glycan array
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Interactions between glycans and glycan binding proteins are essential for numerous processes in all kingdoms of life. Glycan microarrays are an excellent tool to examine protein-glycan interactions. Here, we present a microbe-focused glycan microarray platform based on oligosaccharides obtained by chemical synthesis. Glycans were generated by combining different carbohydrate synthesis approaches including automated glycan assembly, solution-phase synthesis, and chemoenzymatic methods. The current library of more than 300 glycans is as diverse as the mammalian glycan array from the Consortium for Functional Glycomics and, due to its microbial focus, highly complementary. This glycan platform is essential for the characterization of various classes of glycan binding proteins. Applications of this glycan array platform are highlighted by the characterization of innate immune receptors and bacterial virulence factors as well as the analysis of human humoral immunity to pathogenic glycans.
- MeSH
- antigeny bakteriální chemie imunologie MeSH
- CHO buňky MeSH
- Cricetulus MeSH
- druhová specificita MeSH
- glykomika MeSH
- imunitní systém MeSH
- lektiny MeSH
- lidé MeSH
- mikročipová analýza metody MeSH
- oligosacharidy MeSH
- polysacharidy chemie klasifikace imunologie MeSH
- rekombinantní proteiny MeSH
- transportní proteiny chemie MeSH
- vazba proteinů MeSH
- zvířata MeSH
- Check Tag
- lidé MeSH
- zvířata MeSH
- Publikační typ
- časopisecké články MeSH
- práce podpořená grantem MeSH
Aberrant glycosylation of glycoproteins has been linked with various pathologies. Therefore, understanding the relationship between aberrant glycosylation patterns and the onset and progression of the disease is an important research goal that may provide insights into cancer diagnosis and new therapy development. In this study, we use a surface plasmon resonance imaging biosensor and a lectin array to investigate aberrant glycosylation patterns associated with oncohematological disease-myelodysplastic syndromes (MDS). In particular, we detected the interaction between the lectins and glycoproteins present in the blood plasma of patients (three MDS subgroups with different risks of progression to acute myeloid leukemia (AML) and AML patients) and healthy controls. The interaction with lectins from Aleuria aurantia (AAL) and Erythrina cristagalli was more pronounced for plasma samples of the MDS and AML patients, and there was a significant difference between the sensor response to the interaction of AAL with blood plasma from low and medium-risk MDS patients and healthy controls. Our data also suggest that progression from MDS to AML is accompanied by sialylation of glycoproteins and increased levels of truncated O-glycans and that the number of lectins that allow discriminating different stages of disease increases as the disease progresses.
- MeSH
- akutní myeloidní leukemie * MeSH
- biosenzitivní techniky * MeSH
- glykoproteiny metabolismus MeSH
- glykosylace MeSH
- krevní plazma metabolismus MeSH
- lektiny MeSH
- lidé MeSH
- myelodysplastické syndromy * terapie MeSH
- Check Tag
- lidé MeSH
- Publikační typ
- časopisecké články MeSH
- práce podpořená grantem MeSH
Glykomika a glykoproteomika představují relativně nové směry pro analýzy komplexních biologických vzorků a jejich důležitost neustále roste. Tyto oblasti jsou komplementární k dalším zavedeným přístupům, např. ke genomickému profilování a proteomice. Glykoproteiny jsou stále více uznávány jako důležité molekuly účastnící se buněčných interakcí a adhezí. Výskyt strukturních změn v glykanových částech se zdá být typický pro různé typy rakoviny. Následující souhrn se zabývá aktuálními trendy v glykomickém profilování a glykoproteomickém výzkumu biologických tekutin a tkání se zaměřením na rakovinu. Použité metody jsou založeny na principech kapilárních separačních technik, hmotnostní spektrometrie a glykanových a lektinových čipů. Všechny zmíněné metody mají značný potenciál pro využití v diagnostických a prediktivních vyšetřeních.
