (n = 5, 7), crown ethers, cyclic peptides, and an ionophore, were used to assess host-guest inclusion complex formation as well as linkage-specific cIMS-MS trends. Our results indicated a linkage-specific trend for the [M + 2α + 2H]2+ cyclodextrin-based host-guest inclusion complexes where the β1,3 linkage-containing isomers were always higher mobility than the β1,4 linkage-containing ones as well one for the [M + α + β + 2H]2+ complexes where the β1,4 linkage-containing isomers were always higher mobility than the β1,3 linkage-containing ones. We also observed diagnostic mobility fingerprints for the cucurbituril-based complexes. We anticipate that linkage-specific and mobility fingerprint trends can potentially aid in identifying linkage positioning for other HMO isomers as well as in complex human milk samples.

Mass Spectrometry-Based Techniques to Elucidate the Sugar Code.

Cells encode information in the sequence of biopolymers, such as nucleic acids, proteins, and glycans. Although glycans are essential to all living organisms, surprisingly little is known about the “sugar code” and the biological roles of these molecules. The reason glycobiology lags behind its counterparts dealing with nucleic acids and proteins lies in the complexity of carbohydrate structures, which renders their analysis extremely challenging. Building blocks that may differ only in the configuration of a single stereocenter, combined with the vast possibilities to connect monosaccharide units, lead to an immense variety of isomers, which poses a formidable challenge to conventional mass spectrometry. In recent years, however, a combination of innovative ion activation methods, commercialization of ion mobility–mass spectrometry, progress in gas-phase ion spectroscopy, and advances in computational chemistry have led to a revolution in mass spectrometry-based glycan analysis. The present review focuses on the above techniques that expanded the traditional glycomics toolkit and provided spectacular insight into the structure of these fascinating biomolecules. To emphasize the specific challenges associated with them, major classes of mammalian glycans are discussed in separate sections. By doing so, we aim to put the spotlight on the most important element of glycobiology: the glycans themselves.

Toward Sequencing the Human Milk Glycome: High-Resolution Cyclic Ion Mobility Separations of Core Human Milk Oligosaccharide Building Blocks.

Human milk oligosaccharides (HMOs) are an unconjugated class of glycans that have been implicated for their role in promoting the healthy development of the brain-gut axes of infants. Production of HMOs is ever-changing and specifically tailored for each infant in response to various biological factors (e.g., cognitive development, diseases, or allergies). While every HMO consists of up to only five monosaccharides, their structures can be composed of many possible glycosidic linkage positions and corresponding α/β anomericities, linear or branched chains, and potential fucosylation/sialylation modifications, thus leading to a tremendous degree of isomeric heterogeneity. With limited availability of authentic standards for every putative HMO structure (estimated to be >200 total), new analytical methods are needed for their accurate characterization. Complete sequencing of the human milk glycome would enable a better understanding of their infant-specific biological roles and potentially lead to their widespread incorporation into infant formula. Herein, we explore the use of our high-resolution cyclic ion mobility spectrometry-mass spectrometry (cIMS-MS)-based platform for the separation of core disaccharide and trisaccharide isomer building blocks as a first step toward the sequencing of larger HMOs. By utilizing the flexible capabilities of the cIMS array, separation pathlengths were extended up to 40 m, thus enabling the resolution of all seven sets of sialylated, fucosylated galactosyllactose and lactosamine HMO building block isomers. Additionally, we assessed the utility of pre-/post-cIMS tandem mass spectrometry (MS/MS) and tandem cIMS (cIMS/cIMS) for the characterization of HMOs based on their diagnostic fragmentation patterns and mobility fingerprints. We anticipate that our presented cIMS-MS-based methodology will enable the better characterization of larger, unknown HMOs when incorporated into an overall workflow that also includes online liquid chromatography and enzymatic hydrolyses.

