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1.
Angeloni S Ridet JL Kusy N Gao H Crevoisier F Guinchard S Kochhar S Sigrist H Sprenger N 《Glycobiology》2005,15(1):31-41
To facilitate deciphering the information content in the glycome, thin film-coated photoactivatable surfaces were applied for covalent immobilization of glycans, glycoconjugates, or lectins in microarray formats. Light-induced immobilization of a series of bacterial exopolysaccharides on photoactivatable dextran-coated analytical platforms allowed covalent binding of the exopolysaccharides. Their specific galactose decoration was detected with fluorescence-labeled lectins. Similarly, glycoconjugates were covalently immobilized and displayed glycans were profiled for fucose, sialic acid, galactose, and lactosamine epitopes. The applicability of such platforms for glycan profiling was further tested with extracts of Caco2 epithelial cells. Following spontaneous differentiation or on pretreatment with sialyllactose, Caco2 cells showed a reduction of specific glycan epitopes. The changed glycosylation phenotypes coincided with altered enteropathogenic E. coli adhesion to the cells. This microarray strategy was also suitable for the immobilization of lectins through biotin-neutravidin-biotin bridging on platforms functionalized with a biotin derivatized photoactivatable dextran. All immobilized glycans were specifically and differentially detected either on glycoconjugate or lectin arrays. The results demonstrate the feasibility and versatility of the novel platforms for glycan profiling. 相似文献
2.
Rosa M. Espinosa-Marzal Giacomo Fontani Frieder B. Reusch Marcella Roba Nicholas D. Spencer Rowena Crockett 《Biophysical journal》2013
Cells are coated with a glycocalyx—a layer of carbohydrate-containing biomolecules, such as glycoproteins. Although the structure and orientation of the cell-surface glycans are frequently regarded as being random, we have found, using α-1-acid glycoprotein and antitrypsin as model systems for surface glycans, that this is not the case. A glycoprotein monolayer was adsorbed onto hydrophilic and hydrophobic substrates. Surface-force measurements revealed that the orientation of the glycans with respect to the aqueous solution has a profound effect on the structure of vicinal water. The glycan antennae of the surface-adsorbed glycoproteins apparently impose an ordering on the water, resulting in a strong repulsive force over some tens of nanometers with superposed film-thickness transitions ranging from ≈0.7 to 1.8 nm. When the glycan orientation is modified by chemical means, this long-range repulsion disappears. These results may provide an explanation as to why the multiantennary structure is ubiquitous in glycoproteins. Although direct, specific interactions between glycans and other biomolecules are essential for their functionality, these results indicate that glycans’ long-range structuring of water may also influence their ability to interact with biomolecules in their vicinity. 相似文献
3.
Rosa?M. Espinosa-Marzal Giacomo Fontani Frieder?B. Reusch Marcella Roba Nicholas?D. Spencer Rowena Crockett 《Biophysical journal》2013,104(12):2686-2694
Cells are coated with a glycocalyx—a layer of carbohydrate-containing biomolecules, such as glycoproteins. Although the structure and orientation of the cell-surface glycans are frequently regarded as being random, we have found, using α-1-acid glycoprotein and antitrypsin as model systems for surface glycans, that this is not the case. A glycoprotein monolayer was adsorbed onto hydrophilic and hydrophobic substrates. Surface-force measurements revealed that the orientation of the glycans with respect to the aqueous solution has a profound effect on the structure of vicinal water. The glycan antennae of the surface-adsorbed glycoproteins apparently impose an ordering on the water, resulting in a strong repulsive force over some tens of nanometers with superposed film-thickness transitions ranging from ≈0.7 to 1.8 nm. When the glycan orientation is modified by chemical means, this long-range repulsion disappears. These results may provide an explanation as to why the multiantennary structure is ubiquitous in glycoproteins. Although direct, specific interactions between glycans and other biomolecules are essential for their functionality, these results indicate that glycans’ long-range structuring of water may also influence their ability to interact with biomolecules in their vicinity. 相似文献
4.
