Amyloid fibrils formation and amorphous aggregation in concanavalin A

We here report an experimental study on the thermal aggregation process of concanavalin A, a protein belonging to the legume lectins family. The aggregation process and the involved conformational changes of the protein molecules were followed by means of fluorescence techniques, light scattering, circular dichroism, zeta potential measurements and atomic force microscopy. Our results show that the aggregation process of concanavalin A may evolve through two distinct pathways leading, respectively, to the formation of amyloids or amorphous aggregates. The relative extent of the two pathways is determined by pH, as amyloid aggregation is favored at high pH values ( approximately 9), while the formation of amorphous aggregates is favored at low pH ( approximately 5). At difference from amorphous aggregation, the formation of amyloid fibrils requires significant conformational changes on the protein, both at secondary and tertiary structural level. To our knowledge, this is the first observation of amyloid fibrils from concanavalin A.     

Computational methods to predict protein aggregation

In most cases, protein aggregation stems from the establishment of non-native intermolecular contacts. The formation of insoluble protein aggregates is associated with many human diseases and is a major bottleneck for the industrial production of protein-based therapeutics. Strikingly, fibrillar aggregates are naturally exploited for structural scaffolding or to generate molecular switches and can be artificially engineered to build up multi-functional nanomaterials. Thus, there is a high interest in rationalizing and forecasting protein aggregation. Here, we review the available computational toolbox to predict protein aggregation propensities, identify sequential or structural aggregation-prone regions, evaluate the impact of mutations on aggregation or recognize prion-like domains. We discuss the strengths and limitations of these algorithms and how they can evolve in the next future.     

Direct visualization and profiling of protein misfolding and aggregation in live cells

Over the past few years, research tools have been developed to monitor the multistep protein aggregation process in live cells, a process that has been associated with a growing number of human diseases. Herein, we describe recent advances in methods that can either survey the distribution of aggregation at the level of the cellular proteome using mass spectroscopy or discern the multistep aggregation process of specific proteins of interest via fluorescence signals. Future development and application of such technologies are expected to provide insights on mechanisms, diagnosis, and treatment of diseases rooted in protein aggregation.     

Influence of concanavalin A on protein synthesis and protein release in BHK 21 cells

Addition of concanavalin A to baby-hamster kidney cells (strain BHK 21/C13) grown in Eagle's minimal essential medium devoid of serum but supplemented with insulin as growth-promoting factor, caused a marked reduction of the total protein content of the cells: as early as 1 h after treatment, the amount of protein decreased to about 60–70% of the values found in untreated cultures.Pulse-labelling experiments performed with [³H]leucine demonstrated that the uptake and incorporation of the labelled amino acid was not affected by the lectin up to 6 h after treatment. Pulse-chase experiments gave no evidence for an enhanced degradation of proteins.Examination of the supernatants of concanavalin A-treated cultures as well as their controls, pre-labelled with [³H]fucose and [¹⁴C]leucine revealed that the amount of membrane-derived glycoproteins which were shed into the culture medium was considerably higher in concanavalin A-treated cultures.However, the bulk of protein which accounts for the difference between lectin-treated and untreated cultures consists of intracellular material which was released during the cell harvest procedure. The loss of protein was prevented by α-methyl-d-mannoside (10^?2 M).Scanning electron microscopy of concanavalin A-treated cells showed a change from the smooth surface of the fibroblastic cells to a retracted one as early as 30 min after addition of the lectin. The surface of the altered cells was characterized by the presence of numerous bleb-like protuberances.The viability of the cells was not affected by concanavalin A-treatment during the course of the experiments.Experiments performed with variable concentrations of insulin excluded the possibility that the observed effects might be due to a competition between the lectin and the hormone.     

Probing protein ubiquitination in live cells

The reversible attachment of ubiquitin governs the interaction, activity and degradation of proteins whereby the type and target of this conjugation determine the biological response. The investigation of this complex and multi-faceted protein ubiquitination mostly relies on painstaking biochemical analyses. Here, we employ recombinant binding domains to probe the ubiquitination of proteins in living cells. We immobilize GFP-fused proteins of interest at a distinct cellular structure and detect their ubiquitination state with red fluorescent ubiquitin binders. With this ubiquitin fluorescent three-hybrid (ubiF3H) assay we identified HP1β as a novel ubiquitination target of UHRF1. The use of linkage specific ubiquitin binding domains enabled the discrimination of K48 and K63 linked protein ubiquitination. To enhance signal-to-noise ratio, we implemented fluorescence complementation (ubiF3Hc) with split YFP. Using in addition a cell cycle marker we could show that HP1β is mostly ubiquitinated by UHRF1 during S phase and deubiquitinated by the protease USP7. With this complementation assay we could also directly detect the ubiquitination of the tumor suppressor p53 and monitor its inhibition by the anti-cancer drug Nutlin-3. Altogether, we demonstrate the utility of the ubiF3H assay to probe the ubiquitination of specific proteins and to screen for ligases, proteases and small molecules controlling this posttranslational modification.     

