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Serum N-glycan profiling studies during the past decades have shown robust associations between N-glycan changes and various biological conditions, including infections, in humans. Similar studies are scarcer for other mammals, despite the tremendous potential of serum N-glycans as biomarkers for infectious diseases in animal models of human disease and in the veterinary context. To expand the knowledge of serum N-glycan profiles in important mammalian model systems, in this study, we combined MALDI-TOF-MS analysis and HILIC-UPLC profiling of released N-glycans together with glycosidase treatments to characterize the glycan structures present in rhesus macaque serum. We used this baseline to monitor changes in serum N-glycans during infection with Brugia malayi, a parasitic nematode of humans responsible for lymphatic filariasis, in a longitudinal cohort of infected rhesus macaques. Alterations of the HILIC-UPLC profile, notably of abundant structures, became evident as early as 5 weeks post-infection. Given its prominent role in the immune response, contribution of immunoglobulin G to serum N-glycans was investigated. Finally, comparison with similar N-glycan profiling performed during infection with the dog heartworm Dirofilaria immitis suggests that many changes observed in rhesus macaque serum N-glycans are specific for lymphatic filariasis.

Diagnosis of soil-transmitted helminth (STH) and schistosome infections relies largely on conventional microscopy which has limited sensitivity, requires highly trained personnel and is error-prone. Rapid advances in miniaturization of optical systems, sensors and processors have enhanced research and development of digital and automated microscopes suitable for the detection of these diseases in resource-limited settings. While some studies have reported proof-of-principle results, others have evaluated the performance of working prototypes in field settings. The extensive commercialization of these innovative devices has, however, not yet been achieved. This review provides an overview of recent publications (2010-2022) on innovative field applicable optical devices which can be used for the diagnosis of STH and schistosome infections. Using an adapted technology readiness level (TRL) scale taking into account the WHO target product profile (TPP) for these diseases, the developmental stages of the devices were ranked to determine the readiness for practical applications in field settings. From the reviewed 18 articles, 19 innovative optical devices were identified and ranked. Almost all of the devices (85%) were ranked with a TRL score below 8 indicating that, most of the devices are not ready for commercialization and field use. The potential limitations of these innovative devices were discussed. We believe that the outcome of this review can guide the end-to-end development of automated digital microscopes aligned with the WHO TPP for the diagnosis of STH and schistosome infections in resource-limited settings.

Core beta-1,2-xylose and alpha-1,3-fucose are antigenic motifs on schistosome N-glycans, as well as prominent IgE targets on some plant and insect glycoproteins. To map the association of schistosome infection with responses to these motifs, we assessed plasma IgE and IgG reactivity using microarray technology among Ugandans from rural Schistosoma mansoni (Sm)-endemic islands (n = 209), and from proximate urban communities with lower Sm exposure (n = 62). IgE and IgG responses to core beta-1,2-xylose and alpha-1,3-fucose modified N-glycans were higher in rural versus urban participants. Among rural participants, IgE and IgG to core beta-1,2-xylose were positively associated with Sm infection and concentration peaks coincided with the infection intensity peak in early adolescence. Responses to core alpha-1,3-fucose were elevated regardless of Sm infection status and peaked before the infection peak. Among urban participants, Sm infection intensity was predominantly light and positively associated with responses to both motifs. Principal component and hierarchical cluster analysis reduced the data to a set of variables that captured core beta-1,2-xylose- and alpha-1,3-fucose-specific responses, and confirmed associations with Sm and the rural environment. Responses to core beta-1,2-xylose and alpha-1,3-fucose have distinctive relationships with Sm infection and intensity that should further be explored for associations with protective immunity, and cross-reactivity with other exposures.

Contains fulltext : 171068.pdf (Publisher’s version ) (Open Access) Glycan molecules from helminth parasites have been associated with diverse biological functions ranging from interactions with neighbouring host cell populations to down-modulation of specific host immunity. Glycoproteins secreted by the intestinal nematode Heligmosomoides polygyrus are of particular interest as the excretory-secretory products (termed HES) of this parasite contain both heat-labile and heat-stable components with immunomodulatory effects. We used MALDI-TOF-MS and LC-MS/MS to analyse the repertoire of N- and O-linked glycans released from Heligmosomoides polygyrus excretory-secretory products by PNGase A and F, beta-elimination and hydrazinolysis revealing a broad range of structures including novel methylhexose- and methylfucose-containing glycans. Monoclonal antibodies to two immunodominant glycans of H. polygyrus, previously designated Glycans A and B, were found to react by glycan array analysis to a methyl-hexose-rich fraction and to a sulphated LacDiNAc (LDN; GalNAcbeta1-4GlcNAc) structure, respectively. We also analysed the glycan repertoire of a major glycoprotein in Heligmosomoides polygyrus excretory-secretory products, VAL-2, which contains many glycan structures present in Heligmosomoides polygyrus excretory-secretory products including Glycan A. However, it was found that this set of glycans is not responsible for the heat-stable immunomodulatory properties of Heligmosomoides polygyrus excretory-secretory products, as revealed by the inability of VAL-2 to inhibit allergic lung inflammation. Taken together, these studies reveal that H. polygyrus secretes a diverse range of antigenic glycoconjugates, and provides a framework to explore the biological and immunomodulatory roles they may play within the mammalian host.

