Báo cáo khoa học: Mechanistic studies on bovine cytosolic 5¢-nucleotidase II, an enzyme belonging to the HAD superfamily doc

Sequence alignment with members of the P-type ATPases/L-2-halo-acid dehalogenase superfamily identified three highly con-served motifs in cN-II and other cytosolic nucleotidases.. Mutagen

Mechanistic studies on bovine cytosolic 5¢-nucleotidase II, an enzyme belonging to the HAD superfamily

Simone Allegrini1,*, Andrea Scaloni2,*, Maria Giovanna Careddu1, Giovanna Cuccu3, Chiara D’Ambrosio2, Rossana Pesi3,*, Marcella Camici3, Lino Ferrara2and Maria Grazia Tozzi3

1

Dipartimento di Scienze del Farmaco, Universita` di Sassari, Italy;2Proteomics and Mass Spectrometry Laboratory, ISPAAM, National Research Council, Naples, Italy;3Dipartimento di Fisiologia e Biochimica, Universita` di Pisa, Italy

Cytosolic 5¢-nucleotidase/phosphotransferase specific for

6-hydroxypurine monophosphate derivatives (cN-II),

belongs to a class of phosphohydrolases that act through the

formation of an enzyme–phosphate intermediate Sequence

alignment with members of the P-type

ATPases/L-2-halo-acid dehalogenase superfamily identified three highly

con-served motifs in cN-II and other cytosolic nucleotidases

Mutagenesis studies at specific amino acids occurring in

cN-II conserved motifs were performed The modification of

the measured kinetic parameters, caused by conservative and

nonconservative substitutions, suggested that motif I is

involved in the formation and stabilization of the covalent

enzyme–phosphate intermediate Similarly, T249 in motif II

as well as K292 in motif III also contribute to stabilize the

phospho–enzyme adduct Finally, D351 and D356 in motif

III coordinate magnesium ion, which is required for

cata-lysis These findings were consistent with data already

determined for P-type ATPases, haloacid dehalogenases and

phosphotransferases, thus suggesting that cN-II and other mammalian 5¢-nucleotidases are characterized by a 3D arrangement related to the 2-haloacid dehalogenase super-fold Structural determinants involved in differential regu-lation by nonprotein ligands and redox reagents of the two naturally occurring cN-II forms generated by proteolysis were ascertained by combined biochemical and mass spectrometric investigations These experiments indicated that the C-terminal region of cN-II contains a cysteine prone

to form a disulfide bond, thereby inactivating the enzyme Proteolysis events that generate the observed cN-II forms, eliminating this C-terminal portion, may prevent loss of enzymic activity and can be regarded as regulatory pheno-mena

Keywords: catalytic residues; HAD; nucleotidase; regulation; site-directed mutagenesis

Mammalian 5¢-nucleotidases (eN, cN-Ia, cN-Ib, cN-II,

cN-III, cdN and mdN) make up a family of proteins with

different subcellular locations and remarkably low sequence

similarities [1] Besides ectosolic 5¢-nucleotidase, one

mito-chondrial and five cytosolic enzymes have been described

to date According to its substrate specificity and tissue

distribution, each protein seems to play a specific role within

the cell In fact, cN-Is, which is highly expressed in skeletal

muscle, heart and testis, is specific for AMP and seems to be

involved in adenosine production during hypoxia or

ischemia, because it mediates the cell response to low

energy charges [2] On the other hand, cN-II is more specific

for inosine monophosphate (IMP) and GMP, and is a ubiquitous enzyme involved in the regulation of intracellular IMP and GMP concentrations [3] Furthermore, cN-III, which is expressed in red blood cells and is specific for pyrimidines, seems to participate in RNA degradation during erythrocyte maturation [4] Likewise, cytosolic and mitochondrial deoxynucleotidases (cdN and mdN) regulate nucleotide pools in their respective compartments [1] cN-II was the first member of the cytosolic 5¢-nucleotid-ases whose reaction mechanism was elucidated [5] During catalysis, this enzyme was shown to become phosphorylated

