Neurochemistry International 63 (2013) 626–634

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Accumulation of altered aspartyl residues in erythrocyte membraneproteins from patients with sporadic amyotrophic lateral sclerosis

0197-0186/$ - see front matter � 2013 Elsevier Ltd. All rights reserved.http://dx.doi.org/10.1016/j.neuint.2013.09.006

Abbreviations: PIMT, protein L-isoaspartyl/D-aspartyl O-methyltransferase; Ado-Met, S-adenosylmethionine; AdoHcy, S-adenosylhomocisteine.⇑ Corresponding author. Address: Dipartimento di Scienze motorie e del benes-

sere, Università degli Studi di Napoli ‘‘Parthenope,’’ Via Medina n� 40, 80133 Napoli,Italy. Tel.: +39 0815474972.

E-mail address: sdangelo@uniparthenope.it (S. D’Angelo).

Stefania D’Angelo a,b,⇑, Francesca Trojsi c, Anna Salvatore b, Luca Daniele c, Marianna Raimo b,Patrizia Galletti b, Maria Rosaria Monsurrò c

a Department of Motor Sciences and Wellness, Parthenope University, Naples, Italyb Department of Biochemistry, Biophysics and General Pathology, Second University of Naples, Naples, Italyc Department of Neurological Sciences, Second University of Naples, Naples, Italy

a r t i c l e i n f o

Article history:Received 23 April 2013Received in revised form 23 August 2013Accepted 3 September 2013Available online 14 September 2013

Keywords:Amyotrophic lateral sclerosisErythrocytesProtein L-isoaspartyl/D-aspartyl O-methyltransferaseS-AdenosylmethionineProtein deamidationOxidative stress

a b s t r a c t

Spontaneous protein deamidation of labile asparagines (Asn), generating abnormal L-isoaspartyl residues(IsoAsp), is associated with cell aging and enhanced by an oxidative microenvironment. The presence ofisopeptide bonds impairs protein structure/function. To minimize the damage, IsoAsp can be ‘‘repaired’’by the protein L-isoaspartyl/D-aspartyl O-methyltransferase (PIMT) and S-adenosylmethionine (AdoMet)is the methyl donor of this reaction. PIMT is a repair enzyme that initiates the conversion of L-isoAsp (orD-Asp) residues to L-Asp residues. Amyotrophic lateral sclerosis (ALS) is a severe neurodegenerative dis-ease principally affecting motor neurons. The condition of oxidative stress reported in familial and spo-radic forms of ALS prompted us to investigate Asn deamidation in ALS tissue. Erythrocytes (RBCs) wereselected as a model system since they are unable to replace damaged proteins and protein methyleste-rification is virtually the only AdoMet-consuming reaction operating in these cells. Our data show that,in vitro assay, abnormal IsoAsp residues were significantly higher in ALS patients erythrocyte membraneproteins with an increased methyl accepting capability relative to controls (p < 0.05). Moreover, weobserved a reduction in AdoMet levels, while AdoHcy concentration was comparable to that detectedin the control, resulting in a lower [AdoMet]/[AdoHcy] ratio. Then, the accumulation of altered aspartylresidues in ALS patients is probably related to a reduced efficiency of the S-adenosylmethionine(AdoMet)-dependent repair system causing increased protein instability at Asn sites. The increase ofabnormal residues represents a new protein alteration that may be present not only in red blood cellsbut also in other cell types of patients suffering from ALS.

� 2013 Elsevier Ltd. All rights reserved.

1. Introduction

Oxidative stress has been linked to neuronal cell death thatoccurs in various neurodegenerative diseases. Post-mortem braintissues from patients with neurodegenerative disorders, such asParkinson’s disease, Alzheimer’s disease and amyotrophic lateralsclerosis (ALS), showed an increased oxidative stress in specificbrain regions (Andersen, 2004).

ALS is a rare and devastating disease characterized by theprogressive degeneration of motor neurons in the primary motorcortex, brainstem, and spinal cord, which results in muscle atro-phy, paralysis, and death. Although ALS was first described more

than 140 years ago by Charcot, most of the advances in our under-standing of the disease have been made in the past 10–15 years.About 90% of ALS patients are sporadic (sALS), whereas 10% arefamilial (fALS). (Mitchell and Borasio, 2007). In this group, a muta-tion in the SOD1 gene (on chromosome 21) that codes for the Cu,Zn-superoxide dismutase (SOD) has been found in a subset of10% of fALS cases, though the frequency varies depending on thepopulation sampled (Andersen, 2006). It is claimed that this muta-tion is associated with an altered Cu, Zn-SOD activity (Deng et al.,1993) and an oxidative stress status (Simonian and Coyle, 1996).However, familial and sporadic forms of ALS are clinically andpathologically very similar and a bulk of evidence supports theoxidative stress hypothesis also in sporadic ALS (Shaw et al.,1995). Studies on post-mortem tissues from sALS patients pro-vided evidence of accumulation of oxidative damage to proteins,lipids, or DNA (Agar and Durham, 2003; Barber et al., 2006).Biochemical markers of oxidative stress such as malondialdehydeand 8-hydroxy-2-deoxyguanosine (Agar and Durham, 2003) were

S. D’Angelo et al. / Neurochemistry International 63 (2013) 626–634 627

found to be increased in sALS patients compared with normal sub-jects. Moreover, it has been demonstrated that cerebrospinal fluidand plasma protein oxidation clearly occur in ALS patients(Siciliano et al., 2007).

Although it is not yet established whether the oxidative stress isa cause or a consequence of the neurodegenerative process, the dif-ferent capacity of each subject to respond to increased oxidativestress may be accounted to the heterogeneity of the sALS patientsin terms of clinical course, disease duration and response to phar-macological treatment.

Proteins and peptides are susceptible to a variety of chemicalmodifications that can affect their structure and biological func-tions. Among these modifications, the isomerization of asparticacid and deamidation of asparagine occur in proteins and peptidesduring cell aging. Isomerization of L-aspartate and deamidation ofL-asparagine in proteins or peptides dominantly give rise to L-isoas-partate by a non-enzymatic reaction via succinimide as a interme-diate under physiological conditions. The rate at which thesealterations occur and their specific localization within a protein ap-pear to be strongly influenced by specific protein structural deter-minants, as well as by protein microenvironment. Particularly,asparagines (Asn) deamidation is a spontaneous post-biosyntheticmodification, related to cell aging, which substantially alters pro-tein structure and function by introducing a partial geometricalisomerization of the polypeptide backbone (Clarke, 2003;Gallettiet al., 1995).

As summarized in Fig. 1, the mechanism of deamidation impliesthe formation of an unstable cyclic succinimidyl intermediate,yielding in turn either a normal peptide or an atypical isopeptidecontaining a b-linked isoaspartate residue (IsoAsp). The ratio ofthe two species (7:3) is in favor of the ‘‘iso’’ form (Galletti et al.,1995). Succinimide is also prone to spontaneous racemization,generating D-Asp and D-isoAsp residues, even if at a lower rate(Clarke, 2003; Reissner and Aswad, 2003).

The occurrence of such abnormal residues can significantly alterprotein structure and function, as has been demonstrated forepidermal growth factor, calmodulin, tubulin, synapsin, eye lens

Fig. 1. Isoaspartate formation and repair by PIMT deamidation of aspargine (L-Asn) and da-amino group of the C-flanking amino acid. This reaction, which forms a succinimidyl iIsoAsp) and L-Asp. L-Succinimide reversely racemizes to D-succinimide. The D-succinimideto generate D-isoaspartate (D-IsoAsp) and D-aspartate (D-Asp). The free a-carbonyl groumethyl donor. Enzymatic methylation followed by spontaneous ester hydrolysis leadsmixture of L-Asp and L-IsoAsp. The nonenzymatic reactions denoted by the thick aadenosylhomocysteine.

crystallins, Alzheimer’s b-amyloid, tissue plasminogen activator(Clarke, 2003; Reissner and Aswad, 2003), B-cell lymphoma-extralarge (Bcl-xL) protein (Deverman et al., 2002), type I collagen(Lanthier and Desrosiers, 2004), protein kinase A (Pepperkoket al., 2000).

