Oxidative events involving band 3 (Anion Exchanger 1) have been associated with RBC (red blood cell) removal through binding of NAbs (naturally occurring antibodies); however, the underlying mechanism has been only partially characterized. In addition to inducing direct membrane protein oxidative modification, oxidative treatment specifically triggers the phosphorylation of band 3 tyrosine residues. The present study reports that diamide, a thiol group oxidant, induces disulfide cross-linking of poorly glycosylated band 3 and that the oligomerized band 3 fraction is selectively tyrosine phosphorylated both in G6PD (glucose-6-phosphate dehydrogenase)-deficient and control RBCs. This phenomenon is irreversible in G6PD-deficient RBCs, whereas it is temporarily limited in control RBCs. Diamide treatment caused p72 Syk phosphorylation and translocation to the membrane. Diamide also induced p72 Syk co-immunoprecipitation with aggregated band 3. Moreover, following size-exclusion separation of Triton X-100-extracted membrane proteins, Syk was found only in the high-molecular-mass fraction containing oligomerized/phosphorylated band 3. Src family inhibitors efficiently abrogated band 3 tyrosine phosphorylation, band 3 clustering and NAbs binding to the RBC surface, suggesting a causal relationship between these events. Experiments performed with the non-permeant cross-linker BS3 (bis-sulfosuccinimidyl-suberate) showed that band 3 tyrosine phosphorylation enhances its capability to form large aggregates. The results of the present study suggest that selective tyrosine phosphorylation of oxidized band 3 by Syk may play a role in the recruitment of oxidized band 3 in large membrane aggregates that show a high affinity to NAbs, leading to RBC removal from the circulation.
- band 3
- p72 Syk
- tyrosine phosphorylation
- Src family kinase
G6PD (glucose-6-phosphate dehydrogenase) deficiency exerts a protective effect against infection by malaria  and causes haemolytic anaemia  that is triggered by exposure to oxidants. This protection is possibly mediated by increased susceptibility of G6PD-deficient RBCs (red blood cells) to membrane modification, leading to the phagocytosis of parasitized RBCs . To explain the mechanisms of RBC destruction and/or their splenic removal, band 3 modifications have been extensively studied in hereditary haemolytic disorders relating to increases in oxidative membrane damage, such as β-thalassaemia, G6PD deficiency, sickle-cell anaemia and malaria-infected RBCs [4–6]. Band 3 is the major integral protein of the RBC membrane, playing a crucial role in membrane functional organization, stability and RBC volume regulation [7,8].
Previous studies have shown that thiol group reagents, such as diamide, can promote membrane oxidative damage, leading to band 3 dimerization through disulfide cross-linking in a dosedependent manner [9,10]. In addition, diamide-induced oxidized/dimerized band 3 acquires the capability to bind anti-band 3 NAbs (naturally occurring antibodies) [4,10,11]. The same phenomenon was observed in the removal of either senescent RBCs or of pathological RBCs, such as in β-thalassaemias, G6PD deficiency, sickle-cell anaemia and malaria-infected RBCs, all characterized by the presence of denatured haemoglobin products binding to the band 3 cytoplasmic domain and possibly inducing its oxidation [5,6,12–19].
In vitro studies have shown that diamide treatment increases the band 3 tyrosine phosphorylation state, suggesting a possible functional connection between membrane oxidative damage and modulation of signal transduction pathways involving kinases/phosphatases [20,21]. The tyrosine kinases Lyn, from the SFKs (Src-family kinases), and Syk have been seen to functionally interact with the cytoplasmic domain of band 3 in RBCs [21,22]. Syk and Lyn act on different tyrosine residues of the band 3 cytoplasmic domain and kinetic studies suggest that Syk action might be the primary tyrosine phosphorylative event [21–23]. RBC oxidative treatment induces Syk phosphorylation , translocation to the RBC membrane , and its activation and association to band 3 [21,26].
