Chemoenzymatic synthesis and lectin recognition of a selectively fluorinated glycoprotein

A chemoenzymatic glycosylation remodeling method for the synthesis of selectively fluorinated glycoproteins is described. The method consists of chemical synthesis of a fluoroglycan oxazoline and its use as donor substrate for endoglycosidase (ENGase)-catalyzed transglycosylation to a GlcNAc-protein to form a homogeneous fluoroglycoprotein. The approach was exemplified by the synthesis of fluorinated glycoforms of ribonuclease B (RNase B). An interesting finding was that fluorination at the C-6 of the 6-branched mannose moiety in the Man3GlcNAc core resulted in significantly enhanced reactivity of the substrate in enzymatic transglycosylation. A structural analysis suggests that the enhancement in reactivity may come from favorable hydrophobic interactions between the fluorine and a tyrosine residue in the catalytic site of the enzyme (Endo-A). SPR analysis of the binding of the fluorinated glycoproteins with lectin concanavalin A (con A) revealed the importance of the 6-hydroxyl group on the α-1,6-branched mannose moiety in con A recognition. The present study establishes a facile method for preparation of selectively fluorinated glycoproteins that can serve as valuable probes for elucidating specific carbohydrate–protein interactions.     

A new application of ellipsometry: assessment of carcinoembryonic antigen interaction with lectins at a liquid-solid interface

Lectins were specifically adsorbed on a metallized glass slide coated with carcinoembryonic antigen. The degree of interaction was rapidly determined by measuring the thickness of the adsorbed lectin layer with an ellipsometer.     

Computer simulations of extractant primary amine N1923 and N1923 hydrochloride salt at water/chloroform interface

A molecular dynamics (MD) simulation has been carried out to investigate the structural and dynamical properties of extractant methyloctadecyl amine (N1923), and its hydrochloride salt at water/chloroform interface. The simulation results show that both N1923 molecule and protonated N1923 ion can self-assembly form a monolayer at the interface like most non-ionic or cationic surfactants. It is not only observed that the headgroup N atom of protonated N1923 ion arranges more tightly and orderly than that of N1923 molecule but also verified that the protonated headgroup of N1923 ion has stronger hydration ability than the polar headgroup of N1923 molecule.     

Microanalysis of N-linked oligosaccharides in a glycoprotein by capillary liquid chromatography/mass spectrometry and liquid chromatography/tandem mass spectrometry

We have studied rapid and simple sugar mapping using liquid chromatography/electrospray ionization mass spectrometry (LC/MS) equipped with a graphitized carbon column. The oligosaccharide mixture was separated on the basis of the sequence, branching structure, and linkage, and each oligosaccharide was characterized based on its molecular mass. In this study we demonstrated the usefulness of capillary LC/MS (CapLC/MS) and capillary liquid chromatography/tandem mass spectrometry (CapLC/MS/MS) as sensitive means for accomplishing the structural analysis of oligosaccharides in a low-abundance glycoprotein. The carbohydrate heterogeneity and molecular mass information of each oligosaccharide can be readily obtained from CapLC/MS of a small amount of glycoprotein. CapLC/MS/MS provided b-ion series, which is informative with regard to monosaccharide sequence. Exoglycosidase digestion followed by CapLC/MS elucidated a carbohydrate residue linkage. Using this method, we characterized N-linked oligosaccharides in hepatocyte growth factor produced in mouse myeloma NS0 cells as the complex-type bi-, tri-, and tetraantennary terminated with N-glycolylneuraminic acids and alpha-linked galactose residues. Sugar mapping with CapLC/MS and CapLC/MS/MS is useful for monitoring glycosylation patterns and for structural analysis of carbohydrates in a low-abundance glycoprotein and thus will become a powerful tool in biological, pharmaceutical, and clinical studies.     

