Protein methylation as a marker of aspartate damage in glucose-6-phosphate dehydrogenase-deficient erythrocytes Role of oxidative stress Diego Ingrosso 1,2 , Amelia Cimmino 1 , Stefania D’Angelo 1 , Fiorella Alfinito 3 , Vincenzo Zappia 1,2 and Patrizia Galletti 1,2 1 Department of Biochemistry and Biophysics, School of Medicine, Second University of Naples, Italy; 2 Cardiovascular Research Centre, School of Medicine, Second University of Naples, Italy; 3 Department of Hematology, School of Medicine, University of Naples Federico II, Italy The ÔMediterraneanÕ variant of glucose-6-phosphate dehy- drogenase (G6PD) deficiency is due to the C563CT point mutation, leading to replacement of Ser with Phe at position 188, resulting in acute haemolysis triggered by oxidants. Previous work has shown increased formation of altered aspartate residues in membrane proteins during cell ageing and in response to oxidative stress in normal erythrocytes. These abnormal residues are specifically recognized by the repair enzyme L -isoaspartate ( D -aspartate) protein O-methyltransferase (PCMT; EC 2.1.1.77). The aim of this work was to study the possible involve- ment of protein aspartate damage in the mechanism linking the G6PD defect and erythrocyte injury, through oxidative stress. Patients affected by G6PD deficiency (Mediterranean variant) were selected. In situ methylation assays were per- formed by incubating intact erythrocytes in the presence of methyl-labelled methionine. Altered aspartate residues were detected in membrane proteins by methyl ester quantifica- tion. We present here evidence that, in G6PD-deficient ery- throcytes, damaged residues are significantly increased in membrane proteins, in parallel with the decay of pyruvate kinase activity, used as a cell age marker. Erythrocytes from patients were subjected to oxidative stress in vitro,bytreat- ment with t-butylhydroperoxide, monitored by a rise in concentration of both methaemoglobin and thiobarbituric acid-reactive substances. L -Isoaspartate residues increased dramatically in G6PD-deficient erythrocytes in response to such treatment, compared with baseline conditions. The increased susceptibility of G6PD-deficient erythro- cytes to membrane protein aspartate damage in response to oxidative stress suggests the involvement of protein deamidation/isomerization in the mechanisms of cell injury and haemolysis. Keywords: erythrocyte membrane; glucose-6-phosphate (G6PD) deficiency; L -isoaspartate residues; oxidative stress; protein methylation. Several biochemical variants of glucose-6-phosphate dehy- drogenase (G6PD), corresponding to about 100 different point mutations of the gene encoding this protein, have been described [1]. Many of these are associated with chronic or acute haemolysis. The ÔMediterraneanÕ clinical variant, resulting from the C563CT change in the gene sequence, results in the replacement of serine with phenylalanine at position 188. Clinical outcome is characterized by neonatal jaundice and acute haemolysis and haemoglobinuria. Haemolysis is triggered by exposure to oxidants, e.g. fava beans(thediseaseisoftenreferredtoasÔfavismÕ, and acute haemolysis is called Ôfavic crisisÕ) or administration of drugs such as primaquine, nitrofurantoin, and sulfamethoxazole, or infection [1]. G6PD activity is almost undetectable in most patients [2,3]. However, despite the vast amount of data on the characterization of different G6PD variants, the pathophysiological link between the enzyme defect and haemolysis has not been unequivocally elucidated. Among G6PD-deficient erythrocytes, aged cells are the most sensitive to haemolysis, because of age-dependent decay of the activity of several enzymes, including G6PD, and antioxidant systems. There is further evidence that altera- tions in the plasma membrane are central to the mechanism of cell destruction [4,5]. Under normal conditions, G6PD- deficient erythrocytes are removed, mainly by phagocytosis, upon opsonization by autologous immunoglobulins and complement [5]. Haemolysis is in most cases autocompen- sated, as it appears to be limited to the oldest erythrocyte population [5]. These observations indicate that biochemical alterations that occur naturally during erythrocyte ageing are part of the mechanism of haemolysis induced by oxidative stress in these patients. Spontaneous post-biosynthetic modifications of mem- brane proteins have been shown to occur during erythrocyte ageing, in particular deamidation of asparagine residues and Correspondence to D. Ingrosso, Department of Biochemistry and Biophysics, Via Costantinopoli 16, 80138 Napoli, Italy. Fax: + 39 081441688, Tel.: + 39 0815667522, E-mail: diego.ingrosso@unina2.it Abbreviations: G6PD, glucose-6-phosphate dehydrogenase; PCMT, L -isoaspartyl-protein O-methyltransferase; PK, pyruvate kinase; RFLP, restriction fragment length polymorphism; TBARS, thio- barbituric acid-reactive substances; t-BHP, t-butylhydroperoxide; AdoMet, S-adenosylmethionine; AdoHcy, S-adenosylcysteine. Enzyme: L -isoaspartate ( D -aspartate) protein O-methyltransferase (PCMT; EC 2.1.1.77). (Received 24 October 2001, revised 8 February 2002, accepted 15 February 2002) Eur. J. Biochem. 