Watching proteins in the wild: fluorescence methods to study protein dynamics in living cells

The advent of GFP imaging has led to a revolution in the study of live cell protein dynamics. Ease of access to fluorescently tagged proteins has led to their widespread application and demonstrated the power of studying protein dynamics in living cells. This has spurred development of next generation approaches enabling not only the visualization of protein movements, but correlation of a protein's dynamics with its changing structural state or ligand binding. Such methods make use of fluorescence resonance energy transfer and dyes that report changes in their environment, and take advantage of new chemistries for site-specific protein labeling.     

Cooperative conformational change and aggregation of concanavalin A in alkaline solutions

Three properties, the binding activity to Sephadex G-75, conformation, and the extent of aggregation, of concanvalin A. (con A) in alkaline pH solutions were examined with special attention to the time course and their time-independent final values. Highly cooperative conformational changes among four subunits were suggested which were coupled either with protonation in the case of demetallized con A or with metal binding in the case of metal-liganded con A. Midpoints of the conversions of the metal-liganded con A were about pH 8.8, 9.1 and 9.1 with respect to the activity, the conformational change and the aggregation, respectively. These values were about 1 pH higher than the corresponding values of demetallized con A: 7.9, 8.05 and 8.2. Each conversion took place in narrow pH ranges. The pH range for the loss of activity was found to be significantly lower than those of the other two. The aggregation was suggested not to be coupled with the conformational change. Dissociation into subunits did not take place indicating strong interactions among four subunits in the tetramer.     

Hemidesmosome protein dynamics in live epithelial cells

Hemidesmosomes mediate stable anchorage of epithelial cells to laminin-5 in the basement membrane zone and have been likened to spot-welds. Indeed, it has been assumed that hemidesmosomes are not dynamic, at least when compared to other matrix adhesion sites including focal contacts. We tested this notion by monitoring the fate of green fluorescent protein (GFP)-tagged human integrin beta4 subunit (GFP-hbeta4) and GFP-tagged 180-kD human bullous pemphigoid (BP) autoantigen (GFP-BP180) in live cultures of 804G cells that assemble numerous mature hemidesmosomes. In subconfluent 804G cells, both GFP-hbeta4 and GFP-BP180 protein clusters are not stable but assemble into and disassemble out of cat paw-like arrays at a relatively rapid rate. In confluent populations of 804G cells, although some cat paw-like clusters of both GFP-hbeta4 and GFP-BP180 are stable over periods of >60 min, other GFP-hbeta4 and GFP-BP180 protein arrays form and/or disappear during the same time period. Moreover, individual labeled particles show considerable motility in the plane of the membrane. Fluorescence recovery after photobleaching analyses provide a further indication of the dynamics of hemidesmosome proteins. In particular, bleached GFP-hbeta4 protein clusters in confluent cells recover signal within about 30 min, indicating that there is a relatively rapid turnover of hemidesmosome components in protein arrays clustered along the substratum attached surface of a cell. The rate of recovery is dependent on an intact microfilament system. In sharp contrast, bleached GFP-BP180 protein clusters in confluent cells fail to recover signal even when observed for longer than 60 min. To evaluate hemidesmosome protein dynamics in motile cells, we monitored GFP-hbeta4 and GFP-BP180 in 804G cells populating scrape wound sites in vitro. In these migratory cells, which lack mature hemidesmosomes, integrin beta4 subunit and BP180 protein clusters progressively assemble and disassemble into linear and cat-paw arrays. In summary, hemidesmosome protein clusters, like their counterparts in focal contacts, are dynamic. We discuss these results in relation to hemidesmosome functions.     