Increased band 3 tyrosine phosphorylation has been reported in G6PD-deficient RBCs treated with low diamide concentrations , confirming the functional relationship between RBC redox states and the regulation of band 3 tyrosine phosphorylation.
Since the changes in protein tyrosine-phosphorylation state are dependent on the balance between kinases and phosphatases, studies on PTPs (protein tyrosine phosphatases) blocked by thiol group reagents have been carried out, showing that PTPs are relevant to band 3 tyrosine-phosphorylation events following oxidant treatment , band 3 being a substrate of the SHP-2 (Src homology 2 domain-containing PTP 2) tyrosine phosphatase [20,29,30]; however, the relative importance of the oxidantactivated tyrosine kinases and/or PTP inhibition on band 3 tyrosine phosphorylation has not been clarified. Since band 3 possesses two reactive cysteine residues (Cys201 and Cys317) in its cytoplasmic domain, it can be disulfide cross-linked by oxidant RBC treatment. Conformational changes of oxidatively modified band 3 has been hypothesized to explain changes to its cytoskeleton-binding properties . It is of interest to note that, at the diamide concentrations needed to observe changes in band 3 tyrosine phosphorylation , a fraction of total band 3 molecules are oxidized and clustered to form high-molecular-mass aggregates . In the present study, we investigate whether oxidatively induced band 3 tyrosine phosphorylation could be involved in the genesis of the membrane modifications that cause G6PD-deficient RBC removal. In particular we studied the mechanisms that lead to the selective recruitment of oxidized band 3, to form the high-molecular-mass band 3 aggregates, binding NAbs, observed in several haemolytic diseases [5,12–14,16,17,31].
Unless otherwise stated, all materials were obtained from Sigma. SFK inhibitors PP1/PP2 were purchased from Calbiochem. The DC protein assay was purchased from Bio-Rad. The BS3 (bis-sulfosuccinimidyl-suberate) and chemiluminescence kit were purchased from Pierce Biotechnology. Anti-Syk and anti-phosphotyrosine antibodies were purchased from Cell Signaling Technologies.
Treatment of RBCs
Venous blood was taken from six normal and five G6PD-deficient human subjects (Mediterranean variant, type II deficiency). Human experimentation was carried out with the appropriate ethical approval and written informed consent was obtained from all patients. G6PD-deficient RBCs had a residual activity lower than 2%. All samples were genotyped  and presented the mutation 563C>T. Blood was washed three times with PBS-glucose [PBS containing 5 mM glucose (pH 7.4)] to obtain packed RBCs. RBCs were resuspended at 30% haematocrit in PBS-glucose. RBCs were incubated in the presence of 2 mM diamide for 45 min at 37 °C; alternatively a time course from 0 to 90 min was carried out where necessary. Identical aliquots of RBCs were incubated for corresponding incubation periods at 37 °C without diamide as a control. In separate experiments, RBCs were incubated with 1 mM orthovanadate for 1 h at 37 °C. When necessary, RBCs were pretreated (before diamide treatment) with SFK inhibitors (10 μM PP1 and 10 μM PP2) for 1 h at 37 °C in the dark. Each reaction was terminated by three washes with PBS-glucose. Control and diamide-treated RBCs were incubated with increasing concentrations (0, 0.25, 0.50 and 1 mM) of BS3 for 15 min at 37 °C. RBCs were washed three times in PBS containing 40 mM Tris (pH 7.4), and membranes were prepared. Solubilized membranes were frozen at −20 °C.
RBC membrane preparation
Packed RBCs from normal and G6PD-deficient human subjects were lysed in hypotonic buffer [5 mM sodium phosphate and 1 mM EDTA (pH 8.0)] containing protease inhibitor cocktail and, when necessary, phosphatase inhibitor cocktail. Membranes were washed as previously described . Membrane alkali treatment to deplete cytoskeletal proteins was performed as previously described .