269, 2032–2039 (2002) Ó FEBS 2002 doi:10.1046/j.1432-1033.2002.02838.x isomerization of aspartate residues [6]. Major targets of these alterations, also called Ôprotein molecular fatigue damageÕ [7], are cytoskeletal components, such as ankyrin and bands 4.1 and 4.2, as well as the integral membrane protein band 3 (AE1; the anion transporter) [6]. In this respect, asparaginyl deamidation has been shown to play a role in the shift of band 4.1b to 4.1a [8] during erythrocyte ageing. Increased molecular fatigue damage of several cytoskeletal proteins has been found associated not only with erythrocyte ageing, but also with intrinsic defects of erythrocytes [7]. In fact, L -isoaspartate, a major protein fatigue degradation product, is increased in hereditary spherocytosis, and its increase correlates positively with the degree of spectrin deficiency [9]. Erythrocyte passage through the spleen microcirculation has also been found to be a key determinant of this type of protein alteration in spherocytosis [10]. Therefore, deamidation and isomeriza- tion of membrane proteins have been proposed to play a role in spleen conditioning [10]. Major byproducts of protein fatigue at the Asn/Asp (Asx) level are L -isoaspartate residues. These residues are selectively recognized by a specific S-adenosylmethionine (AdoMet)-dependent enzyme, L -isoaspartate ( D -aspartate) protein O-methyltransferase (PCMT; EC 2.1.1.77) [11,12]. Enzymatic methylation of abnormal aspartate residues is physiologically involved in the repair and/or disposal of fatigue damaged proteins [13]. Because of its unusual substrate specificity, methylation can be used to monitor the occurrence of these protein alterations as the erythrocyte ages [7,8]. A number of erythrocyte stress conditions, including oxidation, have been shown to significantly increase isoas- partate content in normal erythrocyte membrane proteins, detected by measuring methylation levels [14,15]. The rationale of this work was to detect the occurrence of such alterations in G6PD deficiency and assess their extent in an oxidative microenvironment. Data presented here show that the effect of oxidative stress on membrane protein deam- idation/isomerization is significantly higher in G6PD-defi- cient erythrocytes (Mediterranean variant) than normal. The implications of these findings in the pathophysiology of haemolysis are discussed. MATERIALS AND METHODS Materials S-Adenosyl[methyl- 14 C]methionine (58 mCiÆmmol )1 )and [methyl- 3 H]methionine (55 CiÆmmol )1 ) were from Amer- sham International. Ready Gel liquid-scintillation cocktail was from Beckman Inc. (Cuppertino, CA, USA). Percoll was purchased from Pharmacia (Uppsala, Sweden). Selec- tographin was obtained from Schering (Berlin, Bergkamen, Germany). Thiobarbituric acid and t-butylhydroperoxide (t-BHP) (70% aqueous solution) were from Sigma Co. (St Louis, MO, USA). Patient enrolment and sample processing All subjects to be enrolled were assessed by a standard screening panel, evaluating blood G6PD activity and the presence of the C563T point mutation. A group of patients and age/sex-matched normal controls were selected. Patients gave informed consent and were made aware of the outcomes of the study. All procedures and manipula- tions, including blood sampling and genetic diagnostics, were subject to authorization by the patients. Experimental design was subject to approval by the bioethics committee, as required. At the time of the study, patients were free of haemolysis and in good clinical condition. Routine bio- chemical blood tests (Hitachi 911 Automatic Analyzer) and a standard haematological screening test for anaemia were performed. For all erythrocyte testing, blood samples were withdrawninEDTA(1mgÆmL )1 blood) and further processed for determination of G6PD activity [16]. Control G6PD activity was 8.34 ± 1.59 UÆg )1 haemoglobin, as defined in the literature [16]. Normal controls showed G6PD activity within this reference range, whereas patients had almost undetectable levels. When