Enhanced Eryptosis in Glucose-6-Phosphate Dehydrogenase Deficiency

Background/Aims: Defects in the Glucose-6-Phosphate Dehydrogenase (G6PD) enzyme enhance cellular oxidative damage, thus impairing erythrocytes and radically shortening their lifespan. We aimed to study programmed erythrocyte cell death in G6PD-deficient patients, describe the molecular genetics basis of G6PD and investigate phenotype-genotype correlations. Methods: We explored eryptosis using the annexin V-binding assay, taken as an indicator of PS exposure at the erythrocyte surface. We assessed reactive oxygen species (ROS) production, intracellular calcium concentrations and ceramide formation at the cell surface. Prior to and following treatments, cells were analyzed by flow cytometry. Finally, we explored G6PD gene mutations through PCR-Sanger sequencing. Results: Before stimulation, PS-exposing erythrocytes were significantly higher in G6PD-deficient patients than in healthy volunteers. This was paralleled by a significant increase in reactive oxygen species production, suggesting that oxidative stress is the main trigger of PS exposure in G6PDdeficient erythrocytes. Five previously described mutations were detected in our patients. Two genotypes correlated with a significantly higher percentage of PS-exposing cells. Conclusion: Our study uncovers a novel effect detected in G6PD-deficient erythrocytes which is cell membrane scrambling with PS translocation to the erythrocyte surface. Our findings shed a light on the mechanisms of premature erythrocyte clearance in G6PD deficiency.

Effect of X-ray radiation on the nanomechanical properties of the erythrocyte surface of rats on a high-cholesterol diet

Objective. To reveal changes in the structural and nanomechanical properties of the erythrocyte surface caused by the action of X-ray radiation in vitro on the whole blood of rats on a high-cholesterol diet using the method of atomic-force microscopy.Materials and methods. The blood of male Wistar rats being on a high-cholesterol diet for two months was exposed to X-ray radiation (320 kV) at doses of 1 and 100 Gy. The structural, elastic and adhesive properties of the surface of isolated and glutaraldehyde-fxed erythrocytes at the nanoscale were studied using the atomic- force microscope BioScope Resolve in PeakForce QNM mode in air.Results. The study has identifed an increase in the stiffness of the erythrocyte surface at a dose of 1 Gy and its decrease to almost control values at a dose of 100 Gy, which was accompanied by an increase in the size of the average cell of the erythrocyte membrane skeleton. At the same time, no signifcant changes in the morphology, adhesive properties and roughness of the relief of erythrocytes have been found.Conclusion. The obtained data indicate that X-ray radiation (1–100 Gy) induces the dose-depending reorganization of the structure and changes in the stiffness of the erythrocyte surface layer at the nanoscale without changing the cell morphology for rats on a high-cholesterol diet.

Effects of trypsin on aggregation, disaggregation and aggregate morphology of red blood cells in autologous plasma and serum

We study the influence of trypsin on aggregation, disaggregation, and aggregate morphology of RBCs in autologous plasma and serum. The effect of trypsin on the surface charge of red blood cells and the aggregation of glutaraldehyde fixed cells after treatment with the enzyme was also studied. RBC aggregation was studied by means of an aggregometer and microscopic observations. The results obtained in this study indicate that trypsin treatment increases RBCs aggregation in autologous plasma and serum. The disaggregation of erythrocytes after trypsin treatment considerably decreased in autologous plasma and serum. Increase in the strength of red blood cell aggregates was observed in autologous plasma and serum. The microscopic images of RBCs aggregates indicate the formation of globular (pathologic) structures of aggregates in autologous plasma and serum. Trypsin decrease the surface charge of RBCs. In autologous plasma and serum, the cup shapes of RBCs appear. The control RBCs fixed with glutaraldehyde were not aggregated after their placement in autologous plasma. At the same time, red blood cells pretreated with trypsin and fixed with glutaraldehyde interact with each other in autologous plasma. The physiological significance of glycoproteins of erythrocyte surface for RBCs aggregation was discussed.