Characterization of glycoprotein digests with hydrophilic interaction chromatography and mass spectrometry

A new hydrophilic interaction chromatography (HILIC) column packed with amide 1.7 μm sorbent was applied to the characterization of glycoprotein digests. Due to the impact of the hydrophilic carbohydrate moiety, glycopeptides were more strongly retained on the column and separated from the remaining nonglycosylated peptides present in the digest. The glycoforms of the same parent peptide were also chromatographically resolved and analyzed using ultraviolet and mass spectrometry detectors. The HILIC method was applied to glyco-profiling of a therapeutic monoclonal antibody and proteins with several N-linked and O-linked glycosylation sites. For characterization of complex proteins with multiple glycosylation sites we utilized 2D LC, where RP separation dimension was used for isolation of glycopeptides and HILIC for resolution of peptide glycoforms. The analysis of site-specific glycan microheterogeneity was illustrated for the CD44 fusion protein.     

In situ measurement of circular dichroism of DNA adsorbing onto a solid surface

The adsorption process of DNA dissolved in aqueous solutions onto the surface of polyethyleneimine (PEI) has been examined by the observation of in situ circular dichroism (CD), including time-resolved measurements, to elucidate the conformation of DNA at the liquid/solid interface. The adsorption process can be characterized by two stages that are characterized in terms of CD. In the first stage, time (t)<700 s, a slight change in the time-resolved CD spectra of DNA is observed, whereas the value of the induced CD of the dye intercalated in DNA is constant. This result can be explained by the interaction between DNA and PEI during the adsorption at the liquid/solid interface. The weakness of the interaction is attributed to the geometrical restriction of this interface. In the second stage, t>700 s, where no further adsorption occurs, a change in the induced CD as well as in the CD of DNA is observed. This change in the induced CD can be interpreted as a significant conformational change of DNA for stabilizing the ion complex with PEI.     

固气界面上花菁染料J-聚集体的研究

研究固气界面上花菁染料J-聚集体的光学性质、形貌特点和形成机制。以花菁染料为研究对象,在云母基底表面制备花菁染料的超分子J-聚集体,根据J-聚集体独特的光学性能,通过紫外吸收光谱,荧光光谱,荧光显微镜等对云母界面上的花菁染料聚集体进行光谱测量和形貌表征。结果表明：云母／空气界面上的花菁染料聚集体在580nm处出现相对于染料单体红移且狭窄的强吸收峰,而在583nm处出现一个伴随微弱Stokes位移的荧光峰,这些结果符合吸附在基质上的J-聚集体的光学特征,同时荧光显微镜观察表明云母基底上分散分布着2～4μm微晶棒状的聚集体。结果证明：花菁染料能够在云母／空气界面生成超分子J-聚集体,其形成机制是通过带正电荷的花菁染料分子与云母基底表面带负电荷的空穴通过外延相互作用形成的。     

Incorporation of beta-lactoglobulin in a lipid/porphyrin monolayer at the air--water interface

A catanionic lipid/porphyrin monolayer was formed at the air-water interface by the tetra-anionic porphyrin, tetra-sodium-meso-tetra(4-sulfonatophenyl)porphine (TSPP), mixed with the cationic lipid dioctadecyldimethylammonium bromide (DODAB) in a 1:4 molar ratio. This binary mixture (TSPP/4DODAB) was used as the incorporation matrix of beta-lactoglobulin (betaLG). Binary and ternary systems (TSPP/4DODAB/zbetaLG, where z stands for the number of protein residues per TSPP) were characterized by surface pressure versus area (pi-A) measurements and by Brewster angle microscopy (BAM) observation at the air-water interface. Pi-A measurements and BAM images show that protein is incorporated in the expanded regime of the monolayer and is gradually expelled upon compression at high surface pressures. The successive compression-expansion cycles indicate that the protein under adsorbed to the floating film is reincorporated after the expansion of the monolayer. At low subphase pH, TSPP tends to aggregate decreasing the interaction with DODAB molecules. Electrostatic and hydrophobic interactions are responsible for the presence of betaLG at the interfacial film.     