Electrophoresis and immunoblotting
The membrane protein content was quantified using the DC protein assay and was solubilized in Laemmli buffer  under reducing [2% (w/v) DTT (dithiothreitol)] or non-reducing conditions at a volume ratio of 1:1. SDS/PAGE was conducted by heating the samples for 5 min at 100 °C and loading 20 μg of membrane proteins on an 8% gel for protein staining by colloidal Coomassie Blue .
For Western blot analysis, 30 μg of protein was loaded in each lane, transferred on to nitrocellulose membranes and immunostained with anti-Syk and anti-phosphotyrosine antibodies, both diluted 1:2000, using anti-mouse peroxidase-labelled secondary antibodies and detected by chemiluminescence according to the manufacturer's protocol (Pierce Biotechnology). For anti-band 3 antibody Western blots, 0.5 μg of protein was loaded and the anti-band 3 antibody was diluted 1:50000 and detected using a chromogenic substrate as described previously .
Anti-Syk antibody immunoprecipitation
RBCs from normal and G6PD-deficient human subjects were treated in the presence or absence of diamide and membranes were prepared as described above. Membrane proteins (200 μl) were solubilized for 15 min at 0 °C with 3 vol. of 1% (v/v) Triton X-100 in hypotonic buffer [5 mM sodium phosphate and 1 mM EDTA (pH 8.0)]. After centrifugation in a refrigerated Eppendorf microfuge at 13000 g, supernatants were collected and incubated with a rabbit anti-Syk antibody cross-linked to Protein A–Sepharose (50 μl) via a bifunctional coupling reagent dimethypimelimidate [34a] for 2 h at 4 °C with gentle mixing. Beads were washed three times with 1% (v/v) Triton X-100 in PBS (pH 7.4). Laemmli sample buffer (3 vol.) containing 2% (w/v) DTT (final concentrations) were added to packed beads, that were heated and subjected to SDS/PAGE and Western blot analysis with mouse anti-band 3 and anti-phosphotyrosine antibodies.
Membrane proteins separated on Sepharose CL-6B
RBCs from normal and G6PD-deficient human subjects were treated in the presence or absence of diamide, and membranes were prepared and fractionated as previously described . With minor modifications, 1 ml of membranes were solubilized in 2 ml of extraction buffer [10 mM Hepes, 130 mM NaCl, 10 mM N-ethylmaleimide, 1 mM EDTA, 1 mM PMSF and 1% (v/v) Triton X-100 (pH 7.4)], gently shaken for 10 min at 20 °C and centrifuged at 21460 g for 5 min in an Eppendorf microfuge. The supernatant was applied to a 40 cm×1 cm column filled with Sepharose CL-6B equilibrated with a solution containing 10 mM Hepes, 50 mM NaCl and 0.1% Triton X-100 (pH 7.4), at a flow rate of 1 ml/min. Constant flow was maintained using an HPLC pump. The effluent was collected in 1 ml fractions. Fractions were concentrated 10-fold and analysed for immunoblotting with anti-band 3, anti-phosphotyrosine and anti-Syk antibodies as described above.
Treatment of control and diamide-treated RBC membranes with control and diamide-treated RBC cytoplasmic fractions
RBCs from six normal and five G6PD-deficient human subjects were treated with or without diamide (see above). Packed RBCs were subjected to haemolysis by adding 5 vol. of 5 mM sodium phosphate (pH 8.0) in the presence of protease inhibitor cocktail, and were centrifuged in a refrigerated Eppendorf microfuge at 13000 g at 4 °C for 10 min, and the supernatant was collected to obtain the cytoplasmic fractions.
RBC membranes were prepared as described above and incubated for 10 min at 4 °C with or without 2 mM diamide. Membranes were washed three times in 5 mM sodium phosphate (pH 8) in the presence of protease inhibitor cocktail at 12000 g at 4 °C for 10 min. Membranes were collected and incubated with the cytoplasmic fractions in the presence of 50 μM ATP for 10 min at 37 °C.