High Antibodies to VAR2CSA in Response to Malaria Infection Are Associated With Improved Birthweight in a Longitudinal Study of Pregnant Women

IntroductionPregnant women have an increased risk of P. falciparum infection, which is associated with low birth weight and preterm delivery. VAR2CSA, a variant surface antigen expressed on the parasitized erythrocyte surface, enables sequestration in the placenta. Few studies have prospectively examined relationships between antibody responses during pregnancy and subsequent adverse birth outcomes, and there are limited data outside Africa.MethodsLevels of IgG against VAR2CSA domains (DBL3; DBL5) and a VAR2CSA-expressing placental-binding P. falciparum isolate (PfCS2-IE) were measured in 301 women enrolled at their first visit to antenatal care which occurred mid-pregnancy (median = 26 weeks, lower and upper quartiles = 22, 28). Associations between antibody levels at enrolment and placental infection, birthweight and estimated gestational age at delivery were assessed by linear and logistic regression with adjustment for confounders. For all outcomes, effect modification by gravidity and peripheral blood P. falciparum infection at enrolment was assessed.ResultsAmong women who had acquired P. falciparum infection at enrolment, those with higher levels of VAR2CSA antibodies (75th percentile) had infants with higher mean birthweight (estimates varied from +35g to +149g depending on antibody response) and reduced adjusted odds of placental infection (aOR estimates varied from 0.17 to 0.80), relative to women with lower levels (25th percentile) of VAR2CSA antibodies. However, among women who had not acquired an infection at enrolment, higher VAR2CSA antibodies were associated with increased odds of placental infection (aOR estimates varied from 1.10 to 2.24).ConclusionsWhen infected by mid-pregnancy, a better immune response to VAR2CSA-expressing parasites may contribute to protecting against adverse pregnancy outcomes.

Antibody Levels to Plasmodium falciparum Erythrocyte Membrane Protein 1-DBLγ11 and DBLδ-1 Predict Reduction in Parasite Density

Plasmodium infection causes devastating disease and high mortality in young children. Immunity develops progressively as children acquire protection against severe disease, although reinfections and recrudescences still occur throughout life in areas of endemicity, partly due to parasite immunoevasion via switching of variant proteins such as Plasmodium falciparum erythrocyte membrane protein 1 (PfEMP1) expressed on the infected erythrocyte surface.

Plasmodium yoelii Erythrocyte Binding Like Protein Interacts With Basigin, an Erythrocyte Surface Protein

Erythrocyte recognition and invasion is critical for the intra-erythrocytic development of Plasmodium spp. parasites. The multistep invasion process involves specific interactions between parasite ligands and erythrocyte receptors. Erythrocyte-binding-like (EBL) proteins, type I integral transmembrane proteins released from the merozoite micronemes, are known to play an important role in the initiation and formation of tight junctions between the apical end of the merozoite and the erythrocyte surface. In Plasmodium yoelii EBL (PyEBL), a single amino acid substitution in the putative Duffy binding domain dramatically changes parasite growth rate and virulence. This suggests that PyEBL is important for modulating the virulence of P. yoelii parasites. Based on these observations, we sought to elucidate the receptor of PyEBL that mediates its role as an invasion ligand. Using the eukaryotic wheat germ cell-free system, we systematically developed and screened a library of mouse erythrocyte proteins against native PyEBL using AlphaScreen technology. We report that PyEBL specifically interacts with basigin, an erythrocyte surface protein. We further confirmed that the N-terminal cysteine-rich Duffy binding-like region (EBL region 2), is responsible for the interaction, and that the binding is not affected by the C351Y mutation, which was previously shown to modulate virulence of P. yoelii. The identification of basigin as the putative PyEBL receptor offers new insights into the role of this molecule and provides an important base for in-depth studies towards developing novel interventions against malaria.