Recently, the prevalence of Fasciola hepatica in some areas has increased considerably and the availability of a vaccine to protect livestock from infection would represent a major advance in tools available for controlling this disease. To date, most vaccine-target discovery research on this parasite has concentrated on proteomic and transcriptomic approaches whereas little work has been carried out on glycosylation. As the F. hepatica tegument (Teg) may contain glycans potentially relevant to vaccine development and the Newly Excysted Juvenile (NEJ) is the first lifecycle stage in contact with the definitive host, our work has focused on assessing the glycosylation of the NEJTeg and identifying the NEJTeg glycoprotein repertoire. After in vitro excystation, NEJ were fixed and NEJTeg was extracted. Matrix-assisted laser desorption ionisation-time of flight-mass spectrometry (MALDI-TOF-MS) analysis of released N-glycans revealed that oligomannose and core-fucosylated truncated N-glycans were the most dominant glycan types. By lectin binding studies these glycans were identified mainly on the NEJ surface, together with the oral and ventral suckers. NEJTeg glycoproteins were affinity purified after targeted biotinylation of the glycans and identified using liquid chromatography and tandem mass spectrometry (LC-MS/MS). From the total set of proteins previously identified in NEJTeg, eighteen were also detected in the glycosylated fraction, including the F. hepatica Cathepsin B3 (FhCB3) and two of the Cathepsin L3 (FhCL3) proteins, among others. To confirm glycosylation of cathepsins, analysis at the glycopeptide level by LC-ESI-ion-trap-MS/MS with collision-induced dissociation (CID) and electron-transfer dissociation (ETD) was carried out. We established that cathepsin B1 (FhCB1) on position N80, and FhCL3 (BN1106_s10139B000014, scaffold10139) on position N153, carry unusual paucimannosidic Man2GlcNAc2 glycans. To our knowledge, this is the first description of F. hepatica NEJ glycosylation and the first report of N-glycosylation of F. hepatica cathepsins. The significance of these findings for immunological studies and vaccine development is discussed., Author Summary Fasciola hepatica is a parasite responsible for the zoonotic disease fasciolosis, prevalence of which has increased in recent years because of the emergence of triclabendazole-resistant strains as well as changing climatic conditions. A number of F. hepatica protein antigens are used for assessing the immune response of the definitive host for the development of recombinant vaccines but no such vaccine has been commercialised yet. Glycans that may contribute to the antigenic, immunological and protective properties in F. hepatica have not been characterised. Using a panel of plant lectins with defined sugar binding specificities alongside mass spectrometric analysis, we found that high mannose and oligomannose N-glycans are the most abundant in the surface of the juvenile fluke and identified eighteen proteins likely to be glycosylated. Additionally, we found that the proteases cathepsin B1 and L3 contain a glycosylation site occupied by an unusually short Man2GlcNAc2 N-glycan. This work is the starting point for understanding how F. hepatica glycans interact with the definitive host at the initiation of infection. Additionally, it provides useful information for including glycans in the design of new vaccine candidates.

Glycans present on glycoproteins from the eggs of the parasite Schistosoma mansoni are mediators of various immune responses of the human host, including T-cell modulation and granuloma formation, and they are the target of glycan-specific antibodies. Here we have analyzed the glycosylation of kappa-5, a major glycoprotein antigen from S. mansoni eggs using a targeted approach of lectin purification followed by mass spectrometry of glycopeptides as well as released glycans. We demonstrate that kappa-5 has four fully occupied N-glycosylation sites carrying unique triantennary glycans composed of a difucosylated and xylosylated core region, and immunogenic GalNAc beta 1-4GlcNAc (LDN) termini. Furthermore, we show that the kappa-5 specific IgE antibodies in sera of S. mansoni-infected individuals are directed against the core region of the kappa-5 glycans. Whereas two previously analyzed immunomodulatory egg glycoproteins, IPSE/alpha-1 and omega-1, both express diantennary N-glycans with a difucosylated core and one or two Gal beta 1-4(Fuc alpha 1-3)GlcNAc (Lewis X) antennae, the kappa-5 glycosylation appears unique among the major soluble egg antigens of S. mansoni. The distinct structural and antigenic properties of kappa-5 glycans suggest a specific role for kappa-5 in schistosome egg immunogenicity. Molecular & Cellular Proteomics 10: 10.1074/mcp.M110.005710, 1-14, 2011.

