Functionalized self-assembled monolayers for measuring single molecule lectin carbohydrate interactions

The specific interactions between sugar-binding proteins (lectins) and their complementary carbohydrates mediate several complex biological functions. There is a great deal of interest in uncovering the molecular basis of these interactions. In this study, we demonstrate the use of an efficient one-step amination reaction strategy to fabricate carbohydrate arrays based on mixed self-assembled monolayers. These allow specific lectin carbohydrate interactions to be interrogated at the single molecule level via AFM. The force required to directly rupture the multivalent bonds between Concanavalin A (Con A) and mannose were subsequently determined by chemical force microscopy. The mixed self-assembled monolayer provides a versatile platform with active groups to attach a 1-amino-1-deoxy sugar or a protein (Con A) while minimizing non-specific adhesion enabling quick and reliable detection of rupture forces. By altering the pH of the environment, the aggregation state of Con A was regulated, resulting in different dominant rupture forces, corresponding to di-, tri- and multiple unbinding events. We estimate the value of the rupture force for a single Con A-mannose bond to be 95 ± 10 pN. The rupture force is consistent even when the positions of the binding molecules are switched. We show that this synthesis strategy in conjunction with a mixed SAM allows determination of single molecules bond with high specificity, and may be used to investigate lectin carbohydrate interactions in the form of carbohydrate arrays as well as lectin arrays.     

Enhanced Iridium Complex Electrochemiluminescence Cytosensing and Dynamic Evaluation of Cell‐Surface Carbohydrate Expression

A newly prepared [(ppy)₂Ir(dcbpy)]⁺?PF₆^? (ppy: 2‐phenylpyridyl; dcbpy: 4,4′‐dicarboxy‐2,2′‐bipyridyl) and gold nanoparticle functionalized mesoporous silica nanoparticle (Au/Ir‐MSN) is reported. Based on the binding between concanavalin A (Con A) and mannose, the novel nanoparticle was applied to an ultrasensitive electrochemiluminescence (ECL) in situ cytosensing strategy and the dynamic evaluation of cell‐surface carbohydrate expression. The ECL activity of the presented Con A@Au/Ir‐MSN nanoprobe was greatly enhanced by employing a functionalized nanoparticle and graphene nanomaterial with an increased surface area and simultaneously improved electron‐transfer efficiency at the electrode interface. Under optimal conditions, the sandwich‐type ECL cytosensor showed a linear response to K562 cells at concentrations ranging from 1.0×10² to 1.0×10⁶ cells mL^?1 and realized a low detection limit of a single cell. The proposed method could also be successfully used for monitoring the dynamic variation of carbohydrate expression in cancer cells in response to external stimulation by an inhibitor.     

Synthesis and Langmuir studies of bivalent and monovalent alpha-D-mannopyranosides with lectin Con A

Highly avid interaction between carbohydrate ligands and lectin receptors nominally requires the ligand presentation in a clustered form. We present herein an approach involving Langmuir monolayer formation of the sugar ligands and the assessment of their lectin binding at the air-water interface. Bivalent alpha-D-mannopyranoside containing the glycolipid ligand was used to study its binding profiles with lectin Con A, in comparison to the corresponding monovalent glycolipid. In addition to the bivalent and monovalent nature of the glycolipid ligands at the molecular level, the ligand densities at the monolayer level were varied with the aid of a nonsugar lipid molecule so as to obtain mixed monolayers with various sugar-nonsugar ratios. Lectin binding of bivalent and monovalent ligands at different ratios was monitored by differential changes in the surface area per molecule of the mixed monolayer, with and without the lectin. The present study shows that maximal binding of the lectin to the bivalent ligand occurs at lower sugar densities at the interface ( approximately 10% sugar in the mixed monolayer) than for that of the monovalent ligand ( approximately 20% sugar in the mixed monolayer). It is observed that complete coverage of the monolayer with only the sugar ligands does not allow all of the sugars to be functionally active.     

