A versatile method for functionalizing surfaces with bioactive glycans

Microarrays and biosensors owe their functionality to our ability to display surface-bound biomolecules with retained biological function. Versatile, stable, and facile methods for the immobilization of bioactive compounds on surfaces have expanded the application of high-throughput "omics"-scale screening of molecular interactions by nonexpert laboratories. Herein, we demonstrate the potential of simplified chemistries to fabricate a glycan microarray, utilizing divinyl sulfone (DVS)-modified surfaces for the covalent immobilization of natural and chemically derived carbohydrates, as well as glycoproteins. The bioactivity of the captured glycans was quantitatively examined by surface plasmon resonance imaging (SPRi). Composition and spectroscopic evidence of carbohydrate species on the DVS-modified surface were obtained by X-ray photoelectron spectroscopy (XPS) and time-of-flight secondary ion mass spectrometry (ToF-SIMS), respectively. The site-selective immobilization of glycans based on relative nucleophilicity (reducing sugar vs amine- and sulfhydryl-derived saccharides) and anomeric configuration was also examined. Our results demonstrate straightforward and reproducible conjugation of a variety of functional biomolecules onto a vinyl sulfone-modified biosensor surface. The simplicity of this method will have a significant impact on glycomics research, as it expands the ability of nonsynthetic laboratories to rapidly construct functional glycan microarrays and quantitative biosensors.     

Trityl-derivatized carbohydrates immobilized on a polystyrene microplate

Carbohydrate biosensors, including carbohydrate arrays, are attracting increased attention for the comprehensive and high-throughput investigation of protein-carbohydrate interactions. Here, we describe an effective approach to fabricating a robust microplate-based carbohydrate array capable of probing protein binding and screening for inhibitors in a high-throughout manner. This approach involves the derivatization of carbohydrates with a trityl group through an alkyl linker and the immobilization of the trityl-derivatized carbohydrates (mannose and maltose) onto microplates noncovalently to construct carbohydrate arrays. The trityl carbohydrate derivative has very good immobilization efficiency for polystyrene microplates and strong resistance to aqueous washing. The carbohydrate arrays can probe the interactions with the lectin Concanavalin A and screen this protein for the well-known inhibitors methyl α-d-mannopyranoside and methyl α-d-glucopyranoside in a high-throughput manner. The method described in this paper represents a convenient way of fabricating robust noncovalent carbohydrate arrays on microplates and offers a convenient platform for high-throughput drug screening.     

The effect of structural differences in the reducing terminus of sugars on the binding affinity of carbohydrates and proteins analyzed using photoaffinity labeling

Because carbohydrates and proteins bind with such low affinity, the nature of their interactions is not clear. Photoaffinity labeling with diazirin groups is useful for elucidating the roles of carbohydrates in these binding processes. However, when carbohydrate probes are synthesized according to this conventional method, the reducing terminus of the sugar is opened to provide an acyclic structure. Because greater elucidation of carbohydrate-protein interactions requires a closed-ring carbohydrate in addition to the photoreactive group, we synthesized new molecular tools. The carbohydrate ligands were synthesized in three steps (glycosylation with allyl alcohol, deprotection, and ozonolysis). Specific binding proteins for carbohydrate ligands were obtained by photoaffinity labeling. Closed ring-type carbohydrate ligands, in which the reducing sugar is closed, bound to lectins more strongly than open ring-type sugars. Carbohydrate to protein binding was observed using AFM.     

Direct immobilization of gangliosides onto gold-carboxymethyldextran sensor surfaces by hydrophobic interaction: applications to antibody characterization

We describe a novel immobilization technique to investigate interactions between immobilized gangliosides (GD3, GM1, and GM2) and their respective antibodies, antibody fragments, or binding partners using an optical biosensor. Immobilization was performed by direct injection onto a carboxymethyldextran sensor chip and did not require derivatization of the sensor surface or the ganglioside. The ganglioside appeared to bind to the sensor surface by hydrophobic interaction, leaving the carbohydrate epitope available for antibody or, in the case of GM1, cholera toxin binding. The carboxyl group of the dextran chains on the sensor surface did not appear to be involved in the immobilization as evidenced by equivalent levels of immobilization following conversion of the carboxyl groups into acyl amino esters, but rather the dextran layer provided a hydrophilic coverage of the sensor chip which was essential to prevent nonspecific binding. This technique gave better reactivity and specificity for anti- ganglioside monoclonal antibodies (anti-GD3: KM871, KM641, R24; and anti-GM2: KM966) than immobilization by hydrophobic interaction onto a gold sensor surface or photoactivated cross-linking onto carboxymethydextran. This rapid immobilization procedure has facilitated detailed kinetic analysis of ganglioside/antibody interactions, with the surface remaining viable for a large number of cycles (>125). Kinetic constants were determined from the biosensor data using linear regression, nonlinear least squares and equilibrium analysis. The values of kd, ka, and KAobtained by nonlinear analysis (KAKM871 = 1.05, KM641 = 1.66, R24 = 0.14, and KM966 = 0.65 x 10(7) M- 1) were essentially independent of concentration and showed good agreement with data obtained by other analytical methods.      