Finally, membranes were washed three times in hypotonic buffer (pH 8), at 13000 g at 4 °C for 10 min in the presence of protease inhibitor cocktail and phosphatase inhibitor cocktail. Membrane proteins were separated on SDS/PAGE and blotted on to nitrocellulose membranes as described above.
Measurement of membrane-bound IgG
Membrane-bound IgG was estimated using an immunoenzymatic assay as previously described . In brief, RBCs were incubated for a short time with an anti-human IgG antibody conjugated to alkaline phosphatase, membranes were prepared as usual and solubilized with 1% (v/v) Triton X-100. Alkaline phosphatase activity was determined by colorimetric detection.
Measurement of RBC GSH concentrations
GSH was measured in packed RBCs by colorimetric detection following protein precipitation as previously described .
Band 3 MS analysis by MALDI TOF (matrix-assisted laser-desorption ionization–time-of-flight)
Band 3 gel slices from one-dimensional Coomassie-stained gels were excised in control, G6PD-deficient and diamide-treated RBC membranes. Pieces of gel were destained by several washes in 5 mM NH4HCO3/acetonitrile (50:50, by vol.) and successively dried with pure acetonitrile. The gel slices were rehydrated for 45 min at 4 °C in 20 μl of a 5 mM NH4HCO3 digestion buffer containing 10 ng/μl trypsin. Excess protease solution was removed and the volume was adjusted with 5 mM NH4HCO3 to cover the gel slices. Digestion was allowed to proceed overnight at 37 °C .
Samples were loaded on to the MALDI target using 1 μl of the tryptic digests mixed 1:1 with a solution of α-cyano-4-hydroxycinnamic acid (10 mg/ml in 0.1% acetonitrile/trifluoroacetic acid; 40:60, by vol.). Band 3 N-terminal phosphorylation was identified operating the instrument in linear modality according to the manufacturer's protocol (Maldi Micro MX). Phosphorylation was confirmed by phosphatase treatment according to a previously described method .
MS analysis of peptides from band 3 fractions sliced from the one-dimensional gel were performed with a MALDI–TOF Micro MX (Micromass), operating the instrument in reflectron modality according to the tuning procedures suggested by the manufacturer. Peak lists were generated with Proteinlynx data preparation using the following parameters: external calibration with lock mass using the mass 2465,1989 Da of the ACTH fragment 18 39 (Sigma; A8346) background subtract with adaptive mode, performing de-isotoping with threshold 3%.
For PMF (peptide mass fingerprinting) analysis of MS spectra were converted into pkl files using Mass Lynx 4.0. Peak lists containing the 20 most intense peaks of the spectrum were subjected to MASCOT PMF search (http://www.matrixscience.com) using the Swiss-Prot database (release 50.0, dating to 30 May 2006). Search settings allowed one missed cleavage with the trypsin enzyme selected, carboxymethylated cysteine as fixed modification and oxidation of methionine as potential variable modification and a peptide tolerance of 50 p.p.m. Only protein identifications with significant Mascot scores (P< 0.05) were taken into consideration.
Selective tyrosine phosphorylation of oxidized/oligomerized band 3 in control and G6PD-deficient RBCs
Although published results report that diamide treatment induces band 3 oligomerization and band 3 tyrosine phosphorylation [20,25], the temporal changes of these modifications and their causal relationships have never been established. Since, GSH plays an important role as an antioxidant system depending on the NADPH function, we evaluated the levels of reduced GSH and band 3 oligomerization states in control and G6PD-deficient RBCs exposed to diamide treatment. In control and G6PD-deficient RBCs GSH rapidly dropped after diamide treatment, but only control RBCs were able to restore basal levels following 90 min of incubation (Figure 1A).