Calorimetric study of concanavalin A binding to saccharides

The binding of concanavalin A in the dimer form to various saccharides has been studied by calorimetry, and estimates of the binding enthalpy and binding constants have been calculated. Methyl α-d-mannoside and methyl α-d-glucoside have a — ΔH⁰ of 21.5 and 11.5 kJ/mol, respectively, at both pH 4 and 4.5. The p-nitrophenyl derivatives react with enthalpic values of 15.6 and 14.6 kJ/mol. The galactosepyranosides show no heat effects during mixing with the protein solutions.The apparent binding enthalpies calculated from the variations of the equilibrium constants with temperature are in good agreement with the values measured experimentally. The two binding sites of the dimer form of concanavalin A are equal and independent, and the low enthalpies obtained do not justify a large conformational change during the reaction.The binding reaction has also been estimated for other sugars normally contained in glycoproteins.     

Disulphide bond formation in food protein aggregation and gelation

In this short review we discuss the role of cysteine residues and cystine bridges for the functional aggregation of food proteins. We evaluate how formation and cleavage of disulphide bonds proceeds at a molecular level, and how inter- and intramolecular disulfide bonds can be detected and modified. The differences between heat-, high-pressure-, and denaturant-induced unfolding and aggregation are discussed. The effect of disulphide bonding between aggregates of proteins and protein mixtures on the functional macroscopic properties of space filling networks in protein gels is briefly presented.     

The attachment to human buccal epithelial cells by Candida albicans: An in vitro kinetic study using concanavalin A

The early in vitro kinetics of Candida albicans attachment to human buccal epithelial cells was studied with the aid of an adhesion assay and solutions of concanavalin A (Con A), a lectin which is capable of inhibiting yeast adhesion. Various saccharides and putative receptor analogues were also tested. Solutions of each single reagent were added to tubes containing aliquots of mucosal cells and germinated yeasts at the beginning of a 1-hour incubation period (time O) or at 10 minute intervals during the assay. The number of yeasts attached to 200 mucosal cells was subsequently determined microscopically. Yeast adhesion remained constant following addition of phosphate-buffered saline (PBS) at time 0 or at any time thereafter. However, addition of Con A at 0, 10 or 20 minutes of incubation decreased adhesion significantly to 38%, 45% and 63% of control values. This inhibitory effect dwindled as time of incubation prior to lectin addition increased and Con A could not inhibit adhesion significantly after twenty minutes. Results obtained with Con A using live germinated yeasts were similar to those obtained with formalin-killed C. albicans. The other reagents tested failed to decrease adhesion significantly. These included the putative receptor analogues fibronectin, N-acetyl-d-glucosamine and d-galactose, and several non-specific saccharides such as -d-methylglucopyranoside, d-ribose and d-xylose. It is suggested that in vitro attachment to human mucosal cells by C. albicans is inhibitable up to a defined point in time by a lectin with affinity for mannosecontaining surface moieties, but becomes non-reversible thereafter. This experimentally-observed irreversibility is independent of yeast cell viability.     

Residue-specific mobility changes in soluble oligomers of the prion protein define regions involved in aggregation

Prion (PrP) diseases are neurodegenerative diseases characterized by the formation of β-sheet rich, insoluble and protease resistant protein deposits (called PrP^Sc) that occur throughout the brain. Formation of synthetic or in vitro PrP^Sc can occur through on-pathway toxic oligomers. Similarly, toxic and infectious oligomers identified in cell and animal models of prion disease indicate that soluble oligomers are likely intermediates in the formation of insoluble PrP^Sc. Despite the critical role of prion oligomers in disease progression, little is known about their structure. In order, to obtain structural insight into prion oligomers, we generated oligomers by shaking-induced conversion of recombinant, monomeric prion protein PrP^c (spanning residues 90–231). We then obtained two-dimensional solution NMR spectra of the PrP^c monomer, a 40% converted oligomer, and a 94% converted oligomer. Heteronuclear single-quantum correlation (¹H–¹⁵N) studies revealed that, in comparison to monomeric PrP^c, the oligomer has intense amide peak signals in the N-terminal (residues 90–114) and C-terminal regions (residues 226–231). Furthermore, a core region with decreased mobility is revealed from residues ~127 to 225. Within this core oligomer region with decreased mobility, there is a pocket of increased amide peak signal corresponding to the middle of α-helix 2 and the loop between α-helices 2 and 3 in the PrP^c monomer structure. Using high-resolution solution-state NMR, this work reveals detailed and divergent residue-specific changes in soluble oligomeric models of PrP.     