Cell surface proteins during early Xenopus development: analysis of cell surface proteins and total glycoproteins provides evidence for a maternal glycoprotein pool

Summary The populations of cell surface proteins and total glycoproteins were investigated in early Xenopus embryos through lectin staining, affinity binding of glycoproteins to lectins, and use of a succinimide ester to biotinylate cell surface molecules. Lectin staining shows that the egg is endowed with a thick layer of surface glycoprotein, and that glycoprotein is immediately detected on the newly formed membranes of nascent blastomeres. The amount of glycoprotein found in eggs and early embryos remains constant, and electrophoretic analysis reveals no changes in abundant lectin-binding glycoproteins through the neurula stage. In contrast, the amount of cell surface protein increases dramatically from the 2-cell to the gastrula stages. Despite this quantiative increase, only a small number of differences in cell surface proteins were detected during this period. A series of bands was detected which appears to be specific to the outer surface of the embryo. Because the populations of surface proteins and of total glycoproteins overlap to a great extent, the increase in cell surface protein, in the absence of a change in total glycoprotein, indicates the presence of a maternal glycoprotein pool in the Xenopus egg, from which the cell surface proteins of embryonic blastomeres are recruited.     

Complex carbohydrate-lectin interaction at the interface: a model for cellular adhesion. II. Reactivity of both the oligosaccharide chain and sugar-binding domain of a glycoprotein lectin.

We describe studies of a new model cell adhesion system involving liposomes bearing lectins and the glycosphingolipid, asialomonosialoganglioside (asialoGM1). The model provides a simple analysis of experimental data to elucidate the mechanism of heterophilic cell-cell adhesion mediated by multiple protein-carbohydrate interactions. Phospholipid vesicles bearing the fatty acid conjugate of a glycoprotein lectin from Ricinus communis (RCAI vesicle) are shown to react with vesicles bearing the fatty acid conjugate of Concanavalin A (Con A) and asialoGM1 (Con A vesicle). The kinetics of aggregation and monosaccharide-induced disaggregation of the two types of vesicles were followed by monitoring the time-dependent change in turbidity. Depending on the surface density of the asialoGM1, 40-60% of the resulting precipitin complex was dissociable only in the presence of both RCAI-specific galactose and Con A-specific alpha-methyl-D-mannoside. Results indicate simultaneous participation of both the saccharide-binding domain and carbohydrate sequence of RCAI, a model cell adhesion molecule, to stabilize the encounter complex by two types of interactions. These findings support the possibility of stable cell-cell adhesion in vivo occurring via interactions between cell adhesion molecules on apposing cell-surface membranes.     

Preparation of ethylenediamine-anchored cellulose and determination of thermochemical data for the interaction between cations and basic centers at the solid/liquid interface

Cellulose was first modified with thionyl chloride, giving 99% substitution at C6, and then reacted with ethylene-1,2-diamine to produce 6-(2'-aminoethylamino)-6-deoxy-cellulose. From the 8.5% of nitrogen incorporated in the polysaccharide backbone, the amount of ethylene-1,2-diamine anchored per gram of modified cellulose was determined to be 3.03+/-0.01mmol. This chemically immobilized surface was characterized by FTIR, TG, (13)C NMR, and SEM techniques. The available basic nitrogen centers covalently bonded to the biopolymer skeleton were studied for copper, cobalt, nickel, and zinc adsorption from aqueous solutions and the respective thermal adsorption effects were determined by calorimetric titration. The ability to adsorb cations gave a capacity order of Co(2+)>Cu(2+)>Zn(2+)>Ni(2+) with affinities of 1.91+/-0.07, 1.32+/-0.07, 1.31+/-0.02, and 1.08+/-0.04mmol/g, respectively. The net thermal effects obtained from calorimetric titration measurements were adjusted to a modified Langmuir equation and the enthalpy of the interaction was calculated to give the following exothermic values: -20.8+/-0.05, -11.72+/-0.03, -7.32+/-0.01, and -6.27+/-0.02kJ/mol for Co(2+), Cu(2+), Zn(2+), and Ni(2+), respectively. With the exception of the entropic value for copper, the other thermodynamic data for these systems are favorable for cation adsorption from aqueous solutions at the solid/liquid interface, suggesting the use of this anchored biopolymer for cation removal from the environment.     