Background: Markers for longevity that reflect the health condition and predict healthy aging are extremely scarce. Such markers are, however, valuable in aging research. It has been shown previously that the N-glycosylation pattern of human immunoglobulin G (IgG) is age-dependent. Here we investigate whether N-linked glycans reflect early features of human longevity. Methodology/Principal Findings: The Leiden Longevity Study (LLS) consists of nonagenarian sibling pairs, their offspring, and partners of the offspring serving as control. IgG subclass specific glycosylation patterns were obtained from 1967 participants in the LLS by MALDI-TOF-MS analysis of tryptic IgG Fc glycopeptides. Several regression strategies were applied to evaluate the association of IgG glycosylation with age, sex, and longevity. The degree of galactosylation of IgG decreased with increasing age. For the galactosylated glycoforms the incidence of bisecting GlcNAc increased as a function of age. Sex-related differences were observed at ages below 60 years. Compared to males, younger females had higher galactosylation, which decreased stronger with increasing age, resulting in similar galactosylation for both sexes from 60 onwards. In younger participants (60 years), decreased levels of non-galactosylated glycoforms containing a bisecting GlcNAc reflected early features of longevity. Conclusions/Significance: We here describe IgG glycoforms associated with calendar age at all ages and the propensity for longevity before middle age. As modulation of IgG effector functions has been described for various IgG glycosylation features, a modulatory effect may be expected for the longevity marker described in this study.

In this study, we show that introduction of human N-acetylglucosaminyltransferase (GnT)-III gene into tobacco plants leads to highly efficient synthesis of bisected N-glycans. Enzymatically released N-glycans from leaf glycoproteins of wild-type and transgenic GnT-III plants were profiled by matrix-assisted laser desorption/ionization time-of-flight mass spectrometry (MALDI-TOF-MS) in native form. After labeling with 2-aminobenzamide, profiling was performed using normal-phase high-performance liquid chromatography with fluorescence detection, and glycans were structurally characterized by MALDI-TOF/TOF-MS and reversephase nano-liquid chromatography-MS/MS. These analyses revealed that most of the complex-type N-glycans in the plants expressing GnT-III were bisected and carried at least two terminal N-acetylglucosamine (GlcNAc) residues in contrast to wild-type plants, where a considerable proportion of N-glycans did not contain GlcNAc residues at the nonreducing end. Moreover, we have shown that the majority of N-glycans of an antibody produced in a plant expressing GnT-III is also bisected. This might improve the efficacy of therapeutic antibodies produced in this type of transgenic plant.

An approach is presented for the determination of the branch location of 1 or 2 extra N-acetyllactosamine units in sialo N-linked carbohydrate chains from glycoproteins. Tetraantennary oligosaccharides containing extra N-acetyllactosamine units were digested with endo-beta-galactosidase, followed by treatment with N-acetyl-beta-glucosaminidase, yielding products which could be analysed by 1H-NMR spectroscopy, thereby giving conclusive data about the location of the extra units in the intact structures.

Three glycoprotein N-glycans, namely, a disialylated diantennary carbohydrate chain linked to Asn. a monosialylated, fucosylated diantennary glycopeptide with bisecting N-acetylglucosamine, and a tetrasialylated, fucosylated tetra-antennary oligosaccharide. have been investigated by two-dimensional NOE and HOHAHA spectroscopy in 1H2O as solvent. The amide protons of all N-acetylglucosamine and sialic acid residues could readily be assigned. The large chemical-shift dispersion of the amide resonances of the N-acetylglucosamine residues, allowed the unambiguous assignment of every N-acetyl methyl signal, via strong NOEs. Subspectra could be obtained of all N-acetylglucosamine residues in HOHAHA spectra. These results have as main implication that several biologically important large glycans, will not become amenable for conformational studies by multidimensional NMR in 1H2O solution

HPLC analysis of sialic acid released from recombinant variants of human tissue plasminogen activator, human chimeric plasminogen activator, human erythropoietin, and human follitropin, expressed in Chinese hamster ovary cells, demonstrates for each glycoprotein the presence of N-acetylneuraminic and N-glycolylneuraminic acid in a ratio of 97:3. Structural analysis by 500 MHz1H-NMR spectroscopy, of the enzymatically released N-linked carbohydrate chains of chimeric plasminogen activator and of erythropoietin, showed that alpha2-3 linked N-glycolylneuraminic acid can occur in different N-acetyllactosamine type antennary structures.

Human β-mannosidosis urine was fractionated by gel permeation chromatography on Bio-Gel P-2 and by high performance liquid chromatography on Partisil 10 SAX. Besides the disaccharide Man β1- 4GlcNAc as the major component, a sialic acid-containing compound was detected in an amount of 10% compared to that of Man β 1-4GlcNAc. Structural characterization of the oligosaccharide and of its reduced analogue by sugar composition analysis, methylation analysis, gas- liquid chromatography-mass spectrometry, and 500-MHz 1H NMR spectroscopy gave conclusive evidence for a novel urinary constituent: NeuAc alpha 2-6Man beta 1-4GlcNAc. This linear trisaccharide can be considered as the result of an alpha 2-6-sialylation of the major accumulating compound, Man beta 1-4GlcNAc. The hitherto unknown linkage between sialic acid and mannose was shown to be susceptible to sialidase digestion.