Role of Surface Electric Charge in Red Blood Cell Interactions

The role of the surface charge of human red blood cells (RBC's) in affecting RBC aggregation by macromolecules was studied by comparing the behavior of normal RBC's with that of RBC's treated with neuraminidase, which removes the sialic acids from the cell membrane and reduces the zeta potential. RBC aggregation in dextrans with different molecular weights (Dx 20, Dx 40, and Dx 80) was quantified by microscopic observation, measurement of erythrocyte sedimentation rate, and determination of low-shear viscosity. Dx 20 did not cause aggregation of normal RBC's, but caused considerable aggregation of neuraminidase-treated RBC's. Neuraminidase-treated RBC's also showed stronger aggregation than normal RBC's in Dx 40 and 80. Together with the electron microscopic findings that the intercellular distance in the RBC rouleaux varies with the molecular size of dextrans used, the present study indicates that the surface charge of RBC's inhibits their aggregation by dextrans and that the electrostatic repulsive force between cell surfaces may operate over a distance of 20 nm.     

用电导法研究悬浮介质中大分子物质对红血球细胞(RBC)聚集的一种抑制效应

通常认为悬浮介质中的大分子物质促进RBC聚集的桥联作用,使RBC聚集,但在高浓度时,大分子物质反而抑制RBC聚集。过去认为主要是高浓度大分子物质影响了RBC表面势能,但我们在研究中发现,在一定条件下低浓度的大分子物质也能抑制红细胞聚集。这说明:大分子物质对RBC聚集的抑制效应,不仅能通过改变RBC膜表面势能实观,还能通过其它途径实现,这种途径之一就是影响RBC膜的力学性质。     

Non-adsorbing macromolecules promote endothelial adhesion of erythrocytes with reduced sialic acids

Background

Abnormal adhesion of red blood cells (RBCs) to vascular endothelium is often associated with reduced levels of sialic acids on RBC membranes and with elevated levels of pro-adhesive plasma proteins. However, the synergistic effects of these two factors on the adhesion are not clear. In this work, we tested the hypothesis that macromolecular depletion interaction originating from non-adsorbing macromolecules can promote the adhesion of RBCs with reduced sialic acid content to the endothelium.

Methods

RBCs are treated with neuraminidase to specifically remove sialic acids from their surface followed by the evaluation of their deformability, zeta potential and membrane proteins. The adhesion of these enzyme-treated RBCs to cultured human umbilical vein endothelial cells (ECs) is studied in the presence of 70 or 500 kDa dextran with a flow chamber assay.

Results

Our results demonstrate that removal of sialic acids from RBC surface can induce erythrocyte adhesion to endothelial cells and that such adhesion is significantly enhanced in the presence of high-molecular weight dextran. The adhesion-promoting effect of dextran exhibits a strong dependence on dextran concentration and molecular mass, and it is concluded to originate from macromolecular depletion interaction.

Conclusion

These results suggest that elevated levels of non-adsorbing macromolecules in plasma might play a significant role in promoting endothelial adhesion of erythrocytes with reduced sialic acids.

General significance

Our findings should therefore be of great value in understanding abnormal RBC–EC interactions in pathophysiological conditions (e.g., sickle cell disease and diabetes) and after blood transfusions.     

Electric field-induced polarization of charged cell surface proteins does not determine the direction of galvanotaxis

Galvanotaxis, that is, migration induced by DC electric fields, is thought to play a significant role in development and wound healing, however, the mechanisms by which extrinsic electric fields orchestrate intrinsic motility responses are unknown. Using mammalian cell lines (3T3, HeLa, and CHO cells), we tested one prevailing hypothesis, namely, that electric fields polarize charged cell surface molecules, and that these polarized molecules drive directional motility. Negatively charged sialic acids, which contribute the bulk of cell surface charge, redistribute preferentially to the surface facing the direction of motility, as measured by labeling with fluorescent wheat germ agglutinin. We treated cells with neuraminidase to remove sialic acids; as expected, this decreased total cell surface charge. We also changed cell surface charge independent of sialic acid moieties, by conjugating cationic avidin to the surface of live cells. Neuraminidase inhibited the electric field-induced directional polarization of membrane ruffling and alpha4 integrin, while avidin treatment actually reversed the directional polarization of sialic acids. Neuraminidase treatment inhibited directionality but did not alter speed of motility. Surprisingly, avidin treatment did not significantly alter either directionality or speed of motility. Thus, our results demonstrate that electric field-induced polarization of charged species indeed occurs. However, polarization of the bulk of charged cell surface proteins is neither necessary nor sufficient to cause motility, thus contradicting the second part of our hypothesis. Because neuraminidase inhibited directional motility, we also conclude that sialic acids are required constituents of some cell surface molecule(s) through which electric fields mount a polarized transmembrane response.     