Protein L-isoaspartyl/(D-asp) methyltransferase (PIMT,EC2.1.1.77), the ‘protein repair enzyme’, specifically methylatesthe isoaspartyl residue generated by the spontaneous deamidationof asparagine, and this methylation is an essential step for convert-ing isoaspartate to aspartate. This enzyme catalyzes the transfer ofan active methyl group from S-adenosyl-L-methionine (AdoMet) tothe a-carboxyl group of atypical L-isoaspartyl and D-aspartyl resi-dues, but not normal L-aspartyl residues, in peptides or proteins(Clarke, 2003; Reissner and Aswad, 2003) (Fig. 1). Lowenson andClarke (1991, 1992) reported that the best substrates for PIMT, atleast in synthetic peptides, contain L-IsoAsp, rather than D-Asp.Continuing cycles of PIMT action efficiently repair L-isoAsp sites,as has been demonstrated with a number of peptides and proteins(Brennan et al., 1994; Galletti et al., 1988; Zhou et al., 2006).

The demethylated product of AdoMet, AdoHcy, is rapidly re-moved by its enzymic hydrolysis to adenosine and homocysteine(De La Haba and Cantoni, 1959; Zappia et al., 1969). The latter en-zyme plays a key role in the methylesterification process, in that allAdoMet-dependent methyltransferases are regulated in vivo by the[AdoMet]/[AdoHcy] ratio (Barber and Clarke, 1984; Cantoni andChiang, 1980). AdoHcy is a powerful competitive inhibitor of allAdoMet-dependent methyltransferases, the extent of inhibitiondepending on both [AdoMet]/[AdoHcy] ratio and Km and Ki valuesof the specific enzyme. The kinetic parameters of PIMT confer tothis enzyme an extreme susceptibility to AdoHcy inhibition(Clarke, 2003; Galletti et al., 1995).

The selective S-adenosylmethionine (AdoMet)-dependentmethylesterification of the a-carboxyl group of L-isoAsp residuesactivates their conversion into L-Asp residues, thus helping to pre-vent the accumulation of dysfunctional proteins (Galletti et al.,1995, 2007; Reissner and Aswad, 2003; Shimuzu et al., 2005; Doyleet al., 2006).

ehydration of aspartic acid (L-Asp) spontaneously occur on nucleophilic attack of thentermediate (L-succinimide), is followed by hydrolyzation to form L-isoaspartate (L-

intermediate also undergoes a rapid hydrolysis at either the a- or b-carbonyl groupp of L-IsoAsp is methylated by PIMT with S-adenosylmethionine (AdoMet) as theto reformation of the L-succinimide intermediate, which is again hydrolyzed to a

rrows are faster than those denoted by the thin arrows. AdoHcy represents S-

628 S. D’Angelo et al. / Neurochemistry International 63 (2013) 626–634

A variety of induced stress conditions, including oxidation, hasbeen shown to significantly increase IsoAsp content in differentcellular models, including red blood (RBCs) and melanoma(D’Angelo et al., 2001; D’Angelo et al., 2005; Ingrosso et al., 2002)cells. Moreover, we have reported a significant accumulation ofabnormal isopeptide bonds in RBC membrane proteins frompatients with Down syndrome (Galletti et al., 2007), and psoriaticpatients (D’Angelo et al., 2012), pathological conditions character-ized by an increased oxidative stress.

As the oxidative status was observed in ALS patients (Bailletet al., 2010; Shaw et al., 1995) we hypothesized that the accumu-lation of L-isoAsp/D-Asp in ALS proteins, potentially responsible forstructural and functional alterations, could be present in thispathology.

Aim of this work is to investigate whether or not an accumula-tion of deamidated proteins occurs in RBCs of sALS patients, as aprobable consequence of the oxidative stress. We selected erythro-cytes as a model system, since these cells, chronically exposed toROS and unable to replace damaged proteins, are especially suit-able to reveal oxidation-dependent molecular alterations. TheRBC was selected as a model system because it is a simplified celltype, lacking the apparatus for protein synthesis and therefore un-able to replace its damaged proteins. Moreover, the enzymes in-volved in methionine metabolism, mainly AdoMet synthetaseand S-adenosylhomocysteine (AdoHcy) hydrolase, are very activein RBCs, whereas protein methylesterification is virtually the onlyAdoMet-consuming reaction operating in these cells (Galletti et al.,1995).

Table 1Detailed patients and controls characteristics.

Clinical features ALS patients (n = 34) Healthy controls(n = 34)

Mean age (years ± SD) (range) 59.6 ± 13.2 (27–73) 47.1 ± 14.4 (32–63)Gender (male:female) 18:16 14:18Mean disease duration 42 ± 40.4 (12–168) –

2. Materials and methods

2.1. Chemicals

S-Adenosyl-L-[methyl-14C]Met (specific activity 58 mCi/mmol),L-[methyl-3H]Met (specific activity 79 Ci/mmol) and L-[methyl-14C]Met (specific activity 55 mC/mmol) were from Amer-sham International, Little Chalfont, UK). Ready Gel liquid scintilla-tion cocktail was from Beckman Inc. (Cuppertino, CA, USA).Acetonitrile and other organic solvents for HPLC were from Merck(Whitehouse Station, NJ, USA) or Carlo Erba (Rodano, Italy). Allother chemicals used in this work were of the purest grade avail-able and were from Sigma Chemical Co. or Carlo Erba.

(months ± SD) (range)Age at onset (mean ± SD)

(range)56.1 ± 13.5 (25–71) –

Classical ALSEl escorial criteria

(probable:definite)19:15 –

Clinical onset (bulbar:upperlimbs:lower limbs)

2:15:17

UMN score (mean ± SD) (range) 8.5 ± 4 (2–15) –ALS FRS-R (mean ± SD) (range) 33.9 ± 8.3 (21–45) –Rate of disease progressiona

(mean ± SD) (range)0.5 ± 0.5 (0.03–2.2) –

FrSBe scale (T-score)b

(mean ± SD) (range)95.5 ± 24 (60–143) –

ComorbiditySubjects with hypertension 11 –Subjects with type 2 diabetes

mellitus2 –

Subjects with hepatitis 2 –Subjects with thyroidal

dysfunction2 –

Subjects with gastritis 2 –Subjects with psychosis 1 –

a Calculated according to Ellis et al. (1999) by the following formula: 48 – ALS-FRS-R/disease duration.

b T-score >65 is defined as impaired behavior and executive functions (Graceet al., 1999).

2.2. Patient enrollment and sample processing

We investigated 34 ALS patients (16 women and 18 men) rang-ing from 27 to 73 years (mean age 59.6 ± 13.2) of age, fulfilling thediagnostic criteria for probable or definite ALS, according to the re-vised El Escorial criteria of the World Federation of Neurology(Brooks et al., 2000). Fifteen out of 34 patients included in thisstudy had ‘‘definite’’ ALS, whereas 19 out of 34 had ‘‘probable’’ALS. According to the revised El Escorial criteria, ALS was diag-nosed as a progressive mixed-lower and upper motor neuron(UMN, LMN) syndrome. Three affected nervous system segmentalregions were defined as definite ALS, two as probable, and one aspossible. The exclusion of neuropathy by nerve conduction studiesand a neurogenic patterns in electromyography confirmed thediagnosis.

The sites of disease onset were upper or lower limbs in 32 pa-tients and oral-pharyngeal (bulbar) site in two patients. All had a‘‘classic’’ phenotype (i.e., mixed UMN and LMN syndrome) withbulbar signs present in 16 patients. We excluded from the studypatients with dominant lower motor neuron (LMN) impairment,such as progressive muscular atrophy and flail leg syndrome orpseudopolyneuritic form, progressive bulbar palsy, primary lateral

sclerosis, postpoliomyelitis ALS and motor neuron disease withmajor cognitive impairment (e.g. ALS–dementia, FTD–ALS). Thetime between the first symptom and the study ranged from1 month to 14 years. All patients were treated with riluzole(50 mg � twice/day). Seven patients were treated with lithium car-bonate (150 mg/day). Two of the 34 cases had disease durations ofmore than 10 years, and were receiving artificial respiratory sup-port (home mechanical non-invasive ventilation; mean duration:68 months). None needed percutaneous endoscopic gastrostomy(PEG) or had other neurological diseases. All patients had no famil-ial ALS or and were not SOD1 mutation positive.