A study on concanavalin a binding to human erythrocytes

Interactions of concanavalin A with human erythrocytes were studied using ¹²⁵I-labelled concanavalin A and a centrifugal technique with dibutyl phthalate which permitted complete separation of bound and free concanavalin A. Binding of ¹²⁵I-labelled concanavalin A to human erythrocytes was dependent on cell concentration, pH and temperature. Specificity of binding was confirmed by inhibition and dissociation studies with sugars and native concanavalin A. Positive cooperative binding of concanavalin A to human erythrocytes was observed at low concanavalin A concentrations (less than 1 μ/ml) in both buffers studied. Positive cooperativity at higher concanavalin A concentrations (more than 100 μ/ml) was seen in Tris-Hepes buffer but not in phosphate-buffered saline. Consistent with this cooperative effect was the observation that although dissociation of ¹²⁵I-labelled concanavalin A from the erythrocytes was complete in the presence of 1 mg/ml of the native lectin, release was inhibited by low concentrations (1 μ/ml). A comparison of concanavalin A binding with hemagglutination studies suggest that the amount of concanavalin A bound determines the rate of erythrocyte agglutination and the size of the aggregates formed.     

Inhibition of conjugation in Tetrahymena pyriformis by concanavalin A : Binding of concanavalin A to material secreted during starvation and to washed cells

A group of glycoproteins which form an insoluble complex with concanavalin A (ConA) are secreted during starvation of the mating types strain, as well as by the non-mating types of Tetrahymena pyriformis. These glycoproteins were isolated by Sepharose-ConA, characterized, and their relevance to the conjugation process studied. The isolated ConA binding proteins (CBM) contain about 26% of their total weight, a phenol sulfuric acid-positive material, presumably carbohydrates, and exhibit about 5–8 major bands by gel electrophoresis analysis. The possibility that ConA inhibits the conjugation process by precipitating with this material was tested. Addition of isolated CBM restored conjugation previously inhibited by ConA. However, incubation of isolated CBM with either of the mating types or with both did not have any effect on the kinetics of the conjugation process. Antibodies prepared against CBM-secreted by both mating types did not prevent conjugation when added to the conjugation medium. The data suggest that CBM does not directly participate in the conjugation process. Inhibition of conjugation by ConA is probably due to its interaction with specific membrane-bound glycoproteins.     

Internalization-competent influenza hemagglutinin mutants form complexes with clathrin-deficient multivalent AP-2 oligomers in live cells

Most membrane proteins are endocytosed through clathrin-coated pits via AP-2 adaptor complexes. However, little is known about the interaction of internalization signals with AP-2 in live cells in the absence of clathrin lattices. To investigate this issue, we employed cells cotransfected with pairs of antigenically distinct influenza hemagglutinin (HA) mutants containing different internalization signals of the YXXZ family. To enable studies on the possible association of the naturally trimeric HAs into higher order complexes via binding to AP-2, we exploited the inability of HAs from different influenza strains to form mutual trimers. Thus, we coexpressed HA pairs from different strains (Japan and X:31) bearing similar cytoplasmic tails mutated to include internalization signals. Using antibody-mediated immunofluorescence co-patching on live cells, we demonstrate that internalization-competent HA mutants form higher order complexes and that this clustering depends on the strength of the internalization signal. The clustering persisted in cells treated with hypertonic medium to disperse the clathrin lattices, as validated by co-immunoprecipitation experiments. The clustering of HAs bearing strong internalization signals appears to be mediated via binding to AP-2, as indicated by (i) the coprecipitation of alpha-adaptin with these HAs, even in hypertonically treated cells; (ii) the co-localization (after hypertonic treatment) of AP-2 with antibody-mediated patches of these mutants; and (iii) the dispersal of the higher order HA complexes following chlorpromazine treatment, which removes AP-2 from the plasma membrane. These results suggest that even in the absence of clathrin lattices, AP-2 exists in multivalent complexes capable of simultaneously binding several internalization signals from the same family.     

The role of Ia-bearing cells in T-cell proliferative response to concanavalin A

Ia-bearing cells are required for the concanavalin A (Con A)-induced T-lymphocyte proliferative response. We attempted to determine how and when the Ia-bearing cells are required in this response. As Ia-bearing cells, mitomycin C-treated nylon wool- and plastic dish-adherent cells (AACmc) were used. AACmc didn't lose their restoring capacity on treatment with anti-Thy-1, anti-Ig, but they did with anti-Ia. Using a Marbrook chamber system, it was shown that cellular contact between T cells and Ia-bearing cells is necessary for restoring the response. Results with AACmc addition after various times from Con A addition, or Ia-bearing cell elimination after various times from Con A addition indicate that the Ia-bearing cells are required for about 12 hr from the start of incubation with Con A. And it was shown that Con A-pretreated AACmc could stimulate Ia-depleted T cells, and allogenic AACmc could replace syngenic ones.