In analogy with GSH level variation, diamide treatment induced band 3 oligomerization that was permanent in G6PD-deficient RBCs and it was reversible in control RBCs (Figure 1B, lanes 1, 2, and 3 compared with lanes 7, 8 and 9). To verify whether a longer time was needed to reduce band 3 in G6PD-deficient RBCs, we incubated the cells for up to 12 h; however, progressive band 3 oligomerization was observed (results not shown). To assess whether changes in band 3 tyrosine phosphorylation might be affected by its oxidative modifications, we performed immunoblot analyses with specific anti-phosphotyrosine antibodies under non-reducing conditions. Diamide treatment caused marked tyrosine phosphorylation in correspondence with the oxidized oligomeric band 3 region both in control and G6PD-deficient RBCs (Figure 1B, lanes 2 and 5 compared with lanes 8 and 11). Only in control RBCs, after incubation for 90 min, was band 3 reduction associated with a net decrease in the band 3 tyrosine phosphorylation state (Figure 1B, lanes 3 and 6 compared with lanes 9 and 12). Reduction of disulfide bridges by DTT caused complete band 3 reduction and a parallel shift of the anti-phosphotyrosine signal to the monomeric band 3 region (Figure 1C), indicating that oxidized band 3 is the site of tyrosine phosphorylation. G6PD-deficient RBCs showed identical behaviour patterns (results not shown).
Since changes in protein phosphorylation states are dependent on the balance between cellular kinases and phosphatases, we evaluated the effect of PTP inhibition by orthovanadate. In control RBCs, orthovanadate treatment caused increases of monomeric band 3 tyrosine phosphorylation (Figure 1D). No changes in the band 3 oxidative state were induced by orthovanadate (results not shown), suggesting that the selective tyrosine phosphorylation of oxidized band 3 induced by diamide cannot be related only to PTP inhibition.
Under our experimental conditions the SFK inhibitors PP1 and PP2 reduced diamide-induced band 3 tyrosine phosphorylation (Figure 1E), showing that SFKs might be involved in band 3 phosphorylation directly or indirectly through the modulation of p72 Syk . G6PD-deficient RBCs had identical behaviour (results not shown).
On the basis of these results, we conclude that, following diamide treatment, only oxidized/oligomerized band 3 is tyrosine phosphorylated. This phenomenon is apparently due to changes of tyrosine kinase activity and/or substrate affinity because, following PTP inhibition, band 3 was phosphorylated irrespective of its oxidation state.
Binding of Syk to modified band 3 in diamide-treated RBCs
Since a small amount of Syk and Lyn had been previously reported to be associated with the RBC membrane in steady-state conditions [21,25], we evaluated the amount of both kinases on the membranes of RBCs treated with diamide. We observed that Syk undergoes membrane translocation following diamide treatment (Figure 2A, lane 2). It is of interest to note that Syk membrane association was evident in immunoblots only when the samples were preliminarily reduced, indicating that, following oxidant treatment, Syk was most probably bound to membrane protein aggregates. In addition, in diamide-treated RBCs, the bound membrane Syk was tyrosine phosphorylated, suggesting that it had been activated (Figure 2A, lane 4). We also blotted Western blot membranes with a specific anti-Lyn antibody, revealing the presence of Lyn constitutively bound to the membrane, but no changes in the amount of Lyn membrane association were observed following diamide treatment (results not shown).