A Simple Electrochemical Aptamer Cytosensor for Direct Detection of Chronic Myelogenous Leukemia K562 Cells

A simple and rapid electrochemical aptamer cytosensor has been developed for direct detection of chronic myelogenous leukemia (CML) K562 cells based on a specific aptamer and a biotin conjugated concanavalin A (bio‐ConA) detection probe. The K562 cell could be specifically recognized by T2‐KK1B10 capture aptamer pre‐immobilized on gold modified electrode surface. Then, bio‐ConA was added in the reaction to identify K562 cell surface mannose, resulting in an aptamer‐K562 cell‐bio‐ConA sandwich complex. Finally, streptavidin conjugated alkaline phosphatase (ST‐ALP) combined with the bio‐ConA to catalyze α‐naphthyl (α‐NP) phosphate to form α‐naphthol which is highly electroactive at an operating voltage of 180 mV (vs. Ag/AgCl). Under optimum conditions, the DPV signals were proportional to the logarithm of K562 cell from 1×10² to 1×10⁷ cells mL⁻¹ with a detection limit of 79 cells mL⁻¹. The cytosensor also exhibited high selectivity, stability and reproducibility. When applied to detect K562 cells in human blood samples, recoveries between 79.6 %–93.3 % were obtained, indicating the developed biosensor would be a potential alternative tool for CML K562 cell detection in real biological samples.     

Lectin arrays for profiling cell surface carbohydrate expression

We present a strategy for the analysis of cell surface carbohydrate expression patterns using lectin arrays fabricated on gold surfaces. Antibody and glycoprotein binding experiments showed that the lectins were effectively immobilized on the surface and retained their carbohydrate-binding specificities. The approach was demonstrated in the analysis of carbohydrate expression on two mammalian cell lines.     

Cell antigens recognized by rabbit antibodies specific for oligomannosyl determinants.

Rabbit antibodies to cell wall mannans of various microbial strains and their mutants were found to be cross-reactive to cell carbohydrates of mammalian sperm and 4-6-days-old blastocysts. Immunochemical studies indicate that oligomers of alpha1 yields to 2, alpha1 yields to 3, alpha1 yields to 6, and probably also alpha yields to 4 linked mannose residues of sperm carbohydrates are available for antibody binding. At least 80 percent of binding activity of a yeast mannan antibody to sperm can be effectively inhibited by specific haptens or digestion with exo-alpha-D-mannosidase, an enzyme activity highest in testicular tissue. In order to determine the role of this enzyme in the metabolism of the cross-reactive mannan antigens of sperm, the relative amount of a specific alpha-linked oligomannosyl determinant of bovine sperm from homozygous normals was compared to that of hetero-zygous carriers of alpha-mannosidase deficiency. Extensive cross-reactivity between the microbial and mammalian oligomannosyl determinants suggest that these are conserved structures in cell carbohydrates, although the organization of these units in the microbial cell wall lipopolysaccharide has very little similarity to the carbohydrate moieties of mammalian glycoproteins.     

Microbial carbohydrate specific antibodies distinguish between different stages of differentiating mouse cerebellum

High titered anticarbohydrate antibodies were used to identify cell surface carbohydrates during different stages in histogenesis of mouse cerebellum in a micro tissue-culture system which mimics selected features of in vivo cerebellum development. Blockage of fiber formation within the first few days in vitro and inhibition of cell migrations by carbohydrate-specific antibodies served as an assay system for possible contributions of surface carbohydrates to the behavior of developing cerebellar cells. Microbial strains were selected on the basis of carbohydrate structures of their cell wall antigens, and anticarbohydrate antibodies were raised against treated whole bacteria and yeast in rabbits. We found that antibodies to mannan were active at all stages of development tested (embryonic day 13, E13; the day of birth, PO; and postnatal day 7, P7). Antibodies to sialic acids prepared against strains B and C of Neisseria meningitidis distinguish different subterminal structures: anti-B reacted with E13 and PO cerebellar cells, and anti-C mostly with cells older than P7. Antifetuin antibody recognized E13 and PO but not P7 cell populations. Pneumococcus C strain R36A-specific antibodies were effective only after coating cells to C type carbohydrate before application of the antibody. The results demonstrate that antimicrobiol carbohydrate antibodies cross-react with mammalian cell surface carbohydrate structures and therefore can be used as a powerful tool in tissue culture to analyse those structures which might control cell behaviors pertinent to cerebellar development.     