Electrophoretic and aggregation behavior of bovine, horse and human red blood cells in plasma and in polymer solutions

The electrophoretic mobility of native and glutaraldehyde-fixed bovine, human, and horse red blood cells (RBC) was investigated as a function of ionic strength (5-150 mM) and concentration of 464 kDa dextran (2 and 3 g/dl); RBC aggregation in autologous plasma and in dextran solutions was also measured. In agreement with previous observations, human and horse RBC form stable rouleaux whereas bovine RBC do not aggregate in either plasma or in dextran 464 kDa solutions. Electrophoretic measurements showed a species-dependent adsorption and depletion of dextran that can be theoretically evaluated. Adsorption of polymer is not a prerequisite for RBC aggregation (bovine RBC show the highest amount of adsorbed dextran yet do not aggregate). Aggregate formation thus occurs as long as the Gibbs free energy difference, given by the osmotic pressure difference between the bulk phase and the polymer-depleted region between two RBC, is larger than the steric and electrostatic repulsive energy contributed by the macromolecules present on the RBC surface. With increasing bulk-phase polymer concentration the depletion layer thickness decreases and the amount of adsorbed macromolecules increases, thereby resulting in an increase of the repulsive component of the interaction energy and decreased aggregation. We thus view electrophoretic measurements of RBC in various media as an important tool for understanding polymer behavior near the red cell surface and hence the mechanisms involved in RBC aggregation.     

Alpha-galactosidase stimulates acetylcholine receptor aggregation in skeletal muscle cells via PNA-binding carbohydrates

Aggregation of nicotinic acetylcholine receptors (AChRs) in skeletal muscle is an essential step in the formation of the mammalian neuromuscular junction. While proteins that bind to myotube receptors such as agrin and laminin can stimulate AChR aggregation in cultured myotubes, removal of cell surface sialic acids stimulates aggregation in a ligand-independent manner. Here, we show that removal of cell surface alpha-galactosides also stimulates AChR aggregation in the absence of added laminin or agrin. AChR aggregation stimulated by alpha-galactosidase was blocked by peanut agglutinin (PNA), which binds to lactosamine-containing disaccharides, but not by the GalNAc-binding lectin Vicia villosa agglutinin (VVA-B4). AChR aggregation stimulated by alpha-galactosidase potentiated AChR clustering induced by either neural agrin or laminin-1 and could be inhibited by muscle agrin. These data suggest that capping of cell surface lactosamines or N-acetyllactosamines with alpha-galactose affects AChR aggregation much as capping with sialic acids does.     

The release of radioactive nucleic acids and mucoproteins by trypsin and ethylenediaminetetra-acetate treatment of baby-hamster cells in tissue culture

