The Band 3 Network in Acanthocytosis

Using an antibody against the cytoplasmic domain of band 3, we found specific immunoblot patterns in the membrane fractions of erythrocytes from patients with ChAc, MLS and HDL2 (Fig. 1). Aberrant immunoblot patterns were also obtained with anti-Diego and anti-phosphotyrosine antibodies [6]. These data suggest that the presence of acanthocytes in patients with various forms of NA is associated with, as well as characterized by, specific changes in the conformation of band 3 [5]. Based on immunoblot data obtained with antibodies of various specificities, these changes are likely to affect not only the interaction between cytoskeleton and lipid bilayer, but also the interaction of band 3 with other integral membrane proteins, and with cytosolic proteins. These changes may affect erythrocyte metabolism, either through the anion transport activity, or through binding and inactivation of enzymes such as aldolase and GAPDH.

Fig. 1 Immunoblots of erythrocyte membranes from HDL2 patients (P and S) show abnormal band 3 patterns with anti-band 3 antisera directed against the cytoplasmic, N-terminal and the membrane domain of band 3, but not with antisera against the C-terminal domain of band 3

Fig. 2 Immunoblots of erythrocyte membranes from age-separated erythrocytes show an aging-related breakdown of band 3 in a control donor (C), and an abnormal band 3 pattern especially in the youngest fraction (fraction I) of a HDL2 patient (NA). I, II and V, erythrocytes of increasing age, consisting of 5%, 15% and 95% acanthocytes, respectively, in the HDL2 patient

Fig. 2 Immunoblots of erythrocyte membranes from age-separated erythrocytes show an aging-related breakdown of band 3 in a control donor (C), and an abnormal band 3 pattern especially in the youngest fraction (fraction I) of a HDL2 patient (NA). I, II and V, erythrocytes of increasing age, consisting of 5%, 15% and 95% acanthocytes, respectively, in the HDL2 patient

In order to determine whether the NA-specific changes in band 3 structure are specific for the acanthocytes or for all erythrocytes of a NA patient, we separated the erythrocytes from an HDL2 patient into different populations using a combination of volume (counterflow elutriation) and subsequent density (Percoll gradient) centrifuga-tion, that has been developed for the isolation of erythrocytes of various ages [7]. The acanthocytes were concentrated in the fraction which, in control donors, consists of the oldest, most dense and smallest erythrocytes (fraction V in [7]). In the samples from the HDL2 patient, this fraction consisted of 95% acanthocytes, whereas the other fractions consisted for maximally 15% erythrocytes with an echinocytic/acanthocytic morphology. This is in agreement with previous analyses showing that acanthocytes from a patient with chorea-acanthocytosis were concentrated in the high-density layers of density gradients [8]. Immunoblot analysis showed an aging-related increase in band 3 degradation in control erythrocytes (Fig. 2), as reported before [7]. The NA-related changes observed in the total erythrocyte populations were observed in all fractions, but especially in the fraction that contains hardly any acanthocytes and that, in control donors, contains the youngest erythrocytes (fraction I in Fig. 2). These data suggest that NA-related alterations in band 3 structure are not associated with the presence of acanthocytic morphology per se. We hypothesize that these alterations may reflect a NA-related imbalance in the erythrocyte membrane stability that, during the lifespan of the erythrocyte, leads to the appearance of acanthocytes.

The red cell membrane stability depends on the maintenance of protein bridges connecting the erythrocyte membrane with the spectrin-based cytoskeleton, which are mainly established by band 3 and ankyrin in the band 3-ankyrin-band 4.2 complex. Additional anchoring sites are organized in the Rh-RhAG-ankyrin complex, and the glycophorin C-band 4.1-p55 complex [2, 18]. The red cell membrane tyrosine phosphorylation pattern is significantly different in ChAc red cells when compared to normal controls [19] (Fig. 3). Using a proteomic approach, we excised the bands in twin gels stained with colloidal Coomassie Blue. We identified some of the proteins that were differently phosphorylated (bands 1 to 4 in Fig. 3) by mass spectro-metric analyses (MALDI-TOF). We confirmed that band 3 tyrosine phosphorylation is increased in ChAc compared to normal controls. We also showed that the tyro-sine phosphorylation of protein 55 kDa, P actin and GAPDH is higher in ChAc than in control erythrocytes (Fig. 3). We also observed differences in the red cell mem-