Membrane trafficking at the ER/Golgi interface: functional implications of RhoA and Rac1

N-WASP and Arp2/3, the components of the actin nucleation/polymerization signaling pathway governed by Cdc42, are located in Golgi membranes and regulate ER/Golgi interface protein transport. In the present study, we examined whether RhoA and Rac1, like Cdc42, are also involved in this early secretory pathway. Unlike Cdc42, RhoA and Rac1 were not observed in the Golgi complex of different clonal cell lines nor were they present in isolated Golgi membranes. Expression of constitutively active or inactive mutants of RhoA or Rac1 proteins in HeLa cells did not alter either the disassembly or the assembly of the Golgi complex following the addition or withdrawal of BFA, respectively, the ER-to-Golgi VSV-G transport or the Sar1(dn)-induced ER accumulation of Golgi proteins. Moreover, unlike Cdc42-expressing cells, the 15 degrees C-induced subcellular redistribution of the KDEL receptor remained unaltered. Only cells that constitutively express the activated Cdc42 mutant (Cdc42Q61L), or that were microinjected with activated Cdc42Q61L protein, exhibited a significant change in Golgi complex morphology. Collectively, our results demonstrate that RhoA and Rac1 are not located in the Golgi complex, nor do they directly or indirectly regulate membrane trafficking at the ER/Golgi interface. This finding, in turn, confirms that Cdc42 is the only Rho GTPase to have a specific function on the Golgi complex.     

Depletion interaction in colloid/polymer mixtures: application of density functional theory

Insert-route density functional approach (IRDFT), modified fundamental measure theory (MFMT) and thermodynamic perturbation theory (TPT1 and TPT2) are combined to study the depletion force between colloidal particles in hard sphere/hard sphere chain mixtures which represent a model of systems containing colloids dispersed in an athermal polymer solution. The predicted results are compared to simulations showing the reliability of the method used which captures the main characteristics of depletion interaction between colloids induced by polymers. Results of TPT2 are slightly more repulsive and better than that of TPT1 especially when the inter-particle distance is small than the diameter of polymer segment indicating the essential influence of the three-body correlations. Effects of the polymer density, polymer chain length and size ratio of colloid to polymer segment on the depletion force are studied in detail. Due to a little deterioration of the prediction in the high density region, further improvement is anticipated to better balance the competition between the excluded-volume effect and the chain connectivity.     

Novel optical PVC probes for on-site detection/determination of fluoroquinolones in a solid/liquid interface: application to the determination of Norfloxacin in aquaculture water

A novel optical disposable probe for screening fluoroquinolones in fish farming waters is presented, having Norfloxacin (NFX) as target compound. The colorimetric reaction takes place in the solid/liquid interface consisting of a plasticized PVC layer carrying the colorimetric reagent and the sample solution. NFX solutions dropped on top of this solid-sensory surface provided a colour change from light yellow to dark orange. Several metals were tested as colorimetric reagents and Fe(III) was selected. The main parameters affecting the obtained colour were assessed and optimised in both liquid and solid phases. The corresponding studies were conducted by visible spectrophotometry and digital image acquisition. The three coordinates of the HSL model system of the collected image (Hue, Saturation and Lightness) were obtained by simple image management (enabled in any computer). The analytical response of the optimised solid-state optical probe against concentration was tested for several mathematical transformations of the colour coordinates. Linear behaviour was observed for logarithm NFX concentration against Hue+Lightness. Under this condition, the sensor exhibited a limit of detection below 50 μM (corresponding to about 16 mg/mL). Visual inspection also enabled semi-quantitative information. The selectivity was ensured against drugs from other chemical groups than fluoroquinolones. Finally, similar procedure was used to prepare an array of sensors for NFX, consisting on different metal species. Cu(II), Mn(II) and aluminon were selected for this purpose. The sensor array was used to detect NFX in aquaculture water, without any prior sample manipulation.     