Monolayers of baby-hamster kidney cells were grown on glass in tissue culture and harvested with trypsin or EDTA in order to investigate the cell surface macromolecules removed by these cell-disaggregating agents. The release of nucleic acids from the cells during the harvesting procedure was monitored by labelling the cellular RNA with [5-(3)H]uridine and the cellular DNA with [2-(14)C]thymidine. Treatment of the cells with EDTA was found to cause an increase in the permeability of the plasma membrane with 7.6% of the cellular RNA, but less than 1% of the cellular DNA, being released. Moreover, 61% of the cells harvested with EDTA were permeable to Trypan Blue. With crude trypsin, lysis of the cell occurred with the release of similar amounts of RNA and DNA amounting to about 11% of the total cellular nucleic acid. In contrast, crystalline trypsin released only 1% of the cellular nucleic acids. Since virtually all the cells (99%) after harvesting in crystalline trypsin were impermeable to Trypan Blue, this method was suitable for obtaining cell surface macromolecules without contamination by intracellular damage. [1-(14)C]Glucosamine was incorporated by the cells only into bound hexosamines and sialic acids. [By monitoring the release of radioactivity in high-molecular-weight material in such experiments a measure of the release of macromolecules containing amino sugars was obtained.] Of the total macromolecules containing amino sugars in the cells 33%, 24% and 13% were released when the cells were harvested with crude trypsin, crystalline trypsin or EDTA respectively. Crystalline trypsin also released 39% of the total sialic acid of the cell, whereas less than 1% of the cellular sialic acid was present in the EDTA-treated fraction. It is concluded that the macromolecules containing amino sugars released with crude trypsin and EDTA are likely to be heavily contaminated with intracellular material. However, the macromolecules released by crystalline trypsin appear to come from the cell surface.     

Effects of red blood cell aggregation on myocardial hematocrit gradient using two approaches to increase aggregation

The normal transmyocardial tissue hematocrit distribution (i.e., subepicardial greater than subendocardial) is known to be affected by red blood cell (RBC) aggregation. Prior studies employing the use of infused large macromolecules to increase erythrocyte aggregation are complicated by both increased plasma viscosity and dilution of plasma. Using a new technique to specifically alter the aggregation behavior by covalent attachment of Pluronic F-98 to the surface of the RBC, we have determined the effects of only enhanced aggregation (i.e., Pluronic F-98-coated RBCs) versus enhanced aggregation with increased plasma viscosity (i.e., an addition of 500 kDa dextran) on myocardial tissue hematocrit in rapidly frozen guinea pig hearts. Although both approaches equally increased aggregation, tissue hematocrit profiles differed markedly: 1) when Pluronic F-98-coated cells were used, the normal transmyocardial gradient was abolished, and 2) when dextran was added, the hematocrit remained at subepicardial levels for about one-half the thickness of the myocardium and then rapidly decreased to the control level in the subendocardial layer. Our results indicate that myocardial hematocrit profiles are sensitive to both RBC aggregation and to changes of plasma viscosity associated with increased RBC aggregation. Furthermore, they suggest the need for additional studies to explore the mechanisms affecting RBC distribution in three-dimensional vascular beds.     

Erythrocyte aggregation: bridging by macromolecules and electrostatic repulsion by sialic acid.

Relation between aggregating force (of fibrinogen and IgG) and disaggregating force (due to electrostatic repulsion among erythrocytes) in erythrocyte aggregation was investigated with a rheoscope combining a video camera, an image analyzer and a computer. (i) Erythrocyte aggregation was augmented with the increase of molecular weight of bridging macromolecules as far as examined for fibrinogen and the degradation products and IgG and the related macromolecules, and the augmentation seemed to be dependent on the molecular length of macromolecules. In accelerating the erythrocyte aggregation, fibrinogen was more effective than IgG, and some interaction between fibrinogen and IgG in their coexistence was suggested. (ii) The decrease of sialic acid content on the erythrocyte surface accelerated IgG-induced erythrocyte aggregation much greater than fibrinogen-induced one. (iii) Counteraction between aggregating force and disaggregating force in leading to erythrocyte aggregation was discussed relating to molecular length of bridging macromolecule and electrostatic repulsive force by sialic acid.     

Comparative analysis of differential expression of sialic acids and adhesion molecules on mononuclear cells of bone marrow and peripheral blood in childhood acute lymphoblastic leukaemia at diagnosis and clinical remission