Manfred Wuhrer Alexandra van Remoortere Crina I.A. Balog André M. Deelder Cornelis H. Hokke 《Analytical biochemistry》2010,406(2):132-140
Characterization of protein-carbohydrate interactions at the molecular level is important for understanding many glycan-mediated processes. Here we present a method for the identification of glycan ligands of carbohydrate-binding proteins. The glycans released from natural sources are labeled with biotinamidocaproyl hydrazide (BACH) and subsequently fractionated by high-performance liquid chromatography. Glycan fractions are screened for binding to carbohydrate-binding proteins (CBPs) using a microtitration plate binding assay; CBPs are immobilized, BACH-glycan fractions are added, and bound BACH-glycans are detected using alkaline phosphatase-conjugated streptavidin. The glycan structures in binding fractions are studied by (tandem) mass spectrometry, exoglycosidase treatment, and rechromatography, thereby revealing the glycan motifs recognized by the CBPs. Subsequent surface plasmon resonance experiments using a reverse setup with immobilization of the BACH-glycan ligands on streptavidin-coated surfaces provide more information on glycan-CBP interactions via association and dissociation curves. The presented method is easy and fast, and the required instrumentation is available in many laboratories. The assay is very sensitive given that both the mass spectrometric analysis and the microtitration plate binding assay can be performed on femtomole amounts of BACH-glycans. This approach should be generally applicable to study and structurally identify carbohydrate ligands of anti-glycan antibodies and lectins. 相似文献
5.
Muñoz FJ Rumbero A Sinisterra JV Santos JI André S Gabius HJ Jiménez-Barbero J Hernáiz MJ 《Glycoconjugate journal》2008,25(7):633-646
The emerging role of glycans as versatile biochemical signals in diverse aspects of cellular sociology calls for establishment
of sensitive methods to monitor carbohydrate recognition by receptors such as lectins. Most of these techniques involve the
immobilization of one of the binding partners on a surface, e.g. atomic force microscopy, glycan array and Surface Plasmon Resonance (SPR), hereby simulating cell surface presentation.
Here, we report the synthesis of fluorescent glycoconjugates, with a functionalization strategy which avoids the frequently
occurring ring opening at the reducing end for further immobilization on a surface or derivatization with biotin. In order
to improve the versatility of these derivatized glycans for biological studies, a new approach for the synthesis of biotinylated
and fluorescent glycans has also been realized. Finally, to illustrate their usefulness the neoglycoconjugates were immobilized
on different surfaces, and the interaction analysis with a model lectin, the toxin from mistletoe, proved them to act as potent
ligands, underscoring the merit of the presented synthetic approach.
Electronic supplementary material The online version of this article (doi:) contains supplementary material, which is available to authorized users. 相似文献
6.
A surface plasmon resonance (SPR) based natural glycan microarray was developed for screening of interactions between glycans
and carbohydrate-binding proteins (CBPs). The microarray contained 144 glycan samples and allowed the real-time and simultaneous
screening for recognition by CBPs without the need of fluorescent labeling. Glycans were released from their natural source
and coupled by reductive amination with the fluorescent labels 2-aminobenzamide (2AB) or anthranilic acid (AA) followed by
high-performance liquid chromatography (HPLC) fractionation making use of the fluorescent tag. The released and labeled glycans,
in addition to fluorescently labeled synthetic glycans and (neo)glycoproteins, were printed on an epoxide-activated chip at
fmol amounts. This resulted in covalent immobilization, with the epoxide groups forming covalent bonds to the secondary amine
groups present on the fluorescent glycoconjugates. The generated SPR glycan array presented a subset of the glycan repertoire
of the human parasite Schistosoma mansoni. In order to demonstrate the usefulness of the array in the simultaneous detection of glycan-specific serum antibodies, the
anti-glycan antibody profiles from sera of S. mansoni-infected individuals as well as from non-endemic uninfected controls were recorded. The SPR screening was sensitive for differences
between infection sera and control sera, and revealed antibody titers and antibody classes (IgG or IgM). All SPR analyses
were performed with a single SPR array chip, which required regeneration and blocking of the chip before the application of
a serum sample. Our results indicate that SPR-based arrays constructed from glycans of natural or synthetic origin, pure or
as mixture, can be used for determining serum antibody profiles as possible markers for the infection status of an individual. 相似文献
7.