Fig. 3 The tyrosine-phosphorylation (P-Tyr) membrane protein pattern was evaluated in normal control and in ChAc erythrocytes. The membrane proteins were solubilized and separated by gel electrophoresis. The gels were then transferred to nitrocellulose membrane and subsequently blotted with anti-phosphotyrosine antibodies (WB-anti-PY). In order to identify the proteins showing a different tyrosine phosphorylation state, the corresponding bands in twin gels were excised and trypsinized before undergoing MALDI-TOF analysis. The bands identified to date are reported in the table (indicated in the figure by an arrow with a corresponding number). To identify the proteins we carried out a database search using the peptide maa volumes against the Swiss-Prot database (taxa human) using the Mascot search engine (Matrix Science Ltd, London, UK). A mass accuracy of 0.3 Da and a single missed cleavage allowed for each matching peptide, Here, we show one Western blot analysis and the corresponding colloidal Coomassie-stained gel that are representative for three experiments with similar results

Fig. 3 The tyrosine-phosphorylation (P-Tyr) membrane protein pattern was evaluated in normal control and in ChAc erythrocytes. The membrane proteins were solubilized and separated by gel electrophoresis. The gels were then transferred to nitrocellulose membrane and subsequently blotted with anti-phosphotyrosine antibodies (WB-anti-PY). In order to identify the proteins showing a different tyrosine phosphorylation state, the corresponding bands in twin gels were excised and trypsinized before undergoing MALDI-TOF analysis. The bands identified to date are reported in the table (indicated in the figure by an arrow with a corresponding number). To identify the proteins we carried out a database search using the peptide maa volumes against the Swiss-Prot database (taxa human) using the Mascot search engine (Matrix Science Ltd, London, UK). A mass accuracy of 0.3 Da and a single missed cleavage allowed for each matching peptide, Here, we show one Western blot analysis and the corresponding colloidal Coomassie-stained gel that are representative for three experiments with similar results

Fig. 4 The tyrosine-phosphorylation (P-Tyr) membrane protein pattern was evaluated in normal control and in MLS erythrocytes as described in the legend to Fig. 3. The bands are now being identified by proteomic analysis. Here, we show one Western blot analysis and the corresponding colloidal Coomassie-stained gel that are representative for four other experiments with similar results

Fig. 4 The tyrosine-phosphorylation (P-Tyr) membrane protein pattern was evaluated in normal control and in MLS erythrocytes as described in the legend to Fig. 3. The bands are now being identified by proteomic analysis. Here, we show one Western blot analysis and the corresponding colloidal Coomassie-stained gel that are representative for four other experiments with similar results

brane tyrosine phosphorylation pattern in MLS erythrocytes compared to normal controls (Fig. 4). In particular, we found increased tyrosine phosphorylation of proteins with molecular weights between 115 and 181 kDa and a band around 82 kDa, without major differences of bands in the lower molecular weight regions. The bands that are differently phosphorylated are now being identified by MALDI-TOF analysis. We have already identified one of the bands as P-spectrin. It is interesting to note that abnormal tyrosine phosphorylation of spectrin is also present in ChAc erythro-cytes (Fig. 3), suggesting a perturbation of the functional connections between the red cell membrane and the cytoskeleton in both disorders. Changes in the tyrosine phos-phorylation state might alter spectrin stability and thereby the spectrin network and cytoskeleton organization [24]. Recently, a three-dimensional computational study of the equilibrium between shape and deformation in red cells, using spectrin-level energetics, has shown that the spectrin network is constantly remodelled in any red cell shape [14, 15]. These data implicate a crucial role for the membrane-anchoring sites, including the band 3-ankyrin bridges between the erythrocyte membrane and the spectrin-based cytoskeleton, in maintaining optimal cell morphology.

These data further support the hypothesis that the generation of acanthocytes is related to a perturbation of the erythrocyte membrane network, associated with abnormalities in the tyrosine phosphorylation state of various proteins, which may result in an abnormal modulation of either membrane protein-protein or membrane protein-lipid bilayer cross-talk.

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