A kinetic model of the agglutination process.

We propose a kinetic model of the aggregation process in a system consisting of two different types of particles. Aggregating particles (cells) are polyvalent and bear on the surface a huge number of binding sites for the other type of particles, ligands. The ligand is bivalent and has two identical active sites for binding to cells. The cross-linking of the cells by the ligands causes the aggregation phenomenon called agglutination. We obtained the analytical solution of this model task describing the time dependence of the aggregate mean size versus the composition of the system. The comparison of the analytical solution with the experimental data for the agglutination of bacterial cells by bivalent antibodies shows that the main factors affecting agglutination were correctly taken into account.     

The C-terminal peptide of thrombospondin-1 stimulates distinct signaling pathways but induces an activation-independent agglutination of platelets and other cells

A peptide from the C-terminal domain of thrombospondin-1 (4N1-1) has been proposed to stimulate platelet aggregation by a novel mechanism involving both an activation-independent agglutination and an activation-dependent, glycoprotein (GP) IIb/IIIa-mediated aggregation which involves GPVI signaling but does not involve CD47. The present study demonstrates that 4N1-1 stimulated a different pattern of signal transduction pathways than the GPVI agonist convulxin. Furthermore, 4N1-1-induced platelet aggregation was activation-independent and not dependent on GPVI or GPIIb/IIIa. Interestingly, 4N1-1 also stimulated activation-independent agglutination of different megakaryocytic and non-megakaryocytic cells. 4N1-1-induced cell agglutination but not platelet signaling was inhibited by anti-CD47 antibodies.     

Aggregation of sponge cells: 22. Species-specific reactivity of a lectin from the sponge Geodia cydonium

Single cells of the marine sponge Geodia cydonium aggregate species-specifically in the presence of a soluble aggregation factor to form large cell clumps. A lectin isolated from the same sponge species does not cause agglutination of Geodia cells but agglutinates only cells from heterologous species (e.g. Tethya lyncurium, Hemimycale columella, Pellina semitubulosa, Cacospongia scalaris, Verongia aerophoba). The process of agglutination is independent of divalent cations (they do not affect the agglutination process at concentrations up to 50 mM), occurs at 2°C, causes a reduction in the viability of the cells and results in an inhibition of programmed syntheses. The observed differences between the properties of cell agglutination (effect of a lectin in a heterologous system) and cell aggregation (effect of an aggregation factor in the homologous system) is discussed. Cell aggregation is dependent upon the presence of an aggregation factor, the presence of cations and an incubation temperature 2̃0°C; cell aggregation results in a stimulation of programmed syntheses. Cell agglutination requires a heterologous macromolecule (e.g. lectin), it is independent of divalent cations and causes inhibition of programmed syntheses in the cells.     

Effects of dextran molecular weight on red blood cell aggregation

The reversible aggregation of human red blood cells (RBC) by proteins or polymers continues to be of biologic and biophysical interest, yet the mechanistic details governing the process are still being explored. Although a depletion model with osmotic attractive forces due to polymer depletion near the RBC surface has been proposed for aggregation by the neutral polyglucose dextran, its applicability at high molecular mass has not been established. In this study, RBC aggregation was measured over a wide range of dextran molecular mass (70 kDa to 28 MDa) at concentrations ≤2 g/dL. Our results indicate that aggregation does not monotonically increase with polymer size; instead, it demonstrates an optimum dextran molecular mass around 200-500 kDa. We used a model for depletion-mediated RBC aggregation to calculate the expected depletion energies. This model was found to be consistent with the experimental results and thus provides new insight into polymer-RBC interactions.     

Exploring New Biological Functions of Amyloids: Bacteria Cell Agglutination Mediated by Host Protein Aggregation

Antimicrobial proteins and peptides (AMPs) are important effectors of the innate immune system that play a vital role in the prevention of infections. Recent advances have highlighted the similarity between AMPs and amyloid proteins. Using the Eosinophil Cationic Protein as a model, we have rationalized the structure-activity relationships between amyloid aggregation and antimicrobial activity. Our results show how protein aggregation can induce bacteria agglutination and cell death. Using confocal and total internal reflection fluorescence microscopy we have tracked the formation in situ of protein amyloid-like aggregates at the bacteria surface and on membrane models. In both cases, fibrillar aggregates able to bind to amyloid diagnostic dyes were detected. Additionally, a single point mutation (Ile13 to Ala) can suppress the protein amyloid behavior, abolishing the agglutinating activity and impairing the antimicrobial action. The mutant is also defective in triggering both leakage and lipid vesicle aggregation. We conclude that ECP aggregation at the bacterial surface is essential for its cytotoxicity. Hence, we propose here a new prospective biological function for amyloid-like aggregates with potential biological relevance.     