on the first aspartate of its DMDYT sequence A similar motif DXDX(T/V) (motif I) is present in all members of the HAD superfamily, where the nucleophilic attack of this aspartate is essential for the catalytic machinery [6–8] P-type ATPase/phosphotransferase members of the HAD superfamily share a similar structural fold and a common reaction mechanism, which requires the formation of a covalent enzyme–phosphate intermediate [8] Furthermore, crystallographic and site-directed mutagenesis studies on these proteins have demonstrated that a series of other common amino acids always occur in their active site [7–9], thus confirming the presence of two additional sequence motifs common to all members of the HAD family [8,9] The first (motif II) is characterized by a threonine/serine residue included in a hydrophobic region; the second (motif III) presents a conserved lysine and a pair of aspartic acid

Correspondence to S Allegrini, Universita` di Sassari, Dipartimento di

Scienze del Farmaco, via Muroni 23/A, 07100 Sassari, Italy.

Fax: +39 079 228708, Tel.: +39 079 228715,

E-mail: enomis@uniss.it

Abbreviations: BPG, 2,3-biphosphoglycerate; CAM,

carboxyamido-methylated; cdN, cytosolic deoxynucleotidase; cN, cytosolic

nucleo-tidase; eN, ectosolic nucleonucleo-tidase; HAD, L-2-haloacid dehalogenase;

IMP, inosine monophosphate; mdN, mitochondrial

deoxynucleoti-dase; PSP, phosphoserine phosphatase.

*Note: These authors contributed equally to the work presented in this

article.

(Received 3 August 2004, revised 11 October 2004,

accepted 25 October 2004)

residues Very recently, the resolution of the crystal structure

of mdN, a dimeric mitochondrial nucleotidase specific for

deoxynucleotides, has been reported, proving that this

enzyme is the first example of a 5¢-nucleotidase belonging to

the HAD superfamily [7] On this basis, a large number

of proteins differing in catalytic activity against various

substrates, polypeptide length (from 200 to 1400 amino

acids), domain arrangement, oligomerization and

conform-ational change following ligand binding, have been related

to the HAD/P-type ATPases/phosphotransferases

super-fold [10] However, no structural data on cN-II are currently

available

Unlike other 5¢-nucleotidases, cN-II activity is

modula-ted by various ligands; it is activamodula-ted by ADP, ATP,

2,3-biphosphoglycerate (BPG) and decavanadate, and is

inhibited by phosphate On the basis of these regulatory

properties, its physiological role has been hypothesized as

being associated with the hydrolysis of excess IMP that has

been newly synthesized or salvaged in the presence of a

high-energy charge [11] The enzyme generates inosine, which, in

turn, can leave the cell and/or be converted into

hypoxan-thine and uric acid However, when IMP accumulates as a

consequence of ATP hydrolysis, cN-II becomes virtually

inactive, allowing the accumulation of the monophosphate

and preventing the loss of precious purine molecules

[3,11–14] Two enzyme forms of bovine cN-II have been

reported, which can be distinguished in terms of

electroph-oretic, chromatographic and regulatory characteristics [13]

The physiological relevance of this observation remains

obscure, together with the nature (either genetic or

regula-tive) of the mechanisms generating these species Moreover,

cN-II presents both phosphatase and phosphotransferase

activities Even though the physiological relevance of the

phosphotransferase activity is not clear, the enzyme has

been demonstrated as being responsible for the

phosphory-lation of nucleoside analogs in use as antineoplastic and

antiviral drugs [15,16] Furthermore, cN-II seems to be

responsible for the resistance to several purine derivative

drugs [17,18] Therefore, it would seem that cN-II plays a

fundamental role in the effectiveness of several purine drugs

and its activity may be predictive of patient survival in acute

myeloid leukaemia [19] Finally, cN-II overactivity has been

demonstrated in Lesch–Nyhan syndrome, which might be

associated with neurological symptoms related to this

disease [20–22]

