Erythrocyte Sialic Acid Research Articles

Evidence for a switching mechanism in the invasion of erythrocytes by Plasmodium falciparum.

The human malaria parasite Plasmodium falciparum demonstrates variability in its dependence upon erythrocyte sialic acid residues for invasion. Some lines of P. falciparum invade neuraminidase-treated or glycophorin-deficient red blood cells poorly, or not at all, while other lines invade such cells at substantial rates. To explore the molecular basis of non-sialic acid dependent invasion, we selected parasite lines from a clone (Dd2) that initially exhibited low invasion of neuraminidase-treated erythrocytes. After maintaining Dd2 for several cycles in neuraminidase-treated erythrocytes, parasite lines were recovered that invaded both untreated and neuraminidase-treated erythrocytes at equivalently high rates (Dd2/NM). The change in phenotype was maintained after removal of selection pressure. Four subclones of Dd2 were isolated and each readily converted from sialic acid dependence to non-sialic acid dependence during continuous propagation in neuraminidase-treated erythrocytes. The neuraminidase-selected lines and the Dd2 clone demonstrated identical restriction fragment length polymorphism markers indicating that the Dd2 clone was not contaminated during the selection process. Parasite proteins that bound to neuraminidase-treated and untreated erythrocytes were indistinguishable among the parent Dd2 clone and the neuraminidase-selected lines. The ability of the Dd2 parasite to change its invasion requirements for erythrocyte sialic acid suggests a switch mechanism permitting invasion by alternative pathways.

Electrophoresis of bovine erythrocyte sialic acids: Existence of additional band in trypanotolerant Ndama cattle

Mild acid-hydrolysis of erythrocyte surface sialic acids of the trypanotolerant Ndama and the trypanosusceptible White/Fulani Zebu breeds of cattle was performed. The cleaved sialic acids from the two breeds of cattle were simultaneously subjected to polyacrylamide gel electrophoresis in the presence of sodium dodecyl sulphate (SDS-PAGE), along with commercial standard N-acetylneuraminic acid (MW 309.28), blank gel and plasma proteins. The cleaved sialic acids migrated in the globulin fractions, as shown by the plasma protein electrophoresis. While the Ndama and the Zebu erythrocytes had one migrating band each of which coincided with the standard N-acetylneuraminic acid, the Ndama had another trailing band of sialic acid, of an estimated molecular weight of 30 kDa, which may account for the higher erythrocyte sialic acid concentrations of the Ndama. This additional band was absent in the Zebu. All these bands were readily reproducible.

Sialic acid in sickle cell disease.

Neuraminic (sialic) acid concentrations in serum from normal and sickle cell (HbSS) subjects were determined for discrete age groups from childhood through adolescence. Values in sickle cell disease were consistently lower over the entire age range. We further investigated the effect of exogenous sialic acid on the rate of sickling reversion of HbSS erythrocytes and demonstrated that this compound in millimole per liter concentrations could revert pre-sickled erythrocytes to their normal morphology in a concentration-dependent manner. When subjected to partial de-sialation with sialidase (EC 3.2.1.18), the HbSS erythrocytes not only sickled faster upon deoxygenation, they also reverted more slowly on treatment with phenylalanine (a more efficient anti-sickling agent than sialic acid) than did untreated cells. We conclude that, in sickle cell disease, erythrocyte sialic acid content could play a significant role, not only in the control of the sickling rate in vivo, but also, after sickling has occurred, in the rate of recovery from a sickling crisis.

Falciparum malaria parasites invade erythrocytes that lack glycophorin A and B (MkMk). Strain differences indicate receptor heterogeneity and two pathways for invasion.

To determine the ligands on erythrocytes for invasion by Plasmodium falciparum, we tested invasion into MkMk erythrocytes that lack glycophorins A and B and enzyme-treated erythrocytes by parasites that differ in their requirement for erythrocyte sialic acid. The 7G8 strain invaded MkMk erythrocytes and neuraminidase-treated normal erythrocytes with greater than 50% the efficiency of normal erythrocytes. In contrast, the Camp strain invaded MkMk erythrocytes at 20% of control and neuraminidase-treated normal erythrocytes at only 1.8% of control. Invasion of MkMk erythrocytes by 7G8 parasites was unaffected by treatment with neuraminidase but was markedly reduced by treatment with trypsin. In contrast, invasion of MkMk cells by Camp parasites was markedly reduced by neuraminidase but was unaffected by trypsin. We conclude that the 7G8 and Camp strains differ in ligand requirements for invasion and that 7G8 requires a trypsin sensitive ligand distinct from glycophorins A and B.

Invasion of mouse erythrocytes by the human malaria parasite, Plasmodium falciparum.