Childhood acute lymphoblastic leukaemia (ALL) is characterized by the neoplasm of immature haematopoietic precursor cells (HPCs). We report significant differences between the expression of sialoglycoproteins and adhesion molecules on mononuclear cells (MNCs) of bone marrow (BM) and peripheral blood (PB) from individual children at diagnosis of the disease. Lymphoblasts in PB predominantly expressed 9-O-acetylated sialoglycoproteins (Neu5,9Ac2-GPs), sialic acid, alpha2-3 linked sialic acid, L- and P-selectins and vascular cell adhesion molecule -1 (VCAM-1) on their surface compared to BM, as determined with selective lectins and monoclonal antibodies (mAbs) by flow cytometric analysis. CD34+CD38+ cells present either in diagnostic PB or BM always showed enhanced expression of both alpha2-3 and alpha2-6 linked sialic acids, Neu5,9Ac2-GPs, L- and P-selectins and VCAM-1, compared to CD34+CD38- population, as confirmed by higher mean fluorescence intensity (MFI). Expression of ICAM-1 was reverse. However, MFI of Neu5,9Ac2-GPs was always higher both in CD34+CD38+ and CD34+CD38- population in PB compared to BM. Diverse trend of these cell surface macromolecules was observed during clinical remission. This is the first comparative study between PB and BM, where significant differential distribution of sialylated macromolecules and adhesion molecules was observed. Hence, supervising these cell surface macromolecules at various stages of treatment might help in minimal residual disease detection, identifying mobilization factor(s) and in isolation of normal HPCs for autologous BM transplantation.     

A STUDY ON THE MECHANISM OF INTERCELLULAR ADHESION : Effects of Neuraminidase, Calcium, and Trypsin on the Aggregation of Suspended HeLa Cells

Aggregation of suspended HeLa cells is increased on removal of cell surface sialic acid. Calcium ions promote aggregation whereas magnesium ions have no effect. The calcium effect is abolished by previous treatment of the cells with neuraminidase. Trypsinization of the HeLa cells followed by thorough washing diminishes the rate of mutual cell aggregation. Subsequent incubation with neuraminidase restores the aggregation rate to the original value before trypsin treatment. Cells which had acquired a greater tendency for aggregation after removal of peripheral sialic acid lose this property when subsequently treated with trypsin. Calcium ions have no aggregative effect on trypsinized cells. In contrast to HeLa cells, aggregation of human erythrocytes was not increased after treatment with neuraminidase or on addition of calcium. The results with HeLa cells are interpreted as follows: (a) Trypsin-releasable material confers adhesiveness upon the cells. (b) The adhesive property of this material is counteracted by the presence of cell surface sialic acids. (c) Calcium ions exert their effect by attenuating the adverse effect of sialic acid.     

Babesia caballi and Babesia equi: implications of host sialic acids in erythrocyte infection

The present study investigated the involvement of host sialic acids in the erythrocyte infection by two equine Babesia parasites, Babesia equi and Babesia caballi. We observed that the in vitro growth of both parasites is influenced by the removal of sialic acids from the surface of equine erythrocytes (RBC). When the parasites were cultured with neuraminidase (Nm, EC 3.2.1.18)-treated RBC, in which alpha2-3-linked sialic acid residues were removed from four membrane proteins of the RBC, B. caballi showed a significant inhibition of the erythrocyte invasion, while the intracellular development of B. equi seemed to be significantly affected. The possible involvement of host sialic acid in the erythrocyte invasion by B. caballi was also supported by a significant reduction in the parasite growth accompanied by an increased number of extracellular merozoites after the addition of exogenous 3'-sialyllactose (Neu5Acalpha(2-3)Galbeta(1-4)Glc) into the culture. These results suggest that the alpha2-3-linked sialic acid residues on host RBC play important roles in the erythrocyte infections by B. caballi and B. equi.     

Depletion-mediated red blood cell aggregation in polymer solutions

Polymer-induced red blood cell (RBC) aggregation is of current basic science and clinical interest, and a depletion-mediated model for this phenomenon has been suggested; to date, however, analytical approaches to this model are lacking. An approach is thus described for calculating the interaction energy between RBC in polymer solutions. The model combines electrostatic repulsion due to RBC surface charge with osmotic attractive forces due to polymer depletion near the RBC surface. The effects of polymer concentration and polymer physicochemical properties on depletion layer thickness and on polymer penetration into the RBC glycocalyx are considered for 40 to 500 kDa dextran and for 18 to 35 kDa poly (ethylene glycol). The calculated results are in excellent agreement with literature data for cell-cell affinities and with RBC aggregation-polymer concentration relations. These findings thus lend strong support to depletion interactions as the basis for polymer-induced RBC aggregation and suggest the usefulness of this approach for exploring interactions between macromolecules and the RBC glycocalyx.     