For these reasons, biochemical studies, aimed at

com-pletely elucidating the cN-II structure with respect to its

functional and regulatory properties, are particularly

important In fact, these investigations will be fundamental

for the design of nucleoside derivatives that could interfere

with enzyme function and stability, thus playing a role both

in the therapy of malignancies and neurological disorders

caused by purine dismetabolisms In this article, we report

the kinetic characterization of a series of cN-II mutants,

designed on the basis of sequence alignment with P-type

ATPases, haloacid dehalogenases and phosphotransferases

Our results indicate that cN-II presents an active site

strongly resembling those present in other members of the

HAD superfamily Furthermore, we investigated the

struc-tural determinants involved in the regulation of cN-II

activity in the presence of ligands or redox reagents by using

a combined biochemical and proteomic approach

Experimental procedures

Materials Talon metal affinity resin was from Clontech Laboratories (Palo Alto, CA, USA) [8-14C]Inosine was purchased from Sigma Chemical Co (St Louis, MO, USA) Thrombin was from Amersham Pharmacia Biotech (Uppsala, Sweden) Poly(vinylidene difluoride) (PVDF) membrane was pur-chased from Millipore Co (Billerica, MA, USA) Goose anticytosolic 5¢-nucleotidase (from pig lung) IgG and rabbit anti-goose IgG serum were kind gifts from R Itoh (Tokyo Kasei Gakuin University, Tokyo, Japan) All other chem-icals were reagent grade All solvents were HPLC grade

Sequence alignment Iterated sequence comparisons and position-specific iterated PSI-BLAST search results, starting from P-type ATPases and HAD, were used as starting multiple alignments [9,23] Several human and bovine 5¢-nucleotidases (cN-Ia, cN-Ib, cN-II, cN-III, cdN and mdN) were aligned by using the same approach These proteins were also analysed for sequence motif by using the MOST program [24] with stringent cut-offs (e.g r¼ 0.0085) Protein secondary

structure was predicted by using thePROF,SCRATCH/SSPRO andPSIPRED programs [25–27] Identified sequence motifs were verified on the basis of the predicted secondary structure All sequences were further aligned by using the MACAWprogram [28], with minor manual adjustments

Site-directed mutagenesis Point mutants were obtained as previously described [6], with minor changes The protocol adopted included two successive PCR reactions In the first, a mutagenic primer was used together with a primer specific for cN-II to amplify

a dsDNA fragment (megaprimer) that contained the desired mutation Each megaprimer, purified from the agarose gel, was used in a second PCR reaction together with a second specific primer, to amplify the final nucleotide fragment, including specific sites for restriction endonucleases at the 5¢ and 3¢ terminus and, in the central part, the mutated triplet Once it had been cleaved, this fragment was used to replace the corresponding one present in the expression plasmid containing bovine wild-type cN-II The specific forward primers used in the PCR reactions were: NheI_F)

to base 14 of the pET28c-cNII construct); and AflII_F) 5¢-CAGTTGACTGGGTTCATT-3¢ (from base 611 to base 628) The specific reverse primers used were: KpnI_R) 5¢-AGTAGACGATGCCATGCT-3¢ (from base 982–965);

(from base 1205–1186) The mutagenic primers used were

as follows (the mutagenic triplette is shown in bold): M53_F) 5¢-TGGGTTTGACANHGATTATACACTTGC TGTGTA-3¢ (from base 147 to base 179) (potentially able

to produce six different mutants: M53I, M53T, M53N, M53K, M53S, M53R); T56_F) 5¢-TGGGTTTGACATG GATTATADNCTTGCTGTGTA-3¢ (from base 147 to base 179) (potentially able to produce six different mutants: T56I, T56M, T56N, T56K, T56S, T56R); T249S_F)

5¢-TTTCTTGCCTCCAACAGTGA-3¢ (from base 736

to base 755); T249V_F) 5¢-TTTCTTGCCGTCAACAG

TGA-3¢ (from base 736 to base 755); S251(T/A)_F)