Glycomics and glycoproteomics represent relatively new directions in detail analyses of complex biological media. These areas of increasing importance to cancer research complement the more established genomic profiling and proteomics. Glycoproteins are being increasingly recognized as important in cellular interactions and adhesion. Structural alterations of their glycan moieties seem to occur in different cancer conditions. We review current directions in glycomic profiling and glycoproteomic investigations of biological fluids and tissues pertaining to cancer. The used methods rely on capillary separation techniques, mass spectrometry, and the glycan and lectin arrays. They all show considerable promise for new diagnostic and prognostic measurements. Key words: glycomics – glycopeptides – cancer – liquid chromatography – mass spectrometry – capillary electrophoresis – glycan profiling – array analysis This work was supported by the European Regional Development Fund and the State Budget of the Czech Republic (RECAMO, CZ.1.05/2.1.00/03.0101) and by MH CZ – DRO (MMCI, 00209805). The authors declare they have no potential conflicts of interest concerning drugs, products, or services used in the study. The Editorial Board declares that the manuscript met the ICMJE “uniform requirements” for biomedical papers. Submitted: 21. 3. 2014 Accepted: 10. 4. 2014
- Klíčová slova
- glykanové profilování,
- MeSH
- chromatografie kapalinová MeSH
- čipová analýza proteinů MeSH
- elektroforéza kapilární MeSH
- glykomika * metody MeSH
- glykopeptidy * analýza MeSH
- glykoproteiny analýza metabolismus MeSH
- glykosylace * MeSH
- hmotnostní spektrometrie MeSH
- lidé MeSH
- nádorové biomarkery * krev MeSH
- nádorové proteiny krev MeSH
- nádory krev MeSH
- Check Tag
- lidé MeSH
- Publikační typ
- práce podpořená grantem MeSH
Protein microarray technology became an important research tool for study and detection of proteins, protein-protein interactions and a number of other applications. The utilization of nanoparticle-based materials and nanotechnology-based techniques for immobilization allows us not only to extend the surface for biomolecule immobilization resulting in enhanced substrate binding properties, decreased background signals and enhanced reporter systems for more sensitive assays. Generally in contemporarily developed microarray systems, multiple nanotechnology-based techniques are combined. In this review, applications of nanoparticles and nanotechnologies in creating protein microarrays, proteins immobilization and detection are summarized. We anticipate that advanced nanotechnologies can be exploited to expand promising fields of proteins identification, monitoring of protein-protein or drug-protein interactions, or proteins structures.
- MeSH
- biologické markery chemie MeSH
- čipová analýza proteinů metody MeSH
- DNA chemie MeSH
- kovové nanočástice chemie MeSH
- lékové transportní systémy MeSH
- lidé MeSH
- mapování interakce mezi proteiny MeSH
- mikroskopie atomárních sil MeSH
- nanočástice chemie MeSH
- nanotechnologie metody MeSH
- polysacharidy chemie MeSH
- povrchová plasmonová rezonance MeSH
- proteiny chemie MeSH
- reportérové geny MeSH
- vazba proteinů MeSH
- zlato chemie MeSH
- Check Tag
- lidé MeSH
- Publikační typ
- časopisecké články MeSH
- práce podpořená grantem MeSH
- přehledy MeSH
Photorhabdus luminescens is known for its symbiosis with the entomopathogenic nematode Heterorhabditis bacteriophora and its pathogenicity toward insect larvae. A hypothetical protein from P. luminescens was identified, purified from the native source, and characterized as an l-fucose-binding lectin, named P. luminescens lectin (PLL). Glycan array and biochemical characterization data revealed PLL to be specific toward l-fucose and the disaccharide glycan 3,6-O-Me2-Glcβ1-4(2,3-O-Me2)Rhaα-O-(p-C6H4)-OCH2CH2NH2 PLL was discovered to be a homotetramer with an intersubunit disulfide bridge. The crystal structures of native and recombinant PLL revealed a seven-bladed β-propeller fold creating seven putative fucose-binding sites per monomer. The crystal structure of the recombinant PLL·l-fucose complex confirmed that at least three sites were fucose-binding. Moreover, the crystal structures indicated that some of the other sites are masked either by the tetrameric nature of the lectin or by incorporation of the C terminus of the lectin into one of these sites. PLL exhibited an ability to bind to insect hemocytes and the cuticular surface of a nematode, H. bacteriophora.
The Photorhabdus species is a Gram-negative bacteria of the family Morganellaceae that is known for its mutualistic relationship with Heterorhabditis nematodes and pathogenicity toward insects. This study is focused on the characterization of the recombinant lectin PLL3 with an origin in P. laumondii subsp. laumondii. PLL3 belongs to the PLL family of lectins with a seven-bladed β-propeller fold. The binding properties of PLL3 were tested by hemagglutination assay, glycan array, isothermal titration calorimetry, and surface plasmon resonance, and its structure was determined by X-ray crystallography. Obtained data revealed that PLL3 binds similar carbohydrates to those that the other PLL family members bind, with some differences in the binding properties. PLL3 exhibited the highest affinity toward l-fucose and its derivatives but was also able to interact with O-methylated glycans and other ligands. Unlike the other members of this family, PLL3 was discovered to be a monomer, which might correspond to a weaker avidity effect compared to homologous lectins. Based on the similarity to the related lectins and their proposed biological function, PLL3 might accompany them during the interaction of P. laumondii with both the nematode partner and the insect host.