Blood donor variability is a modulatory factor for P. falciparum invasion phenotyping assays

Human erythrocytes are indispensable for Plasmodium falciparum development. Unlike other eukaryotic cells, there is no existing erythroid cell line capable of supporting long-term P. falciparum in vitro experiments. Consequently, invasion phenotyping experiments rely on erythrocytes of different individuals. However, the contribution of the erythrocytes variation in influencing invasion rates remains unknown, which represents a challenge for conducting large-scale comparative studies. Here, we used erythrocytes of different blood groups harboring different hemoglobin genotypes to assess the relative contribution of blood donor variability in P. falciparum invasion phenotyping assays. For each donor, we investigated the relationship between parasite invasion phenotypes and erythrocyte phenotypic characteristics, including the expression levels of surface receptors (e.g. the human glycophorins A and C, the complement receptor 1 and decay accelerating factor), blood groups (e.g. ABO/Rh system), and hemoglobin genotypes (e.g. AA, AS and AC). Across all donors, there were significant differences in invasion efficiency following treatment with either neuraminidase, trypsin or chymotrypsin relative to the control erythrocytes. Primarily, we showed that the levels of key erythrocyte surface receptors and their sensitivity to enzyme treatment significantly differed across donors. However, invasion efficiency did not correlate with susceptibility to enzyme treatment or with the levels of the selected erythrocyte surface receptors. Furthermore, we found no relationship between P. falciparum invasion phenotype and blood group or hemoglobin genotype. Altogether, our findings demonstrate the need to consider erythrocyte donor uniformity and anticipate challenges associated with blood donor variability in early stages of large-scale study design.

Sickle Cell Trait Modulates the Proteome and Phosphoproteome of Plasmodium falciparum-Infected Erythrocytes

The high prevalence of sickle cell disease in some human populations likely results from the protection afforded against severe Plasmodium falciparum malaria and death by heterozygous carriage of HbS. P. falciparum remodels the erythrocyte membrane and skeleton, displaying parasite proteins at the erythrocyte surface that interact with key human proteins in the Ankyrin R and 4.1R complexes. Oxidative stress generated by HbS, as well as by parasite invasion, disrupts the kinase/phosphatase balance, potentially interfering with the molecular interactions between human and parasite proteins. HbS is known to be associated with abnormal membrane display of parasite antigens. Studying the proteome and the phosphoproteome of red cell membrane extracts from P. falciparum infected and non-infected erythrocytes, we show here that HbS heterozygous carriage, combined with infection, modulates the phosphorylation of erythrocyte membrane transporters and skeletal proteins as well as of parasite proteins. Our results highlight modifications of Ser-/Thr- and/or Tyr- phosphorylation in key human proteins, such as ankyrin, β-adducin, β-spectrin and Band 3, and key parasite proteins, such as RESA or MESA. Altered phosphorylation patterns could disturb the interactions within membrane protein complexes, affect nutrient uptake and the infected erythrocyte cytoadherence phenomenon, thus lessening the severity of malaria symptoms.

Human erythrocyte surface fucose expression increases with age and hyperglycemia

Background: Reactive oxygen species and other free radicals, together with glucose and its metabolites are believed to play important roles in the aging process. The carbohydrate components of glycosylated proteins are important in mediating cell-cell interactions and a role has been suggested for them in the aging process. Erythrocytes are critical cells in the human body, heavily glycosylated and relatively easily available and so are good candidates to yield insights into how patterns of glycosylation change with age and disease. It has been claimed, based on a periodic acid Schiff assay, that human aging is associated with a decline of erythrocyte surface sialic acids. Plant lectins allow for more specific assays for glycans, including determining the linkage of sialic acids and analysis of single cells by flow cytometry. Methods: Plant lectins, including Maackia amurensis lectin II (MAL), binding to α-2,3 linked sialic acids and Sambucus nigra (SNA), α-2,6 sialic acids, were used in flow cytometry and western blot of erythrocyte surface membrane. N-glycomics mass spectrometry determines glycan structures. Donors varying in age and hyperglycemia, as indicated by HbA1c were analysed. Results: Erythrocyte surface sialic acids have no significant associations with donor age. A combination of storage and cellular aging produces a specific loss of α-2,6 sialic acids. By contrast, erythrocyte surface terminal fucoses increase significantly with donor age. In order to determine which aspects of aging are important in determining this change, we investigated whether this novel human aging biomarker is associated with higher plasma glucose values, assessed by glycated hemoglobin (HbA1c) and reactive oxygen species (ROS) generation. Fucose levels were associated with HbA1c levels, but not ROS generation. Conclusion: Our study identifies novel glycan-based biomarkers for human aging and disease. The simplicity of lectin-based assays provide an attractive cellular tool to study aging and disease processes.