Plasmodium falciparum malaria merozoites require erythrocyte sialic acid for optimal invasion of human erythrocytes. Since mouse erythrocytes have the form of sialic acid found on human erythrocytes (N-acetyl neuraminic acid), mouse erythrocytes were tested for invasion in vitro. The Camp and 7G8 strains of P. falciparum invaded mouse erythrocytes at 17-45% of the invasion rate of human erythrocytes. Newly invaded mouse erythrocytes morphologically resembled parasitized human erythrocytes as shown on Giemsa-stained blood films and by electron microscopy. The rim of parasitized mouse erythrocytes contained the P. falciparum 155-kD protein, which is on the rim of ring-infected human erythrocytes. Camp but not 7G8 invaded rat erythrocytes, indicating receptor heterogeneity. These data suggest that it may be possible to adapt the asexual erythrocytic stage of P. falciparum to rodents. The development of a rodent model of P. falciparum malaria could facilitate vaccine development.

Erythrocyte sialic acid in human sickle-cell disease

Membrane-bound erythrocyte surface sialic acid concentrations in sickle-cell human patients (SS) and in control patients with sickle-cell trait (AS) and normal genes (AA) were assayed. The erythrocyte sialic acid concentration was significantly ( P < 0·001) higher in sickle-cell patients than in non-sicklers and although the concentration was higher in AS than AA individuals the difference was not significant ( P > 0·05). The anaemic state of the SS patients was shown by a significantly ( P < 0·001) lower PCV than in non-sicklers. This finding is discussed in relation to membrane toughness and subsequent membrane loss leading to irreversible sickling of red cells in the deoxygenated state.

Invasion of erythrocytes by malaria parasites: a cellular and molecular overview.

Studies on the morphology, cell biology, and immunology of invasion have characterized events that are now being studied at the molecular level. The initial events of invasion are receptor-specific. A determinant associated with Duffy blood group antigens is involved in the invasion of human erythrocytes by P. knowlesi and P. vivax. The Duffy Fya antigen has recently been identified and further characterization of its role in reception and invasion should now be possible. P. falciparum utilizes erythrocyte ligands that differ from those of P. knowlesi and P. vivax. Sialic acid and a trypsin-sensitive erythrocyte membrane component are important for invasion by P. falciparum parasites. There is evidence that at least two ligands are involved in invasion. For P. knowlesi there is a ligand for attachment, common to both Duffy-negative and Duffy-positive human erythrocytes, and a second ligand for invasion, which is found only on Duffy-positive human erythrocytes. P. vivax also appears to utilize two ligands, a Duffy-associated ligand and a ligand specific for reticulocytes. P. falciparum binds to sialic acid-dependent and sialic acid-independent trypsin-sensitive ligands. P. falciparum merozoites require erythrocyte sialic acid to varying degrees in order to invade; this indicates heterogeneity of the receptor mechanism. Monoclonal antibodies and recombinant DNA technology have greatly facilitated the identification, isolation, and characterization of proteins that may be involved in invasion. Molecules that may have invasion-related functions include those whose antibodies block invasion, those that bind to erythrocyte ligands important for invasion, those that appear on the merozoite surface, and those that appear to be inserted into the erythrocyte membrane at the time of invasion. It has not been possible to identify a definite function for any of the molecules identified thus far. No monoclonal or polyclonal monospecific antibody has been identified that reacts specifically over the surface of the apical region of the merozoite where junction formation occurs. Identification of molecules responsible for apical attachment and junction formation will be important for our understanding of invasion. In terms of vaccine development, it is not yet known whether any of the molecules discussed here will prove to be effective immunogens. It is clear from the data obtained with the 140-kd protein of P. knowlesi that antigenic variation poses a potential problem for vaccine development. As the molecular events responsible for invasion become better understood, novel ways may be devised to interfere with the process and prevent the disease.

Incidence of T activation in a hospital population.

The incidence of T activation in a hospital population has been studied. T poly-agglutinability likely to cause serological problems was found in only 1:10,000 but minor degrees of T activation occurred in 1:200. In a number of cases, severe infections were accompanied by T activation, but we were unable to establish any primary association between T activation and anaemia. The findings suggested that individuals whose erythrocyte sialic acid levels are slightly lower than normal are those who, when hospital patients, are likely to develop various degrees of T activation.

Incidence of T Activation in a Hospital Population

The incidence of T activation in a hospital population has been studied. T polyagglutinability likely to cause serological problems was found in only 1:10,000 but minor degrees of T activation occurred in 1:200. In a number of cases, severe infections were accompanied by T activation, but we were unable to establish any primary association between T activation and anaemia. The findings suggested that individuals whose erythrocyte sialic acid levels are slightly lower than normal are those who, when hospital patients, are likely to develop various degrees of T activation.