In all patients we assessed: the ALS Functional Rating Scale-re-vised (ALSFRS-R), for evaluating and monitoring ALS-related dis-ability (Cedarbaum et al., 1999); the disease progression rate,derived from the formula 48-ALSFRS-R/ months of disease duration(Ellis et al., 1999); the Frontal Systems Behaviour (FrSBe) Scale, forestimating the degree of frontal dysfunction through the evalua-tion of total or T-score, derived from the caregiver form and refer-ring to the time of observation (Grace et al., 1999); and the UMNscore, a measure of pyramidal dysfunction which evaluates thenumber of pathologic reflexes, elicited from 15 body sites (i.e., gla-bellum, orbicularis oris, masseter, biceps, triceps and finger jerksbilaterally, and knee, ankle, and Babinski responses bilaterally)(Turner et al., 2004) (for more details about these measures inour population view Table 1).

Our study population comprised 34 healthy controls (14 males,18 females, average age 47 ± 14.4). Pregnancy and lactation, malig-nancies, skin infections and inflammatory diseases were exclusioncriteria. All the participants in this study signed the consent form.

Heparinized fresh human blood was obtained from donors re-cruited from staff, following informed consent. After centrifugationfor 10 min at 1000g, plasma and buffy coat were removed and redblood cells were washed three times with isotonic buffer saline(NaCl 0.9%) (Ingrosso et al., 2000).

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2.3. In vitro evaluation of L-isoAsp residues in RBC membrane proteins

To evaluate L-isoAsp residues in membrane proteins, RBCs werehemolyzed in hypotonic buffer (5 mM sodium phosphate, pH 8.0,containing 25 lM phenylmethanesulfonyl fluoride). Membraneswere then washed twice with the same hypotonic solution. Twentymicroliters of resuspended cell membranes was incubated with 28U of recombinant PIMT in 0.1 M sodium citrate (pH 6.0), containing30 lM S-adenosyl-L-[methyl-14C]Met in a 40 ll volume at 37 �C for60 min. At the end of incubation, the reaction was quenched byadding an equal volume (40 ll) of 0.2 M NaOH and 1% (w/v) SDS.Radioactivity due to methyl incorporation was determined as pre-viously described (Galletti et al., 2007). Results are expressed aspmol methyl ester formed/mg proteins. Protein concentrationwas estimated using the method of Bradford, by means of theBio-Rad (Hercules, CA, USA) protein assay kit.

2.4. Methyl esterification of membrane proteins in intact RBCs: in situassay

Methyl esterification of membrane proteins was investigatedhere in intact RBCs through an in situ protein methylation ap-proach, closely mimicking the in vivo pathway. Intact erythrocyteswere incubated with methyl-labeled methionine, the in vivo pre-cursor of AdoMet (Ingrosso et al., 2000); 250 ll of packed erythro-cytes were resuspended in an equal volume of 5 mM Tris/HClbuffer (pH 7.4), containing 160 mM NaCl, 0.96 mM MgCl2 and2.8 mM glucose. Then, 0.93 nmol L-[methyl-3H]methionine(15 lCi) were added and the mixture incubated at 37 �C for60 min. Cells were then hemolysed in hypotonic buffer (5 mM so-dium phosphate, pH 8.0, containing 25 mM phenylmethanesulfo-nyl fluoride). Membranes were then washed twice with the samehypotonic solution at decreasing pH (7.2 and 6.2) to preservemethyl ester stability. Radioactivity incorporated as protein methylesters was determined after solubilization of 10 ll membranepreparation in 125 ll of 10 mM acetic acid/2.5% SDS. Protein con-centration was determined as described previously (D’Angeloet al., 2012). RBC samples from each individual subject were pro-cessed and assayed in duplicate.

2.5. Electrophoretic analysis of membrane proteins

Membrane proteins were methyl esterified in intact RBCs fromsALS patients. After hypotonic hemolysis, membranes were iso-lated and protein species were separated by sodium dodecylsulfatepolyacrylamide gel electrophoresis (SDS–PAGE) performed follow-ing the method of Fairbanks et al. (1971), with modifications. Gels,1.5-mm thick, containing acrylamide mix 5.6% (w/v), in the pres-ence of 1% SDS (pH 7.4) were used. Membrane RBC samples wererun in duplicate, so that control and patient samples were analyzedin 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 CoomassieBrilliant Blue to visualize protein bands and densitometricallyscanned for area quantification (Ingrosso et al., 1995). The otherhalf was used for methyl ester quantification; therefore, lanes weresliced into 2-mm fractions and the incorporated radioactivity wasdetermined after elution of proteins from each slice (Galletti et al.,1983, 2007).

2.6. PIMT activity in RBCs and L-[methyl-14C]Met uptake

Endogenous PIMT specific activity was determined, in vitro, inthe cytosol of RBCs obtained by rapid freeze–thawing after dilutingRBCs 10-fold with a stabilizing solution (2.7 mM EDTA, pH 7.0,0.7 mM 2-mercaptoethanol). Membranes were removed by centri-fugation at 10,000g for 20 min using an Avant™ J-301 centrifuge

and JA-30.50Ti rotor (Beckman Instruments, Palo Alto, CA, USA).The assay mixture contained, in a final volume of 40 ll, 1.6 lg ofovalbumin as methyl acceptor, 2.8 mg of cytosolic proteins, 0.1 Msodium citrate buffer at pH 6.0, and 30 lM S-adenosyl-L-[methyl-14C]Met as methyl donor. After incubation at 37 �C for10 min, the reaction was quenched by adding an equal volume(40 ll) of 0.2 m NaOH and 1% (w/v) SDS. Radioactivity due tomethyl incorporation was determined as previously described (In-grosso et al., 2000). Results are expressed as enzyme units (pmol ofmethyl esters formed per minute) per milligram of haemoglobin(Hb). Hb concentration was determined spectrophotometrically.

Met transport was determined in RBCs as described in Ingrossoet al. (2000). RBCs (250 ll) were suspended in an equal volume of5 mM Tris/HCl (pH 7.4), 160 mM NaCl, 0.96 mM MgCl2, 2.8 mMglucose, and 50 mM cycloleucine. L-[methyl-14C]Met(150,000 d.p.m.) was added, and the mixture was incubated at37 �C. Fifty-microliter aliquots were withdrawn at 5, 15, 25, 35and 45 min, centrifuged using a 5810R centrifuge and F45-30-11rotor (Eppendorf) at 10,000 g, and rapidly hemolyzed in 200 ll ofice-cold distilled water. Proteins were pelleted with 100 ll of40% trichloroacetic acid, and the radioactivity in the supernatantwas determined.

2.7. Measurement of AdoMet and AdoHcy intracellular content

Intracellular concentrations of AdoMet and AdoHcy were deter-mined by HPLC in a perchloric acid-soluble fraction of RBC cytosol.All samples were filtered through a 0.2 lm pore filter before beinginjected onto a 25 cm � 4.6 mm Zorbax Eclipse C8 reverse-phasecolumn (Agilent Technologies, Wilmington, DE, USA), equilibratedwith buffer A (50 mM NaH2PO4/10 mM heptane sulfonic acid buf-fer, pH 3.2), containing 4% (w/v) acetonitrile. Nucleosides wereeluted with a 15 min linear gradient from 4% to 20% acetonitrile,followed by a 10 min linear gradient from 20% to 25% acetonitrile,at a flow rate of 1 ml/min. AdoMet and AdoHcy peak areas wereinterpolated on a calibration curve of external authentic AdoMetand AdoHcy, injected separately (D’Angelo et al., 2012; Gallettiet al., 2007).

2.8. Statistical analysis

Statistical analysis was performed by Student’s t test. The re-sults are presented as mean ± SD values. Differences were consid-ered to be significant at p < 0.05.

Pearson correlation coefficients were calculated to evaluatethe relationship between RBC content of L-IsoAsp residues in sALSpatients and clinical measures of disease duration, age, UMNinvolvement, disability, disease progression and behavioural dys-function. p Values <0.05 were considered statistically significant,after correction for multiple comparisons with the Bonferronimethod.