741 to base 762); K292(R/M)_F) 5¢-GCACGGAKGC

CACTGTTCT-3¢ (from base 868 to base 886); D351E_R)

TGTGCTCTCCAATA-3¢ (from base 1065 to base 1044);

(from base 1071 to base 1050); D356N_R) 5¢-AATGTTCC

CAAAAATGTGATCT-3¢ (from base 1071 to base 1050)

Table 1 shows the primer couples used to produce the

mutants described in this article

The PCR mixtures and cycling conditions were as

follows First PCR mixture: 50 lL containing 7.5 ng of

pET28c-cNII DNA as template, 2 lMof mutagenic primer,

1 lMof specific primer, 200 lM of dNTP, 1 mM MgSO4

and 1.25 U of Platinum Pfx DNA polymerase in PCR

reaction buffer The first PCR cycling conditions were:

2 min at 94C; 15 s at 94 C; 30 s at 50–60 C (depending

on the couple of primers used); and 30 s at 68C Steps 2–4

were repeated 30 times The second PCR mixture was:

25 lL containing 10 ng of pET28c-cNII DNA, all the

megaprimers recovered after purification from the agarose

gel (usually 0.5–0.8 lM), 3 lM specific primer, 200 lM

dNTP, 1 mM MgSO4 and 0.7 U of Platinum Pfx DNA

polymerase in PCR reaction buffer The cycling conditions

in the second PCR were the same as those used in the first

PCR, but in the second PCR, the annealing temperature

was always 60C

Expression of the recombinant proteins

Bovine wild-type and recombinant cN-II mutants were

prepared and purified as previously described [29] At the N

terminus all the recombinant products presented an

addi-tional MGSSHHHHHHSSGLVPRGSHMAS sequence

(whose amino acids were numbered with negative values)

containing the histidine tag and the thrombin cleavage site

The protein concentration was determined according to

Bradford [30], using BSA as a standard The molar

concentration of the enzymes was determined by using the calculated subunit molecular mass (67 300 Da)

Electrophoresis and immunoblotting Electrophoresis under denaturing conditions was performed

on 12% polyacrylamide gels, according to Laemmli [31] After electrophoresis, proteins were blotted onto a PVDF membrane Immunostaining with specific antibody was carried out as previously described [5]

Enzyme assays Unless stated otherwise, the nucleotidase activity of cN-II and its mutants was measured as the rate of [8-14C]inosine formation from 2 mM[8-14C]IMP in the presence of 1.4 mM inosine, 20 mMMgCl2, 4.5 mMATP and 5 mM dithiothre-itol, as previously described [11] Phosphotransferase activ-ity was measured as the rate of [8-14C]IMP formation from 1.4 mM[8-14C]inosine, in the presence of 2 mMIMP, 20 mM MgCl2, 4.5 mMATP and 5 mMdithiothreitol, as previously described [11] For the determination of kinetic parameters (Kmand kcat) the concentration of the labelled substrates ranged from 0.02 to 4 mM A curve of dependence of the rate of phosphotransferase activity on MgCl2concentration was used to determine K50for MgCl2

Under these experimental conditions, the accumulation

of radiolabeled inosine (nucleotidase activity) represents the sum of the phosphatase and the phosphotransferase activ-ities It has previously been reported that, at a concentration close to the Kmvalue (1.4 mM), inosine reduces phosphatase activity to 50% without affecting the Vmax for both reactions [11] Thus, the expected value of 2 was determined for the ratio between nucleotidase and phosphotransferase activities, under the experimental conditions used for the wild-type recombinant cN-II assay Accordingly, an alter-ation of this ratio for a mutant was considered as being caused either by an alteration of the Kmvalue for one of the two substrates or by a variation of the kcatvalue for one of the two activities