Enhanced concanavalin a agglutination of trypsinised erythrocytes is due to a specific class of aggregation

Using the output of a rotational viscometer as a continuous index of aggregation, we have shown previously that the concanavalin A agglutination of native human erythrocytes can be resolved into three distinct classes of aggregation, static, type I and type II. Static aggregation occurs in the absence of shear forces while both type I and II aggregations are shear-induced. We now report that the increased concanavalin A agglutination of trypsinised erythrocytes is attributable to a specific enhancement in the development of type II aggregation. While type II formation in native cell suspensions requires high concanavalin A concentrations and continual shearing, an indistinguishable type of aggregation develops in suspensions of trypsinised red cells at considerably lower lectin concentrations and in the absence of applied shear forces.     

Inhibition of spontaneous aggregation by wheat germ agglutinin in suspensions of BALB/c-3T3 and BHK21 tissue culture fibroblasts

A quantitative method of measuring cytoaggregation based on the Coulter electronic cell counter has been applied to the agglutination of BALB/c-3T3 and BHK21 tissue culture fibroblasts by wheat germ agglutinin. When agglutinin is added to transformed or trypsinized cell suspensions high aggregation rates are seen, and the results obtained are in close agreement with the well-known sensitivity of transformed cells to agglutination by lectins.In the absence of agglutinin, a small but reproducible amount of spontaneous aggregation can also be detected. It is related in some way to growth, since it is absent in suspension prepared from confluent (density-inhibited) cultures and is induced by either transfer to low density, additional serum, or transformation by viruses. Under conditions favouring growth, both BALB/c-3T3 and BHK21 cells show aggregation indices close to 25, corresponding to 10% of the maximum aggregation rate seen.This spontaneous aggregation is susceptible to inhibition by agglutinin. Inhibition occurs at low concentration (about 10 μg/ml) and aggregation rates thus pass through a minimum as the concentration of agglutinin is increased. Among the four different cell lines studied, sensitivity to inhibition is inversely related to agglutination. Thus 3T3 cells, which are barely agglutinated by 1 mg/ml of agglutinin, show 90% inhibition; polyoma virus-transformed BHK cells, which are agglutinated by 10 μg of agglutinin, show no inhibition at all.It is suggested that the agglutination of transformed cells is a consequence of their failure to respond to an inhibitory effect exerted by lectins upon an intrinsic adhesive property of the cell surface.     

Identification and characterization of Myxococcus xanthus mutants deficient in calcofluor white binding.

Calcofluor white is a fluorescent dye that binds to glycans and can be used to detect extracellular polysaccharide in Myxococcus xanthus and many other bacteria. We observed that an esg mutant showed less binding to calcofluor white than wild-type cells. Unlike S-motility mutants that share this phenotypic characteristic, the esg mutant exhibited S motility. This led us to identify a collection of nine new transposon insertion mutants, designated Cds (for calcofluor white binding deficient and S motile), which exhibited a phenotype similar to that of the esg strain. The Cds phenotype was found in 0.6% of the random insertion mutants that were screened. The Cds mutants were also found to be defective in cell-cell agglutination and developmental aggregation. Extracellular matrix fibrils composed of roughly equal amounts of polysaccharide and protein have been shown to be involved in agglutination, and electron microscopic examination showed that esg and the other Cds mutants lack the wild-type level of fibrils. Analysis of total M. xanthus carbohydrate demonstrated that polysaccharide content increased by about 50% when wild-type cells entered stationary phase. This induction was reduced or eliminated in all of the Cds mutants. The degree of polysaccharide deficiency in the Cds mutants correlated with the degree of loss of agglutination and dye binding as well as with the severity of the developmental aggregation defect. Preliminary genetic characterization demonstrated that the transposon insertion mutations in three of the Cds mutants (SR53, SR171, and SR200) were loosely linked. The results of this study suggest that many genes are involved in the production of calcofluor white binding polysaccharide material found in the extracellular matrix and that the polysaccharide is fibrillar. These results are also consistent with the findings of earlier studies which indicated that fibrils function to join agglutinating cells and to form multicellular fruiting aggregates.     