A new immobilization method for immunoaffinity (IA) biosensors that ensures the high surface density and the stability of the IA layer was developed. For the immobilization of biomolecules, the molecular recognition protein was first thiolated by covalent conjugation of mercaptopropionic acid, and then the thiolated protein was attached on the gold surface of the transducer. In this work, horseradish peroxidase (HRP) and its antibody were used as a model antigen-antibody, and the following properties of the IA layer prepared by thiolated protein were estimated: (i) biological integrity of HRP after the immobilization process by using activity assay, (ii) charge transfer resistance by immobilization, (iii) mass loading by the surface plasmon resonance (SPR) biosensor, (iv) number of binding sites, and (v) feasibility test for the measurement of capacitive change by the antigen-antibody interaction. Based on these parameters, the immobilization method by using thiolated protein was determined to be feasible for application to IA biosensors. 相似文献
8.
Sibel Cetinel H. Burak Caliskan Deniz T. Yucesoy A. Senem Donatan Esra Yuca Mustafa Urgen Nevin G. Karaguler Prof. Candan Tamerler 《Biotechnology journal》2013,8(2):262-272
Successful nanobiotechnology implementation largely depends on control over the interfaces between inorganic materials and biological molecules. Controlling the orientations of biomolecules and their spatial arrangements on the surface may transform many technologies including sensors, to energy. Here, we demonstrate the self-organization of L -lactate dehydrogenase (LDH), which exhibits enhanced enzymatic activity and stability on a variety of gold surfaces ranging from nanoparticles to electrodes, by incorporating a gold-binding peptide tag (AuBP2) as the fusion partner for Bacillus stearothermophilus LDH (bsLDH). Binding kinetics and enzymatic assays verified orientation control of the enzyme on the gold surface through the genetically incorporated peptide tag. Finally, redox catalysis efficiency of the immobilized enzyme was detected using cyclic voltammetry analysis in enzyme-based biosensors for lactate detection as well as in biofuel cell energy systems as the anodic counterpart. Our results demonstrate that the LDH enzyme can be self-immobilized onto different gold substrates using the short peptide tag under a biologically friendly environment. Depending on the desired inorganic surface, the proposed peptide-mediated path could be extended to any surface to achieve single-step oriented enzyme immobilization for a wide range of applications. 相似文献
9.
Carbohydrate and protein immobilization onto solid surfaces by sequential Diels-Alder and azide-alkyne cycloadditions 总被引:1,自引:0,他引:1
We demonstrate the applicability of sequential Diels-Alder and azide-alkyne [3 + 2] cycloaddition reactions (click chemistry) for the immobilization of carbohydrates and proteins onto a solid surface. An alpha,omega-poly(ethylene glycol) (PEG) linker carrying alkyne and cyclodiene terminal groups was synthesized and immobilized onto an N-(epsilon-maleimidocaproyl) (EMC)-functionalized glass slide via an aqueous Diels-Alder reaction. In the process, an alkyne-terminated PEGylated surface was provided for the conjugation of azide-containing biomolecules via click chemistry, which proceeded to completion at low temperature and in aqueous solvent. As anticipated, alkyne, azide, cyclodiene, and EMC are independently stable and do not react with common organic reagents nor functional groups in biomolecules. Given an appropriate PEG linker, sequential Diels-Alder and azide-alkyne [3 + 2] cycloaddition reactions provide an effective strategy for the immobilization of a wide range of functionally complex substances onto solid surfaces. 相似文献
10.