Purification and properties of a Ca2+-independent sialic acid-binding lectin from human placenta with preferential affinity to O-acetylsialic acids

A Ca2+-independent sialic acid-specific lectin from two developmental stages of human placenta was similarly purified to apparent homogeneity by DEAE-cellulose chromatography, affinity chromatography on bovine submaxillary mucin, and gel filtration. Sodium dodecyl sulfate-polyacrylamide gel electrophoresis and gel filtration disclosed a molecular mass of 53 kDa. The specificity of the lectin for O-acetylsialic acids was substantiated by the dependence of hemagglutination on the presence of acetylated sialic acids on the surface of mammalian erythrocytes of various sources, by hapten inhibition in hemagglutination assays with protease-treated rabbit erythrocytes and by hapten inhibition of binding of labeled N-acetylneuraminic acid-bovine serum albumin to the lectin in a solid-phase assay. Bovine and equine submaxillary mucins that contain 9(7,8)-O-acetyl and 4-O-acetylsialic acids were potent inhibitors in contrast to the non-acetylated sialic acids of ovine submaxillary mucin. Absence of inhibitory efficiency of other negatively charged substances like phosphorylated sugars, glucuronic acid, heparin, or oligodeoxynucleotides emphasized the importance of structural features instead of simple ionic interaction. In the presence of acetylation, the pattern of inhibition by gangliosides in the solid-phase assay indicated a preference to alpha-2,8- or alpha-2,6-linked sialic acids in comparison to alpha-2,3-linked moieties. Chemical modification of the lectin by group-specific reagents allowed to emphasize the role of primarily lysine residues, but also, although less pronounced, arginine, tryptophan, and carboxyl groups for ligand binding and/or maintenance of the active conformational state. Application of reagents, specific for histidine or tyrosine residues, failed to affect lectin activity.     

“Just a spoonful of sugar...”: import of sialic acid across bacterial cell membranes

Eukaryotic cell surfaces are decorated with a complex array of glycoconjugates that are usually capped with sialic acids, a large family of over 50 structurally distinct nine-carbon amino sugars, the most common member of which is N-acetylneuraminic acid. Once made available through the action of neuraminidases, bacterial pathogens and commensals utilise host-derived sialic acid by degrading it for energy or repurposing the sialic acid onto their own cell surface to camouflage the bacterium from the immune system. A functional sialic acid transporter has been shown to be essential for the uptake of sialic acid in a range of human bacterial pathogens and important for host colonisation and persistence. Here, we review the state-of-play in the field with respect to the molecular mechanisms by which these bio-nanomachines transport sialic acids across bacterial cell membranes.     

A novel cell surface trans-sialidase of Trypanosoma cruzi generates a stage-specific epitope required for invasion of mammalian cells.

When trypomastigotes of T. cruzi emerge from cells of the mammalian host, they contain little or no sialic acids on their surfaces. However, rapidly upon entering the circulation, they express a unique cell surface trans-sialidase activity. This enzyme specifically transfers alpha (2-3)-linked sialic acid from extrinsic host-derived macromolecules to parasite surface molecules, leading to the assembly of Ssp-3, a trypomastigote-specific epitope. The T. cruzi trans-sialidase does not utilize cytidine 5' monophospho-N-acetylneuraminic acid as a donor substrate, but readily transfers sialic acid from exogenously supplied alpha (2-3)-sialyllactose. Monoclonal antibodies that recognize sialic acid residues of Ssp-3 inhibit attachment of trypomastigotes to host cells, suggesting that the unusual trans-sialidase provides Ssp-3 with structural features required for target cell recognition.     