Identification of the Glycan Binding Profile of Human and Rodent Plasmodium Sporozoites

The transmission of Plasmodium spp. sporozoites to the mammalian host is the first step in the initiation of the mosquito-borne disease known as malaria. The exact route of transmission from the bloodstream to the liver is still not clearly elucidated, and identification of the host glycan structures bound by the sporozoites may inform as to which host cells are involved. Here, we provide a comprehensive analysis of the glycan structures that sporozoites from the human pathogen, P. falciparum, and the rodent pathogen, P. yoelii, recognize and bind. Glycan array analysis was used to profile the glycans bound by the sporozoites, and the binding affinities of these sporozoite-glycan interactions were then determined by surface plasmon resonance. Data showed that the different Plasmodium spp. bind different classes of glycans. P. falciparum was observed to bind to glycans with terminal N-acetylgalactosamine (GalNAc) or Galactose (Gal) linked to a GalNAc, and the highest-affinity observed was with the GalNAc monosaccharide (12.5 nM). P. yoelii bound glycosaminoglycans, mannosyl glycans, Gal linked to N-acetylglucosamine structures, and the αGal epitope. The highest-affinity interaction for P. yoelii was with the αGal epitope (31.4 nM). This is the first study to identify the key host glycan structures recognized by human and rodent Plasmodium spp. sporozoites. An understanding of how Plasmodium sporozoites interact with the specific glycan structures identified here may provide further insight into this infectious disease that could help direct the design of an effective therapeutic.

The cell surface protein MUL_3720 confers binding of the skin pathogen Mycobacterium ulcerans to sulfated glycans and keratin.

Mycobacterium ulcerans is the causative agent of the chronic, necrotizing skin disease Buruli ulcer. Modes of transmission and molecular mechanisms involved in the establishment of M. ulcerans infections are poorly understood. Interactions with host glycans are often crucial in bacterial pathogenesis and the 22 kDa M. ulcerans protein MUL_3720 has a putative role in host cell attachment. It has a predicted N-terminal lectin domain and a C-terminal peptidoglycan-binding domain and is highly expressed on the surface of the bacilli. Here we report the glycan-binding repertoire of whole, fixed M. ulcerans bacteria and of purified, recombinant MUL_3720. On an array comprising 368 diverse biologically relevant glycan structures, M. ulcerans cells showed binding to 64 glycan structures, representing several distinct classes of glycans, including sulfated structures. MUL_3720 bound only to glycans containing sulfated galactose and GalNAc, such as glycans known to be associated with keratins isolated from human skin. Surface plasmon resonance studies demonstrated that both whole, fixed M. ulcerans cells and MUL_3720 show high affinity interactions with both glycans and human skin keratin extracts. This MUL_3720-mediated interaction with glycans associated with human skin keratin may contribute to the pathobiology of Buruli ulcer.

Mucin-Mimetic Glycan Arrays Integrating Machine Learning for Analyzing Receptor Pattern Recognition by Influenza a Viruses

Influenza A viruses (IAVs) exploit host glycans in airway epithelial mucosa to gain entry and initiate infection. Rapid detection of changes in IAV specificity towards host glycan classes can provide early indication of virus transmissibility and infection potential. IAVs use hemagglutinins (HA) to bind sialic acids linked to larger glycan structures and a switch in HA specificity from α2,3- to α2,6-linked sialoglycans is considered a prerequisite for viral transmission from birds to humans. While the changes in HA structure associated with the evolution of binding phenotype have been mapped, the influence of glycan receptor presentation on IAV specificity remains obscured. Here, we describe a glycan array platform which uses synthetic mimetics of mucin glycoproteins to model how receptor presentation in the mucinous glycocalyx, including glycan type and valency of the glycoconjugates and their surface density, impact IAV binding. We found that H1N1 virus produced in embryonated chicken eggs, which recognizes both receptor types, exclusively engaged mucin-mimetics carrying α2,3-linked sialic acids in their soluble form. The virus was able to utilize both receptor structures when the probes were immobilized on the array; however, increasing density in the mucin-mimetic brush diminished viral adhesion. Propagation in mammalian cells produced a change in receptor pattern recognition by the virus, without altering its HA affinity, toward improved binding of α2,6-sialylated mucin mimetics and reduced sensitivity to surface crowding of the probes. Application of a support-vector machine (SVM) learning algorithm as part of the glycan array binding analysis efficiently characterized this shift in binding preference and may prove useful to study the evolution of viral responses to a new host.