CD4-gp120 interaction interface - a gateway for HIV-1 infection in human: molecular network,modeling and docking studies

The major causative agent for Acquired Immune Deficiency Syndrome (AIDS) is Human Immunodeficiency Virus-1 (HIV-1). HIV-1 is a predominant subtype of HIV which counts on human cellular mechanism virtually in every aspect of its life cycle. Binding of viral envelope glycoprotein-gp120 with human cell surface CD4 receptor triggers the early infection stage of HIV-1. This study focuses on the interaction interface between these two proteins that play a crucial role for viral infectivity. The CD4–gp120 interaction interface has been studied through a comprehensive protein–protein interaction network (PPIN) analysis and highlighted as a useful step towards identifying potential therapeutic drug targets against HIV-1 infection. We prioritized gp41, Nef and Tat proteins of HIV-1 as valuable drug targets at early stage of viral infection. Lack of crystal structure has made it difficult to understand the biological implication of these proteins during disease progression. Here, computational protein modeling techniques and molecular dynamics simulations were performed to generate three-dimensional models of these targets. Besides, molecular docking was initiated to determine the desirability of these target proteins for already available HIV-1 specific drugs which indicates the usefulness of these protein structures to identify an effective drug combination therapy against AIDS.     

Effects of interaction with sulphur compounds and free volume in imidazolium-based ionic liquid on desulphurisation: a molecular dynamics study

Interaction energy with sulphur compounds and free volume in imidazolium-based ionic liquid were calculated by molecular dynamics (MD) simulations to examine their effects on desulphurisation. From microstructure analysis and energy contribution calculation, it is found that an increasing fractional free volume in ionic liquid and an enhancement of interaction with solute by tuning the structure of ionic liquid or oxidising sulphur compounds are favourable for desulphurisation, which allows more efficient packing of sulphur compounds in ionic liquids and more easily extraction of sulphur compounds from fuel. The MD results are in good agreement with experimental desulphurisation performance.     

Origin of cortical microtubules organized at M/G1 interface: recruitment of tubulin from phragmoplast to nascent microtubules

The origin of cortical microtubules (CMTs) was investigated in transgenic BY-2 cells stably expressing a GFP (green fluorescent protein) -tubulin fusion protein (BY-GT16). In a previous study, we found that CMTs were initially organized in the perinuclear regions but then elongated to reach the cell cortex where they formed bright spots, and that the appearance of parallel MTs from the bright spots was followed by the appearance of transverse MTs (Kumagai et al., Plant Cell Physiol. 42, 723-732, 2001). In this study, we investigated the migration of tubulin to the reorganization sites of CMTs at the M/G1 interface. After synchronization of the BY-GT16 cells by aphidicolin, the localization of GFP-tubulin was monitored and analyzed by deconvolution microscopy. GFP-tubulin was found to accumulate on the nuclear surface near the cell plate at the final stage of phragmoplast collapse. Subsequently, GFP-tubulin accumulated again on the nuclear surface opposite the cell plate, where nascent MTs elongated to the cell cortex. The significance of these observations on the mode of CMT organization is discussed.     

