SIgA Binding to Mucosal Surfaces Is Mediated by Mucin-Mucin Interactions

The oral mucosal pellicle is a layer of absorbed salivary proteins, including secretory IgA (SIgA), bound onto the surface of oral epithelial cells and is a useful model for all mucosal surfaces. The mechanism by which SIgA concentrates on mucosal surfaces is examined here using a tissue culture model with real saliva. Salivary mucins may initiate the formation of the mucosal pellicle through interactions with membrane-bound mucins on cells. Further protein interactions with mucins may then trigger binding of other pellicle proteins. HT29 colon cell lines, which when treated with methotrexate (HT29-MTX) produce a gel-forming mucin, were used to determine the importance of these mucin-mucin interactions. Binding of SIgA to cells was then compared using whole mouth saliva, parotid (mucin-free) saliva and a source of purified SIgA. Greatest SIgA binding occurred when WMS was incubated with HT29-MTX expressing mucus. Since salivary MUC5B was only able to bind to cells which produced mucus and purified SIgA showed little binding to the same cells we conclude that most SIgA binding to mucosal cells occurs because SIgA forms complexes with salivary mucins which then bind to cells expressing membrane-bound mucins. This work highlights the importance of mucin interactions in the development of the mucosal pellicle.     

M-ORBIS: Mapping of mOleculaR Binding sItes and Surfaces

M-ORBIS is a Molecular Cartography approach that performs integrative high-throughput analysis of structural data to localize all types of binding sites and associated partners by homology and to characterize their properties and behaviors in a systemic way. The robustness of our binding site inferences was compared to four curated datasets corresponding to protein heterodimers and homodimers and protein–DNA/RNA assemblies. The Molecular Cartographies of structurally well-detailed proteins shows that 44% of their surfaces interact with non-solvent partners. Residue contact frequencies with water suggest that ∼86% of their surfaces are transiently solvated, whereas only 15% are specifically solvated. Our analysis also reveals the existence of two major binding site families: specific binding sites which can only bind one type of molecule (protein, DNA, RNA, etc.) and polyvalent binding sites that can bind several distinct types of molecule. Specific homodimer binding sites are for instance nearly twice as hydrophobic than previously described and more closely resemble the protein core, while polyvalent binding sites able to form homo and heterodimers more closely resemble the surfaces involved in crystal packing. Similarly, the regions able to bind DNA and to alternatively form homodimers, are more hydrophobic and less polar than previously described DNA binding sites.     

Location of Ferritin-Labeled Concanavalin A Binding to Influenza Virus and Tumor Cell Surfaces

Concanavalin A (Con-A) was linked to ferritin with glutaraldehyde and chromatographed on Sepharose 6B to separate unconjugated Con-A and ferritin from covalently cross-linked molecules. Ehrlich ascites tumor cells were infected with WSA influenza virus, stained at intervals with the ferritin-labeled Con-A and examined by electron microscopy. The surfaces of most mature viruses were specifically stained, providing direct evidence that influenza viruses maturing in this cell type have exposed Con-A receptor sites. The ferritin cores of the staining reagent were found at an average distance of 21.3 nm from the virus membrane and 10.8 nm from the uninfected cell membrane. This finding was interpreted to mean that the population of Con-A receptor sites on influenza virus particles is located at an average distance from the virus membrane twice that of the population of Con-A receptor sites found on uninfected cells. The structural elements of viral membranes can provide a reliable means for evaluating electron microscopy staining reagents, thereby enhancing their usefulness as probes for the study of membrane relationships.     

Atomistic Simulation of Mineral Surfaces

Abstract

Atomistic simulation techniques are now able to model the structure of mineral surfaces at the atomic level. In this paper we begin to address the question of whether surface reactivity can be studied reliably by modelling the surface reactivity of calcite, fluorite and forsterite under aqueous conditions. We first used energy minimisation techniques to investigate the interaction between the minerals calcite and fluorite with water and methanoic acid. The relative adsorption energies suggest that methanoic acid preferentially adsorbs onto fluorite surfaces, while water adsorbs preferentially onto calcite as inferred from experiments on mineral separation. Molecular Dynamics simulations were also used to model the effect of temperature on the adsorption of water on the calcite {1014} and fluorite {111} surfaces. Furthermore we used these techniques to model point defect formation at surfaces. We are also interested in modelling the competition between associative and dissociative adsorption on mineral surfaces. Simulations of adsorption of water on the low-index forsterite surfaces have predicted the adsorption energies and equilibrium morphology. The calculated equilibrium morphology adequately reproduces the experimental morphology of forsterite suggesting that the relative stabilities of the surfaces, both unhydrated and hydroxylated, are calculated correctly.     