Glycan Node Analysis of Plasma-Derived Extracellular Vesicles.

Blood plasma is a readily accessible source of extracellular vesicles (EVs), i.e., cell-secreted nanosized carriers that contain various biomolecules, including glycans. Previous studies have demonstrated that glycans play a major role in physiological and pathological processes, and certain plasma glycans have been associated with disease conditions. However, glycome studies have been limited by a lack of analytical techniques with the throughput capacity necessary to study hundreds of clinical samples. This study is the first to characterize the EV plasma glycome based on all major glycan classes. The results based on glycan node analysis revealed, as expected, that plasma-derived EVs have distinct glycan features from donor-matched whole plasma. Specifically, glycan nodes corresponding to those observed in chondroitin sulfate, dermatan sulfate, type I keratan sulfate, and type II keratan sulfate were enriched on EVs. The identification of specific differences in glycan features in plasma vs. plasma-derived EVs is relevant for understanding the physiological role of EVs and as a reference for future diagnostic studies. Additionally, the results indicate that EV glycan nodes do not substantially differ among a small set of healthy donors. These results lay the framework for the further evaluation of all EV glycan classes as diagnostic markers, therapeutic targets, and biologically active components in health and disease.

N-glycomic profiling of colorectal cancer according to tumor stage and location.

Alterations in glycosylation are seen in many types of cancer, including colorectal cancer (CRC). Glycans, the sugar moieties of glycoconjugates, are involved in many important functions relevant to cancer and can be of value as biomarkers. In this study, we have used mass spectrometry to analyze the N-glycan profiles of 35 CRC tissue samples and 10 healthy tissue samples from non-CRC patients who underwent operations for other reasons. The tumor samples were divided into groups depending on tumor location (right or left colon) and stage (II or III), while the healthy samples were divided into right or left colon. The levels of neutral and acidic N-glycan compositions and glycan classes were analyzed in a total of ten different groups. Surprisingly, there were no significant differences in glycan levels when all right- and left-sided CRC samples were compared, and few differences (such as in the abundance of the neutral N-glycan H3N5) were seen when the samples were divided according to both location and stage. Multiple significant differences were found in the levels of glycans and glycan classes when stage II and III samples were compared, and these glycans could be of value as candidates for new markers of cancer progression. In order to validate our findings, we analyzed healthy tissue samples from the right and left colon and found no significant differences in the levels of any of the glycans analyzed, confirming that our findings when comparing CRC samples from the right and left colon are not due to normal variations in the levels of glycans between the healthy right and left colon. Additionally, the levels of the acidic glycans H4N3F1P1, H5N4F1P1, and S1H5N4F1 were found to change in a cancer-specific but colon location-nonspecific manner, indicating that CRC affects glycan levels in similar ways regardless of tumor location.

0058 Effects of Acute Total Sleep Deprivation on Human Plasma N-glycans

Abstract Introduction There is a need for a novel biomarker that can be used to measure sleep sufficiency as it pertains to fitness for duty. As glycans (polysaccharides) are known to be involved in modifying protein effectiveness, we are exploring these as biomarkers that may be sensitive to differences between sleep deprivation and normal healthy adult sleep duration. We have measured one major class of glycans, called N-glycans, which are covalently linked to asparagine residues of polypeptide chains of membrane-bound and secreted proteins. We compared the plasma N-glycan profiles of participants before and after they participated in a total sleep deprivation protocol. Methods 10 healthy participants (6 male, 4 female) aged 30–44 went through 88 hours of total sleep deprivation. Hourly blood draws were taken via forearm catheter throughout the protocol. N-glycan analysis was performed using plasma samples collected at 17:35 prior to the first night of sleep deprivation and at 17:35 following 82.5 hours of continuous wakefulness. N-glycans were first cleaved from peptides and isolated from plasma, and profiles were then measured using Matrix-Assisted Laser Desorption/Ionization-Time of Flight (MALDI-TOF) mass spectrometry. Results 66 N-glycans were observed in our profiles. Of these, the relative abundance of 17 N-glycans were significantly different following sleep deprivation (paired t-test, 13 with p<0.05, 4 with p<0.01). In each case, the relative abundance was lower in the sleep deprivation time point. We found two structures, Hex6HexNAc5NeuAc3 and Hex7HexNAc6NeuAc2, which were also significant in one of our previous chronic sleep restriction protocols. Conclusion While we observed that many N-glycans decreased in relative abundance, it is unclear whether these changes represent a shift in glycan synthesis or result from decreased expression of the proteins they are bound to. Our next steps involve exploring the functions of the proteins associated with Hex6HexNAc5NeuAc3 and Hex7HexNAc6NeuAc2, and measuring their expression levels. Support NIH/HL75501; NIH/National Center for Research Resources UL1-RR02758 and M01-RR01032 to the Harvard Clinical and Translational Science Center.