Immunoassay of P-glycoprotein on single cell by capillary electrophoresis with laser induced fluorescence detection

The cellular mechanism based on P-glycoprotein (PGP) for its drug pump function has become very important in multidrug resistance (MDR) research. A method has been established to characterize PGP on single K562 cell by coupling capillary electrophoresis with laser induced fluorescence detection. A permeable intact cell after the immunoassay binding with fluorescence labeling antibody was injected into the capillary and directly separated without lysis. It was found that once 5-10 optional cells were detected in batch, the PGP amount on this cell line could be outlined and calculated clearly. The PGP amount on K562 MDR cell line is 3.88 times higher than that on K562 sensitive cell line. These two cell lines with immunoassay binding were also analyzed by injection of multi-cells in order to improve the throughput. A resistance factor so called multidrug resistance multiple (MRM) was introduced to evaluate the MDR difference between cell lines. The MRM values of the cell line K562 measured by single cell analysis are well correlated with those by flow cytometry, which also prove the validity of our method in single cell analysis for the possibility of cancer diagnosis, pharmacokinetics and drug screening in future.     

Differences in the redistribution of concanavalin A and wheat germ agglutinin binding sites on mouse neuroblastoma cells.

Concanavalin A (Con A), wheat germ agglutinin (WGA), and Ricinus communis agglutinin (RCA) bound with either 125I, fluorescent dyes, or fluorescent polymeric microspheres were used to quantitate and visualize the distribution of lectin binding sites on mouse neuroblastoma cells. As viewed by fluorescent light and scanning electron microscopy, over 10(7) binding sites for Con A, WGA, and RCA appeared to be distributed randomly over the surface of differentiated and undifferentiated cells. An energy-dependent redistribution of labeled sites into a central spot occurred when the cells were labeled with a saturating dose of fluorescent lectin and maintained at 37 degrees C for 60 min. Reversible labeling using appropriate saccharide inhibitors indicated that the labeled sites had undergone endocytosis by the cell. A difference in the mode of redistribution of WGA or RCA and Con A binding sites was observed in double labeling experiments. When less than 10% of the WGA or RCA lectin binding sites were labeled, only these labeled sites appeared to be removed from the cell surface. In contrast, when less than 10% of the Con A sites were labeled, both labeled and unlabeled Con A binding sites were removed from the cell surface. Cytochalasin B uncoupled the coordinate redistribution of labeled and unlabeled Con A sites, suggesting the involvement of microfilaments. Finally, double labeling experiments employing fluorescein-tagged Con A and rhodamine-tagged WGA indicate that most Con A and WGA binding sites reside on different membrane components and redistribute independenty of each other.     

Protein-directed assembly of binary monolayers at the interface and surface patterns of protein on the monolayers

Ferritin-directed assembly of binary monolayers of zwitterionic dipalmitoylphosphatidylcholine and cationic dioctadecyldimethylammonium bromide (DOMA) at the interface and surface patterns of ferritin on the monolayers have been investigated using a combination of infrared reflection absorption spectroscopy, surface plasmon resonance, and atomic force microscopy. Ferritin binding to the binary monolayers at the air-water interface at the surface pressure 30 mN/m, primarily driven by the electrostatic interaction, gives rise to a change in tilt angle of hydrocarbon chains from 15 degrees +/- 1 degrees to 10 degrees +/- 1 degrees with respect to the normal of the monolayer at the mole fraction of DOMA (XDOMA) of 0.1. The chains at XDOMA = 0.3 are oriented vertical to the water surface before and after protein binding. A new mechanism for protein binding to the binary monolayers is proposed. The secondary structures of the adsorbed ferritin are prevented from changing to some extent due to the existence of the monolayers. The amounts of the bound protein on the monolayers at the air-water interface are increased in comparison with those on the pre-immobilized monolayers at low XDOMA. The increased amounts and different patterns of the adsorbed protein at the monolayers are mostly attributed to the formation of multiple binding sites available for ferritin, which is due to the lateral reorganization of the lipid components in the monolayers induced by the protein in the subphase. The created multiple binding sites on the monolayer surfaces through the protein-directed assembly can be preserved for subsequent protein binding.     