3. Results

3.1. Overall assessment of the repair process of IsoAsp sites in intactRBCs

3.1.1. IsoAsp content of RBC membranes in sALS patients: in vitro assayThe IsoAsp content of RBC membrane proteins has been quanti-

fied using human recombinant PIMT as a specific enzymatic probe.The purified enzyme has been widely employed to tag these abnor-mal residues in protein extracts from various experimental models,including PIMT knockout mice, cell cultures, as well as purifiedproteins and peptides.

Table 2Protein methyl esterification in RBC membranes: in vitro assay and in situ assay. In vitro assay: the evaluation of L-isoAsp sites in membraneproteins was accomplished in the RBC membrane isolated from sALS and control donors. Membrane proteins were methyl esterified in vitroin the presence of human recombinant PIMT and S-adenosyl-L-[methyl-14C]Met as described in ‘‘Section 2.3’’. Values refer to the mean ofeach duplicate determination. ⁄p < 0.001. In situ assay: the extent of methyl esterification of membrane proteins was evaluated in intactRBCs by incubating the RBCs in the presence of [methyl-3H]Met, as described in ‘‘Section 2.4’’. RBC samples from each individual subjectwere processed and assayed in duplicate.

In vitro assay In situ assayDamaged amino acid residues(pmol methyl groups/mg proteins)

Methylesters (DPM/mg proteins)

sALS (n = 34) 865.0 ± 87.0* 115.35 ± 64.63Control (n = 34) 472.0 ± 71.0 100.01 ± 44.67

* p < 0.001.

MW 1 2

200,000

97,400

43,000

29,000

Fig. 2. Electropherogram of Coomassie Brilliant Blue-stained gels for analysis ofmembrane proteins in RBCs from sALS. Membrane proteins were methyl esterifiedin intact RBCs from ALS patients and controls as reported in ‘‘Section 2.5’’. Afterhypotonic hemolysis, membranes were isolated and protein species were separatedby SDS/PAGE. Duplicate samples were run in parallel for the analysis of proteincomposition and determination of incorporated radioactivity of each individualband, respectively. Lane 1: membrane proteins from control RBCs. Lane 2:membrane proteins from sALS RBCs.

630 S. D’Angelo et al. / Neurochemistry International 63 (2013) 626–634

According to this method, IsoAsp residues were quantified inthe purified membranes from freshly isolated RBCs, from both sALSpatients (n = 34) and healthy controls (n = 34). As reported inTable 2, the L-isoAsp content in RBC membrane proteins of sALSpatients largely exceeds the corresponding level detected in a com-parable healthy population (865.0 ± 87.0 pmol methyl groups/mgproteins vs 472.0 ± 71.0 pmol methyl groups/mg proteins,p < 0.001).

No significant correlations were found between RBC content ofL-isoAsp in sALS patients and clinical measures of age, pyramidalimpairment (UMN score), disease duration, disability (ALSFRS-R),progression rate (48-ALSFRS-R/disease duration) and behaviouraldysfunction (FrSBe scale).

We have observed a comparable increase of membrane proteinmethylation in human erythrocytes treated with tert-butyl hydro-peroxide, an oxidizing agent (data not shown).

3.1.2. IsoAsp content of RBC membranes in sALS patients: in situ assayMethyl esterification of membrane proteins was measured by

the in situ assay in freshly RBCs. Protein methyl esterification isquantitatively the most important AdoMet-consuming reaction inhuman erythrocytes, accounting for the bulk of the utilization ofthis thioether (Oden and Clarke, 1983). The overall efficiency ofthe PIMT-dependent methylation repair system was directly as-sessed by means of the in situ assay, which closely mimics thein vivo pathway (Ingrosso et al., 2000, 2002). This was performedby incubating intact RBCs with labeled Met, as described under‘‘Section 2.4’’. Under these conditions, the amino acid is rapidly ta-ken up and converted into AdoMet by the cytosolic AdoMet syn-thetase (Galletti et al., 1995). Endogenous proteins are thenmethylesterified by cytoplasmic PIMT, and AdoHcy hydrolase en-sures rapid removal of the thioether through its cleavage intoadenosine and Hcy, thus preventing product inhibition of themethyltransferase. Under these conditions, no significant differ-ence (p = 0.13) was detected between sALS patients(115.35 ± 64.63 DPM/mg proteins; n = 34) and the relevant con-trols (100.01 ± 44.67 DPM/mg proteins; n = 34).

The electrophoretic pattern of membrane proteins from sALSRBCs was similar to that of controls (Fig. 2). The analysis of methylesterified membrane proteins in intact RBCs was performed bySDS/PAGE, as reported in ‘‘Section 2.4’’. The electropherograms ofCoomassie Blue-stained profiles of sALS and control samples weresuperimposable, indicating that, in sALS membranes, neither pro-tein fragmentation nor polymerization occurred (Fig. 2). Moreover,the major methyl-accepting proteins, both in patients than in con-trols, included ankyrin, band 4.1, band 4.2, and the integral mem-brane protein band 3.

3.2. Functional evaluation of the RBC biochemical machinery requiredfor protein methylation

The L-isoAsp content of the RBC membrane proteins depends,under steady-state conditions, on the rate of spontaneous Asn

deamidation and the efficiency of the enzymatic PIMT-dependentrepair. The effectiveness of the methylation process was evaluatedin intact RBCs obtained from both sALS subjects and the controlgroup.

To monitor the biochemical integrity of the methylation path-way, the following individual steps were taken into account: (a)methionine transport; (b) PIMT specific activity and (c) intracellu-lar concentration of the AdoMet-methyl donor and of its demethy-lated product AdoHcy.

3.2.1. Methionine transportMet transport was evaluated as described under ‘Materials and

methods’. No significant difference was detected between sALS pa-tients and the relevant controls. Equilibrium was reached within25 min, and the kinetics were strictly comparable in both casesand controls (data not shown).

3.2.2. Determination of PIMT activityIn order to check whether a possible impairment of PIMT activ-

ity might account for the reported increase of IsoAsp levels in sALSsubjects, the in vitro specific activity of the enzyme in the RBC cyto-solic extracts was measured.

The data are shown in Table 3 and clearly indicate that PIMTspecific activity did not vary significantly among the two groups,allowing us to rule out any contribution of this factor to the in-creased methyl incorporation observed in RBC membrane proteins.

3.2.3. AdoMet and AdoHcy intracellular concentrationsWhen AdoMet is used for the methylation reaction, AdoHcy is

formed. The AdoMet/AdoHcy ratio is considered an indicator ofthe flow of methyl groups from AdoMet to methyl acceptors inthe cells, and has thus been called ‘‘methylation potential’’. AdoHcyis the natural by-product of the methyl transfer reaction from

Table 3PIMT activity, AdoMet and AdoHcy concentrations in sASL RBCs. PIMT specific activity was measured, in vitro, in RBC cytosol by radiochemical enzyme assay, as indicated in‘‘Section 2.6’’. Results are expressed as enzyme units per milligram of Hb. AdoMet and AdoHcy intracellular concentrations were determined by HPLC in the trichloroacetic acid-soluble fraction obtained from RBC lysates, as described in ‘‘Section 2.7 section’’. Micromolar concentrations of compounds are referred to 1 L of packed cells.

PIMT specific activity (U/mg Hb) AdoMet (lM) AdoHcy (lM) AdoMet/AdoHcy

sALS (n = 21) 2.2 ± 0.7 1.4 ± 0.3* 0.7 ± 0.2 2.3 ± 0.8**

Control (n = 19) 2.4 ± 0.5 2.6 ± 0.8 0.9 ± 0.4 3.4 ± 1.9

* p < 0.001.** p < 0.02.

S. D’Angelo et al. / Neurochemistry International 63 (2013) 626–634 631

AdoMet to methyl accepting proteins, and also a potent inhibitor ofAdoMet-dependent reactions.

AdoMet and AdoHcy intracellular concentrations were assayedin RBCs from healthy, age-matched control subjects (n = 19) andsALS patients (n = 21). The results are presented in Table 3. andshow that the concentration of AdoMet is reduced by half in sALSpatients with respect to control. The level of AdoMet in the controlRBC was 2.6 ± 0.8 lM and in the patient group was significantlylower, 1.4 ± 0.3 lM (p < 0.001). With regard to the AdoHcy levels,no difference was observed between patient and control groups(Table 3). Because the AdoHcy inhibitory mechanism is competi-tive with respect to AdoMet, changes in relative concentrationsof the two compounds can actually determine the alteration ofreaction. We have calculated AdoMet/AdoHcy ratio (the trans-methylation potential) and, as shown in Table 3 it was significantlylower in sALS group (p < 0.02).