Chemically induced fusion of fresh human erythrocytes.

The fusion of fresh human erythrocytes was shown to be induced by calcium and phosphate ions. Prior treatment of erythrocytes with phosphate ion was a pre-requisite for the calcium-induced fusion. ATP levels in cells incubated with phosphate and calcium decreased 46 fold while cell-associated calcium increased 70 fold during 1 hour of incubation at 37°C as compared to cells which were incubated with calcium in saline. Our results suggest that a phosphate complex formed bridges between adjacent erythrocytes causing agglutination followed by aggregation of membrane proteins leading to protein-free areas of lipids. Where these protein-free areas are in close contact fusion may occur.     

The Complexity and Reversibility of the Timer for the Onset of Aggregation in Dictyostelium

A simple conditional method is used to probe the complexity of the rate limiting pathway, or timer, for the onset of aggregation in the cellular slime mold Dictyostelium discoideum . The method depends upon a difference in the time required for the onset of aggregation under two sets of environmental conditions in which one parameter (ionic strength) is varied, and involves reciprocal shifts between the two sets of conditions at short time intervals. Evidence is presented that the timer for the onset of aggregation is composed of at least two components operating in sequence. In addition, a shift in one direction during the progress of the initial component of the timer causes the timer to be reset to zero. Our results also indicate that resetting the timer delays the acquisition of several aggregation associated changes, including chemotactic responsiveness, cAMP binding sites on the cell surface, and EDTA-resistant agglutination.     

Identification and purification of an endogenous receptor for the lectin pallidin from Polysphondylium pallidum

We report the identification and purification of an endogenous carbohydrate-containing receptor of pallidin, the cell surface lectin implicated in mediating cell-cell adhesion in the cellular slime mold Polysphondylium pallidum. The receptor is identified in an aqueous extract of crude P. pallidum membranes as a potent inhibitor of the hemagglutination activity of pallidin. The inhibitor is purified to apparent homogeneity by affinity precipitation with pallidin followed by fractionation of the solubilized precipitate on Sepharose 4B. The hemagglutination inhibitor (HAI) is metabolically radiolabeled, indicating that it is a biosynthetic product of the amoebae and not an ingested food substance. The HAI is released into the extracellular medium by living, differentiated amoebae. This release is markedly facilitated by the addition of D-galactose, a specific saccharide that binds to pallidin. Hence, the HAI appears to have an in situ association with pallidin at the cell surface. Exogenously added HAI promotes the agglutination of differentiated amoebae in a gyrated suspension at very low concentrations. The results are consistent with a model of cell-cell adhesion in which the HAI is a multivalent, extracellular aggregation factor that is recognized by pallidin molecules on adjacent cells. The HAI would then be analogues to the aggregation factors identified in marine sponges.     

Cloning of the gene for myxobacterial hemagglutinin and isolation and analysis of structural gene mutations.

Myxobacterial hemagglutinin (MBHA) is a major developmentally induced protein that accumulates during the period of cellular aggregation in the bacterium Myxococcus xanthus. It has been shown that this lectin is targeted to the cell surface and periplasmic space of developmental cells, suggesting that it may play a role in cell-cell recognition or agglutination. We have cloned the structural gene for MBHA by using synthetic deoxyoligonucleotides containing sequences deduced from the amino acid sequence of MBHA and have used the cloned gene to construct strains of M. xanthus that cannot synthesize MBHA. We found that although the MBHA-deficient strains are delayed in their developmental time course, they are otherwise able to aggregate and sporulate normally. Our results suggest that MBHA may function to increase the efficiency of fruiting-body formation but is not a critical component of cellular aggregation.     

Simple method of recording the kinetics of cell agglutination and aggregation

The light scattering measure method for studying the kinetics of cell agglutination and aggregation under conditions of continuous and uniform stirring of suspension has been suggested.     