One-step synthesis of biotinyl photoprobes from unprotected carbohydrates

A simple and versatile approach for the preparation of carbohydrate photoprobes has been developed. By a single-step reaction at 37 degrees C, a biotinylated carbene-generating unit was introduced to the reducing end of unprotected carbohydrates. Micromole quantities of N-acetyllactosamine, Lewis X trisaccharide, and sialyl Lewis X tetrasaccharide were easily converted to their biotinylated photoreactive analogues, which enabled the nonradioisotopic chemiluminescent detection of the photolabeled products. Thus, a sequence of lectin photoaffinity labeling, from the probe synthesis to the detection of labeled protein, was readily accomplished within one week. Our strategy may be applicable to any aldehyde-bearing ligand.     

Carbohydrate arrays for the evaluation of protein binding and enzymatic modification

This paper reports a chemical strategy for preparing carbohydrate arrays and utilizes these arrays for the characterization of carbohydrate-protein interactions. Carbohydrate chips were prepared by the Diels-Alder-mediated immobilization of carbohydrate-cyclopentadiene conjugates to self-assembled monolayers that present benzoquinone and penta(ethylene glycol) groups. Surface plasmon resonance spectroscopy showed that lectins bound specifically to immobilized carbohydrates and that the glycol groups prevented nonspecific protein adsorption. Carbohydrate arrays presenting ten monosaccharides were then evaluated by profiling the binding specificities of several lectins. These arrays were also used to determine the inhibitory concentrations of soluble carbohydrates for lectins and to characterize the substrate specificity of beta-1,4-galactosyltransferase. Finally, a strategy for preparing arrays with carbohydrates generated on solid phase is shown. This surface engineering strategy will permit the preparation and evaluation of carbohydrate arrays that present diverse and complex structures.     

Surface plasmon resonance imaging studies of protein-carbohydrate interactions

Carbohydrate arrays fabricated on gold films were used to study carbohydrate-protein interactions with surface plasmon resonance (SPR) imaging. An immobilization scheme consisting of the formation of a surface disulfide bond was used to attach thiol-modified carbohydrates onto gold films and to fabricate carbohydrate arrays. The carbohydrate attachment steps were characterized using polarization modulation Fourier transform infrared reflection absorption spectroscopy; and poly(dimethylsiloxane) microchannels were used to immobilize probe compounds at discrete locations on a gold film. The binding of the carbohydrate-binding proteins concanavalin A (ConA) and jacalin to arrays composed of the monosaccharides mannose and galactose was monitored with SPR imaging. SPR imaging measurements were employed to accomplish the following: (i) construct adsorption isotherms for the interactions of ConA and jacalin to the carbohydrate surfaces, (ii) monitor protein binding to surfaces presenting different compositions of the immobilized carbohydrates, and (iii) measure the solution equilibrium dissociation constants for ConA and jacalin toward mannose and galactose, respectively. Adsorption coefficients (K(ADS)) of 2.2 +/- 0.8 x 10(7) M(-)(1) and 5.6 +/- 1.7 x 10(6) M(-)(1) were obtained for jacalin adsorbing to a galactose surface and ConA adsorbing to a mannose surface, respectively. The solution equilibrium dissociation (K(D)) constant for the interaction of jacalin and galactose was found to be 16 +/- 5 microM, and for ConA and mannose was found to be 200 +/- 50 microM.     

Design of biomolecular interface for detecting carbohydrate and lectin weak interactions

A hybrid functional biomolecular interface designed at a molecular size level is very effective at capturing an analyte with high sensitivity even if the interaction is very weak, as when detecting proteins with carbohydrate. We designed and processed a protein (lectin) recognition molecular interface taking the following points into consideration: (1) the height (molecular length) difference between the capturing and spacer molecules; (2) the ratio of capturing molecules in the recognition interface. When the height difference between the maltoside part (Concanavalin A (Con A) recognition group) and the OH group terminated spacer molecules exceeded (>(CH(2))(6)), the association rate constant (k(a)) became larger (k(a)(1/Ms): ～2.6 times) and the dissociation constant (K(D)) became much smaller (K(D)(M): 1.0 × 10(-6): ～0.17 times) compared with the similar heights (lengths) of both molecular interfaces. With regard to maltoside density, a 100% maltoside monolayer was unsuitable for detecting Con A. We constructed a nanostructured recognition site with a maltoside part of 10%, which was the most suitable ratio for Con A detection. The binding interaction between Con A and the maltoside group was changed from monovalent binding to bivalent binding when the maltoside part was diluted in the recognition interface. From electrochemical measurements, even though there was a small amount of maltoside component on the suitable recognition monolayer, quality similar to that of 100% maltoside was observed.     