The Binding of Cations to the Surfaces of Cells from Early Chick Blastoderms

Cells of the early chick blastoderms are either preparing for or undergoing regulated morphogenetic movements which culminate in the formation of a three-layered embryo. Information on the changes in the physical-chemical properties of cell surfaces may help in the understanding of this process. The binding of magnesium, manganese, strontium, barium and lanthanum to surfaces of early embryonic cells was estimated by the changes induced by these cations in the cells' electrophoretic mobilities (EPM). Cells show a positive EPM at concentrations of MgCl₂ and MnCl₂ at 3 × 10⁻² M while SrCl₂, and BaCl₂ were not able to reverse the cells' charge at concentrations up to 6 × 10⁻² M. CaCl₃ reversed the cells' EPM at concentrations as low as 5 × 10⁻³ M.
Our results suggest that the surfaces of early embryonic cells have a high affinity for Mg and Mn. This is indicated by a reversal of polarity which cannot be detected in cells of differentiating or adult tissues at the cation concentrations used in these experiments.     

Fractal Binding and Dissociation Kinetics of Heart-Related Compounds on Biosensor Surfaces

A fractal analysis is presented for the binding and dissociation of different heart-related compounds in solution to receptors immobilized on biosensor surfaces. The data analyzed include LCAT (lecithin cholesterol acyl transferase) concentrations in solution to egg white apoA-I rHDL immobilized on a biosensor chip surface (), native, mildly oxidized, and strongly oxidized LDL in solution to a heparin-modified Au-surface of a surface plasmon resonance (SPR) biosensor (), and TRITC-labeled HDL in solution to a bare optical fiber surface (). Single-and dual-fractal models were used to fit the data. Values of the binding and the dissociation rate coefficient(s), affinity values, and the fractal dimensions were obtained from the regression analysis provided by Corel Quattro Pro 8.0 (). The binding rate coefficients are quite sensitive to the degree of heterogeneity on the sensor chip surface. Predictive equations are developed for the binding rate coefficient as a function of the degree of heterogeneity present on the sensor chip surface and on the LCAT concentration in solution and for the affinity as a function of the ratio of fractal dimensions present in the binding and the dissociation phases. The analysis presented provided physical insights into these analyte-receptor reactions occurring on different biosensor surfaces.     

Errors Introduced in Radioligand Binding Studies Due to Displaceable,Cation Dependent, [3H]prazosin Binding to Glass-Fibre Filters and Glass Surfaces

Abstract

[³H]prazosin not only specifically and homogeneously labels α₁-adrenoceptors, but also binds to glass surfaces and non-linearly to the glass-fibre filters, commonly used in radioligand binding experiments. Binding to filters can be modulated by unlabeled α-adrenergic compounds and cations. If no correction is applied for displaceable filter binding, analysis of [³H]prazosin binding experiments leads to erroneous results. Analysis of [³H]prazosin saturation experiments on guinea-pig cerebral cortex membranes with correction for filter binding before the non-linear fit procedure indicated that [³H]prazosin labels a homogeneous population of α₁-adrenoceptors (R_tot: 8.33 fmol˙mg^?1 wet tissue) with a dissociation constant of 1.28×10^?10 M. However, analysis of the same data after correction for non-specific binding, (determined in parallel experiments by adding 10 μM phentolamine to the incubation medium) resulted in a best fit to a model in which [³H]prazosin labels two α₁-adrenoceptor subpopulations (R₁: 15.0 fmol˙mg^?1 and R₂: 14.6 fmol˙mg^?1 wet tissue) with dissociation constants of respectively 1.78×10^?10 and 5.63×10^?9 M. The discrepancy between the two methods of analysis is due to displacement of the radioligand from the filters by phentolamine.