Effect of anionic amphophiles on erythrocyte properties.

This preliminary study describes effects of two pharmacologic agents on erythrocyte behavior. Increased erythrocyte aggregation has been proposed as important in the pathogenesis of a number of disorders, but the exact mechanism by which it plays a role in disease production remains unclear. Several anionic amphophiles have been reported to benefit diabetic vascular disease and atherosclerosis. If anionic amphophiles enter the erythrocyte plasma membrane they can increase its negative charge, reducing the energy of attraction between red blood cells and diminishing erythrocyte aggregation. Erythrocytes were studied after suspension in phosphate-buffered saline containing dextran as an aggregation-promoting agent. A marginal reduction of the suspension's viscosity was found at low shear rate when 2,5- dihydroxybenzene sulfonate was added. Additionally, erythrocyte sedimentation rate was marginally influenced. Both dihydroxybenzene sulfonate and acetylsalicylate protected human erythrocytes from hemolysis at concentrations from 10(-3) to 10(-5) M. The removal of erythrocyte sialic acid using neuraminidase to reduce surface negative charge led to unequivocal interference with aggregation (MAI technique of CHIEN et al., J. Gen. Physiol., 1973) by both anionic amphophiles were studied. Dihydroxybenzene sulfonate and actylsalicylate reduced the aggregation propensity of sialic-free erythrocytes, suggesting that the effect on the low shear rate viscosity of sialic acid-containing erythrocytes, though modest, is real.

Binding and phagocytosis of sialidase-treated rat erythrocytes by a mechanism independent of opsonins.

Rat peritoneal macrophages bind and phagocytoze homologous sialidase-treated erythrocytes at a rate which is dependent on the amount of sialic acid that has been removed from the cells. Increased binding of erythrocytes is observed after the removal of 10-20% of membrane sialic acid, while for phagocytosis at least 30-40% of this substance must be removed. With Vibrio cholerae sialidase only a partial (80%) hydrolysis of rat erythrocyte sialic acid is possible, whereas Arthrobacter ureafaciens sialidase leads to complete desialylation and therefore causes stronger binding and phagocytosis of the erythrocytes than the V. cholerae enzyme. Preincubation of peritoneal macrophages with sialidase impairs binding and phagocytosis. Experiments were performed to account for the stimulation of binding and phagocytosis observed in the presence of native, homologous serum. However, an involvement of immunoglobulins and complement factors of the classical and alternative pathway in the engulfment process has been excluded. Fibronectin, tuftsin and substance P have no influence, either. On the other hand, peanut agglutinin and Erythrina crystagalli agglutinin are potent stimulators of binding and phagocytosis of sialidase-treated erythrocytes, whereas soybean agglutinin has only little and limulin no influence at all. It is concluded that sialidase-treated erythrocytes, having been bound to the beta-galactose-specific lectin on the macrophage surface, are phagocytozed as a function of their number and binding strength to the macrophages. The influence of native serum and especially of the plant lectins on this process is discussed.

Natural immunity to Sindbis virus is influenced by host tissue sialic acid content.

Recent studies have shown that the sialic acid content of Sindbis virus influences both its ability to active the alternative pathway in vitro and its susceptibility to complement dependent clearance from the bloodstream in vivo. Other studies have shown that the sialic acid content of Sindbis virus is determined by the host in which it is propagated. Because individuals vary in their cell surface sialic acid content, it is possible they also vary in their ability to defend themselves against Sindbis virus infection by virtue of their ability to modify the virus sialic acid content and thereby the capacity of the virus to activate the alternative pathway. To test this hypothesis, outbred Swiss mice were injected subcutaneously with Sindbis virus. There was a significant positive correlation between the level of viremia 18 hr after infection and the sialic acid content of the host's erythrocytes. In addition, animals with erythrocyte sialic acid levels equal to or greater than the mean had a higher level of viremia than animals with erythrocyte sialic acid levels less than the mean. Finally, animals that had muscle sialic acid levels equal to or greater than the mean had a higher incidence of viremia than animals with muscle sialic acid levels less than the mean. These studies suggest that the amount of tissue sialic acid in an individual host influences its ability to resist Sindbis virus infection.

An autosomal dominant gene regulates the extent of 9-O-acetylation of murine erythrocyte sialic acids. A probable explanation for the variation in capacity to activate the human alternate complement pathway.

Nydegger et al. (4) have reported that the difference in susceptibility of erythrocytes from different inbred murine strains to lysis by the human alternate complement pathway is determined by an autosomal locus. We have found a good correlation between the degree of O-acetylation of the erythrocyte sialic acid residues and the susceptibility to complement lysis, whereas there was no correlation between total erythrocyte sialic acid content and complement sensitivity. The major O-acetylated species in all the murine strains is 9-O-acetyl-N-acetylneuraminic acid. We propose that the autosomal dominant locus, which determines complement sensitivity, acts by influencing the extent of 9-O-acetylation of the erythrocyte sialic acid residues. By using recombinant inbred strains, we determined that this genetic locus is probably located on chromosome 9. The nature of the gene product remains unknown.