Lactobacillus helveticus glycosyltransferases: from genes to carbohydrate synthesis

Bioactive carbohydrates are crucial in mediating essential biological processes, and their biosynthesis is an essential aspect to develop for a global view of their biological functions. Lactic acid bacteria display an array of diverse and complex carbohydrates and, therefore, are of particular interest. Here we present the identification of a novel exocellular polysaccharide structure and the corresponding gene cluster from Lactobacillus helveticus NCC2745. The development of a glycosyltransferase-specific enzymatic assay allowed the assignment of sugar specificities, which as a general approach will for the future permit a faster and more direct characterization of glycosyltransferase specificities. A model of the biosynthesis of the repeating unit is proposed. EpsE is a phosphoglucosyltransferase initiating the repeating unit biosynthesis by linking a glucose residue to a membrane-associated lipophilic acceptor. EpsF elongates the carbohydrate chain by forming an alpha(1,3)-Glcp linkage onto the first Glcp, whereas EpsG adds a backbone alpha(1,6)-Galp onto alpha-Glcp and EpsH attaches a alpha(1,6)-Glcp branch onto the first glucose residue. Finally, EpsI would add a beta(1,6)-Galp linkage onto alpha-Glcp terminating the sidechain and EpsJ would terminate the synthesis of the polysaccharides' repeating unit by forming a beta(1,3)-Galp linkage onto alpha-Galp.     

Human cytomegalovirus detection by a quartz crystal microbalance immunosensor

A piezoelectric affinity sensor has been developed to detect distinctive antigens of the human cytomegalovirus. Either the specific antibodies or the antigen were immobilized on the gold electrode. To develop a rapid immunoassay, various assay formats were tested in relation with the different antigen composition. First, a direct assay was carried out immobilizing the specific antibody on the crystal surface by passive adsorption. Next, Protein A, thiol/poly L-lysine mixed self-assembled monolayers were tested as methods of gold modification. A competitive format was exploited by immobilization of the antigen onto the crystal activated by SAM and poly L-lysine. This procedure yielded a preliminary calibration curve. A linear range between 2.5 and 5 μg/ml of gB epitope in solution and a detection limit of 1 μg/ml were measured.     

Photons to illuminate the universe of sugar diversity through bioarrays

In this mini-review, we summarize the photochemical approaches for developing high-throughput carbohydrate microarray technologies. Newly established methods for photo-immobilizing unmodified monosaccharides, oligosaccharides and polysaccharides onto photoactive surfaces and coupling of photoactive carbohydrates onto polymer surfaces are reviewed.     

Orientation of proteins on the sensor surface and optimization of their immobilization by prior protection of the active center

When studying biospecific interactions with application of surface plasmon resonance, one of the main problems is reagent proper orientation to the sensor surface. Due to rather high chemical activity of molecular receptor sites, the interaction between these areas and surface may become predominant. Here we propose a technique for prevention of such orientation of bioreceptors using soybean trypsin inhibitor STI as an example. To obtain oriented STI immobilization on a modified gold surface its active site has been previously blocked through interaction with its specific partner trypsin. After conjugate immobilization on the sensor surface the components were separated using a glycine buffer (pH 2.2).     

Fabrication of carbohydrate chips and their use to probe protein-carbohydrate interactions

Carbohydrate microarrays have received considerable attention as an advanced technology for the rapid analysis of carbohydrate-protein interactions. This protocol provides detailed procedures for the preparation of carbohydrate microarrays by immobilizing hydrazide-conjugated carbohydrates on epoxide-derivatized glass slides. In addition, we describe the use we make of these microarrays in glycomics research. Unlike other techniques that require large amounts of samples and long assay times, carbohydrate microarrays are used to carry out the rapid assessment of a number of carbohydrate-recognition events with tiny amounts of carbohydrate samples. Furthermore, the microarray technology is also utilized for the rapid assay of enzyme activities. We are able to routinely prepare carbohydrate microarrays within 12 h by using hydrazide-conjugated carbohydrates and apply these microarrays for the studies of glycan-protein interactions within 8 h.     

Man alpha1-2 Man alpha-OMe-concanavalin A complex reveals a balance of forces involved in carbohydrate recognition.