- MeSH
- bakteriální proteiny chemie genetika metabolismus MeSH
- fruktosa metabolismus MeSH
- kalorimetrie MeSH
- krystalografie rentgenová MeSH
- lektiny chemie genetika metabolismus MeSH
- Photorhabdus metabolismus MeSH
- povrchová plasmonová rezonance MeSH
- rekombinantní proteiny chemie metabolismus MeSH
- sekundární struktura proteinů MeSH
- vazebná místa MeSH
- Publikační typ
- časopisecké články MeSH
Lectins, a distinct group of glycan-binding proteins, play a prominent role in the immune system ranging from pathogen recognition and tuning of inflammation to cell adhesion or cellular signalling. The possibilities of their detailed study expanded along with the rapid development of biomaterials in the last decade. The immense knowledge of all aspects of glycan-lectin interactions both in vitro and in vivo may be efficiently used in bioimaging, targeted drug delivery, diagnostic and analytic biological methods. Practically applicable examples comprise photoluminescence and optical biosensors, ingenious three-dimensional carbohydrate microarrays for high-throughput screening, matrices for magnetic resonance imaging, targeted hyperthermal treatment of cancer tissues, selective inhibitors of bacterial toxins and pathogen-recognising lectin receptors, and many others. This review aims to present an up-to-date systematic overview of glycan-decorated biomaterials promising for interactions with lectins, especially those applicable in biology, biotechnology or medicine. The lectins of interest include galectin-1, -3 and -7 participating in tumour progression, bacterial lectins from Pseudomonas aeruginosa (PA-IL), E. coli (Fim-H) and Clostridium botulinum (HA33) or DC-SIGN, receptors of macrophages and dendritic cells. The spectrum of lectin-binding biomaterials covered herein ranges from glycosylated organic structures, calixarene and fullerene cores over glycopeptides and glycoproteins, functionalised carbohydrate scaffolds of cyclodextrin or chitin to self-assembling glycopolymer clusters, gels, micelles and liposomes. Glyconanoparticles, glycan arrays, and other biomaterials with a solid core are described in detail, including inorganic matrices like hydroxyapatite or stainless steel for bioimplants.
- MeSH
- Bacteria chemie MeSH
- biokompatibilní materiály chemie normy MeSH
- cukry chemie MeSH
- galektiny chemie metabolismus MeSH
- lektiny typu C chemie metabolismus MeSH
- lektiny chemie metabolismus MeSH
- molekuly buněčné adheze metabolismus MeSH
- receptory buněčného povrchu metabolismus MeSH
- Publikační typ
- časopisecké články MeSH
- přehledy MeSH
The opportunistic pathogen Burkholderia cenocepacia expresses several soluble lectins, among them BC2L-C. This lectin exhibits two domains: a C-terminal domain with high sequence similarity to the recently described calcium-dependent mannose-binding lectin BC2L-A, and an N-terminal domain of 156 amino acids without similarity to any known protein. The recombinant N-terminal BC2L-C domain is a new lectin with specificity for fucosylated human histo-blood group epitopes H-type 1, Lewis b, and Lewis Y, as determined by glycan array and isothermal titration calorimetry. Methylselenofucoside was used as ligand to solve the crystal structure of the N-terminal BC2L-C domain. Additional molecular modeling studies rationalized the preference for Lewis epitopes. The structure reveals a trimeric jellyroll arrangement with striking similarity to TNF-like proteins, and to BclA, the spore protein from Bacillus anthracis which may play an important role in bioadhesion of anthrax spores in human lungs.
- MeSH
- antigeny krevních skupin chemie imunologie metabolismus MeSH
- bakteriální proteiny chemie metabolismus MeSH
- Burkholderia chemie metabolismus MeSH
- epitopy chemie imunologie metabolismus MeSH
- fukosa chemie metabolismus MeSH
- kvarterní struktura proteinů MeSH
- lektiny chemie metabolismus MeSH
- lidé MeSH
- molekulární modely MeSH
- molekulární sekvence - údaje MeSH
- sekvence aminokyselin MeSH
- sekvenční seřazení MeSH
- vazebná místa MeSH
- Check Tag
- lidé MeSH
- Publikační typ
- časopisecké články MeSH
- práce podpořená grantem MeSH
Aspergillus fumigatus is an important allergen and opportunistic pathogen. Similarly to many other pathogens, it is able to produce lectins that may be involved in the host-pathogen interaction. We focused on the lectin AFL, which was prepared in recombinant form and characterized. Its binding properties were studied using hemagglutination and glycan array analysis. We determined the specificity of the lectin towards l-fucose and fucosylated oligosaccharides, including α1-6 linked core-fucose, which is an important marker for cancerogenesis. Other biologically relevant saccharides such as sialic acid, d-mannose or d-galactose were not bound. Blood group epitopes of the ABH and Lewis systems were recognized, Le(Y) being the preferred ligand among others. To provide a correlation between the observed functional characteristics and structural basis, AFL was crystallized in a complex with methyl-α,L-selenofucoside and its structure was solved using the SAD method. Six binding sites, each with different compositions, were identified per monomer and significant differences from the homologous AAL lectin were found. Structure-derived peptides were utilized to prepare anti-AFL polyclonal antibodies, which suggested the presence of AFL on the Aspergillus' conidia, confirming its expression in vivo. Stimulation of human bronchial cells by AFL led to IL-8 production in a dose-dependent manner. AFL thus probably contributes to the inflammatory response observed upon the exposure of a patient to A. fumigatus. The combination of affinity to human epithelial epitopes, production by conidia and pro-inflammatory activity is remarkable and shows that AFL might be an important virulence factor involved in an early stage of A. fumigatus infection.