A self-assembled monolayer based electrochemical immunosensor for detection of leukemia K562A cells

A novel strategy to quantify the cell number of leukemia K562A cells using electrochemical immunosensor was developed by effective surface immunoreaction between P-glycoprotein (P-gp) on cell membrane and P-gp mouse monoclonal antibody bound on an epoxysilane monolayer modified glassy carbon electrode. The surface morphologies of the epoxysilane monolayer and the bound antibodies were studied with atomic force microscopy. The binding of target K562A cells onto the immobilized antibodies increased the electron-transfer impedance of electrochemical probe, which depended linearly on the cell concentration in the range of 5.0 × 10⁴–1.0 × 10⁷ cells mL⁻¹. The detection limit of the immunosensor was 7.1 × 10³ cells mL⁻¹. The proposed strategy showed acceptable reproducibility with an RSD of 3.4% for the linear slope and good precision with the RSD of 3.7% and 3.0% examined at the cell concentrations of 2.0 × 10⁶ and 1.0 × 10⁷ cells mL⁻¹.     

Electrospray mass spectrometry of neuac oligomers associated with the c fragment of the tetanus toxin

The Clostridial neurotoxins, botulinum and tetanus, gain entry into motor neurons by binding to the sialic or N-acetylneuraminic acid (NeuAc) residues of gangliosides and specific protein receptors attached to the cell's surface. While the C-fragment of tetanus toxin (TetC) has been identified to be the ganglioside binding domain, remarkably little is known about how this domain discriminates between the structural features of different gangliosides. We have used electrospray ionization mass spectrometry (ESI-MS) to examine the formation of complexes between TetC and carbohydrates containing NeuAc groups to determine how NeuAc residues contribute to ganglioside binding. ESI-MS was used to obtain an estimate of the dissociation constants (KD values) for TetC binding to a number of related NeuAc-containing carbohydrates (sialyllactose and disialyllactose), as well as six (NeuAc)n oligomers (n = 1-6). KD values were found to range between approximately 10-35 microM. The strength of the interactions between the C fragment and (NeuAc)n are consistent with the topography of the targeting domain of tetanus toxin and the nature of its carbohydrate binding sites. These results suggest that the targeting domain of tetanus toxin contains two binding sites that can accommodate NeuAc (or a dimer) and that NeuAc may play an important role in ganglioside binding and molecular recognition, a process critical for normal cell function and one frequently exploited by toxins, bacteria, and viruses to facilitate their entrance into cells.     

Carbohydrate–protein interactions and their biosensing applications

Carbohydrate recognition is clearly present throughout nature, playing a major role in the initial attachment of one biological entity to another. The important question is whether these prevalent interactions could provide a real suitable alternative to the use of antibodies or nucleic acid for detection and identification. Currently, examples of carbohydrates being employed in biological detection systems are limited. The challenges of using carbohydrate recognition for detection mainly come from the weak affinity of carbohydrate–protein interactions, the lack of versatile carbohydrate scaffolds with well-defined structures, and the less developed high-information-content, real-time, and label-free assay technology. In this review, we focus on discussing the characteristics of carbohydrate–protein interactions in nature and the methods for carbohydrate immobilization based on surface coupling chemistry in terms of their general applicability for developing carbohydrate- and lectin-based label-free sensors. Furthermore, examples of innovative design of multivalent carbohydrate–protein interactions for sensor applications are given. We limit our review to show the feasibility of carbohydrate and lectin as recognition elements for label-free sensor development in several representative cases to formulate a flexible platform for their use as recognition elements for real-world biosensor applications.     

The potent anti-HIV protein cyanovirin-N contains two novel carbohydrate binding sites that selectively bind to Man(8) D1D3 and Man(9) with nanomolar affinity: implications for binding to the HIV envelope protein gp120.