We have determined the crystal structure of the methyl glycoside of Man alpha1-2 Man in complex with the carbohydrate binding legume lectin concanavalin A (Con A). Man alpha1-2 Man alpha-OMe binds more tightly to concanavalin A than do its alpha1-3 and alpha1-6 linked counterparts. There has been much speculation as to why this is so, including a suggestion of the presence of multiple binding sites for the alpha1-2 linked disaccharide. Crystals of the Man alpha1-2 Man alpha-OMe-Con A complex form in the space group P2(1)2(1)2(1) with cell dimensions a = 119.7 A, b = 119.7 A, c = 68.9 A and diffract to 2. 75A. The final model has good geometry and an R factor of 19.6% (Rfree= 22.8%). One tetramer is present in the asymmetric unit. In three of the four subunits, electron density for the disaccharide is visible. In the fourth only a monosaccharide is seen. In one subunit the reducing terminal sugar is recognized by the monosaccharide site; the nonreducing terminal sugar occupies a new site and the major solution conformation of the inter-sugar glycosidic linkage conformation is adopted. In contrast, in another subunit the non reducing terminal sugar sits in the so called monosaccharide binding site; the reducing terminal sugar adopts a different conformation about its inter-sugar glycosidic linkage in order for the methyl group to access a hydrophobic pocket. In the third subunit, electron density for both binding modes is observed. We demonstrate that an extended carbohydrate binding site is capable of binding the disaccharide in two distinct ways. These results provide an insight in to the balance of forces controlling protein carbohydrate interactions.     

伴刀豆球蛋白A-糖复合物的模拟分析

主要分析ConA与不同的糖特异性结合时其活性位点构象变化的特征。模拟分析了ConA糖结合活性中心氨基酸残基结构特征,同时对相应残基原子可及性表面进行了计算和分析。结果表明：（1）ConA在和不同的糖结合时,存在不同的结合方式;（2）不管ConA和什么糖结合,主要的作用是由活性中心的Tyr12、Asn14、Asp208和Arg228提供的;（3）无论是结合单糖还是寡糖,活性中心总是与第一个糖环起主要的结合作用。     

Impedance-based detection of DNA sequences using a silicon transducer with PNA as the probe layer

Electrochemical impedance measurements were used for the detection of single-strand DNA sequences using a peptide nucleic acid (PNA) probe layer immobilized onto Si/SiO2 chips. An epoxysilane layer is first immobilized onto the Si/SiO2 surface. The immobilization procedure consists of an epoxide/amine coupling reaction between the amino group of the PNA linker and the epoxide group of the silane. A 20-nucleotide sequence of PNA was used. Impedance measurements allow for the detection of the changes in charge distribution at the oxide/solution interface following modifications to the oxide surface. Due to these modifications, there are significant shifts in the semiconductor's flat-band potential after immobilization and hybridization. The results obtained using this direct and rapid approach are supported by fluorescence measurements according to classical methods for the detection of nucleic acid sequences.     

Proteins that bind high-mannose sugars of the HIV envelope

A broad range of proteins bind high-mannose carbohydrates found on the surface of the envelope protein gp120 of the human immunodeficiency virus and thus interfere with the viral life cycle, providing a potential new way of controlling HIV infection. These proteins interact with the carbohydrate moieties in different ways. A group of them interacts as typical C-type lectins via a Ca2+ ion. Another group interacts with specific single, terminal sugars, without the help of a metal cation. A third group is involved in more intimate interactions, with multiple carbohydrate rings and no metal ion. Finally, there is a group of lectins for which the interaction mode has not yet been elucidated. This review summarizes, principally from a structural point of view, the current state of knowledge about these high-mannose binding proteins and their mode of sugar binding.     

Affibody protein capture microarrays: synthesis and evaluation of random and directed immobilization of affibody molecules

Affibody molecules, 58-amino acid three-helix bundle proteins directed to different targets by combinatorial engineering of staphylococcal protein A, were used as capture ligands on protein microarrays. An evaluation of slide types and immobilization strategies was performed to find suitable conditions for microarray production. Two affibody molecules, Z(Taq) and Z(IgA), binding Taq DNA polymerase and human IgA, respectively, were synthesized by solid phase peptide synthesis using an orthogonal protection scheme, allowing incorporation of selective immobilization handles. The resulting affibody variants were used for random surface immobilization (through amino groups) or oriented surface immobilization (through cysteine or biotin coupled to the side chain of Lys58). Evaluation of the immobilization techniques was carried out using both a real-time surface plasmon resonance biosensor system and a microarray system using fluorescent detection of Cy3-labeled target protein. The results from the biosensor analyses showed that directed immobilization strategies significantly improved the specific binding activity of affibody molecules. However, in the microarray system, random immobilization onto carboxymethyl dextran slides and oriented immobilization onto thiol dextran slides resulted in equally good signal intensities, whereas biotin-mediated immobilization onto streptavidin-coated slides produced slides with lower signal intensities and higher background staining. For the best slides, the limit of detection was 3 pM for IgA and 30 pM for Taq DNA polymerase.     