A glycan microarray was developed by using 2,6-diaminopyridine (DAP) as a fluorescent linker and printing of the glycan-DAP
conjugates (GDAPs) on epoxy-activated glass slides. Importantly, all coupled GDAPs showed a detectable level of concentration-dependent
GDAP fluorescence under blue laser excitation (495 nm) that can be used for both grid location and on-slide quantification.
A glycan array including a large number of GDAP’s derived from natural and commercially available free glycans was constructed
and glycan interactions with various plant lectins were investigated. In addition, binding parameters of lectins to glycans
were obtained by varying both the amount of GDAPs on the array and the lectin concentration in analyses. These data demonstrate
the general utility of GDAP microarrays for functional glycomic analyses and for determining binding parameters of glycan
binding proteins (GBPs). 相似文献
11.
Uwe B. Sleytr Margit Sra Paul Messner Dietmar Pum 《Journal of cellular biochemistry》1994,56(2):171-176
Two-diminsional crystalline surface layers (S-layers) composed of prtein or glucoprotein subunits are one of the most commonly observed prokaryotic cell envelope structures. lsolated S-layer Subunits are endowed with the ability to assemble into monomolecular arrays in suspension, on surfaces or interface by an entropy-driven process. S-layer lattices are isoporous structures with functional groups located on the surface in an identical position and orientation. These characteristic featupes have alreadu led to applicatioinns of S-layers as (1) ultrafilration membranes with well-defiled mmlecular weight cut -ooffs and excellent antifouling characteristics, (2) immobilization matrices for functional molecules as required for affiviy and enzyme memberanes, affiniy micricarriers and biosensors, (3) conjugate vaaines, (4) carriers for Langmuir-Blodgett films and reconstituted biological memberanes, and (5) patterning elements in molecular nanotechnology. 相似文献
12.
Hizukuri Y Yamanishi Y Nakamura O Yagi F Goto S Kanehisa M 《Carbohydrate research》2005,340(14):2270-2278
There have been almost no standard methods for conducting computational analyses on glycan structures in comparison to DNA and proteins. In this paper, we present a novel method for extracting functional motifs from glycan structures using the KEGG/GLYCAN database. First, we developed a new similarity measure for comparing glycan structures taking into account the characteristic mechanisms of glycan biosynthesis, and we tested its ability to classify glycans of different blood components in the framework of support vector machines (SVMs). The results show that our method can successfully classify glycans from four types of human blood components: leukemic cells, erythrocyte, serum, and plasma. Next, we extracted characteristic functional motifs of glycans considered to be specific to each blood component. We predicted the substructure alpha-D-Neup5Ac-(2-->3)-beta-D-Galp-(1-->4)-D-GlcpNAc as a leukemia specific glycan motif. Based on the fact that the Agrocybe cylindracea galectin (ACG) specifically binds to the same substructure, we conducted an experiment using cell agglutination assay and confirmed that this fungal lectin specifically recognized human leukemic cells. 相似文献
13.
Glycan array development is limited by the complexity of efficiently generating derivatives of free reducing glycans with primary amines or other functional groups. A novel bi-functional spacer with selective reactivity toward the free glycan and a second functionality, a primary amine, was synthesized. We demonstrated an efficient one-step derivatization of various glycans including naturally isolated N-glycans, O-glycans, milk oligosaccharides, and bacterial polysaccharides in microgram scale. No protecting group manipulations or activation of the anomeric center was required. To demonstrate its utility for glycan microarray fabrication, we compared glycans with different amine-spacers for incorporation onto an amine-reactive glass surface. Our study results revealed that glycans conjugated with this bi-functional linker were effectively printed and detected with various lectins and antibodies. 相似文献
14.