Prazosin and oxymetazoline are also able to displace filter-bound [³H]prazosin. The extent to which displaceable filter binding distorts the proper results depends on the actual magnitude of the error and also on the method of analysis.     

Production,Binding Characteristics and Protective Immunity of Monoclonal Antibody to Pneumococcal Type-9V Conjugate

A monoclonal antibody (MAb) to pneumococcal type-9V polysaccharide (PS) was produced using PS conjugated to inactivated pneumolysin as the immunogen. The MAb to 9V PS was of the IgG₁ subclass. The antigen-antibody reaction increased rapidly at low concentrations and reached a plateau at 10 μg PS/ml as measured by nephelometry of the group 9 PS against 9V MAb binding. In contrast, the binding of group 9 PS against rabbit 9V antiserum (AS) increased proportionally and continued to increase up to the highest concentration of PS tested (20 μg PS/ml). The 9V MAb reacted with all group 9 PSs (9A, 9L, 9N and 9V) by immunodiffusion. In the homologous 9V Ag-MAb reaction, there were marked differences in the inhibition of binding by the cross-reactive 9L PS (19.2% inhibition) and the 9N PS (0.2%). In contrast, inhibition of the homologous 9V Ag-rabbit AS binding by cross-reactive 9L and 9N PSs ranged from 57.8 to 62.7%. Removal of the O-acetyl group from 9V PS by alkali hydrolysis resulted in decreased binding with rabbit 9V AS. However, the binding reaction with 9V MAb was less affected by the loss of O-acetyl content. The 9V MAb was both opsonic and passively protected young mice against challenge with type-9V pneumococci.     

Conformational Antibody Binding to a Native,Cell-Free Expressed GPCR in Block Copolymer Membranes

G-protein coupled receptors (GPCRs) play a key role in physiological processes and are attractive drug targets. Their biophysical characterization is, however, highly challenging because of their innate instability outside a stabilizing membrane and the difficulty of finding a suitable expression system. We here show the cell-free expression of a GPCR, CXCR4, and its direct embedding in diblock copolymer membranes. The polymer-stabilized CXCR4 is readily immobilized onto biosensor chips for label-free binding analysis. Kinetic characterization using a conformationally sensitive antibody shows the receptor to exist in the correctly folded conformation, showing binding behaviour that is commensurate with heterologously expressed CXCR4.     

Theory and Simulations of Adhesion Receptor Dimerization on Membrane Surfaces

The equilibrium constants of trans and cis dimerization of membrane bound (2D) and freely moving (3D) adhesion receptors are expressed and compared using elementary statistical-thermodynamics. Both processes are mediated by the binding of extracellular subdomains whose range of motion in the 2D environment is reduced upon dimerization, defining a thin reaction shell where dimer formation and dissociation take place. We show that the ratio between the 2D and 3D equilibrium constants can be expressed as a product of individual factors describing, respectively, the spatial ranges of motions of the adhesive domains, and their rotational freedom within the reaction shell. The results predicted by the theory are compared to those obtained from a novel, to our knowledge, dynamical simulations methodology, whereby pairs of receptors perform realistic translational, internal, and rotational motions in 2D and 3D. We use cadherins as our model system. The theory and simulations explain how the strength of cis and trans interactions of adhesive receptors are affected both by their presence in the constrained intermembrane space and by the 2D environment of membrane surfaces. Our work provides fundamental insights as to the mechanism of lateral clustering of adhesion receptors after cell-cell contact and, more generally, to the formation of lateral microclusters of proteins on cell surfaces.     