Nanoflow liquid chromatography coupled to matrix-assisted laser desorption/ionization mass spectrometry: sample preparation, data analysis, and application to the analysis of complex peptide mixtures

We report the development of a robust interface for off-line coupling of nano liquid chromatography (LC) to matrix-assisted laser desorption/ionisation-mass spectrometry (MALDI-MS) and its application to the analysis of proteolytic digests of proteins, both isolated and in mixtures. The interface makes use of prestructured MALDI sample supports to concentrate the effluent to a small sample plate area and localize the MALDI sample to a predefined array, thereby enriching the analyte molecules and facilitating automated MALDI-MS analysis. Parameters that influence the preparation of MALDI samples from the LC effluent were evaluated with regard to detection sensitivity, spectra quality, and reproducibility of the method. A procedure for data processing is described. The presented nano LC MALDI-MS system allowed the detection of several peptides from a tryptic digest of bovine serum albumin, at analyzed amounts corresponding to one femtomole of the digested protein. For the identification of native proteins isolated from mouse brain by two-dimensional gel electrophoresis, nano LC MALDI-MS increased the number of detected peptides, thereby allowing identification of proteins that could not be identified by direct MALDI-MS analysis. The ability to identify proteins in complex mixtures was evaluated for the analysis of Escherichia coli 50S ribosomal subunit. Out of the 33 expected proteins, 30 were identified by MALDI tandem time of flight fragment ion fingerprinting.     

Enzymatic synthesis of green notes with hydroperoxide-lyase from olive leaves and alcohol-dehydrogenase from yeast in liquid/gas reactor

Hydroperoxide-lyase is a suitable enzyme for the biocatalytic production of six-carbon aldehydes which are responsible for the “green notes” in many fruits and vegetables. The hydroperoxide-lyase isolated from olive leaves is used in this work. The effects of pH, temperature, and substrate and enzyme concentrations on hexenals generation were optimized. The main objective of this paper consists on the elaboration of a biocatalytic procedure for the synthesis of flavors with green odor characteristics. For this purpose an enzymatic liquid/gas reactor, where the synthesis of C6-compounds was coupled to their extraction, has been proposed. Hexenals were produced in two steps: (1) 13 hydroperoxy-linolenic acid was produced from hydrolyzed linseed oil in presence of soybean lipoxygenase. (2) 3Z- and 2E-hexenals (up to 0.36 g kg⁻¹ of reaction medium) were produced from 13 hydroperoxy-linolenic acid in presence of olive hydroperoxide-lyase at 50% yield. The hexenals were successfully reduced into their corresponding alcohols by adding yeast cells Saccharomyces cerevisiae containing alcohol-dehydrogenase activity to the same reactor. Significant amounts of 3Z-hexenol (up to 3.54 g kg⁻¹ of olive leaves) were produced and extracted at a yield of 47.7% and with high purity when permeabilized yeast cells were used.     

Antigen-antibody interface properties: Composition, residue interactions, and features of 53 non-redundant structures

The structures of protein antigen-antibody (Ag-Ab) interfaces contain information about how Ab recognize Ag as well as how Ag are folded to present surfaces for Ag recognition. As such, the Ab surface holds information about Ag folding that resides with the Ab-Ag interface residues and how they interact. In order to gain insight into the nature of such interactions, a data set comprised of 53 non-redundant 3D structures of Ag-Ab complexes was analyzed. We assessed the physical and biochemical features of the Ag-Ab interfaces and the degree to which favored interactions exist between amino acid residues on the corresponding interface surfaces. Amino acid compositional analysis of the interfaces confirmed the dominance of TYR in the Ab paratope-containing surface (PCS), with almost two fold greater abundance than any other residue. Additionally TYR had a much higher than expected presence in the PCS compared to the surface of the whole antibody (defined as the occurrence propensity), along with aromatics PHE, TRP, and to a lesser degree HIS and ILE. In the Ag epitope-containing surface (ECS), there were slightly increased occurrence propensities of TRP and TYR relative to the whole Ag surface, implying an increased significance over the compositionally most abundant LYS > ASN > GLU > ASP > ARG. This examination encompasses a large, diverse set of unique Ag-Ab crystal structures that help explain the biological range and specificity of Ag-Ab interactions. This analysis may also provide a measure of the significance of individual amino acid residues in phage display analysis of Ag binding.