indicated, erythrocytes were separated according to density as previously described [17]. The two most abundant age-density fractions were used for the enzyme assays, and referred to as Ôbuoyant fractionÕ and Ôdense fractionÕ. Pyruvate kinase (PK) activity was measured, as a cell-age marker, in individual erythrocyte populations. Diagnostic evaluation and identification of the G6PD gene defect The study investigated 15 unrelated, G6PD-deficient men. The molecular defect was assessed as described previously [1]. Briefly, lymphocytes from peripheral blood were isolated on a Ficoll-Telepaque gradient; DNA was extracted [18], and G6PD exon 1 was amplified by PCR and isolated by agarose gel electrophoresis. As shown in Fig. 1, the C563T point mutation, responsible for the Mediterranean clinical variant, was then identified by digestion of PCR-amplified product with MboII [19]. A complete haematological screening of these subjects revealed no other alteration. For control purposes we also investigated 14 healthy men of the same age. Erythrocyte oxidative treatment Erythrocytes were subjected to oxidative stress as described previously [15], with modifications. Cells, prepared as described above, were filtered through nylon net and thoroughly washed with phosphate buffered saline, spH 7.4, before the oxidative treatment. This was per- formed by incubating the cells at 37 °C in a shaking water bath, in the presence of t-BHP at the indicated concentra- tion, in 25-mL flasks (final haematocrit 10%). After incubation, supernatants were used to determine levels of thiobarbituric acid-reactive substances (TBARS) as described below. Erythrocytes were washed seven times with isotonic buffer to remove t-BHP. Evaluation of oxidation markers Determination of methaemoglobin and oxyhaemoglo- bin. Methaemoglobin and oxyhaemoglobin contents were determined by a spectrophotometric method [20]. Briefly, 5 lL packed oxidized erythrocytes were mixed with 995 lL stabilizing solution (2.7 m M EDTA, pH 7.0, and 0.7 m M 2-mercaptoethanol). After shaking, oxyhaemoglobin and methaemoglobin concentrations were measured spectro- photometrically. Ó FEBS 2002 Protein methylation in G6PD-deficient erythrocytes (Eur. J. Biochem. 269) 2033 Evaluation of lipid peroxidation. Lipid peroxidation was evaluated by detecting the amount of TBARS, mainly malondialdehyde, as described previously [21]. Briefly, 2 mL of the supernatant of oxidized erythrocyte pellet was mixed with 1 mL 30% (w/v) trichloroacetic acid and centrifuged at 5000 g for 15 min. A 2-mL aliquot of supernatant was added to 0.5 mL 1% (w/v) thiobarbituric acid in 0.05 M NaOH and heated in a boiling-water bath for 10 min. The absorbance of the developing pink chromophore was measured at 532 nm. Methyl esterification of membrane proteins in intact erythrocytes ( in situ assay) Every day at least one patient and one control sample was processed in parallel. When oxidative stress was applied, control and patient samples were treated at the same time. Intact erythrocytes were incubated with methyl-labelled methionine, the in vivo precursor of AdoMet [6]. First, 250 lL packed erythrocytes were resuspended in an equal volume of 5 m M Tris/HCl buffer (pH 7.4), containing 160 m M NaCl, 0.96 m M MgCl 2 ,and2.8m M glucose. Then 0.93 nmol L -[methyl- 3 H]methionine (15 lCi) was added and the mixture incubated at 37 °C for 60 min. Cells were then haemolysed in hypotonic buffer (5 m M sodium phosphate, pH 8.0, containing 25 m M phenylmethanesulfonyl fluoride). Membranes were then washed twice with the same hypotonic solution at decreasing pH (7.2 and 6.2) to preserve methyl ester stability. Radioactivity incorporated as protein methyl esters was determined after solubilizationof10 lLmembrane preparation in 125 lL10 m M acetic acid/2.5% SDS. Protein concentration was determined as described previously [6]. Electrophoretic analysis of membrane proteins SDS/PAGE of membrane erythrocytes was performed by method of Fairbanks et al.