4. Discussion

Amyotrophic lateral sclerosis is one of the more prevalentadult-onset neurodegenerative diseases and despite extensive re-search, the cause of this disease is unknown in the majority ofcases, and the mechanisms of motor neuron injury are complexand incompletely understood. Studied carried out on the post-mortem tissue from sALS and fALS patients clearly show an accu-mulation of oxidative damage to proteins, lipids and DNA indicat-ing a direct role of oxidative stress in ALS (Agar and Durham,2003; Barber et al., 2006). In order to better understand the ALSpathogenesis, most of the recent studies have focused on rodentmouse models that express the mutant human SOD1 forms(Wang et al., 2011; Liu et al., 2012; Pickles et al., 2013). This evi-dence on animal models has shown age-dependent motor neurondegeneration with cellular and biochemical damage to nerve fi-bres and spinal cord tissue as well as increased protein and lipidoxidation.

Among current findings from the mutSOD1 mouse model, themost accepted theory is that the different point mutations createa misfolding defect, leading to small amyloid-like aggregates thatappear in late stages of the disease (Kabashi et al., 2007; Bendottiet al., 2012). These misfolded aggregated proteins could producetoxic effect towards neurons, similar to the neurotoxicity thatarises in other amyloidoses. Misfolded aggregates are insolubleand are not cleared by proteasomal degradation, therefore theyeventually impair and ultimately overwhelm the system (Bendottiet al., 2012).

A lot of data suggest that both a toxic gain of function due toprotein aggregation/mislocation together with a loss of normalbiological function upon mutations contribute to diseasepathogenesis.

Several evidences underlined the role of reactive oxygenspecies and nitric oxide in neurodegenerative mechanism (Shawet al., 1995; Simonian and Coyle, 1996; Andersen, 2004). Methyltransfer reactions mediated by AdoMet are involved in oxidativedamage repair (Ingrosso et al., 2000). Furthermore, previous

studies relevant that increased formation of L-isoaspartyl resi-dues is one of the major structural alterations occurring inerythrocyte membrane protein as a result of oxidative stress (In-grosso et al., 2000, 2002; Shimuzu et al., 2005; Galletti et al.,2007). These abnormal residues are physiologically convertedinto normal L-aspartyl residues by PIMT, which methylates thealpha-carboxyl group of atypical L-isoaspartyl residues. PIMT isubiquitous and its repair function is particularly crucial in eryth-rocytes, as a consequence of molecular ageing and oxidativestress.

The results from the present study concern the demonstrationof enhanced deamidation/isomerization of RBC membrane pro-teins in sALS, and the possible impact of this phenotype on thepathophysiology of the disease.

Asn deamidation is a spontaneous post-biosynthetic modifica-tion, related to cell aging, which typically occurs at protein siteswhere certain amino acids, such as small, non bulky residues, areadjacent to Asn (Clarke, 2003). The reaction is both non enzymaticand slow, and it has therefore been proposed that labile Asn resi-dues may represent specific biological clocks and/or signals forthe commitment to protein degradation (Robinson, 2002). Indeed,the accumulation of isoAsp residues substantially alters proteinstructure and function by introducing negatively charged carbox-ylic acid side chains and partial geometrical isomerization of thepolypeptide backbone (Clarke, 2003; Reissner and Aswad, 2003).As expected, these events affect tertiary protein structure and areusually detrimental to protein function, inducing protein inactiva-tion, autoimmunity, and aggregation (Doyle et al., 2006; Shimuzuet al., 2005) potentially involved in neurotoxicity mechanisms(Reissner et al., 2006; Zhou et al., 2006). In particular, increasedlevels of atypical L-isoAsp residues, derived from impairment ofPIMT enzymatic activity, may induce abnormalities of intracellularsignal pathways through hyperphosphorylation of several mem-bers of mitogen-activated protein kinase cascade (Kosugi et al.,2008). Furthermore, some studies on PIMT deficient mice modelshave revealed accumulation of L-isoAsp in several tissues, includ-ing brain, showing neurodegenerative pathology of hyppocampuswith alterations of pre-synaptic proteins, such as synapsin I, andearly development of fatal epileptic seizures (Reissner et al.,2006; Zhou et al., 2006).

Potentially labile Asn residues are generally present in nativeproteins in conformations where the peptide-bond nitrogen cannotapproach the side chain carbonyl to form the succinimide ring(Clarke, 2003; Reissner and Aswad, 2003). Nevertheless, cell stressor pathological conditions may enhance protein deamidation byincreasing polypeptide flexibility and/or by inducing transient pro-tein unfolding (Clarke, 2003).

This ubiquitous enzymatic post-translational modification hasbeen examined in detail in human RBCs, where some critical mem-brane protein components are substrates of PIMT (Freitag andClarke, 1981; Ingrosso and Clarke, 1991). This represents, by far,the most prominent methyltransferase reaction that takes placein red cells (Freitag and Clarke, 1981). Thus, RBCs represent anideal model system to study this reaction (Ingrosso and Clarke,1991). It has been demonstrated that specific erythrocyte

632 S. D’Angelo et al. / Neurochemistry International 63 (2013) 626–634

membrane proteins, among which, in particular, cytoskeletal com-ponents bands 2.1 (ankyrin) and 4.1 (the same proteins that pro-vide some rigidity to the membrane) are substrates for themethyltransferase (Freitag and Clarke, 1981; Galletti et al., 1983;Barber and Clarke, 1983).

In the present work, we reported a significant accumulation ofabnormal isoaspartyl residues, in the vitro assay, in RBC membraneproteins from ALS patients independently from clinical characteris-tics of the examined patients, such as age, pyramidal impairment,disease duration, disability, progression rate and behavioural dys-functions. This result may be interpreted as a compensatory re-sponse to the enhanced protein deamidation, presumably relatedto the oxidative microenvironment featuring ALS patients in sev-eral stages of disease. Subjects under study include patients withvarying degrees of sALS and a control group of healthy subjectsage and sex matched to patients. Our data are also in line withthe evidence, previously obtained by our group, that exposure ofRBCs to an exogenous or endogenous oxidative stress is able to in-duce the accumulation of isoAsp residues both in normal and path-ological conditions. Also, the formation of isoAsp residues is aprotein alteration, which is added to those already discovered(Shaw et al., 1995; Siciliano et al., 2007). However, the lack of sig-nificant correlations between RBC increase of abnormal isoaspartylresidues and clinical parameters may depend from the limitednumbers of patients studied, the heterogeneity of their clinical pre-sentations, with predominantly UMN or LMN signs and differentdisease duration and disability, and the current lack of clinicalscores highly specific for monitoring UMN, LMN or behaviouralimpairment.

Moreover, we also detected increased methyl esterification ofmembrane proteins in vitro assay, but not in intact RBCs sALS pa-tients (in situ assay). Granted that the sample is small enoughand extremely heterogeneous (high standard deviations), and boththe well known natural heterogeneity of ALS patients, the not sig-nificant increase protein methylation observed in the in situ assaymay be interpreted as a consequence of the overall repair system isnot fully operative.

The analysis of methyl esterified membrane proteins in intactRBCs was performed by SDS/PAGE, as reported in Experimentalprocedures. The electropherograms of Coomassie Blue-stained pro-files of sALS and control samples were superimposable, indicatingthat, in sALS membranes, neither protein fragmentation nor poly-merization occurred (Fig. 2). Moreover, the major methyl-accept-ing proteins are the same in both patients and controls, andincluded ankyrin, band 4.1, band 4.2, and the integral membraneprotein band 3 (data not shown).