Sensitivity enhancement of surface plasmon resonance biosensor with colloidal gold

We enhanced the sensitivity of surface plasmon resonance biosensor by the conversion of the real-time direct binding immunoassay into the sandwich immunoassay, in which colloidal gold particles coated with anti-mouse IgG was used. By the immobilization of anti-mouse IgG onto the carboxymethyl dextran surface of thin gold film, the direct binding of analyte (mouse IgG) onto the sensor chip, and the injection of colloidal gold particles coated with antimouse IgG, about 100 times of sensitivity enhancement was obtained. This result suggests that nanoparticles, which has a high refractive index, homogeneous ultrafine structure and capability of size control, would be applicable for the detection of very small quantity of biomaterial.     

Matter-tag: A universal immobilization platform for enzymes on polymers,metals, and silicon-based materials

Enzyme immobilization is extensively studied to improve enzyme properties in catalysis and analytical applications. Here, we introduce a simple and versatile enzyme immobilization platform based on adhesion-promoting peptides, namely Matter-tags. Matter-tags immobilize enzymes in an oriented way as a dense monolayer. The immobilization platform was established with three adhesion-promoting peptides; Cecropin A (CecA), liquid chromatography peak I (LCI), and Tachystatin A2 (TA2), that were genetically fused to enhanced green fluorescent protein and to two industrially important enzymes: a phytase (from Yersinia mollaretii) and a cellulase (CelA2 from a metagenomic library). Here, we report a universal and simple Matter-tag–based immobilization platform for enzymes on various materials including polymers (polystyrene, polypropylene, and polyethylene terephthalate), metals (stainless steel and gold), and silicon-based materials (silicon wafer). The Matter-tag–based enzyme immobilization is performed at ambient temperature within minutes (<10 min) in an aqueous solution harboring the phytase or cellulase by immersing the targeted material. The peptide LCI was identified as universal adhesion promoter; LCI immobilized both enzymes on all investigated materials. The attachment of phytase-LCI onto gold was characterized with surface plasmon resonance spectroscopy obtaining a dissociation constant value (K_D) of 2.9·10⁻⁸ M and a maximal surface coverage of 504 ng/cm².     

Carbohydrate accumulation and partitioning in Pinus banksiana seedlings and seedling cuttings

Tests were conducted to identify possible relations between carbohydrates and callusing-rooting of Pinus banksiana Lamb, cuttings. Terminals, upper stems, and basal (1 cm) stems of 90-day-old untreated seedlings and seedling cuttings were analyzed for sucrose, total soluble reducing sugar, starch and total non-structural carbohydrate during propagation. Seedlings were evaluated in order to determine whether data for cuttings alone properly described carbohydrate-callusing-rooting relations under conditions where stock plants and cuttings were propagated in different environments. Results indicated that seedling terminals and upper stems, but not basal stems, accumulated the measured carbohydrates much like cuttings, though to lesser concentrations. Thus, carbohydrate accumulation by cutting terminals and upper stems would have been overestimated, based on cutting data alone. In terms of rooting, results indicated that: 1) Total carbohydrate accumulation in cutting basal stems was related to callusing-rooting, but a cause-effect relation was not established; 2) The positive relation between callusing-rooting and total carbohydrate accumulation was primarily due to accumulation of reducing sugar and starch, with reducing sugar predominant. 3) Reducing sugar/starch concentration ratios were the most sensitive and convenient indicators of specific carbohydrate differences within and between seedlings and cuttings.     

Conjugation of oligosaccharides by reductive amination to amine modified chondroitin oligomer and γ-cyclodextrin

Carbohydrates present on cell surfaces participate in numerous biological recognition phenomena including cell–cell interactions, cancer metastasis and pathogen invasion. Therefore, synthetic carbohydrates have a potential to act as pharmaceutical substances for treatment of various pathological phenomena by inhibiting specifically the interaction between cell surface carbohydrates and their protein receptors (lectins). However, the inherently low affinity of carbohydrate-protein interactions has often been an obstacle for successful generation of carbohydrate based pharmaceuticals. Multivalent glycoconjugates, i.e. structures carrying several copies of the active carbohydrate sequence in a carrier molecule, have been constructed to overcome this problem. Here we present two novel types of multivalent carbohydrate conjugates based on chondroitin oligomer and cyclodextrin carriers. These carriers were modified to express primary amino groups, and oligosaccharides were then bound to carrier molecules by reductive amination. Multivalent conjugates were produced using the human milk type oligosaccharides LNDFH I (Lewis-b hexasaccharide), LNnT, and GlcNAcβ1-3Galβ1-4GlcNAcβ1-3Galβ1-4Glc.     

Addressable self-immobilization of lactate dehydrogenase across multiple length scales

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.