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

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

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

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

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.

• MeSH
• bakteriální proteiny chemie   izolace a purifikace   MeSH
• fukosa chemie   MeSH
• krystalografie rentgenová MeSH
• kvarterní struktura proteinů MeSH
• lektiny chemie   izolace a purifikace   MeSH
• Photorhabdus chemie   MeSH
• proteinové domény MeSH
• Publikační typ
• časopisecké články MeSH

Lectins are a group of ubiquitous proteins which specifically recognize and reversibly bind sugar moieties of glycoprotein and glycolipid constituents on cell surfaces. The mutagenesis approach is often employed to characterize lectin binding properties. As lectins are not enzymes, it is not easy to perform a rapid specificity screening of mutants using chromogenic substrates. It is necessary to use different binding assays such as isothermal titration calorimetry (ITC), surface plasmon resonance (SPR), microscale thermophoresis (MST), enzyme-linked lectin assays (ELLA), or glycan arrays for their characterization. These methods often require fluorescently labeled proteins (MST), highly purified proteins (SPR) or high protein concentrations (ITC). Mutant proteins may often exhibit problematic behaviour, such as poor solubility or low stability. Lectin-based cell agglutination is a simple and low-cost technique which can overcome most of these problems. In this work, a modified method of the agglutination of human erythrocytes and yeast cells with microscopy detection was successfully used for a specificity study of the newly prepared mutant lectin RS-IIL_A22S, which experimentally completed studies on sugar preferences of lectins in the PA-IIL family. Results showed that the sensitivity of this method is comparable with ITC, is able to determine subtle differences in lectin specificity, and works directly in cell lysates. The agglutination method with microscopy detection was validated by comparison of the results with results obtained by agglutination assay in standard 96-well microtiter plate format. In contrast to this assay, the microscopic method can clearly distinguish between hemagglutination and hemolysis. Therefore, this method is suitable for examination of lectins with known hemolytic activity as well as mutant or uncharacterized lectins, which could damage red blood cells. This is due to the experimental arrangement, which includes very short sample incubation time in combination with microscopic detection of agglutinates, that are easily observed by a small portable microscope.

• MeSH
• aglutinace účinky léků   MeSH
• aglutinační testy * metody   MeSH
• bakteriální proteiny farmakologie   MeSH
• erytrocyty cytologie   účinky léků   MeSH
• hemaglutinace účinky léků   MeSH
• hemolýza účinky léků   MeSH
• kvasinky cytologie   účinky léků   MeSH
• lektiny farmakologie   MeSH
• lidé MeSH
• mikroskopie MeSH
• povrchová plasmonová rezonance MeSH
• proteiny z Escherichia coli farmakologie   MeSH
• Saccharomyces cerevisiae cytologie   účinky léků   MeSH
• Check Tag
• lidé MeSH
• Publikační typ
• časopisecké články MeSH
• práce podpořená grantem MeSH

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

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