Role of sialic acid in erythrocyte survival

The role of membrane sialic acid in erythrocyte survival is unclear, although there is evidence for a reduction in sialic acid and surface charge in older erythrocytes. We reduced the surface charge of human, rat, and rabbit erythrocytes by removing sialic acid with neuraminidase.Reduction in sialic acid correlated with decreases in electrophoretic mobility and loss of PAS staining of membrane glycoproteins on polyacrylamide gels. No changes in ATP levels or deformability were found. 51Cr erythrocyte survivals in rats and rabbits showed rapid clearance of desialylated erythrocytes with sequestration by the liver.These results suggest that reduction in erythrocyte sialic acid is a mechanism of erythrocyte destruction and may be important in erythrocyte senescence.

Role of Sialic Acid in Erythrocyte Survival

The role of membrane sialic acid in erythrocyte survival is unclear, although there is evidence for a reduction in sialic acid and surface charge in older erythrocytes. We reduced the surface charge of human, rat, and rabbit erythrocytes by removing sialic acid with neuraminidase. Reduction in sialic acid correlated with decreases in electrophoretic mobility and loss of PAS staining of membrane glycoproteins on polyacrylamide gels. No changes in ATP levels or deformability were found. 51Cr erythrocyte survivals in rats and rabbits showed rapid clearance of desialylated erythrocytes with sequestration by the liver. These results suggest that reduction in erythrocyte sialic acid is a mechanism of erythrocyte destruction and may be important in erythrocyte senescence.

Relative susceptibilities to neuraminidase of the glycoproteins of the human erythrorcyte

Release of sialic acid from the glycoproteins of the normal human erythrocyte surface by neuraminidase was investigated. The glycoproteins of the membrane were separated by electrophoresis in sodium dodecylsulfate polyacrylamide gels. Sialic acid was determined in the sliced gel by a modification of the 2-thiobarbituric acid method, revealing three sialic acid-containing glycoproteins. Treatment of intact erythrocytes with neuraminidase to remove varying amounts of sialic acid indicates that all the glycoproteins are essentially equally accessible to the neuraminidase when 20%–60% of the sialic acid is removed. Similar but not quite identical results were obtained with isolated erythrocyte membranes. Treatment of intact cells with the lectins concanavalin A or phytohemagglutinin-P resulted in shielding of about 25% and 50%, respectively, of the sialic acid from neuraminidase. Concanavalin A blocked sialic acid release over long time periods and with high concentrations of neuraminidase. In contrast, the sialic acid shielding by phytohemagglutinin-P can be overcome by high concentrations of neuraminidase. Both lectins were found to shield the various glycoproteins selectively, with different patterns of shielding. Wheat germ agglutinin exhibited no detectable effect on the susceptibility of the erythrocyte sialic acid to neuraminidase.

Sialic acid in erythrocytes of patients with amaurotic idiocy and Huntington's chorea.

Total erythrocyte sialic acid and lipid-extractable sialic acid were estimated in material obtained from 14 normal subjects, and from 8 parents and 1 sibling of patients suffering from late infantile or juvenile forms of amaurotic idiocy, from 1 patient in the terminal phase of the disease and 2 patients with Huntington's chorea. In contrast to a previous report no increase in lipid-sialic acid was found, nor were the total sialic acid values in the “pathological” material raised. The cause of these different results is still obscure. The possible role of lipid-peroxidation products is discussed.

Boar seminal haemagglutinin. II. Combination with red cells and spermatozoa.

Summary. The haemagglutinins in boar seminal plasma are absorbed by ejaculated washed motile ram, bull and rabbit spermatozoa. Washed epididymal spermatozoa also absorb the haemagglutinin, but less avidly. Considerable motility of the heterologous spermatozoa is still present after 15 min contact with boar seminal plasma. Mixed cell agglutination of bull spermatozoa and red cells gives a reticulate agglutination visible microscopically, and tail to tail agglutination occurs. The haemagglutinins are absorbed at least as well by washed pig red cell ghosts as by the intact washed erythrocytes. The absorptive power of the ghosts is not destroyed by boiling them in n-NaOH or n-HCl for one hour. It is presumed that a negatively charged `receptor' group on the surface of the red cell is responsible for the absorption. The receptor does not appear to be connected with erythrocyte sialic acid removable by receptor-destroying enzyme, trypsin or by 0·1 n-HCl.