Influence of dendrimers on red blood cells

Dendrimers, highly branched macromolecules with a specific size and shape, provide many exciting opportunities for biomedical applications. However, most dendrimers demonstrate toxic and haemolytic activity because of their positively charged surface. Masking the peripheral cationic groups by coating them with biocompatible molecules is a method to reduce it. It was proven that modified dendrimers can even diminish haemolytic activity of encapsulated drugs. Experiments confirmed that anionic dendrimers are less haemotoxic than cationic ones. Due to the high affinity of dendrimers for serum proteins, presence of these components in an incubation buffer might also influence red blood cell (RBC)-dendrimer interactions and decrease the haemolysis level. Generally, haemotoxicity of dendrimers is concentration-, generation-, and time-dependent. Various changes in the RBCs’ shape in response to interactions with dendrimers have been observed, from echinocytic transformations through cell aggregation to cluster formation, depending on the dendrimer’s type and concentration. Understanding the physical and chemical origins of dendrimers’ influences on RBCs might advance scientists’ ability to construct dendrimers more suitable for medical applications.     

Sulfated N-linked oligosaccharides in mammalian cells. III. Characterization of a pancreatic carcinoma cell surface glycoprotein with N- and O-sulfate esters on asparagine-linked glycans

In the preceding two papers, we described two new classes of sulfated N-linked oligosaccharides isolated from total cellular 35SO4-labeled macromolecules of different mammalian cell lines. The first class carries various combinations of sialic acids and 6-O-sulfate esters on typical complex-type chains, while the second carries heparin and heparan-like sequences. In this study, we have characterized a sulfophosphoglycoprotein of 140 kDa from FG-Met-2 pancreatic cancer cells whose oligosaccharides share some properties of both these classes. The molecule was localized to the cell surface by electron microscopy using a monoclonal antibody (S3-53) and by cell surface 125I-labeling. Metabolic labeling of the cells with radioactive glucosamine, methionine, inorganic sulfate, or phosphate all demonstrated a single 140-kDa molecule. Pulse-chase analysis and tunicamycin treatment indicated the glycosylation of a putative primary translation product of 110 kDa via an intermediate (120 kDa) to the mature form (140 kDa). Digestion with peptide:N-glycosidase F (PNGaseF) indicated a minimum of four N-linked glycosylation sites. PNGaseF released more than 90% of the [6-3H]GlcNH2 label and 40-70% of 35SO4 label from the immunoprecipitated 140-kDa molecule. The isolated oligosaccharides were characterized as described in the preceding two papers. The majority of [6-3H]GlcNH2-labeled molecules were susceptible to neuraminidase. More than 50% of the 35SO4 label was associated with only 5-10% of the 3H-labeled chains. Some of the sulfated chains were partly sialylated molecules with four to five negative charges. Treatment with nitrous acid released about 25% of the 35SO4 label as free sulfate, together with 6% of the [6-3H]GlcNH2 label, indicating the presence of N-sulfated glucosamine residues. Some of these oligosaccharides were degraded by heparinase and heparitinase. Therefore, while they are not as highly charged as typical heparin or heparan chains, they must share structural features that permit recognition by the enzymes. Thus, this 140-kDa glycoprotein contains at least four asparagine-linked chains substituted with a heterogeneous mixture of sulfated sequences. The heterogeneity of these molecules is as extensive as that described for whole-cell sulfated N-linked oligosaccharides in the preceding two papers.     

蛋白质转导及其内在化机制

蛋白质转导是新近发展起来的向细胞内快速输送外源性大分子或高极性分子的有效途径。它实质上是一些蛋白质,尤其是病毒蛋白上被称为蛋白质转导区(PTD)的小片段,蛋白质和其他物质,如DNA、脂质体、纳米颗粒、环孢素A等与之结合后,即能够被携带进入细胞或穿过血脑屏障。蛋白质转导的内在化机制目前尚不清楚,可能与带正电荷(富Arg)的PTD肽与细胞膜上带负电荷的硫酸乙酰肝素有关,但不排除其他内在化机制。     

Synthetically functionalized retroviruses produced from the bioorthogonally engineered cell surface

Conjugation of desired molecules onto retroviral surfaces through the ease of the bioorthogonal functionalization method was demonstrated. Oxidation of surface sialic acids using periodate and further p-anisidine-catalyzed conjugation with aminooxy-bearing molecules were used to directly label retroviral envelope with a fluorescent dye. The retroviral particles that were produced from a bioorthogonally functionalized virus producing cell surface and further tethered with magnetic nanoparticles were efficiently purified by simple magnetic column separation and capable of magnet-directed transduction.