[22] with modifications [9]. The gels were 1.5 mm thick and contained acrylamide mix 5.6% (mass/vol), in the presence of 1% SDS, at pH 7.4. All samples were run in duplicate so that one control and one treated (and/or patient) samples were analysed in parallel on each gel half of the same gel. At the end of the run, gels were cut into half, and one half was stained with Coomassie Brilliant Blue to visualize protein bands and densitometri- cally scanned for area quantification [9]. The other half was used for methyl ester quantification. For this, lanes were sliced into 2-mm fractions and the incorporated radioacti- vity was determined after elution of proteins from each slice [6,9]. Radioactivity was expressed as d.p.m./band area. Determination of AdoMet and S -adenosylhomocysteine intracellular content Intracellular concentrations of AdoMet and S-adenosyl- homocysteine were determined by HPLC in a perchloric acid-soluble fraction of erythrocyte cytosol [23]. All samples were filtered through a 0.2-lm pore filter before injection on to a Zorbax C8 reverse-phase column (25 cm · 4 mm; Du Pont-New England Nuclear, Boston, MA, USA), equili- brated with buffer A (50 m M NaH 2 PO 4 /10 m M heptanesulf- onic acid buffer, pH 3.2), containing 4% (w/v) acetonitrile. Nucleosides were eluted with a 15-min linear gradient of 4–20% acetonitrile, followed by a 10-min linear gradient of 20–25% acetonitrile, at a flow rate of 1 mLÆmin )1 . Enzyme assays PCMT specific activity was determined, in vitro, in the cytosol of erythrocytes subjected to oxidative stress, as previously described [9,24]. Erythrocyte lysates were obtained by rapid freeze–thawing after 10-fold dilution of oxidized erythrocytes with a stabilizing solution (2.7 m M EDTA, pH 7.0, and 0.7 m M 2-mercaptoethanol) [16]. Wild type AB Mediterranean 417 120 120100317 1a 1b 2 417 1 2 3 4 5 6 7 8 9 10 11 St 317 120 100 Fig. 1. Diagnostic assessment of molecular defect in G6P-deficient patients. Patient selection, sampling and DNA extraction were as described in Materials and Methods. The C563T mutation, associated with the Mediterranean variant was identified after PCR amplification of exon 5 and 6, followed by digestion with MboII restriction enzyme. (A) Schematic representation of the expected restriction fragment length polymorphism (RFLP) in wild-type and Mediterranean mutants, where the latter show an additional MboII site. (B) RFLP analysis of some patients and controls. 1, 3, 10, Mediterranean variant male patients; 4, 5, heterozygous females; 2, 7, 9, 11, normal controls. Mutants are characterized by sensitivity to MboII digestion of PCR amplified fragments, yielding additional bands of 317 and 100 bp, respectively. 2034 D. Ingrosso et al.(Eur. J. Biochem. 269) Ó FEBS 2002 Membranes were removed by centrifugation at 10 000 g for 20 min. The assay mixture contained, in a final volume of 40 lL, 1.6 mg ovalbumin as the methyl acceptor, 2.8 mg cytosolic proteins, 0.1 M sodium citrate buffer, pH 6.0, and 30 l M (final concentration) S-adenosyl- L -[methyl- 14 C]- methionine as the methyl donor. After incubation at 37 °C for 10 min, the reaction was quenched by adding an equal volume (40 lL) of 0.2 M NaOH/1% (w/v) SDS. Radioactivity due to methyl incorporation was determined as previously described [9]. Results are expressed as enzyme units (pmol methyl ester formedÆmin )1 ) per mg haemoglo- bin. Haemoglobin concentration was determined spectro- photometrically [24]. PK activity was evaluated as a cell-age marker by the method of Beutler et al.[16]. Statistical analysis Statistical analysis was performed by Student’s paired or unpaired t-test. Results are presented as the mean ± SE. Differences were considered significant at P < 0.05. RESULTS AND DISCUSSION Methyl esterification of membrane proteins is increased particularly in the G6PD-deficient ‘dense’ erythrocyte fraction There is evidence from at least three independent labora- tories that methyl esterification of membrane proteins, catalysed by PCMT, is increased as erythrocytes age in the circulation [6,25,26]. This has been related to an increased number of abnormal aspartate residues, spontaneously arising from L -asparaginyl deamidation and/or L -aspartyl isomerization reactions [7]. In addition it has been reported that isoaspartate residues, detected by the PCMT in situ assay, increase in membrane proteins of normal erythro- cytes subjected to oxidative stress [14], suggesting that susceptibility to oxidative damage may render these mem- brane protein components more prone to deamidation/ isomerization. We measured methyl esterification of membrane proteins in G6PD-deficient erythrocytes, to establish if abnormal isoaspartate residues occur, in this condition, at a higher rate than normal, while in the circulation. To this end, cells were fractionated according to density, the two most abundant, intermediate fractions being used in the subse- quent procedure. Cell recovery in these fractions, with respect to the total amount of cells loaded on to the gradient, was 70.3 ± 5.1% (control) vs. 80.9 ± 3.8% (G6PD). Cell percentages were 46.6 ± 4.1% (buoyant fraction) vs. 23.7 ± 1.9 (dense fraction) for the control samples, and 61.0 ± 3.2% (buoyant fraction) vs. 19.9 ± 2.3 (dense fraction) for the G6PD samples. An equal number of cells from each fraction was incubated with methyl-labelled methionine, the in vivo AdoMet precursor. PK activity was measured in parallel, as a cell age marker, in cytosolic extracts of the same erythrocyte fractions. PK activities in the dense cell fraction, in both normal and G6PD-deficient cells, were always significantly lower than in the corresponding buoyant cell fraction (Fig. 2A), confirm- ing that PK is a suitable cell age marker in the G6PD- deficient as well the normal erythrocyte. Moreover PK activity in the G6PD-deficient erythrocytes was significantly higher than in the corresponding control cell populations (Fig. 2A). This is consistent with G6PD-deficient erythro- cytes having a reduced half-life in the circulation. Each cell fraction from individual patient populations was then subjected to the in situ methylation assay. A general increase in methyl ester formation with cell age was found in membrane proteins of both pathological and normal erythrocytes. However, this age-dependent increase in methylation (i.e. protein damage) was significantly more marked in the membrane of G6PD-deficient erythrocytes than controls (Fig. 2B), despite the fact that the control cells were older (i.e. their life span was prolonged) according to PK activity. As a whole, the results show that, in G6PD deficiency, erythrocyte membrane proteins have an increased tendency to isoaspartate formation, in spite of the reduced half-life of the circulating erythrocyte population. In other words, the increase in altered residues resulting from protein deamida- tion/isomerization reactions show a premature onset with erythrocyte ageing, in the G6PD deficiency Mediterranean variant. Oxidative stress increases membrane protein ‘fatigue’ damage in G6PD-deficient erythrocytes It has been reported that oxidation, induced by cell treatment with t-BHP, leads to significant membrane alterations, including the occurrence of deamidated/isomer- ized Asx residues of membrane-cytoskeletal proteins [15]. Fig. 2. Membrane protein methylation levels and PK activity of density- fractionated G6PD-deficient erythrocytes. (A) PK activity, as a cell-age marker, was determined in erythrocyte cytosol, as detailed in Materials and methods. (B) Membrane protein methylation levels were deter- mined in two different erythrocyte age/density fractions obtained by isopycnic centrifugation on a Percoll gradient. Methyl esterification in intact erythrocytes was assayed by incubating them in the presence of [ 3 H]methionine according to the in situ procedure (see Materials and methods). Ó FEBS 2002 Protein methylation in G6PD-deficient erythrocytes (Eur. J. Biochem. 269) 2035 We were therefore intrigued to investigate whether the abnormal susceptibility of G6PD-deficient erythrocytes to oxidative stress could be responsible for their increased tendency to isoaspartate formation in membrane proteins. Therefore we monitored development of this alteration to evaluate its pathophysiological meaning in the mechanism of cell damage, using the in situ methylation assay, in isolated G6PD-deficient erythrocytes subjected to oxidative stress. Erythrocytes from both normal control and G6PD- deficient patients were subjected to oxidant treatment, with t-BHP, before the in situ methylation assay. To limit possible interference of cell manipulation with the oxidative stress conditions, cells were not fractionated according to density. The effects of reactive oxygen species on erythrocyte membranes were assessed by measuring lipid peroxidation products (Fig. 3A), which showed a dramatic rise. Proteins were also generally affected by the oxidative treatment, as demonstrated by the increase in methaemoglobin concen- tration (Fig. 3B). As expected, the effects of the oxidant treatment were much more dramatic on G6PD-deficient erythrocytes than on normal cells. We next evaluated the effects of such treatment on isoaspartate formation, by measuring the levels of mem- brane protein methyl esters by the in situ methylation assay. PK activity was also measured in parallel, to assess the loss of the older cell fractions as the result of possible haemol- ysis. Figure 4A shows that erythrocyte exposure to oxida- tive conditions resulted in higher intracellular PK activity, probably due to haemolysis, which affected the oldest, intrinsically less resistant cells, so that the remaining erythrocyte population was significantly younger. This effect was clearly more pronounced in G6PD-deficient than normal erythrocytes. Methyl esterification of membrane proteins