Aggregation of human RBC in binary dextran-PEG polymer mixtures

The present study was prompted by prior reports suggesting that small polymers can affect RBC aggregation induced by large macromolecules. Human RBC were washed and re-suspended in isotonic buffer solutions containing 72.5 kDa dextran (DEX 70, 2 g/dl) or 35.0 kDa poly(ethylene glycol) (PEG 35, 0.35 g/dl), then tested for aggregation in these solutions with and without various concentrations of smaller dextrans (10.5 and 18.1 kDa) or PEGs (3.35, 7.5 and 10.0 kDa). RBC aggregation was measured at stasis and at low shear using a photometric cone-plate system (Myrenne Aggregometer) and RBC electrophoretic mobility (EPM) in the various polymer solutions via an automated system (E4, HaSoTec GmbH). Our results indicate: (1) a heterogeneous effect with greater reduction of aggregation for small PEGs added to DEX 70 or for small dextrans added to PEG 35 than for small polymers of the same species; (2) for cells in DEX 70, aggregation decreased with increasing molecular mass and concentration of the small dextrans or PEGs; (3) for cells in PEG 35, small dextrans decreased aggregation with increasing molecular mass and concentration, whereas small PEGs had minimal effects with a minor influence of concentration and an inverse association between molecular mass and inhibition of aggregation. RBC EPM results indicated the expected polymer depletion for cells in DEX 70 or PEG 35, and that small PEGs yielded greater EPM values than small dextrans for cells in PEG 35 whereas the opposite was true for cells in DEX 70. Interpretation of our results in terms of the depletion model for RBC aggregations appears appropriate, and our findings are consistent with the assumption that inhibition of aggregation occurs because of an increase of small molecules in the depletion region. Our results thus suggest the merit of further studies of red blood cell aggregation in binary polymer systems.     

Effect of polyene antibiotics on the lectin-induced agglutination of transformed and untransformed cell lines.

Treatment of transformed Py3T3, SV101-3T3, and L1210 cells, as well as mitotic and Pronase-treated untransformed 3T3 cells, with the polyene antibiotics filipin, nystatin, and amphotericin B inhibited agglutination by wheat germ agglutinin. The effect of polyene antibiotic treatment was lectin and cell specific. Concanavalin A induced agglutination was not inhibited, wheat germ agglutination induced agglutination of untransformed 3T3 interphase cells was not influenced, and other aggregation phenomena, including those of erythrocytes with blood group specific antibodies or divalent cations, were unaffected by polyene treatments. This suggests that the formation of polyene-cholesterol complexes in transformed and erythrocyte cell membranes may specifically affect wheat germ agglutinin receptors and/or secondary events necessary for wheat germ agglutinin induced agglutination. Fluorescence studies of membrane filipin-cholesterol complexes showed that pretreating the cells with wheat germ agglutinin, but not concanavalin A, perturbed the fluorescence properties of filipin. Electron spin resonance studies with spin-labeled fatty acids revealed at best only a slight decrease in fatty acyl chain flexibility following filipin treatment. These studies indicate that there are not only quantitative differences between the agglutinability of transformed and untransformed cells with wheat germ agglutinin but that qualitative differences exist as well.     

Nonideality and protein thermal denaturation

We studied the thermal denaturation of eglin c by using CD spectropolarimetry and differential scanning calorimetry (DSC). At low protein concentrations, denaturation is consistent with the classical two-state model. At concentrations greater than several hundred microM, however, the calorimetric enthalpy and the midpoint transition temperature increase with increasing protein concentration. These observations suggested the presence of intermediates and/or native state aggregation. However, the transitions are symmetric, suggesting that intermediates are absent, the DSC data do not fit models that include aggregation, and analytical ultracentrifugation (AUC) data show that native eglin c is monomeric. Instead, the AUC data show that eglin c solutions are nonideal. Analysis of the AUC data gives a second virial coefficient that is close to values calculated from theory and the DSC data are consistent with the behavior expected for nonideal solutions. We conclude that the concentration dependence is caused by differential nonideality of the native and denatured states. The nondeality arises from the high charge of the protein at acid pH and is exacerbated by low buffer concentrations. Our conclusion may explain differences between van't Hoff and calorimetric denaturation enthalpies observed for other proteins whose behavior is otherwise consistent with the classical two-state model.     

