Plasma biomarkers of hemoglobin loss in Plasmodium falciparum-infected children identified by quantitative proteomics

Anemia is common among young children infected with Plasmodium falciparum (Pf) and severe malarial anemia (SMA) is a major cause of their mortality. Two major mechanisms cause malarial anemia: hemolysis of uninfected as well as infected erythrocytes and insufficient erythropoiesis. In a longitudinal birth cohort in Mali, we commonly observed marked hemoglobin reductions during Pf infections with a small proportion that progressed to SMA. We sought biomarkers of these processes using quantitative proteomic analysis on plasma samples from 9 P. falciparum-infected children, comparing those with reduced hemoglobin (with or without SMA) versus those with stable hemoglobin. We identified higher plasma levels of circulating 20S proteasome and lower IGF-1 levels in children with reduced hemoglobin. We confirmed these findings in independent ELISA-based validation studies of subsets of children from the same cohort (20S proteasome, N=71; IGF-1, N=78). We speculate that circulating 20S proteasome plays a role in digesting erythrocyte membrane proteins modified by oxidative stress, resulting in hemolysis, while decreased IGF-1, a critical factor for erythroid maturation, might contribute to insufficient erythropoiesis. Quantitative plasma proteomics identified soluble mediators that may contribute to the major mechanisms underlying malarial anemia.

Novel pathogenic XK mutations in McLeod syndrome and interaction between XK protein and chorein

ObjectiveTo identify XK pathologic mutations in 6 patients with suspected McLeod syndrome (MLS) and a possible interaction between the chorea-acanthocytosis (ChAc)- and MLS-responsible proteins: chorein and XK protein.MethodsErythrocyte membrane proteins from patients with suspected MLS and patients with ChAc, ChAc mutant carriers, and normal controls were analyzed by XK and chorein immunoblotting. We performed mutation analysis and XK immunoblotting to molecularly diagnose the patients with suspected MLS. Lysates of cultured cells were co-immunoprecipitated with anti-XK and anti-chorein antibodies.ResultsAll suspected MLS cases were molecularly diagnosed with MLS, and novel mutations were identified. The average onset age was 46.8 ± 8 years, which was older than that of the patients with ChAc. The immunoblot analysis revealed remarkably reduced chorein immunoreactivity in all patients with MLS. The immunoprecipitation analysis indicated a direct or indirect chorein-XK interaction.ConclusionsIn this study, XK pathogenic mutations were identified in all 6 MLS cases, including novel mutations. Chorein immunoreactions were significantly reduced in MLS erythrocyte membranes. In addition, we demonstrated a possible interaction between the chorein and XK protein via molecular analysis. The reduction in chorein expression is similar to that between Kell antigens and XK protein, although the chorein-XK interaction is a possibly noncovalent binding unlike the covalent Kell-XK complex. Our results suggest that reduced chorein levels following lack of XK protein are possibly associated with molecular pathogenesis in MLS.

Beneficial impact of human placenta extracts on erythrocyte membrane thermostability

PURPOSE: To study the influence of human placenta extract (HPE) and its individual fractions on the thermal stability of human erythrocyte membrane. METHODS: HPE fractions were isolated by gel chromatography. Thermal hemolysis of erythrocytes, exposed to 55°C was measured spectrophotometrically. Cytosol microvscosity and barrier function of erythrocyte membranes at hyperthermia were investigated by EPR spin probe TEMPON. Thermal denaturation of erythrocyte membrane proteins were studied by differential scanning calorimetry. RESULTS: Pre-treatment of erythrocytes with HPE or its fractions inhibited thermal hemolysis. Low-molecular fractions (below 4 kDa and 12-20 kDa) were the most effective in thermal hemolysis inhibition ((31.7±3.3) % and (31.5±3.2) %, respectively). The latter fractions markedly reduced the hyperthermia (55°C)-induced permeability of erythrocytes for ferricyanide ions and inhibited the thermo-induced structural transitions in erythrocyte membrane between 40 and 50°C, which are associated with cytoskeletal proteins. HPE fractions reversibly increased the denaturation temperatures of erythrocyte membrane proteins, except that of spectrin, and enlarged the enthalpy of denaturation of all membrane proteins. CONCLUSIONS: HPE and its individual fractions increased the thermal stability of erythrocyte membranes and erythrocytes. This effect was attributed to the reversible binding of some low molecular ingredient of HPE to the integral proteins and consequent stabilization of their interaction with under-membrane cytoskeleton.

Hereditary Spherocytosis in the Neonatal Period: A Case Report

Hereditary spherocytosis (HS) is the third most common yet most frequently underrecognized, congenitally acquired hemolytic disease of the neonate. Hereditary spherocytosis is caused by a defect of one or more erythrocyte membrane proteins, which leads to an increased rate of destruction of circulating red blood cells. The HS spectrum of symptoms is varied from asymptomatic to intrauterine hydrops. Diagnostic tests range from a complete blood count (CBC) analysis to deoxyribonucleic acid (DNA) sequencing. Management in the neonatal period focuses primarily on associated comorbidities, including the prevention of severe hyperbilirubinemia and anemia. Life span implications of HS include hemolysis, jaundice, anemia, splenomegaly, and periodic gallstones. Early identification and diagnosis of HS is essential to ensure proper monitoring and medical management throughout infancy, childhood, and adulthood.

An Improved 2-Dimensional Gel Electrophoresis Method for Resolving Human Erythrocyte Membrane Proteins

The 2-dimensional gel electrophoresis (2-DE) technique is widely used for the analysis of complex protein mixtures extracted from biological samples. It is one of the most commonly used analytical techniques in proteomics to study qualitative and quantitative protein changes between different states of a cell or an organism (eg, healthy and diseased), conditionally expressed proteins, posttranslational modifications, and so on. The 2-DE technique is used for its unparalleled ability to separate thousands of proteins simultaneously. The resolution of the proteins by 2-DE largely depends on the quality of sample prepared during protein extraction which increases results in terms of reproducibility and minimizes protein modifications that may result in artifactual spots on 2-DE gels. The buffer used for the extraction and solubilization of proteins influences the quality and reproducibility of the resolution of proteins on 2-DE gel. The purification by cleanup kit is another powerful process to prevent horizontal streaking which occurs during isoelectric focusing due to the presence of contaminants such as salts, lipids, nucleic acids, and detergents. Erythrocyte membrane proteins serve as prototypes for multifunctional proteins in various erythroid and nonerythroid cells. In this study, we therefore optimized the selected major conditions of 2-DE for resolving various proteins of human erythrocyte membrane. The modification included the optimization of conditions for sample preparation, cleanup of protein sample, isoelectric focusing, equilibration, and storage of immobilized pH gradient strips, which were further carefully examined to achieve optimum conditions for improving the quality of protein spots on 2-DE gels. The present improved 2-DE analysis method enabled better detection of protein spots with higher quality and reproducibility. Therefore, the conditions established in this study may be used for the 2-DE analysis of erythrocyte membrane proteins for different diseases, which may help to identify the proteins that may serve as markers for diagnostics as well as targets for development of new therapeutic potential.