Simultaneous Binding of the Anti-Cancer IgM Monoclonal Antibody PAT-SM6 to Low Density Lipoproteins and GRP78

The tumour-derived monoclonal IgM antibody PAT-SM6 specifically kills malignant cells by an apoptotic mechanism linked to the excessive uptake of plasma lipids. The mechanism is postulated to occur via the multi-point attachment of PAT-SM6 to the unfolded protein response regulator GRP78, located on the surface of tumour cells, coupled to the simultaneous binding of plasma low density lipoprotein (LDL). We prepared and characterised LDL and oxidized LDL using sedimentation velocity and small-angle X-ray scattering (SAXS) analysis. Enzyme-linked immunosorbent (ELISA) techniques indicated apparent dissociation constants of approximately 20 nM for the binding of LDL or oxidized LDL to PAT-SM6. ELISA experiments showed cross competition with LDL inhibiting PAT-SM6 binding to immobilised GRP78, while, in the reverse experiment, GRP78 inhibited PAT-SM6 binding to immobilized LDL. In contrast to the results of the ELISA experiments, sedimentation velocity experiments indicated relatively weak interactions between LDL and PAT-SM6, suggesting immunoabsorbance to the microtiter plate is driven by an avidity-based binding mechanism. The importance of avidity and the multipoint attachment of antigens to PAT-SM6 was further investigated using antigen-coated polystyrene beads. Absorption of GRP78 or LDL to polystyrene microspheres led to an increase in the inhibition of PAT-SM6 binding to microtiter plates coated with GRP78 or LDL, respectively. These results support the hypothesis that the biological action of PAT-SM6 in tumour cell apoptosis depends on the multivalent nature of PAT-SM6 and the ability to interact simultaneously with LDL and multiple GRP78 molecules clustered on the tumour cell surface.     

Identification of Distant Drug Off-Targets by Direct Superposition of Binding Pocket Surfaces

Correctly predicting off-targets for a given molecular structure, which would have the ability to bind a large range of ligands, is both particularly difficult and important if they share no significant sequence or fold similarity with the respective molecular target (“distant off-targets”). A novel approach for identification of off-targets by direct superposition of protein binding pocket surfaces is presented and applied to a set of well-studied and highly relevant drug targets, including representative kinases and nuclear hormone receptors. The entire Protein Data Bank is searched for similar binding pockets and convincing distant off-target candidates were identified that share no significant sequence or fold similarity with the respective target structure. These putative target off-target pairs are further supported by the existence of compounds that bind strongly to both with high topological similarity, and in some cases, literature examples of individual compounds that bind to both. Also, our results clearly show that it is possible for binding pockets to exhibit a striking surface similarity, while the respective off-target shares neither significant sequence nor significant fold similarity with the respective molecular target (“distant off-target”).     

Investigate the Binding of Catechins to Trypsin Using Docking and Molecular Dynamics Simulation

To explore the inhibitory mechanism of catechins for digestive enzymes, we investigated the binding mode of catechins to a typical digestive enzyme-trypsin and analyzed the structure-activity relationship of catechins, using an integration of molecular docking, molecular dynamics simulation and binding free energy calculation. We found that catechins with different structures bound to a conservative pocket S1 of trypsin, which is comprised of residues 189–195, 214–220 and 225–228. In the trypsin-catechin complexes, Asp189 by forming strong hydrogen bonding, and Gln192, Trp215 and Gly216 through hydrophobic interactions, all significantly contribute to the binding of catechins. The number and the position of hydroxyl and aromatic groups, the structure of stereoisomers, and the orientation of catechins in the binding pocket S1 of trypsin all affect the binding affinity. The binding affinity is in the order of Epigallocatechin gallate (EGCG) > Epicatechin gallate (ECG) > Epicatechin (EC) > Epigallocatechin (EGC), and 2R-3R EGCG shows the strongest binding affinity out of other stereoisomers. Meanwhile, the synergic conformational changes of residues and catechins were also analyzed. These findings will be helpful in understanding the knowledge of interactions between catechins and trypsin and referable for the design of novel polyphenol based functional food and nutriceutical formulas.     

Covalent Binding of BMP-2 on Surfaces Using a Self-assembled Monolayer Approach

Bone morphogenetic protein 2 (BMP-2) is a growth factor embedded in the extracellular matrix of bone tissue. BMP-2 acts as trigger of mesenchymal cell differentiation into osteoblasts, thus stimulating healing and de novo bone formation. The clinical use of recombinant human BMP-2 (rhBMP-2) in conjunction with scaffolds has raised recent controversies, based on the mode of presentation and the amount to be delivered. The protocol presented here provides a simple and efficient way to deliver BMP-2 for in vitro studies on cells. We describe how to form a self-assembled monolayer consisting of a heterobifunctional linker, and show the subsequent binding step to obtain covalent immobilization of rhBMP-2. With this approach it is possible to achieve a sustained presentation of BMP-2 while maintaining the biological activity of the protein. In fact, the surface immobilization of BMP-2 allows targeted investigations by preventing unspecific adsorption, while reducing the amount of growth factor and, most notably, hindering uncontrolled release from the surface. Both short- and long-term signaling events triggered by BMP-2 are taking place when cells are exposed to surfaces presenting covalently immobilized rhBMP-2, making this approach suitable for in vitro studies on cell responses to BMP-2 stimulation.     