- MeSH
- Aspergillus fumigatus chemie MeSH
- aspergilóza imunologie MeSH
- bronchy cytologie mikrobiologie MeSH
- epitopy chemie MeSH
- faktory virulence chemie MeSH
- fukosa chemie MeSH
- galaktosa chemie MeSH
- genom fungální MeSH
- hemaglutinace MeSH
- interakce hostitele a patogenu MeSH
- interleukin-8 metabolismus MeSH
- kyselina N-acetylneuraminová chemie MeSH
- lektiny chemie MeSH
- lidé MeSH
- mannosa chemie MeSH
- molekulární sekvence - údaje MeSH
- oligosacharidy chemie MeSH
- sekvence aminokyselin MeSH
- sekvenční homologie aminokyselin MeSH
- sekvenční seřazení MeSH
- spory hub chemie MeSH
- vazebná místa MeSH
- Check Tag
- lidé MeSH
- Publikační typ
- časopisecké články MeSH
- práce podpořená grantem MeSH
- Research Support, N.I.H., Extramural MeSH
Galectin-4 and its homologue galectin-6 are members of the tandem-repeat subfamily of monomer divalent galectins. Expression of mouse galectin-4 and galectin-6 by RT-PCR using primers designed to distinguish both galectin transcripts indicates that both are expressed in the small intestine, colon, liver, kidney, spleen and heart and P19X1 cells while only galectin-4 is expressed in BW-5147 and 3T3 cell lines. In situ hybridization confirmed the presence of galectin-4/-6 transcripts in the liver and small intestine. Galectin-4 is expressed in spermatozoons and oocytes and its expression during early mouse emryogenesis appears in 8-cell embryos and remains in later stages, as tested by RT-PCR. To study the role of carbohydrate recognition domains (CRDs) in oligosaccharide binding and epitope recognition, we cloned mouse full-length galectin-4 and galectin-6 cDNA and constructed bacterial expression vectors producing histidin-tagged recombinant galectin-4 and its truncated CRD1 and CRD2 forms. Oligosaccharide binding profile for all recombinant forms was assessed using Glycan Array available through the Consortium for Functional Glycomics. Acquired data indicate that mGalectin-4 binds to alpha-GalNAc and alpha-Gal A and B type structures with or without fucose. While the CRD2 domain has a high specificity and affinity for A type-2 alpha-GalNAc structures, the CRD1 domain has a broader specificity in correlation to the total binding profile. These data suggest that CRD2 might be the dominant binding domain of mouse galectin-4. Mapping of epitopes reactive for biotinylated his-tagged CRD1, CRD2 and mGalectin-4 performed on mouse cryosections showed that all three forms bind to alveolar macrophages, macrophages of red pulp of the spleen and proximal tubuli of the kidney and this binding was inhibited by 5 mM lactose. Interestingly, mGalectin-4, but not CRD forms, binds to the suprabasal layer of squamous epithelium of the tongue, suggesting that the link region also plays an important role in ligand recognition.
- MeSH
- buňky 3T3 MeSH
- epitopy chemie metabolismus MeSH
- financování organizované MeSH
- galektin 4 genetika chemie MeSH
- galektiny genetika chemie MeSH
- hybridizace in situ metody MeSH
- imunohistochemie MeSH
- komplementární DNA genetika MeSH
- molekulární sekvence - údaje MeSH
- myši inbrední C57BL MeSH
- myši MeSH
- nádorové buněčné linie MeSH
- oligosacharidy chemie metabolismus MeSH
- rekombinantní proteiny biosyntéza chemie MeSH
- sacharidové sekvence MeSH
- stanovení celkové genové exprese MeSH
- vazba proteinů MeSH
- vazebná místa MeSH
- zvířata MeSH
- Check Tag
- myši MeSH
- zvířata MeSH