To evaluate whether native or oxidatively activated Syk possesses different degrees of affinity to native or oxidized band 3, we compared control or diamide-treated membranes with the cytoplasmic RBC fractions obtained from control or diamide-treated RBCs. Syk from untreated RBC cytoplasm was able to bind to diamide-treated membranes (Figure 2B, lane 2) and at a much lower extent to control membranes (Figure 2B, lane 1). The addition of diamide-treated RBC cytoplasm caused a further increase in Syk binding to the diamide-treated membranes (Figure 2B, lane 4) and, to a minor extent, also to control membranes (Figure 2B, lane 3). Under the same experimental conditions, we also measured band 3 and Syk tyrosine-phosphorylation states. In parallel with Syk membrane translocation, we observed intense band 3 tyrosine phosphorylation only in diamide-treated membranes (Figure 2B, lanes 6 and 8). Following cytoplasm diamide treatment, membrane association of tyrosine-phosphorylated Syk was seen to increase (Figure 2B, lanes 7 and 8), but Syk binding to untreated membranes caused small changes in band 3 tyrosine phosphorylation (Figure 2B, lane 7), indicating that Syk requires oxidized band 3 to show its maximal affinity and/or catalytic activity.
To further validate this hypothesis, we immunoprecipitated Syk with a specific anti-Syk antibody from solubilized RBC membrane proteins. As shown in Figure 2(C) (lanes 2 and 4), in diamide-treated RBCs, Syk was immunoprecipitated in association with phosphorylated band 3. It is interesting to note that when sample reduction was omitted, both immunoprecipitated Syk and band 3 were present only in high-molecular-mass protein aggregates localized at the top of the gel (results not shown). Following a 90 min incubation time we observed complete reversal of the diamide-induced effects in control RBCs, but not in G6PD-deficient RBCs (results not shown).
On the basis of these results, we conclude that selective phosphorylation of oxidized/oligomerized band 3 could be due to increased Syk affinity toward modified band 3.
Mapping of band 3 phosphorylation sites in diamide-treated RBCs
Since Syk has been reported to specifically phosphorylate the tyrosine residues 8 and 21 of the band 3 cytoplasmic domain , we mapped the band 3 tyrosine phosphorylation sites in RBCs treated with diamide. Figure 3 shows the occurrence of a double-tyrosine-phosphorylated (+160 Da) band 3 N-terminal tryptic peptide. Since Tyr8 and Tyr21 are the only two tyrosine residues contained in the 6538 N-terminal tryptic peptide, the 160 Da mass shift indicates their phosphorylation. MALDI–TOF analysis had to be performed by linear modality owing to the high-molecular-mass of the N-terminal peptide. The double phosphorylated 6698 Da peptide was absent in band 3 isolated from untreated RBCs (Figure 3, CTRL) and in diamide-treated RBCs in the presence of either the SFK inhibitors PP1 and PP2, and after phosphatase treatment of the sample (results not shown). The presence of the tyrosine-phosphorylated tryptic peptide containing the putative Lyn substrate Tyr359 (from Tyr347 to Lys360) was also detected but, under our experimental conditions, its corresponding signal was weak and sporadic (results not shown). On the basis of these results, the involvement of Syk on the phosphorylation of oxidized band 3 was substantiated.
Preferential clustering of oxidized/hyperphosphorylated band 3
Previously, studies have shown that in diamide-treated RBCs oxidized band 3 preferentially clusters in high-molecular-mass aggregates , but the band 3 phosphorylation state was not taken into account in the formation of this aggregate. In the present study, size-exclusion chromatographic fractions of solubilized membrane proteins were subsequently subjected to immunoblot analysis to evaluate the occurrence of tyrosine-phosphorylated band 3 and Syk in the high-molecular-mass fractions (Figure 4A, fractions 3–5). We observed a net prevalence of tyrosine-phosphorylated band 3 and Syk in the high-molecular-mass fraction, suggesting preferential aggregation of tyrosine phosphorylated band 3. The relevance of band 3 tyrosine phosphorylation for band 3 clustering was also supported by the observation that treatment with the SFK inhibitors PP1 and PP2, in addition to inhibiting band 3 phosphorylation, also caused a decrease in diamide-induced high-molecular-mass band 3 aggregates (Figure 4A). Marked differences were detectable after 90 min of incubation, when band 3-containing aggregates regained basal levels in control RBCs, but not in G6PD-deficient RBCs (Figure 4B).