We have observed a low efficiency of the PIMT-mediated repairsystem. In fact AdoMet concentration was about 50% lower in ALSpatients erythrocytes, while AdoHcy levels were equivalent to thecontrols, measuring a lower AdoMet/AdoHcy ratio in ALS patients(Table 2). The erythrocyte AdoMet concentration, ranging from2.5 lM (Perna et al., 1993) to 3.5 lM (Barber et al., 1986), is inthe same order of magnitude as the apparent Km value of PIMTfor the methyl donor substrate (2 lM) (Kim, 1973). This relativelylow intracellular AdoMet concentration, compared with other tis-sues, is balanced by the fact that the enzymic methyl esterificationof proteins represents virtually the only AdoMet-dependent reac-tion operative in mature erythrocytes, accounting for more than90% of utilization of the sulphonium compound (Barber et al.,1986). However, it is possible to hypothesize that any situationleading to a depletion of AdoMet and/or an increase of AdoHcycould affect the PIMT-catalyzed reaction, by altering the [Ado-Met]/[AdoHcy] ratio. It has been proposed as an alteration of theintracellular [AdoMet]/[AdoHcy] ratio in patients with Parkinson’sdisease is probably able to interfere seriously with methyl transferreactions (De Bonis et al., 2010). Therefore, if the transmethylation

potential is altered, the efficiency of the PIMT-mediated repair sys-tem is probably altered as well.

The significant reduction of AdoMet concentration in sALSerythrocytes could be linked with altered activity of L-methio-nine-S-adenosyltransferase (EC 2.5.1.6; MAT), a regulatory enzymeof S-adenosylmethionine biosynthesis. AdoMet is formed frommethionine and ATP in a reaction catalyzed by MAT. AbnormalMAT activity has been previously found in patients withAlzheimer’s diseases (Gomes-Trolin et al., 1996), Parkinson’sdiseases (Cheng et al., 1997) and in ASL (Ekegren et al., 1999). Infact, Ekegren et al. (1999) have demonstrated a 41% decrease inthe affinity of MAT for methionine in male sALS patients, withoutbeing able to explain the cause. Anyway, the decrease of the affin-ity of MAT for methionine activity suggests an alteration of thereaction catalyzed by this enzyme, which might be reflected onthe erythrocyte AdoMet concentration in sALS patients.

Methyl accepting capability in erythrocyte of membrane pro-teins of ALS patients is in fact significantly higher than the valueobserved in healthy subjects. This isoAsp-residues increase isattributable both to an increase of the damage to cargo proteins,presumably consequence of the oxidative stress due to the micro-environment in which sRBC are exposed, as to the incomplete effi-ciency of the repair system caused by altered ratio oftransmethylation potential.

Then, the results reported in this paper may be partly connectedand give an explanation to the data of Suchy et al. (2010), on theassumption that a dietary supplementation with S-adenosylmethionine delays the onset of motor neuron pathology in a mur-ine model of amyotrophic lateral sclerosis, and support thehypothesis that AdoMet supplementation maybe useful as part ofa comprehensive therapeutic approach for sALS. Moreover, severalmolecules able to prevent or reduce the intracellular accumulationof abnormal IsoAsp residues, such as lithium (Lamarre and Desro-siers, 2008) and valproic acid (Cournoyer and Desrosiers, 2009),have been experimented as possible therapeutic approaches inmotor neuron disorders, but they have showed conflicting effectson survival and disease progression (Rouaux et al., 2007; Fenget al., 2008; Fornai et al., 2008; Chiò and Mora, 2013). Future ther-apeutic perspectives might be offered by supplementing riluzole,the most effective anti-degenerative drug in ALS, with other anti-oxidant molecules able to prevent the pathological intracellular in-crease of L-IsoAsp residues, such as betaine (Kharbanda et al., 2007)and carnosine (Hipkiss, 2009), both characterized by documentedneuroprotective properties (Chiu et al., 2010; Ignacio et al., 2005).

Our findings, demonstrating an impairment of membrane pro-tein methyl esterification in RBCs accompanied by low intracellularAdoMet, may not be limited to erythrocytes alone. S-adenosylme-thionine is a versatile molecule used in many biological reactions.It is a commonly used methyl donor in numerous biologicallyimportant methylation reactions, including DNA methylation,RNA methylation, and protein methylation (Grillo and Colombatto,2008). It is worthwhile noting that Cantoni et al. (1979) have ob-served that an altered effect on a number of methyl transfer reac-tions can be calculated when the [AdoMet]/[AdoHcy] ratio drops to1.6. Our paper shows already alteration of methylation reactionseven at a value equal to 2.3 (Table 2).

In conclusion, our results suggest an injured action of PIMT inALS patients erythrocytes, with accumulation of L-isoaspartyl res-idues, notably responsible of abnormal protein conformation. Theincrease of abnormal residues represents a new protein alterationthat may be present not only in red blood cells but also in other celltypes of patients suffering from ALS. These considerations suggestthe possibility that reduced AdoMet concentration, if present inother cells as well, could affect the function of other methyltrans-ferases. If indeed such alterations occur, this could provide at leastpartial explanation for some of the several cell dysfunctions

S. D’Angelo et al. / Neurochemistry International 63 (2013) 626–634 633

present in sALS. Such studies in other cells are beyond the scope ofour investigation and require additional exploration.

References

Agar, J., Durham, H., 2003. Relevance of oxidative injury in the pathogenesis ofmotor neuron diseases. Amyotroph. Lateral Scler. Other Motor Neuron. Disord.4, 232–242.

Andersen, J.K., 2004. Oxidative stress in neurodegeneration: cause or consequence?Nat. Med. 10 (Suppl. 1), S18–S25.

Andersen, P.M., 2006. Amyotrophic lateral sclerosis associated with mutations inthe CuZn superoxide dismutase gene. Curr. Neurol. Neurosci. Rep. 6, 37–46.

Baillet, A., Chanteperdrix, V., Trocme, C., Casez, P., Garrel, C., Besson, G., 2010. Therole of oxidative stress in amyotrophic lateral sclerosis and Parkinson’s disease.Neurochem. Res. 35, 1530–1537.

Barber, J.R., Clarke, S., 1983. Membrane protein carboxyl methylation increases withhuman erythrocytes age. J. Biol. Chem. 258, 1189–1196.

Barber, J.R., Clarke, S., 1984. Inhibition of protein carboxyl methylation by S-adenosyl-L-homocysteine in intact erythrocytes. Physiological consequences. J.Biol. Chem. 10, 7115–7122.

Barber, J.R., Morimoto, B.H., Brunauer, L.S., Clarke, S., 1986. Metabolism of S-adenosyl-L-methionine in intact human erythrocytes. Biochim. Biophys. Acta.29, 361–372.

Barber, S.C., Mead, R.J., Shaw, P.J., 2006. Oxidative stress in ALS: A mechanism ofneurodegeneration and a therapeutic target. Biochim. Biophys. Acta 1762,1051–1067.

Bendotti, C., Marino, M., Cheroni, C., Fontana, E., Crippa, V., Poletti, A., De Biasi, S.,2012. Dysfunction of constitutive and inducible ubiquitin-proteasome systemin amyotrophic lateral sclerosis: implication for protein aggregation andimmune response. Prog. Neurobiol. 97, 101–126.

Brennan, T.V., Anderson, J.W., Jia, Z., Waygood, E.B., Clarke, S., 1994. Repair ofspontaneously deamidated HPr phosphocarrier protein catalyzed by the L-isoaspartate-(D-aspartate) O-methyltransferase. J. Biol. Chem. 269, 24586–24595.

Brooks, B.R., Miller, R.G., Swash, M., Munsat, T.L., 2000. World Federation ofNeurology Research Group on Motor Neuron Diseases. El Escorial revisited:revised criteria for the diagnosis of amyotrophic lateral sclerosis. Amyotroph.Lateral Scler. Other Motor Neuron. Disord. 1, 293–299.

Cantoni, G.L., Chiang, P.K., 1980. The role of the S-adenosylhomocysteine and S-adenosylhomocysteine idrolase in the control of biological methylations. In:Cavallini, D., Gaull, G.E., Zappia, V. (Eds.), Natural Sulfur Compounds: NovelBiochemical and Structural Aspects. Plenum Press, New York, pp. 67–80.

Cantoni, G.L., Richards, H.H., Chiang, P.K., 1979. Inhibitors of S-adenosylhomocysteine hydrolase and their role in the regulation of biologicalmethylation. In: Usdin, E., Borchart, R.T., Creveling, C.R. (Eds.),Transmethylation. Elsevier, North Holland/Amsterdam, pp. 155–164.