Evidence for multiple mechanisms of interaction between wheat germ agglutinin and human platelets

Wheat germ agglutinin induced aggregation and secretion of serotonin from human platelets in plasma. This aggregation of platelets was blocked by ethylenediaminetetraacetate, azide or prostaglandin E₁. The secretion of serotonin was not affected by ethylenediaminetetraacetate but was inhibited by progstaglin E₁. Thus, wheat germ agglutinin acts on platelets in plasma as a true aggregating agent.Washed platelets showed increased light transmission when treated with the lectin which was not blocked by ethylenediaminetetraacetate or prostaglandin E₁. The capacity to inhibit platelet clumping by the above agents was restored if plasma was added back to the cell suspension. Washed platelets did not release serotonin under the conditions of cell clumping. Thus, in contrast to platelets in plasma, washed platelets are agglutinated by the lection.Platelets fixed in formaldehyde were not agglutinated by the lectin in the aggregometer but agglutination was observed in the microtiter plate. This agglutination may be mediated by interplatelet bridging. These results show that the same agent may act on platelets by different mechanisms depending on the state of the cell and its environment.     

On the Characterization of Intermediates in the Isodesmic Aggregation Pathway of Hen Lysozyme at Alkaline pH

Protein aggregation leading to formation of amyloid fibrils is a symptom of several diseases like Alzheimer’s, type 2 diabetes and so on. Elucidating the poorly understood mechanism of such phenomena entails the difficult task of characterizing the species involved at each of the multiple steps in the aggregation pathway. It was previously shown by us that spontaneous aggregation of hen-eggwhite lysozyme (HEWL) at room temperature in pH 12.2 is a good model to study aggregation. Here in this paper we investigate the growth kinetics, structure, function and dynamics of multiple intermediate species populating the aggregation pathway of HEWL at pH 12.2. The different intermediates were isolated by varying the HEWL monomer concentration in the 300 nM—0.12 mM range. The intermediates were characterized using techniques like steady-state and nanosecond time-resolved fluorescence, atomic force microscopy and dynamic light scattering. Growth kinetics of non-fibrillar HEWL aggregates were fitted to the von Bertalanffy equation to yield a HEWL concentration independent rate constant (k = (6.6±0.6)×10⁻⁵ s⁻¹). Our results reveal stepwise changes in size, molecular packing and enzymatic activity among growing HEWL aggregates consistent with an isodesmic aggregation model. Formation of disulphide bonds that crosslink the monomers in the aggregate appear as a unique feature of this aggregation. AFM images of multiple amyloid fibrils emanating radially from amorphous aggregates directly confirmed that on-pathway fibril formation was feasible under isodesmic polymerization. The isolated HEWL aggregates are revealed as polycationic protein nanoparticles that are robust at neutral pH with ability to take up non-polar molecules like ANS.     

How native proteins aggregate in solution: a dynamic Monte Carlo simulation

Aggregation of native proteins in solution is of fundamental importance with regard to both the processing and the utilization of proteins. In the present work, a dynamic Monte Carlo simulation has been performed to give a molecular insight into the way in which native proteins aggregate in solution and to explore means of suppressing aggregation, using two proteins of different compositions and conformations represented by a two-dimensional (2D) lattice model (HP model). It is shown that the native HP protein with accessible hydrophobic beads on its surface is prone to aggregation. The aggregation of this protein is intensified when the solution conditions favor the partially unfolded conformation as opposed to either the native or fully unfolded conformations. In this case, the partially unfolded proteins form the cores of aggregates, which may also encapsulate the native protein. One way to inhibit protein aggregation is to introduce polymers of appropriate hydrophobicity and chain length into the solution, such that these polymer molecules wrap around the hydrophobic regions of both the unfolded and folded proteins, thereby segregating the protein molecules. Our simulation is consistent with experimental observations reported elsewhere and provides a molecular basis for the behavior of proteins in liquid environments.     

F Pilus as f+ Antigen

Specific aggregate formation of F pili was observed, by electron microscopy, in a mixture of male Escherichia coli (or of isolated F pili) and anti-f(+) serum. Cellular appendages other than F pili never showed such aggregation when mixed with anti-f(+) serum. The f(+) agglutinability of male cells, as well as F piliation, was sensitive to mechanical agitation. The f(+) agglutination was inhibited when appropriate numbers of phage M12, capable of attaching to F pili, were mixed with the male culture before the addition of anti-f(+) serum. Correlation between f(+) agglutinability and the extent of F piliation was observed. It was concluded that the F pilus is the structure of the f(+) antigen and is responsible for f(+) agglutination.