The efficient surface patterning of oligonucleotides was accomplished onto the inner wall of fused-silica capillary tubes as well as on the surface of glass slides through oxime bond formation. The robustness of the method was demonstrated by achieving the surface immobilization of up to three different oligonucleotide sequences inside the same capillary tube. The method involves the preparation of surfaces grafted with reactive aminooxy functionalities masked with the photocleavable protecting group, 2-(2-nitrophenyl) propyloxycarbonyl group (NPPOC). Briefly, NPPOC-aminooxy silane 1 was prepared and used to silanize the glass surfaces. The NPPOC group was cleaved under brief irradiation to unmask the reactive aminooxy group on surfaces. These reactive aminooxy groups were allowed to react with aldehyde-containing oligonucleotides to achieve an efficient surface immobilization. The advantage associated with the present approach is that it combines the high-coupling efficiency of oxime bond formation with the convenience associated with the use of photolabile groups. The present strategy thus offers an alternative approach for the immobilization of biomolecules in the microchannels of "labs on a chip" devices. 相似文献
15.
《Expert review of proteomics》2013,10(5):761-774
Glycan microarrays are emerging as increasingly used screening tools with a high potential for unraveling protein–carbohydrate interactions: probing hundreds or even thousands of glycans in parallel, they provide the researcher with a vast amount of data in a short time-frame, while using relatively small amounts of analytes. Natural glycan microarrays focus on the glycans’ repertoire of natural sources, including both well-defined structures as well as still-unknown ones. This article compares different natural glycan microarray strategies. Glycan probes may comprise oligosaccharides from glycoproteins as well as glycolipids and polysaccharides. Oligosaccharides may be purified from scarce biological samples that are of particular relevance for the carbohydrate-binding protein to be studied. We give an overview of strategies for glycan isolation, derivatization, fractionation, immobilization and structural characterization. Detection methods such as fluorescence analysis and surface plasmon resonance are summarized. The importance of glycan density and multivalency is discussed. Furthermore, some applications of natural glycan microarrays for studying lectin and antibody binding are presented. 相似文献
16.
Sercan Karav Joshua L. Cohen Daniela Barile Juliana Maria Leite Nobrega de Moura Bell 《Biotechnology progress》2017,33(1):104-112
Glycans play important biological roles in cell‐to‐cell interactions, protection against pathogens, as well as in proper protein folding and stability, and are thus interesting targets for scientists. Although their mechanisms of action have been widely investigated and hypothesized, their biological functions are not well understood due to the lack of deglycosylation methods for large‐scale isolation of these compounds. Isolation of glycans in their native state is crucial for the investigation of their biological functions. However, current enzymatic and chemical deglycosylation techniques require harsh pretreatment and reaction conditions (high temperature and use of detergents) that hinder the isolation of native glycan structures. Indeed, the recent isolation of new endoglycosidases that are able to cleave a wider variety of linkages and efficiently hydrolyze native proteins has opened up the opportunity to elucidate the biological roles of a higher variety of glycans in their native state. As an example, our research group recently isolated a novel Endo‐β‐N‐acetylglucosaminidase from Bifidobacterium longum subsp. infantis ATCC 15697 (EndoBI‐1) that cleaves N‐N′‐diacetyl chitobiose moieties found in the N‐linked glycan (N‐glycan) core of high mannose, hybrid, and complex N‐glycans. This enzyme is also active on native proteins, which enables native glycan isolation, a key advantage when evaluating their biological activities. Efficient, stable, and economically viable enzymatic release of N‐glycans requires the selection of appropriate immobilization strategies. In this review, we discuss the state‐of‐the‐art of various immobilization techniques (physical adsorption, covalent binding, aggregation, and entrapment) for glycosidases, as well as their potential substrates and matrices. © 2016 American Institute of Chemical Engineers Biotechnol. Prog., 33:104–112, 2017 相似文献
17.