Presence of N-acetylgalactosamine/galactose residues on bronchioloalveolar cells during rat postnatal development.

In mammals, the alveolarization process develops predominantly after birth. Airway cells display a complex assemblage of glycans on their surface. These glycans, particularly terminal glycan extensions, are important effective carriers of information that change during the differentiation process. Nevertheless, few systematic data are reported about the cell surface sugar residue content during postnatal lung development. In the present work, we aimed to identify and semi-quantify N-acetylgalactosamine (GalNAc)/galactose (Gal) residues on the bronchioloalveolar cell surface in rat lung sections from 1-, 4-, 8- day old and adult animals and link these data with the lung glycocalyx composition. Horseradish peroxidase- conjugated lectin from Glycine max (soybean agglutinin, SBA) was used, and light microscopy methodologies were performed. SBA labelling intensity was studied before and after sialidase pre-treatment, in 1-, 4-, and 8-day-old animals and adult animals. For semi-quantitative evaluation of SBA binding intensity, two investigators performed the analysis independently, blinded to the type of experiment. Reactivity of the lectin was assessed in bronchiolar and respiratory portion/alveolar epithelial cell surfaces. We evidenced a stronger positive reaction when lung sections were pre-treated with neuraminidase before incubation with the lectin in 1- and 4-day-old animals and adult animals. These results were not so manifest in 8- day-old animals. This binding pattern, generally points towards the presence of terminal but mainly sub-terminal GalNAc/Gal residues probably capped by sialic acids on the rat bronchiolar/respiratory tract epithelial cells. As this glycan extension is common in Oand N-glycans, our results suggest that these glycan classes can be present in bronchioloalveolar cells immediately after birth and exist during the postnatal period. The results observed in eight-day-old rat lung sections may be due to the dramatic lung morphologic changes and the possible underlying biological mechanisms that occur during this age-moment.

Identification and analysis of cell wall glycan epitopes and polyphenol oxidase in pawpaw (Asimina triloba [L.] Dunal) fruit pulp as affected by high pressure processing and refrigerated storage.

This research explores the cell wall composition and polyphenol oxidase activity of two pawpaw (Asimina triloba) fruit varieties, Susquehanna and Green River Belle, that were subjected to high pressure processing and 45 days of refrigerated storage. We hypothesize that high pressure processing may inhibit enzymatic action responsible for pawpaw's deleterious postharvest tissue softening and browning. Glycome profiling uses mAb groupings that recognize 19 groups of glycan epitopes present in most major classes of cell wall glycans and was used to determine cell wall composition. Results show that both varieties have typical type I primary cell walls of flowering dicots. However, differences in the fine cell wall structure between the varieties can be inferred and the varieties behaved differently during refrigerated storage, likely indicating of a difference in cell wall-modifying enzymes present in the primary cell walls. High pressure processing treatment does not seem to be effective at eliminating polyphenol oxidase activity.

Complex N-glycan breakdown by gut Bacteroides involves an extensive enzymatic apparatus encoded by multiple co-regulated genetic loci.