was mea- sured in parallel, in order to monitor isoaspartate forma- tion. Figure 4B shows that such abnormal residues increased in membrane proteins in response to oxidants in both normal and pathological erythrocytes, but to different extents. This effect was in fact significantly more marked in G6PD deficiency, particularly when we consider that, in this disease, the erythrocyte population that survived the in vitro oxidative stress was younger than the controls (compare Figs 4A and 4B). No significant differences were noted in the AdoMet and AdoHcy concentrations, as well as in PCMT specific activity, after oxidative treatment, confirm- ing our previous findings [15]. As a whole, the results indicate that the lack of reducing power is a crucial element in conditioning erythrocyte susceptibility to undergo membrane protein damage in the form of Asx deamidation/isomerization. Isoaspartate formation may also be one of the ultimate events in cell destruction. Evidence shows that erythrocyte removal during cell ageing or after oxidative damage is mediated by binding of band 3 antibodies to band 3 antigenic sites [5]. Therefore, the occurrence of altered aspartate residues in band 3 of normal and abnormal erythrocytes during ageing [6,7] or oxidative stress [15] may be relevant to the fact that the same protein becomes a major site of new antigen generation under the same conditions. It should be pointed out, in this respect, that erythrocyte ageing was initially believed to be the main determinant of isoaspartate formation in membrane proteins [6]. Our results are in line with a different interpretation, which underscores the equally important role played by cell stress in the occurrence of such protein damage. This may be particularly relevant to pathological conditions, such as Fig. 3. Evaluation of oxidation markers in G6PD-deficient erythrocytes subjected to oxidative stress. Measurements were performed on both G6PD-deficient and normal control erythrocytes after exposure to oxidative stress with t-BHP. (A) TBARS evaluation of incubation medium; (B) methaemoglobin content in erythrocyte cytosol. Fig. 4. Membrane protein methylation levels and PK activity of G6PD- deficient erythrocytes subjected to oxidative stress. (A) PK activity, as a cell age marker, was measured in parallel in the cytosolic fraction of the same cell preparations. (B) Membrane protein methylation levels were evaluated by the in situ methylation assay. 2036 D. Ingrosso et al.(Eur. J. Biochem. 269) Ó FEBS 2002 spherocytosis or G6PD deficiency, in which erythrocytes are intrinsically altered, so that they are more prone to this type of protein alteration than normal. Protein methyl esterification as an adaptive response to cell exposure to damaging conditions Different spontaneous post-biosynthetic modifications have been shown to occur during erythrocyte senescence. For example nonenzymatic glycosylation of haemoglobin (gly- cation) has been shown to increase in the course of mismanaged hyperglycaemia in diabetes [27]. Haemoglobin is a useful protein marker of this kind of damage, although a number of other protein molecules are known to be affected by this alteration, with unpredictable functional conse- quences. Deamidation/isomerization of Asx residues has been shown to occur in haemoglobin a chain [28]. Haemoglobin mutations have been also shown to increase its susceptibility to deamidation, such as in the case of haemoglobin ÔProvidenceÕ [29]. Nevertheless the major targets of Asx deamidation/isomerization during cell ageing are several constituents of the membrane-cytoskeletal network. The electrophoretic shift of protein 4.1 has been shown to occur in aged erythrocytes, and it is due to deamidation of sensitive asparagine residues [8]. This protein is involved in the maintenance of erythrocyte shape and deformability, by stabilizing interactions between the spectrin–actin network and integral membrane proteins glycophorin C and band 3 (AE1) [30]. The functional consequences of deamidation/isomeriza- tion have often been investigated under near-pathological conditions. Homozygous knockout mice for PCMT are affected by growth retardation, and die prematurely with tonic-clonic seizures [31,32]. In these animals, isomerized proteins accumulate in all organs and tissues, indicating lack of PCMT-driven repair activity [31,32]. However, the functional outcome of such alterations on individual proteins is still uncertain, although the biological activity of different proteins appears to be compromised in vitro by deamidation and isomerization. Previous experience with several