To investigate the hypothesis that band 3 phosphorylation may promote its clustering by weakening its cytoskeletal connections, we incubated diamide-treated RBCs with increasing concentrations of the poorly membrane-permeating cross-linkers BS3. Figure 5 shows that diamide pre-treatment caused enhancement of band 3 cross-linking even at low BS3 concentrations. As expected, at longer incubation times this phenomenon was reversed in control RBCs. On the basis of these results, we conclude that tyrosine-phosphorylated band 3 is selectively recruited in high-molecular mass aggregates due to an apparent increase in its lateral mobility.
Naturally occurring IgG membrane binding
As previous studies had demonstrated that NAb binding to the RBC surface depends on the band 3 oxidation/oligomerization state [10,36], we tested the effect of SFK inhibitors on NAb binding. Diamide-treated RBCs were incubated with autologous serum to allow naturally occurring IgG binding to the RBC surface. As shown in Figure 6, membrane IgG binding was occasional and transient in control RBCs, in contrast, IgG binding was more sustained and progressively increased in G6PD-deficient RBCs. Following 45 min of diamide treatment, PP1 and PP2 treatment caused an average 1.92±0.66-fold decrease in IgG binding in control RBCs and a 1.89±0.78-fold decrease in G6PD-deficient RBCs. These results indicate that tyrosine phosphorylation apparently also plays a role in the formation of band 3 antigenic complexes.
Selective recruitment of poorly glycosylated band 3 in diamide-induced oligomers
As shown in the present study (Figure 1B) and in a previous study , the SDS/PAGE faster-migrating band 3 fraction is seen to disappear following diamide treatment. To investigate the hypothesis that band 3 is preferentially aggregated we developed a method to estimate the rate of glycosylation of the SDS/PAGE-separated band 3 by measuring the relative amount of the tryptic fragments bound to the glycosidic moiety. Band 3 trypsin cleavage theoretically originates from different fragments containing the Arg646 residue linked to the glycosidic moiety. MS analysis showed the net prevalence of the 1564 Da peptide from Leu632 to Arg646 (LSVPDGFKVSNSSAR). The mass of this peptide should increase following its glycosylation and it should be out-of-scale of the analytical setting used. Therefore the amount of the 1564 Da peptide should be inversely related to the amount of glycosylated band 3 molecules. The band 3 gel region was cut into three parts (Figure 7, fractions A, B and C). The ratio between the 1564 Da peptide and the 1490 Da band 3 peptide (from amino acids 234 to 246, chosen as an internal reference) decreased in tandem with the band electrophoretic mobility in membranes of control RBCs shifting from 0.21 to 0.74. The 1564/1490 ratio shifted from the average ratio of 0.42±0.08 in monomeric band 3, to 0.80±0.13 in oligomeric band 3 in membranes of diamide-treated RBCs (Figure 7, Diamide), indicating the preferential oligomerization of the less glycosylated band 3 fraction. No significant differences were observed between control and G6PD-deficient RBCs incubated with diamide for 45 min (results not shown).
Physiologically senescent RBCs or pathological RBCs such as in G6PD deficiency, β-thalassaemias and malaria are possibly removed by the peripheral circulation following the binding of denatured haemoglobin molecules (haemichromes) to the cytoplasmic domain of the integral membrane protein band 3 [4–6]. Interestingly, G6PD deficiency, as well as β-thalassaemias, provide protection against malaria and are characterized by enhanced haemichrome formation induced by malaria parasite growth . Haemichromes produced in different pathological situations are able to bind and oxidize the band 3 cytoplasmic domain inducing the formation of large band 3 clusters . It must be underlined that band 3 clusters cause RBC recognition by way of a mechanism involving anti-band 3 NAbs binding to the RBC surface [4,37,38].