Hu H Armstrong PC Khalil E Chen YC Straub A Li M Soosairajah J Hagemeyer CE Bassler N Huang D Ahrens I Krippner G Gardiner E Peter K 《PloS one》2011,6(4):e19190
Background
Staphylococcus aureus (S. aureus) is a common pathogen capable of causing life-threatening infections. Staphylococcal superantigen-like protein 5 (SSL5) has recently been shown to bind to platelet glycoproteins and induce platelet activation. This study investigates further the interaction between SSL5 and platelet glycoproteins. Moreover, using a glycan discovery approach, we aim to identify potential glycans to therapeutically target this interaction and prevent SSL5-induced effects.Methodology/Principal Findings
In addition to platelet activation experiments, flow cytometry, immunoprecipitation, surface plasmon resonance and a glycan binding array, were used to identify specific SSL5 binding regions and mediators. We independently confirm SSL5 to interact with platelets via GPIbα and identify the sulphated-tyrosine residues as an important region for SSL5 binding. We also identify the novel direct interaction between SSL5 and the platelet collagen receptor GPVI. Together, these receptors offer one mechanistic explanation for the unique functional influences SSL5 exerts on platelets. A role for specific families of platelet glycans in mediating SSL5-platelet interactions was also discovered and used to identify and demonstrate effectiveness of potential glycan based inhibitors in vitro.Conclusions/Significance
These findings further elucidate the functional interactions between SSL5 and platelets, including the novel finding of a role for the GPVI receptor. We demonstrate efficacy of possible glycan-based approaches to inhibit the SSL5-induced platelet activation. Our data warrant further work to prove SSL5-platelet effects in vivo. 相似文献18.
N-linked glycans with similar location in the fusion protein head modulate paramyxovirus fusion
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N-linked glycans not only orchestrate the folding and intracellular transport of viral glycoproteins but also modulate their function. We have characterized the three glycans attached to fusion (F) proteins of the morbilliviruses canine distemper virus and measles virus. The individual Morbillivirus glycans have similar functional properties: the glycan at position 68 is essential for protein transport, and those at positions 36 and 75 modulate fusion (numbering according to the Newcastle disease virus [NDV] F protein sequence). Based on the crystal structure of the NDV F protein, we then predicted the locations of the Morbillivirus glycans: the glycan at position 36 is located in the F protein head, and those at positions 68 and 75 are located near the neck-stalk interface. NDV position 36 is not occupied by a glycan; the only glycan in that F protein head also has a fusion control function and grows from residue 366, located only 6 A from residue 36. We then exchanged the glycan at position 36 with the glycan at position 366 and showed functional complementation. Thus, structural information about the F proteins of Paramyxoviridae coupled with functional analysis disclosed a location in the protein head into which fusion-modulating glycans independently evolved. 相似文献
19.
The new field of functional glycomics encompasses information about both glycan structure and recognition by carbohydrate-binding proteins (CBPs) and is now being explored through glycan array technology. Glycan array construction, however, is limited by the complexity of efficiently generating derivatives of free, reducing glycans with primary amines for conjugation. Here we describe a straightforward method to derivatize glycans with 2,6-diaminopyridine (DAP) to generate fluorescently labeled glycans (glycan-DAP conjugates or GDAPs) that contain a primary amine for further conjugation. We converted a wide variety of glycans, including milk sugars, N-glycans, glycosaminoglycans and chitin-derived glycans, to GDAPs, as verified by HPLC and mass spectrometry. We covalently conjugated GDAPs to N-hydroxysuccinimide (NHS)-activated glass slides, maleimide-activated protein, carboxylated microspheres and NHS-biotin to provide quantifiable fluorescent derivatives. All types of conjugated glycans were well-recognized by appropriate CBPs. Thus, GDAP derivatives provide versatile new tools for biologists to quantify and covalently capture minute quantities of glycans for exploring their structures and functions and generating new glycan arrays from naturally occurring glycans. 相似文献
20.
David W. Ralin Shane C. Dultz Judd E. Silver Jeffrey C. Travis Majlinda Kullolli William S. Hancock Marina Hincapie 《Clinical proteomics》2008,4(1-2):37-46