Cyanovirin-N (CVN) is a monomeric 11 kDa cyanobacterial protein that potently inactivates diverse strains of human immunodeficiency virus (HIV) at the level of cell fusion by virtue of high affinity interactions with the surface envelope glycoprotein gp120. Several lines of evidence have suggested that CVN-gp120 interactions are in part mediated by N-linked complex carbohydrates present on gp120, but experimental evidence has been lacking. To this end we screened a comprehensive panel of carbohydrates which represent structurally the N-linked carbohydrates found on gp120 for their ability to inhibit the fusion-blocking activity of CVN in a quantitative HIV-1 envelope-mediated cell fusion assay. Our results show that CVN specifically recognizes with nanomolar affinity Man(9)GlcNAc(2) and the D1D3 isomer of Man(8)GlcNAc(2). Nonlinear least squares best fitting of titration data generated using the cell fusion assay show that CVN binds to gp120 with an equilibrium association constant (K(a)) of 2.4 (+/- 0.1) x 10(7) M(-1) and an apparent stoichiometry of 2 equiv of CVN per gp120, Man(8)GlcNAc(2) D1D3 acts as a divalent ligand (2 CVN:1 Man(8)) with a K(a) of 5.4 (+/- 0.5) x 10(7) M(-1), and Man(9)GlcNAc(2) functions as a trivalent ligand (3 CVN:1 Man(9)) with a K(a) of 1.3 (+/- 0.3) x 10(8) M(-1). Isothermal titration calorimetry experiments of CVN binding to Man(9)GlcNAc(2) at micromolar concentrations confirmed the nanomolar affinity (K(a) = 1.5 (+/- 0.9) x 10(8) M(-1)), and the fitted data indicated a stoichiometry equal to approximately one (1 Man(9):1 CVN). The 1:1 stoichiometry at micromolar concentrations suggested that CVN has not only a high affinity binding site-relevant to the studies at nM concentrations-but a lower affinity site as well that facilitates cross-linking of CVN-oligomannose at micromolar concentrations or higher. The specificity of CVN for Man(8) D1D3 and Man(9) over the D1D2 isomer of Man(8) indicated that the minimum structure required for high affinity binding comprises Manalpha1 --> 2Manalpha. By following the (1)H-(15)N correlation spectrum of (15)N-labeled CVN upon titration with this disaccharide, we unambiguously demonstrate that CVN recognizes and binds to the disaccharide Manalpha1 --> 2Manalpha via two distinct binding sites of differing affinities located on opposite ends of the protein. The high affinity site has a K(a) of 7.2 (+/- 4) x 10(6) M(-1) and the low affinity site a K(a) of 6.8 (+/- 4) x 10(5) M(-1) as determined by isothermal titration calorimetry. Mapped surfaces of the carbohydrate binding sites are presented, and implications for binding to gp120 are discussed.     

Glycomimetics and glycodendrimers as high affinity microbial anti-adhesins

Adhesion to epithelial surface is often the first step in bacterial and viral infection. In this process, the microbes use a variety of proteins for interaction with host carbohydrates presented as glycoconjugates on cell surfaces. Crystal structures of adhesin and lectin binding sites in complexes with oligosaccharide open the route for design and synthesis of glycomimetics, glycodendrimers, and glycopolymers that are able to block infection at an early stage.     

Molecular insight for the role of key residues of calreticulin in its binding activities: A computational study

Calreticulin (CRT) is localized to and has functions in multiple cellular compartments, including the cell surface, the endoplasmic reticulum, and the extracellular matrix. Mutagenesis studies have identified several residues on a concave β-sheet surface of CRT critical for CRT binding to carbohydrate and other proteins/peptides. How the mutations of these key residues in CRT affect the conformation and dynamics of CRT, further influencing CRT binding to carbohydrates and other proteins to signal the important biological activities remain unknown. In this study, we investigated the effect of three key point mutations (C105A, C137A and W319A) on CRT conformation and dynamics via atomistic molecular dynamics simulations. Results show that these three key residues mutations induced the changes of CRT local backbone flexibility and secondary structure of CRT N-domain, which could further affect CRT’s binding activity. C137A mutation led to dramatic decrease of the overall size of CRT due to the P-domain fold back to the globular domain and formed new inter-domain contacts, which can cause blockage of CRT’s binding with other large substrates. Furthermore, for CRT concave β-strand surface patch containing lectin binding site, CRT C105A, C137A and W319A point mutation resulted in the changes in solvent accessible surface area, key residues’ side chain atom positions and dynamical correlated motions between residues. All these changes could directly affect CRT binding behavior. Results of this study provide molecular and structural insights into understanding the role of key residues of CRT in its binding behavior.