Since band 3 is normally linked to the cytoskeleton by junctional complexes, the formation of large band 3 aggregates should be restrained by such interactions unless they are weakened by regulatory mechanisms. As a matter of fact, ankyrin docks in close proximity to the band 3 cytoplasmic domain tyrosine-phosphorylation sites, and non-phosphorylated band 3 appears to be less prone to forming high-molecular-mass aggregates [39,40]. Furthermore, hyperphosphorylated band 3 has been found to be associated with G6PD deficiency , deoxygenated sickle cells [26,41] and senescent RBCs , all clinical conditions characterized by increased RBC oxidative stress and band 3 cluster formation.
In the present study, we observed selective phosphorylation of oxidized/oligomeric band 3 both in diamide-treated control and G6PD-deficient RBCs. Control and G6PD-deficient RBCs, showed differential band 3 phosphorylation time courses in close correspondence with GSH.
The band 3 cytoplasmic domain appears to be one of the principal substrates of tyrosine kinases, such as the SFKs Lyn and Syk . Even if possible inhibition of PTPs might contribute to enhancing band 3 tyrosine phosphorylation [29,40], in the present study we show that diamide causes selective tyrosine phosphorylation of the oxidized/oligomerized band 3 fraction, whereas PTP inhibition by orthovanadate causes widespread band 3 tyrosine phosphorylation irrespective of its oxidation state [29,41]. These findings indicate a primary increase of tyrosine kinase activity and/or affinity for oxidatively modified band 3, whereas phosphatase inhibition seems to play a secondary role in enhancing kinase-selective action on oxidized band 3.
Naturally occurring IgG binding to diamide-treated RBCs proceeded in a parallel fashion with band 3 phosphorylation and clustering in control and G6PD-deficient RBCs. Band 3 phosphorylation, clustering and IgG binding were all inhibited by SFK inhibitors PP1 and PP2, indicating a causal role of band 3 tyrosine phosphorylation.
In diamide-treated RBCs, we observed the preferential oligomerization/phosphorylation of the less-glycosylated band 3 fraction, supporting the hypothesis that, in RBCs, only a fraction of band 3 molecules are prone to being oxididatively cross-linked and tyrosine phosphorylated. Previous reports have indicated that band 3 shows an increased tendency to cluster in congenital dyserythropoietic anaemia type 2 which is characterized by band 3 under-glycosylation and haemolytic anaemia [42,43].
Our results in the present study therefore indicate that two band 3 post-translational modifications modulate the clusterization capability of band 3: glycosylation appears to be a restraint to its oxidative cross-linking, whereas tyrosine phosphorylation appears to facilitate oxidatively modified band 3 clusterization. However, it is of importance to note that, in the present study, we only studied the early events that followed thiol group oxidation, whereas, in pathological conditions, more complex membrane modifications, involving haemoglobin denaturation products and additional phosphorylative events, may occur. Our results also highlight the need to further investigate the molecular details of the RBC tyrosine kinase oxidative activation and phosphorylative regulation of band 3 cytoskeleton interactions.
In summary, the findings of the present study provide new evidence to explain the formation of band 3 clusters observed in different physiological and pathological situations associated with RBC removal, shifting from the classical view of a passive role of damaged membrane components to the activation of a regulatory mechanism involving Src kinase as an upstream regulator of Syk through its affinity change to modified band 3 (Figure 8). Taking into account that haemichrome-bound band 3 molecules become quickly oxidized , band 3 tyrosine phosphorylation may represent a mechanism for their selective recruitment in large membrane clusters.
This work was supported by Telethon [grant number GGP04242].
Abbreviations: BS3, bis-sulfosuccinimidyl-suberate; DTT, dithiothreitol; G6PD, glucose-6-phosphate dehydrogenase; MALDI TOF, matrix-assisted laser-desorption ionization–time-of-flight; NAb, naturally occurring antibody; PMF, peptide mass fingerprinting; PTP, protein tyrosine phosphatase; RBC, red blood cell; SFK, Src-family kinase
- © The Authors Journal compilation © 2009 Biochemical Society