The oxidative inhibitory effect was measured by incuba-ting the enzyme with CuCl2(final concentration 1–250 lM)

in 50 mM Tris/HCl, pH 7.4, for 10 min Enzyme was quickly measured for nucleotidase activity, before and after the addition of 5 mM dithiothreitol to the incubation mixture Parallel experiments were also performed by incubating cN-II with or without 20 lM 5,5¢-dithiobis-(2-nitro-benzoic acid), in 50 mMTris/HCl, pH 7.4, at room temperature At different time-points, samples were with-drawn and assayed for nucleotidase activity After 80 min, Ellman’s reagent treated-cN-II was added with 5 mM dithiothreitol and assayed for nucleotidase activity

Structural characterization of 5¢-nucleotidase samples Purified wild-type recombinant 5¢-nucleotidase samples (100 lg), obtained by treatment with or without 5 mM dithiothreitol, and with or without thrombin (1 lg), in

50 mMTris/HCl, pH 7.4, were alkylated with 1.1M iodo-acetamide in 0.25MTris/HCl, 1.25 mMEDTA, containing

6Mguanidinium chloride, pH 7.0, at room temperature for

1 min in the dark Proteins were freed from salt and excess

Table 1 Primers used in PCR reactions for the production of cytosolic

nucleotidase-II (cN-II) point mutants Mut Pr., mutant primer; Sp Pr.,

specific primer; MP, megaprimer.

Mutant

First PCR Second PCR

Mut Pr Sp Pr MP (bp) Sp Pr.

M53(I/N) M53_F + KpnI_R fi 836 + NheI_F

T56R T56_F + KpnI_R fi 836 + NheI_F

T249S T249S_F + Csp45I_R fi 470 + NheI_F

T249V T249V_F + Csp45I_R fi 470 + NheI_F

S251(T/A) S251(T/A)_F + Csp45I_R fi 465 + NheI_F

K292(R/M) K292(R/M)_F + Csp45I_R fi 338 + NheI_F

D351E D351E_R + AflII_F fi 455 + NheI_F

D351N D351N_R + AflII_F fi 455 + NheI_F

D356E D356E_R + AflII_F fi 461 + NheI_F

D356N D356N_R + AflII_F fi 461 + NheI_F

reagents by passing the reaction mixtures through PD10

columns (Amersham Pharmacia Biotech), as previously

reported [32] Protein samples were manually collected,

lyophilized and analysed/concentrated by SDS/PAGE

under nonreducing conditions

Bands from SDS/PAGE were excised from the gel,

triturated and washed with water Proteins were in-gel

digested with trypsin or 2% (v/v) formic acid, as previously

described [33] Digest aliquots were removed and subjected to

a desalting/concentration step on ZipTipC18devices

(Milli-pore Corp., Bedford, MA, USA) before analysis by

MALDI-TOF-MS Peptide mixtures were eluted from the

ZipTipC18 in a stepwise manner, using an increasing

concentration of acetonitrile in the elution solution, and

loaded directly on the MALDI target by using the dried

droplet technique and a-cyano-4-hydroxycinnamic as

mat-rix Samples were analysed on a Voyager-DE PRO mass

spectrometer (Applied Biosystems, Framingham, MA,

USA) Assignments of the recorded mass values to individual

peptides were performed on the basis of their molecular mass

and proteolytic agent specificity, as previously described [6]

Peptide mixtures were also fractionated by RP-HPLC on

a Vydac 218TP52 column (250· 2.1 mm), 5 lm, 300 A˚

pore size (The Separation Group, Hesperia, CA, USA) by

using a linear, 5–60% gradient of acetonitrile in 0.1% (v/v)

trifluoroacetic acid over 60 min, at a flow rate of

0.2 mLÆmin)1 Individual components were collected

manu-ally Disulfide-containing peptides were identified on the

basis of their mass value Sequence analysis was performed

by using a Procise 491 protein sequencer (Applied

Biosys-tems) equipped with a 140C microgradient HPLC and a

785 A UV detector (Applied Biosystems) for the identifica-tion of PTH amino acids

Results

cN-II and HAD superfamily CN-II chemical labelling and site-directed mutagenesis experiments identified D52 as a residue that is essential for enzyme activity and involved in phosphate-adduct forma-tion [6] This amino acid occurs in a sequence region similar