Cedarbaum, J.M., Stambler, N., Malta, E., Fuller, C., Hilt, D., Thurmond, B., Nakanishi,A., 1999. The ALSFRS-R: a revised ALS functional rating scale that incorporatesassessments of respiratory function: BDNF ALS study group (phase III). J. Neurol.Sci. 169, 13–21.

Cheng, H., Gomes-Trolin, C., Aquilonius, S.M., Steinberg, A., Lofberg, C., Ekblom, J.,Oreland, L., 1997. Levels of L-methionine S-adenosyltransferase activity inerythrocytes and concentrations of S-adenosylmethionine and S-adenosylhomocysteine in whole blood of patients with Parkinson’s disease.Exp. Neurol. 145, 580–585.

Chiò, A., Mora, G., 2013. The final chapter of the ALS lithium saga. Lancet Neurol. 12,324–325.

Chiu, K., Zhou, Y., Yeung, S.C., Lok, C.K., Chan, O.O., Chang, R.C., So, K.F., Chiu, J.F.,2010. Up-regulation of crystallins is involved in the neuroprotective effect ofwolfberry on survival of retinal ganglion cells in rat ocular hypertension model.J. Cell. Biochem. 110, 311–320.

Clarke, S., 2003. Aging as war between chemical and biochemical processes: proteinmethylation and the recognition of age-damaged proteins for repair. AgeingRes. Rev. 2, 263–285.

Cournoyer, P., Desrosiers, R.R., 2009. Valproic acid enhances protein L-isoaspartylmethyltransferase expression by stimulating extracellular signal-regulatedkinase signaling pathway. Neuropharmacology 56, 839–848.

D’Angelo, S., Ingrosso, D., Perfetto, B., Baroni, A., Zappia, M., Lo bianco Lubrano, L.,Tufano, M.A., Galletti, P., 2001. UVA irradiation induces L-isoaspartyl formationin melanoma cell proteins. Free Radical Biol. Med. 31, 1–9.

D’Angelo, S., Ingrosso, D., Migliardi, V., Sorrentino, A., Donnarumma, G., Baroni, A.,Masella, L., Tufano, M.A., Zappia, M., Galletti, P., 2005. Hydroxytyrosol, a naturalantioxidant from olive oil, prevents protein damage induced by long-waveultraviolet radiation in melanoma cells. Free Radical Biol. Med. 38, 908–919.

D’Angelo, S., Lembo, S., Flora, F., De Bonis, M.L., Balato, A., Ayala, F., Balato, N.,Galletti, P., Zappia, V., 2012. Abnormal isoaspartyl residues in erythrocytemembranes from psoriatic patients. Arch. Dermatol. Res. 304, 475–479.

De Bonis, M.L., Tessitore, A., Pellecchia, M.T., Longo, K., Salvatore, A., Russo, A.,Ingrosso, D., Zappia, V., Barone, P., Galletti, P., Tedeschi, G., 2010. Impairedtransmethylation potential in Parkinson’s disease patients treated with L-Dopa.Neurosci. Lett. 14, 287–291.

De La Haba, G., Cantoni, G.L., 1959. The enzymatic synthesis of S-adenosyl-L-homocysteine from adenosine and homocysteine. J. Biol. Chem. 234, 603–608.

Deng, H.X., Hentati, A., Tainer, J.A., Iqbal, Z., Cayabyab, A., Hung, W.Y., Getzoff, E.D.,Hu, P., Herzfeldt, B., Roos, R.P., 1993. Amyotrophic lateral sclerosis andstructural defects in Cu, Zn superoxide dismutase. Science 261, 1047–1050.

Deverman, B.E., Cook, B.L., Manson, S.R., Niederhoff, R.A., Langer, E.M., Rosova, I.,Kulans, L.A., Fu, X., Weinberg, J.S., Heinecke, J.W., Roth, K.A., Weintraub, S.J.,2002. Bcl-xL deamidation is a critical switch in the regulation of the response toDNA damage. Cell 111, 51–62.

Doyle, H.A., Zhou, J., Wolff, M.J., Harvey, B.P., Roman, R.M., Gee, R.J., Koski, R.A.,Mamula, M.J., 2006. Isoaspartyl post-translational modification triggers anti-tumor T and B lymphocyte immunity. J. Biol. Chem. 281, 32676–32683.

Ekegren, T., Askmark, H., Aquilonius, S.M., Gomes-Trolin, C., 1999. Methionineadenosyltransferase activity in erythrocytes and spinal cord of patients withsporadic amyotrophic lateral sclerosis. Exp. Neurol. 158, 422–427.

Ellis, C.M., Simmons, A., Jones, D.K., Bland, J., Dawson, J.M., Horsfield, M.A., Williams,S.C., Leigh, P.N., 1999. Diffusion tensor MRI assesses corticospinal tract damagein ALS. Neurology 53, 1051–1058.

Fairbanks, G., Steck, T.L., Wallach, D.F.H., 1971. Electrophoretic analysis of the majorpolypeptides of the human erythrocyte membrane. Biochemistry 10, 2606–2616.

Feng, H.L., Leng, Y., Ma, C.H., Zhang, J., Ren, M., Chuang, D.M., 2008. Combinedlithium and valproate treatment delays disease onset, reduces neurologicaldeficits and prolongs survival in an amyotrophic lateral sclerosis mouse model.Neuroscience 155, 567–572.

Fornai, F., Longone, P., Cafaro, L., Kastsiuchenka, O., Ferrucci, M., Manca, M.L.,Lazzeri, G., Spalloni, A., Bellio, N., Lenzi, P., Modugno, N., Siciliano, G., Isidoro, C.,Murri, L., Ruggieri, S., Paparelli, A., 2008. Lithium delays progression ofamyotrophic lateral sclerosis. Proc. Natl. Acad. Sci. USA 105, 2052–2057.

Freitag, C., Clarke, S., 1981. Reversible methylation of cytoskeletal and membraneproteins in intact human erythrocytes. J. Biol. Chem. 256, 6102–6108.

Galletti, P., Ingrosso, D., Nappi, A., Gragnaniello, V., Lolascon, A., Pinto, L., 1983.Increased methyl esterification of membrane proteins in aged red blood cells:preferential esterification of ankyrin and band-4 I cytoskeletal proteins. Eur. J.Biochem. 135, 25–31.

Galletti, P., Ciardiello, A., Ingrosso, D., Di Donato, A., 1988. Repair of isopeptidebonds by protein carboxyl O-methyltransferase: seminal ribonuclease as amodel system. Biochemistry 27, 1752–1757.

Galletti, P., Ingrosso, D., Manna, C., Clemente, G., Zappia, V., 1995. Protein damageand methylation-mediated repair in the erythrocyte. Biochem. J. 306, 313–325.

Galletti, P., De Bonis, M.L., Sorrentino, A., Raimo, M., D’Angelo, S., Scala, I., Andria, G.,D’Aniello, A., Ingrosso, D., Zappia, V., 2007. Accumulation of altered aspartylresidues in erythrocyte proteins from patients with Down’s syndrome. FEBS J.27, 5263–5277.

Gomes-Trolin, C., Gottfries, C.G., Regland, B., Oreland, L., 1996. Influence of vitaminB12 on brain methionine adenosyltransferase activity in senile dementia of theAlzheimer’s type. J. Neural. Transm. Gen. Sect. 103, 861–872.

Grace, J., Stout, J.C., Malloy, P.F., 1999. Assessing frontal lobe behavioral syndromeswith the frontal lobe personality scale. Assessment 6, 269–284.

Grillo, M.A., Colombatto, S., 2008. S-adenosylmethionine and its products. AminoAcids 34, 187–193.

Hipkiss, A.R., 2009. On the enigma of carnosine’s anti-ageing actions. Exp. Gerontol.44, 237–242.

Ignacio, S., Moore, D.H., Smith, A.P., Lee, N.M., 2005. Effect of neuroprotective drugson gene expression in G93A/SOD1 mice. Ann. N.Y. Acad. Sci. 1053, 121–136.

Ingrosso, D., Clarke, S., 1991. Human erythrocyte D-aspartyl/L-isoaspartylmethyltransferase: enzymes that recognize age-damaged proteins. In:Magnani, M., De Flora, A. (Eds.), Red Blood Cell Aging. Plenum Press, NewYork, pp. 263–276.

