Sickle cell disease (SCD) is characterized by the presence of sickle hemoglobin (HbS), which has the unique property of polymerizing when deoxygenated and thereby triggering erythrocyte sickling and dehydration. Repeated HbS polymerization is associated with generation of dense, distorted red cells characterized by an extensive membrane oxidative damage and abnormal activation of membrane cation transport pathways. The recently developed proteomic technologies represent a useful tool for identification of new functional pathways in sickle red cells. We studied the membrane proteoma of sickle red cells from homozygous patients (SS), not carrying alpha-thalassemia, not undergoing hydroxyurea treatment and not transfused at least three months before the study. The buffy-coat was removed and the red cells were separated into two fractions corresponding to the reticulocytes enriched fraction and to the dense red cell fraction (n= 8). Following osmotic lysis, the red cell ghost proteins were separated by one- dimensional or by two-dimensional gel electrophoresis (1-DE, 2-DE). Using the ImageMaster 2D Platinum (5.0 Amersham-Biosciences) software the 2-DE gels from control and sickle cells were compared, allowing us to identify spots differently expressed in sickle cells compared to normal cells. The selected spots were excised and trypsinized before mass spectrometric analysis and compared with the SWISS-PROT and NCBI non-redundant databases. In sickle reticulocytes enriched fraction compared to the control one, we selectively identified the following cystokeleton proteins: four spots for beta actin, two spots for myosin light chain and a train of four proteins identified as protein 4.1, one of which was phosphorylated on Tyr13, a protein similar to flotillin 2, carbonic anhydrase I and the following stress-response proteins: heat shock protein 70 (HSP70) protein 9B, HSPC108 and peroxiredoxin II (PrxII), which was phosphorylated in Thr142. In dense sickle red cell fraction compared to normal one we selectively identified a train of two spots for 4.1R, band 3 and aldolase and two spots identified as HSP70 protein 5.
The identified proteins were then confirmed by western-blotting analysis.
These data suggest that in sickle red cells HSP70, HSP108 function may be required by the extensive assistance in the refolding of misfolded proteins most likely related to abnormal sickle red cell oxidative environment as also supported by the presence of phosphorylated peroxiredoxin II, which has been recently proposed as regulator of redox-sensitive signaling in other cell types.