to motif I, which is common to all members of the HAD superfamily [8] Iterated sequence comparisons and posi-tion-specific iterated searches starting from bovine cN-II

or different phosphomonoesterases, phosphotransferases, phosphomutases and dehalogenases were used to identify,

in the cN-II primary structure, the remaining two motifs already reported for these proteins Similarly to HAD superfamily members, these motifs should contain amino acids hypothetically present in the enzyme active site which are essential for metal ion coordination, nucleophilic attack

to substrate and stabilization of an excess of negative charge

in the reaction intermediate Furthermore, analysis of HAD superfamily members demonstrated that conserved residues from each of the motifs appear to occur at specific positions

in the succession of secondary structure elements [9] For this reason, the sequence of cN-II was also analysed in order

to predict the secondary structure of the protein In addition

to the already identified motif I, this investigation highligh-ted two separate regions in the cN-II primary structure as being associated with motif II and motif III (Fig 1) As

Fig 1 Multiple alignment of mammalian 5¢-nucleotidases and members of the P-type ATPase-L-2-haloacid dehalogenase (ATPase-HAD) super-family Proteins are listed under their SWISS-PROT codes Bb, Bos bovis; Ec, Escherichia coli; Eh, Enterococcus hirae; Hs, Homo sapiens; Mg, Mycoplasma genitalium; Psp, Pseudomonas sp.; Sa, Staphylococcus aureus; Sc, Saccharomyces cerevisiae; and Sp, Schizosaccharomyces pombe Only the three common sequence motifs are reported The numbers indicate the distances to the N terminus of each protein and the sizes of the gaps between aligned segments The upper, middle and lower block of sequences include mammalian 5¢-nucleotidases, members of the HAD superfamily and P-type ATPases, respectively Blue shading indicates conserved amino acid residues required for catalytic activity Red shading indicates conserved amino acids alternatively present in motif I Yellow shading indicates uncharged amino acid residues Common secondary structure elements are indicated as a-helices, b-strands and l (loop) regions.

clearly illustrated in the reported multiple alignment,

conserved amino acids in each of the motifs were inserted

in regions that always presented uncharged residues at

specific positions and with a well-defined secondary

struc-ture content Moreover, on the basis of the recent

obser-vation that mdN also belongs to the HAD superfamily, the

above reported sequence analysis was extended to all

mammalian 5¢-nucleotidases Iterated sequence

compari-sons and position-specific iterated searches identified the

three motifs in all of these proteins, except for eN, (Fig 1)

suggesting that cytosolic and mitochondrial 5¢-nucleotidases

and deoxynucleotidases present a structural arrangement

related to the HAD superfamily fold

Site-directed mutagenesis

To verify the predicted role for the conserved residues

reported in Fig 1, 13 mutated cN-II products were

constructed and expressed Extracts were prepared 16 h

after the addition of isopropyl thio-b-D-galactoside (IPTG)

and recombinant wild-type and cN-II mutants were purified

as previously reported [6] In all cases, SDS/PAGE analysis

showed a single component migrating with an apparent

molecular mass of 60 kDa (data not shown) Purified

proteins were used in kinetic measurements, and their

parameters are reported in Table 2 The fact that all the

expressed proteins conserved the same chromatographic

behaviour (data not shown) indicated that observed changes

of activity were not caused by gross folding problems [34]

In a previous work [6], we demonstrated that both

conservative and nonconservative substitution of D52 and

D54 (motif I) completely abolished both enzyme activity

and formation of the cN-II–phosphate intermediate

Replacement at other positions of this motif strongly

affected how cN-II functioned In fact, substitution at

position 56 (mutant T56R) resulted in a protein devoid of

nucleotidase and phosphotransferase activities On the other

hand, the mutagenesis of M53 had a less severe effect However, while nonconservative substitutions (M53N) resulted in a strong decrease in catalytic efficiency, conser-vative substitution (M53I) led to effects on both enzyme activity and affinity towards IMP This latter mutant exhibited the lowest nucleotidase vs phosphotransferase activity ratio; it also showed a sigmoid dependence on