Ingrosso, D., D’Angelo, S., Perna, A.F., Iolascon, A., Miraglia del Giudice, E., Perrotta,S., Zappia, V., Galletti, P., 1995. Increased membrane-protein methylation inhereditary spherocytosis. A marker of cytoskeletal disarray. Eur. J. Biochem.288, 894–898.

Ingrosso, D., D’Angelo, S., di Carlo, E., Perna, A.F., Zappia, V., Galletti, P., 2000.Increased methyl esterification of altered aspartyl residues in erythrocytemembrane proteins in response to oxidative stress. Eur. J. Biochem. 267, 4397–4405.

Ingrosso, D., Cimmino, A., D’Angelo, S., Alfinito, F., Zappia, V., Galletti, P., 2002.Protein methylation as a marker of aspartate damage in glucose-6-phosphatedehydrogenase-deficient erythrocytes: role of oxidative stress. Eur. J. Biochem.269, 2032–2039.

Kabashi, E., Valdmanis, P.N., Dion, P., Rouleau, G.A., 2007. Oxidized/misfoldedsuperoxide dismutase-1: the cause of all amyotrophic lateral sclerosis? Ann.Neurol. 62, 553–559.

Kharbanda, K.K., Mailliard, M.E., Baldwin, C.R., Sorrell, M.F., Tuma, D.J., 2007.Accumulation of proteins bearing atypical isoaspartyl residues in livers ofalcohol-fed rats is prevented by betaine administration: effects on protein-L-isoaspartyl methyltransferase activity. J. Hepatol. 46, 1119–11125.

Kim, S., 1973. Purification and properties of protein methylase II. Arch. Biochem.Biophys. 157, 476–484.

Kosugi, S., Furuchi, T., Katane, M., Sekine, M., Shirasawa, T., Homma, H., 2008.Suppression of protein L-isoaspartyl (D-aspartyl) methyltransferase results inhyperactivation of EGF-stimulated MEK-ERK signaling in cultured mammaliancells. Biochem. Biophys. Res. Commun. 371, 22–27.

Lamarre, M., Desrosiers, R.R., 2008. Up-regulation of protein L-isoaspartylmethyltransferase expression by lithium is mediated by glycogen synthasekinase-3 inactivation and b-catenin stabilization. Neuropharmacology 55, 669–676.

634 S. D’Angelo et al. / Neurochemistry International 63 (2013) 626–634

Lanthier, J., Desrosiers, R.R., 2004. Protein L-isoaspartyl methyltransferase repairsabnormal aspartyl residues accumulated in vivo in type-I collagen and restorescell migration. Exp. Cell Res. 293, 96–105.

Liu, H.N., Tjostheim, S., Dasilva, K., Taylor, D., Zhao, B., Rakhit, R., Brown, M.,Chakrabartty, A., McLaurin, J., Robertson, J., 2012. Targeting of monomer/misfolded SOD1 as a therapeutic strategy for amyotrophic lateral sclerosis. J.Neurosci. 32, 8791–8799.

Lowenson, J.D., Clarke, S., 1991. Structural elements affecting the recognition of L-isoaspartyl residues by the L-isoaspartyl/D-aspartyl protein methyltransferaseImplications for the repair hypothesis. J. Biol. Chem. 266 (29), 19396–19406.

Lowenson, J.D., Clarke, S., 1992. Recognition of D-aspartyl residues in polypeptidesby the erythrocyte L-isoaspartyl/D-aspartyl protein methyltransferaseImplications for the repair hypothesis. J. Biol. Chem. 25267 (9), 5985–5995.

Mitchell, J.D., Borasio, G.D., 2007. Amyotrofic lateral sclerosis. Lancet 369, 2031–2041.

Oden, K.L., Clarke, S., 1983. S-adenosyl-L-methionine synthetase from humanerythrocytes: role in the regulation of cellular S-adenosylmethionine levels.Biochemistry 22, 2978–2986.

Pepperkok, R., Hotz-Wagenblatt, A., Konig, N., Girod, A., Bossemeyer, D., Kinzel, V.,2000. Intracellular distribution of mammalian protein kinase A catalyticsubunit altered by conserved Asn2 deamidation. J. Cell. Biol. 148, 715–726.

Perna, A.F., Ingrosso, D., Zappia, V., Galletti, P., Capasso, G., De Santo, N.G., 1993.Enzymatic methyl esterification of erythrocyte membrane proteins is impairedin chronic renal failure. Evidence for high levels of the natural inhibitor S-adenosylhomocysteine. J. Clin. Invest. 91 (6), 2497–2503.

Pickles, S., Destroismaisons, L., Peyrard, S.L., Cadot, S., Rouleau, G.A., Brown, R.H. Jr,Julien, J.P., Arbour, N., Vande Velde, C., 2013. Mitochondrial damage revealed byimmunoselection for ALS-linked misfolded SOD1. Hum. Mol. Genet. 22(19),2013, 3947–59.

Reissner, K.J., Aswad, D.W., 2003. Deamidation and isoaspartate formation inproteins: unwanted alterations or surreptitious signals? Cell Mol. Life Sci. 60,1281–1295.

Reissner, K.J., Paranandi, M.V., Luc, T.M., Doyle, H.A., Mamula, M.J., Lowenson, J.D.,Aswad, D.W., 2006. Synapsin I is a major endogenous substrate for protein L-

isoaspartyl methyltransferase in mammalian brain. J. Biol. Chem. 281, 8389–8398.

Robinson, N.E., 2002. Protein deamidation. Proc. Natl. Acad. Sci. USA 99, 5283–5288.Rouaux, C., Panteleeva, I., René, F., Gonzalez de Aguilar, J.L., Echaniz-Laguna, A.,

Dupuis, L., Menger, Y., Boutillier, A.L., Loeffler, J.P., 2007. Sodium valproateexerts neuroprotective effects in vivo through CREB-binding protein-dependentmechanisms but does not improve survival in an amyotrophic lateral sclerosismouse model. J. Neurosci. 27, 5535–5545.

Shaw, P.J., Ince, P.G., Falkous, G., Mantle, D., 1995. Oxidative damage to protein insporadic motor neuron disease spinal cord. Ann. Neurol. 38, 691–695.

Shimuzu, T., Matsuoka, Y., Shirasawa, T., 2005. Biological significance of isoaspartateand its repair system. Biol. Pharm. Bull. 28, 1590–1596.

Siciliano, G., Piazza, S., Carlesi, C., Del Corona, A., Franzini, M., Pompella, A., Malvaldi,G., Mancuso, M., Paolicchi, A., Murri, L., 2007. Antioxidant capacity and proteinoxidation in cerebrospinal fluid of amyotrophic lateral sclerosis. J. Neurol. 254,575–580.

Simonian, N.A., Coyle, J.T., 1996. Oxidative stress in neurodegenerative diseases.Annu. Rev. Pharmacol. Toxicol. 36, 83–106.

Suchy, J., Sangmook, L., Ahmed, A., Shea, T.B., 2010. Dietary supplementation with S-adenosyl methionine delays the onset of motor neuron pathology in a murinemodel of amyotrophic lateral sclerosis. Neurol. Med. 12, 86–97.

Turner, M.R., Cagnin, A., Turkheimer, F.E., Miller, C.C., Shaw, C.E., Brooks, D.J., Leigh,P.N., Banati, R.B., 2004. Evidence of widespread cerebral microglial activation inamyotrophic lateral sclerosis: an [(11)C](R)-PK11195 positron emissiontomography study. Neurobiol. Dis. 15, 601–609.

Wang, L., Popko, B., Roos, R.P., 2011. The unfolded protein response in familialamyotrophic lateral sclerosis. Hum. Mol. Genet. 20, 1008–1015.

Zappia, V., Zydek-Cwick, R., Schlenk, F., 1969. The specificity of S-adenosylmethionine derivatives in methyl transfer reactions. J. Biol. Chem.244 (16), 4499–4509.

Zhou, J.X., Doyle, H.A., Mamula, M.J., Aswad, D.W., 2006. Protein repair in the brain,proteomic analysis of endogenous substrates for protein L-isoaspartylmethyltransferase in mouse brain. J. Biol. Chem. 281, 33802–33813.