Regulatory T-cells (Treg) are essential for maintaining immune homeostasis and tolerance. Surface glycosylation is ubiquitous on mammalian cells and regulates diverse biological processes. While it ...is currently well accepted that surface glycan expression influences multiple aspects of T-cell function, little is known about the relevance of glycosylation to Treg biology. This study aimed to profile the surface glycosylation characteristics of Treg in various lymphoid compartments of mouse and in human peripheral blood with comparison to non-regulatory, conventional CD4
T-cells (Tconv). It also sought to determine the relationship between the surface glycosylation characteristics and suppressive potency of Treg. Lectin-based flow cytometric profiling demonstrated that Treg surface glycosylation differs significantly from that of Tconv in the resting state and is further modified by activation stimuli. In mouse, the surface glycosylation profiles of FoxP3
Treg from spleen and lymph nodes were closely comparable but greater variability was observed for Treg in thymus, bone marrow, and blood. Surface levels of tri/tetra-antennary
glycans correlated with expression of proteins known to be involved in Treg suppressive functions, including GITR, PD-1, PD-L1, CD73, CTLA-4, and ICOS. In coculture experiments involving purified Treg subpopulations and CD4
or CD8
Tconv, higher surface tri/tetra-antennary
glycans was associated with greater Treg suppressive potency. Enzymatic manipulation of mouse Treg surface glycosylation resulting in a temporary reduction of surface
-glycans significantly reduced Treg capacity to suppress Tconv activation through contact-dependent mechanisms. Overall, these findings demonstrate that Treg have distinctive surface glycan characteristics that show variability across anatomical locations and are modulated by activation events. They also provide evidence of an important role for surface glycosylation in determining Treg phenotype and suppressive potency. These insights may prove relevant to the analysis of Treg in disease settings and to the further development of Treg-based immunotherapies.
Mucin glycosylation is the key facilitator of microbial attachment and nutrition and it varies according to biological location, health and disease status, microbiome composition, infection, and ...multiple other factors. Mucin glycans have also been reported to attenuate pathogen virulence and mediate biofilm dispersal. With the labor intensive and time-consuming purification required for natural mucins and their low quantitative yield from biological sources, natural mucin microarrays provide a convenient and multiplexed platform to study mucin glycosylation and interactions. In this chapter we describe the purification of natural mucins, using sputum as an example biological source, and the printing of natural mucin microarrays.
Fasciola hepatica, commonly known as liver fluke, is a trematode that causes Fasciolosis in ruminants and humans. The outer tegumental coat of F. hepatica (FhTeg) is a complex metabolically active ...biological matrix that is continually exposed to the host immune system and therefore makes a good vaccine target. F. hepatica tegumental coat is highly glycosylated and helminth-derived immunogenic oligosaccharide motifs and glycoproteins are currently being investigated as novel vaccine candidates. This report presents the first systematic characterization of FhTeg glycosylation using lectin microarrays to characterize carbohydrates motifs present, and lectin histochemistry to localize these on the F. hepatica tegument. We discovered that FhTeg glycoproteins are predominantly oligomannose oligosaccharides that are expressed on the spines, suckers and tegumental coat of F. hepatica and lectin blot analysis confirmed the abundance of N- glycosylated proteins. Although some oligosaccharides are widely distributed on the fluke surface other subsets are restricted to distinct anatomical regions. We selectively enriched for FhTeg mannosylated glycoprotein subsets using lectin affinity chromatography and identified 369 proteins by mass spectrometric analysis. Among these proteins are a number of potential vaccine candidates with known immune modulatory properties including proteases, protease inhibitors, paramyosin, Venom Allergen-like II, Enolase and two proteins, nardilysin and TRIL, that have not been previously associated with F. hepatica. Furthermore, we provide a comprehensive insight regarding the putative glycosylation of FhTeg components that could highlight the importance of further studies examining glycoconjugates in host-parasite interactions in the context of F. hepatica infection and the development of an effective vaccine.
A compact biosensor for a label-free, rapid (<80 s) detection of glycan-lectin interactions using ac impedance measurements was developed for the first time. A galactose-binding peanut agglutinin ...(PNA) and sialic acid-binding Sambucus nigra agglutinin (SNA) were covalently surface-immobilized on the layered Cu/Ni/Au printed circuit board (PCB) electrodes. Samples of artificial and natural glycoconjugates consisting of (1) gold glyconanoparticles encapsulated with approximately 90-100 copies of TF-antigen disaccharide Galalpha1-3GalNAc (TF-AuNP), (2) asialofetuin (ASF) containing both LacNAc (Galbeta1-4GlcNAc) and TF-antigen, and (3) fetuin (FET), the sialylated glycoform of ASF. The samples were run separately on PNA- and SNA-immobilized PCB electrodes. Our results indicate that TF-AuNP could be rapidly and reliably detected up to 1 pg/mL (13 fM) concentration on PNA electrode but, as expected, yielded no response on the SNA electrode. ASF and FET glycocoproteins were unambiguously detectable up to 10 pg/mL (150 fM) on PNA and SNA electrodes, respectively. Moreover, the technique allowed us to observe glyco-microheterogeneity of FET as well as establish the presence of two isoforms of SNA lectin, SNA-I and SNA-II, in one of the vendor's formulations. Further elaboration of the described technology into novel electrochemically driven lectin arrays may find applications in diagnosis of cancer and other diseases with multiple glycobiomarkers or as a rapid low-cost bioanalytical tool for glycoproteome analyses.