Glycans are the major carbon sources available to the human colonic microbiota. Numerous N-glycosylated proteins are found in the human gut, from both dietary and host sources, including immunoglobulins such as IgA that are secreted into the intestine at high levels. Here, we show that many mutualistic gut Bacteroides spp. have the capacity to utilize complex N-glycans (CNGs) as nutrients, including those from immunoglobulins. Detailed mechanistic studies using transcriptomic, biochemical, structural and genetic techniques reveal the pathway employed by Bacteroides thetaiotaomicron (Bt) for CNG degradation. The breakdown process involves an extensive enzymatic apparatus encoded by multiple non-adjacent loci and comprises 19 different carbohydrate-active enzymes from different families, including a CNG-specific endo-glycosidase activity. Furthermore, CNG degradation involves the activity of carbohydrate-active enzymes that have previously been implicated in the degradation of other classes of glycan. This complex and diverse apparatus provides Bt with the capacity to access the myriad different structural variants of CNGs likely to be found in the intestinal niche.

A preliminary study of the heterogeneity in endometrial morphology and glycosylation in the uterine horns of the non-pregnant impala (Aepyceros melampus)

Placentation commences only in the right uterine horn in impala (Aepyceros melampus). To investigate possible differences in morphology or glycosylation between the two horns, right and left uterine horns from six non-pregnant, wild impala were examined morphometrically and histochemically using a panel of 23 lectins and an avidin-biotin revealing system. The presence of ovarian 3ß hydroxysteroid dehydrogenase (3ßHSD) and aromatase was also investigated using immunocytochemistry. There were few detectable differences in morphology and glycosylation between right and left uterine horns in five of the specimens, but the sixth had deep clefts and plentiful exocrine secretions in the right horn, and not the left. Heavily glycosylated clusters of supranuclear granules were present in the epithelial cells, which had many classes of O-linked glycans. The serum progestagen was not markedly different, however, from that of the other specimens. In five of the six specimens, the height of luminal epithelium was greater on the right than that on the left, and the height of the gland epithelium was also greater on the right side in four of these. The 3ßHSD and aromatase activities were present in the ovaries and were similar in impala with or without progestagen concentrations >1 ng/ml in peripheral blood. No corpus haemorrhagicum or corpus luteum could be discerned. These findings indicate there are morphological and biochemical differences between right and left uterine horns in the impala and further studies are needed on both impala and other species in which placentation commences only in one uterine horn, to establish the cyclical hormone changes which induce them.

Post-Column Make-Up Flow (PCMF) Enhances the Performance of Capillary-Flow PGC-LC-MS/MS-Based Glycomics.

Deep characterization of biologically relevant glycans remains challenging. Porous graphitized carbon-liquid chromatography tandem mass spectrometry (PGC-LC-MS/MS) enables the quantitative elucidation of glycan fine structures. However, the early PGC-LC elution of smaller glycans (tri-, tetra-, and pentasaccharides) at low organic solvent content hampers their detection. In efforts to improve the glycan profiling sensitivity and accuracy, we present a new capillary-flow PGC-LC-MS/MS-based configuration comprising a post-column make-up flow (PCMF) that supplies an ion-promoting organic solvent to separated glycans prior to their detection by MS. The analytical performance of this setup was systematically evaluated against our existing capillary-flow PGC-LC-MS/MS platform (Jensen et al., Nat. Protoc. 2012, 7, 1299). Specifically, the ion intensities and signal-to-noise ratios of various classes of nonderivatized glycans from N- and O-glycoproteins and fructooligosaccharide mixtures were compared using methanol (MeOH)-, isopropanol (IPA)-, and acetonitrile (ACN)-based PCMF at various concentrations. In particular, ACN- and IPA-based PCMF dramatically increased the signal response across all glycan types (30- to 100-fold), improved the MS/MS spectral quality, and reduced the quantitative glycoprofile variation between replicates. In particular, the detection of the early eluting glycans benefitted from the PCMF. The highest sensitivity gains were achieved with the supplements of 100% ACN and IPA (equating to 57% (v/v) net concentration at the ion source) while neither compromising the favorable PGC-LC properties including the high peak capacity and glycan isomer separation nor changing the MS detection behavior. In conclusion, PCMF-based PGC-LC-MS/MS dramatically improves the glycomics sensitivity, coverage, and quantitative accuracy not least for the difficult-to-detect early eluting and low-abundance glycans detached from N- and O-glycoproteins.