cell models has shown that the isoaspartate content of intracellular proteins is increased as the result of heat shock [33] as well as of UVA irradiation [34]. As far as the Fig. 5. SDS/PAGE profile of membrane proteins from G6PD-deficient and normal erythrocytes subjected to oxidative stress. Oxidative stress was induced, where indicated, by t-BHP treatment. Lane 1, nonoxi- dizednormalerythrocyte;lane2,oxidizednormalerythrocyte;lane3, nonoxidized G6PD-deficient erythrocyte; lane 4, oxidized G6PD- deficient erythrocyte. Fig. 6. Schematic representation of the overall hypothesis on the relationships between oxidative stress and isoaspartate formation in G6PD deficiency. G6PD-deficient erythrocytes are intrinsically less resistant to subliminal oxidant levels, so that protein deamidation/isomerization products (i.e. isoaspartate residues) tend to accumulate despite the fact that the life span of these cells is, on average, shorter than normal. In other words, they reach levels of aspartate damage that are typical of a much older normal erythrocyte population. Exposure to certain foods or drugs (fava beans, nonsteroidal anti-inflammatory drugs, antimalaria drugs, chemotherapeutics, etc.) trigger the haemolytic crisis, which is also associated with a further increase in the levels of deamidated/isomerized proteins. The mechanism linking oxidation to haemolysis involves membrane alterations. Ó FEBS 2002 Protein methylation in G6PD-deficient erythrocytes (Eur. J. Biochem. 269) 2037 erythrocyte is concerned, there is evidence that deamida- tion/isomerization of Asx residues, monitored by methyla- tion, is significantly increased under cell stress conditions. This occurs when erythrocytes are subjected to haemody- namic shear forces in a metabolically hostile microenviron- ment, such as the spleen microcirculation [35]. These results support the role of protein fatigue damage in the mechanism of spleen conditioning, in haemocatheresis [35,36]. It has also been shown that membrane–cytoskeletal proteins of resealed/engineered erythrocytes display increased suscepti- bility to Ômolecular fatigueÕ, detected by methyl esterifica- tion, after repeated osmotic stress [14]. In a previous report on the effects of oxidative stress on normal erythrocytes, we found that treatment with t-BHP increased isoaspartate occurrence in membrane proteins [15]. Conversely, we did not observe under such conditions any of the extensive membrane alterations described by others, including formation of protein aggregates with haemoglobin [15]. The electrophoretic pattern of membrane proteins from G6PD-deficient cells, both treated and untreated with oxidants, was similar to that of controls (Fig. 5). This allows us to conclude that molecular alterations, in the form of isoaspartate residues, take place on t-BHP treatment, before and not as a consequence of massive alterations of membrane protein composition. The results indicate that protein damage at the aspartate level is a sensitive and early marker of erythrocyte exposure to oxidants, before the appearance of more extensive damage of morphological relevance [15]. In conclusion, the data presented here show that G6PD deficiency, which renders erythrocyte adaptation to an oxidative microenvironment more difficult, makes mem- brane proteins more prone to isoaspartate formation, both during cell ageing and, even more so, under stress conditions (see scheme in Fig. 6). Taken as a whole, the results support the role of this post-biosynthetic protein modification in the mechanism of haemolysis in G6PD deficiency. ACKNOWLEDGEMENTS Genetic testing of patients was accomplished at the International Institute of Genetic and Biophysics (I.I.G.B.) of the National Research Council, Naples, Italy, under the supervision of Dr Giuseppe Martini and Stefania Filosa. The work was supported in part by research grants from Ministero dell’Istruzione, dell’Universita ` e della Ricerca, Progetti di Rilevante Interesse Nazionale (M.I.U.R. P.R.I.N., 1999): ÔExtra and intracellular nucleotide and nucleoside: chemical signals, metabolic regulators and potential drugsÕ and ÔHyperhomocysteinemia as a cardiovascular risk factor: biochemical mechanism(s)Õ. REFERENCES 1. Luzzatto, L., Mehta, A. & Vulliamy, T. (2001) Glucose 6-phos- phate dehydrogenase deficiency. In The Metabolic and Molecular Bases of Inherited Disease (Scriver,C.R.,Beaudet,A.L.,Sly,W.S. & Valle, D., eds), vol. 3, pp. 4517–4553. McGraw-Hill, New York. 2. Mason, J.P. (1996) New insights into G6PD deficiency. Br. J. Haematol. 94, 585–591. 3. 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