Parasitic helminths resort to various mechanisms to evade and modulate their host's immune response, several of which have been described for
. We recently reported the presence of sialic acid ...residues on the surface of adult
extracellular vesicles (EVs). We now report that these sialylated molecules are mammalian serum proteins. In addition, our data suggest that most sialylated EV-associated proteins do not elicit a humoral response upon injection into mice, or in sera obtained from infected animals. Sialic acids frequently terminate glycans on the surface of vertebrate cells, where they serve important functions in physiological processes such as cell adhesion and signalling. Interestingly, several pathogens have evolved ways to mimic or utilise host sialic acid beneficially by coating their own proteins, thereby facilitating cell invasion and providing protection from host immune effectors. Together, our results indicate that
EVs are coated with host glycoproteins, which may contribute to immune evasion by masking antigenic sites, protecting EVs from removal from serum and aiding in cell adhesion and entry to exert their functions.
The field of biosensor development now encompasses several areas specifically geared toward the rapid and sensitive detection, identification, and quantification of target analytes. In contrast to ...the more mature research and development of nucleic acid and protein biosensors, the development of 'glyco-biosensors' for detecting carbohydrates and conjugates of carbohydrates (glycoconjugates) is at a relatively nascent stage. The application of glyco-biosensors aims to open novel analytical and diagnostic avenues, encompassing industrial bioprocesses, biomedical and clinical applications. This area of research has been greatly aided by advancement brought by interdisciplinary mergers of engineering, biology, chemistry and physical sciences and enabling the miniaturization of detection platforms. In this review, we briefly introduce the need for glyco-biosensors, discuss current analytical technologies, and examine advances in glyco-biosensor approaches aimed at the detection and/or quantification of glycoconjugates or carbohydrates derived from glycoconjugates since 2005.
•Gram-negative Campylobacter jejuni pathogenic in humans but commensal in chickens.•Cell surface glycosylation is a virulence factor which may alter with temperature.•Surface glycosylation of two ...strains compared between human and avian temperatures.•LPS-like and N-linked structures dominant at 37°C and LPS reduced for 81116 at 42°C.•Relative distribution of CPS and LOS structures altered for 81-176 at 42 from 37°C.
Gram-negative Campylobacter jejuni is the leading cause of bacterial gastroenteritis in humans worldwide and the most frequently identified infectious trigger in patients developing Guillain–Barré syndrome (GBS). While C. jejuni is pathogenic in humans, it is a commensal in avian hosts. Bacterial cell surface carbohydrates are important virulence factors and play roles in adherence, colonisation and infection. The mechanisms leading to infection or persistent colonisation of C. jejuni are not well understood but host temperature may provide an important stimulus for specific adaptation. Thus, examination of the modulation of the total surface glycome of C. jejuni in response to temperature may help shed light on commensal and pathogenic mechanisms for this species. C. jejuni strains 81116 and 81-176 were cultured at 37 and 42°C to simulate human and avian host conditions, respectively, and whole cells were profiled on lectin microarrays constructed to include a wide range of binding specificities. C. jejuni 81116 profiles indicated that the previously characterised lipopolysaccharide (LPS)-like molecule and N-linked glycans were the predominantly recognised cell surface structures while capsular polysaccharide (CPS), lipooligosaccharides (LOS) and N-linked glycosylation were best recognised for strain 81-176 at 37°C. The profiles of both strains varied and were distinguishable at both temperatures. At the higher temperature, reduced dominance of the LPS-like structure was associated with strain 81116 and a change in the relative distribution of CPS and LOS structures was indicated for strain 81-176. This change in LOS molecular mass species distribution between temperatures was confirmed by SDS–PAGE analysis. Additionally, opposite behaviour of certain lectins was noted between the plate agglutination assay and the microarray platform. Insights into the important glycosylation involved in C. jejuni host cell tropism at different growth temperatures were gained using the lectin microarray platform.
Glycobiomimics and glycobiosensors Gerlach, Jared Q; Cunningham, Stephen; Kane, Marian ...
Biochemical Society transactions
38, Številka:
5
Journal Article
Recenzirano
Following steady advances in analytical technologies, our knowledge in glycomics is now increasing rapidly. Over the last decade, specific glycans have been described that are associated with a range ...of diseases, such as cancer and inflammation, with host-pathogen interactions and with various stages during stem cell development and differentiation. Simultaneously, deeper structural insight has been gained on glycosylated biopharmaceutical protein therapeutics manufactured in CHO (Chinese-hamster ovary) and other cell systems. This glycomic information is highly relevant for clinicians and biomanufacturing industries as a new class of glycobiomarkers emerges. However, current methods of glycoanalysis are primarily research tools and are not suitable for point-of-care on-site detection and analysis, or sensor devices. Lectin-based glycan detection provides the most promising approach to fill these gaps. However, the limited availability of lectins with high specificity and sensitivity for specific glycan motifs presents one of the main challenges in building reliable glycobiosensors. Recent reports have demonstrated the use of recombinant protein engineering, phage display and aptamer technologies in the production of lectin mimics, as well as the construction of biosensors that are capable of rapidly detecting glycan motifs at low levels in both a labelled and label-free manner. These are primarily proof-of-principle reports at this stage, but some of the approaches, either alone or in combination, will lead to functional glycobiosensors in the coming years which will be valuable tools for the clinical, biopharmaceutical and life science research communities.
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