Fractal Analysis of Binding and Dissociation Kinetics and Interactions of Cancer Markers on Biosensor Surfaces

A fractal analysis is presented for the binding and dissociation of different cancer markers on biosensor surfaces. The data analyzed include putrescine in solution to PDDA/APTES/MWCNT/Puo-modified GCE (glassy carbon electrode) () and vascular endothelial growth factor (VEGF) in solution to the soluble form of the VEGF receptor (SFlt-1 or VEGF-1) immobilized on a sensor chip (). Single- and dual-fractal models were used to fit the data. Values of the binding and dissociation rate coefficient(s), affinity values, and the fractal dimensions were obtained from the regression analysis provided by Corel Quattro Pro 8.0 (). The binding rate coefficients and the affinity values are sensitive to the degree of heterogeneity on the sensor chip surface. Predictive equations are developed for the binding rate coefficient as a function of the heterogeneity present on the biosensor chip surface. The analysis presented provides physical insights into these cancer biomarker-receptor reactions occurring on the different biosensor surfaces.     

Specific Fluorine Labeling of the HyHEL10 Antibody Affects Antigen Binding and Dynamics

To more fully understand the molecular mechanisms responsible for variations in binding affinity with antibody maturation, we explored the use of site specific fluorine labeling and (19)F nuclear magnetic resonance (NMR). Several single-chain (scFv) antibodies, derived from an affinity-matured series of anti-hen egg white lysozyme (HEL) mouse IgG1, were constructed with either complete or individual replacement of tryptophan residues with 5-fluorotryptophan ((5F)W). An array of biophysical techniques was used to gain insight into the impact of fluorine substitution on the overall protein structure and antigen binding. SPR measurements indicated that (5F)W incorporation lowered binding affinity for the HEL antigen. The degree of analogue impact was residue-dependent, and the greatest decrease in affinity was observed when (5F)W was substituted for residues near the binding interface. In contrast, corresponding crystal structures in complex with HEL were essentially indistinguishable from the unsubstituted antibody. (19)F NMR analysis showed severe overlap of signals in the free fluorinated protein that was resolved upon binding to antigen, suggesting very distinct chemical environments for each (5F)W in the complex. Preliminary relaxation analysis suggested the presence of chemical exchange in the antibody-antigen complex that could not be observed by X-ray crystallography. These data demonstrate that fluorine NMR can be an extremely useful tool for discerning structural changes in scFv antibody-antigen complexes with altered function that may not be discernible by other biophysical techniques.     

Crowded waters: short-term response of invertebrate drift to water abstraction

Water abstraction modifies the environmental conditions of stream ecosystems, which can affect invertebrate assemblages by altering drift. We examined this issue with a before–after–control–impact design experiment in which we diverted 90% of the natural flow from a headwater stream. We measured flow-reduction effects on drift densities (animals/m³), total drift rates (animals leaving the reach per hour) and net balance of invertebrates entering or leaving the Impact reach. We also identified the specific taxa and traits that drove these responses. The sudden decrease in flow promoted a 12-fold increase in overall drift density at the Impact reach, which was primarily driven by filterers, shredders and taxa associated with fast velocities, such as simulids. By contrast, drift densities of other abundant taxa, such as Baetis, Esolus and chironomids, increased less than what could be expected from the magnitude of flow reduction. While drift rates remained unchanged after water abstraction, the Impact reach became a net invertebrate exporter indicating that many taxa drifted actively as a response to stressful conditions rather than passively, which would be reduced by water abstraction. Therefore, our results suggest that water abstraction influences drift, with potentially important consequences for the invertebrate assemblages and ecosystem processes further downstream.