Mg2+concentration These results indicate the essential role

of these residues for proper D52 and D54 orientation and effective cN-II catalysis, confirming the function of motif I,

as deduced by previous chemical labelling and site-directed mutagenesis experiments

The conserved amino acid present in motif II, which is common to all HAD superfamily members, is always a serine or a threonine residue This amino acid is important for a correct orientation of the substrate within the active site through specific hydrogen bonding The alignment reported in Fig 1 shows that T249 is an essential residue for cN-II activity However, another amino acid (S251), with similar properties, occurs closely in motif II In order to unambiguously identify the residue present in motif II, conservative and nonconservative mutants of both amino acids were prepared Mutant T249V showed a strongly reduced enzyme activity and an alteration of Kmvalues for both substrates On the other hand, a conservative substi-tution (mutant T249S) yielded a kinetic behaviour more similar to that of wild-type enzyme In addition, the effect

on enzyme activity exerted by a nonconservative mutation

of residue 251 (mutant S251A) was far less pronounced than that produced by the conservative S251T mutation These results demonstrated the essential role of the T249 hydroxyl group for cN-II catalysis, thus confirming the nature of motif II deduced by multiple sequence alignments Motif III in the HAD superfamily is characterized by the presence of a conserved lysine residue and two negatively charged residues which are involved in stabilizing the negatively charged reaction intermediate and metal ion

Table 2 Effect of point mutations on various kinetic parameters of bovine recombinant cytosolic nucleotidase-II (cN-II) Nucleotidase and phos-photransferase activities were measured as described in the Experimental procedures The results reported are the average of at least three independent assays IMP, inosine monophosphate; nd, not detectable; ND, not determined; WT, wild type.

Mutant

k cat Phosphotransferase

(s)1)

Nucleotidase/

phosphotransferase

K m IMP (m M )

K m inosine (m M ) k cat /K m inosine

K 50 MgCl 2

(m M )

WT 40.0 ± 13 1.8 ± 0.1 0.1 ± 0.04 1.1 ± 0.3 36.0 ± 2.4 1.8 ± 0.6 Motif I

M53I 3.7 ± 2.1 0.9 ± 0.05 1.0 ± 0.4 0.8 ± 0.05 4.5 ± 2.3 3.0 ± 1.0 sigmoid M53N 0.6 ± 0.1 2.4 ± 0.1 0.1 ± 0.01 1.1 ± 0.4 0.6 ± 0.1 ND

Motif II

T249S 19.8 ± 1.9 3.4 ± 0.2 0.2 ± 0.06 0.9 ± 0.1 22.7 ± 1.2 4.1 ± 1.1 T249V 0.3 ± 0.15 1.5 ± 0.1 0.4 ± 0.25 0.25 ± 0.07 1.1 ± 0.3 4.4 ± 1.2 S251T 3.4 ± 0.6 6.7 ± 0.3 0.3 ± 0.15 1.5 ± 0.5 2.3 ± 0.4 3.5 ± 1.8 S251A 12.3 ± 1.3 3.7 ± 0.2 0.1 ± 0.01 1.0 ± 0.15 11.8 ± 0.5 3.5 ± 1.3 Motif III

K292M < 0.1 1.8 ± 0.1 ND 0.5 ± 0.1 < 0.2 ND

D351E < 0.1 1.5 ± 0.1 ND 0.9 ± 0.2 < 0.1 > 30

D356E 2.2 ± 1.0 2.5 ± 0.1 0.2 ± 0.1 1.0 ± 0.25 2.2 ± 0.8 15.0 ± 4

D356N 1.4 ± 0.1 3.2 ± 0.2 0.2 ± 0.06 10.2 ± 0.8 0.1 ± 0.02 > 30

coordination, respectively As expected, on the basis of the

proposed alignment, the mutation of K292 (mutant K292R

and K292M) strongly affected cN-II activity Similarly, the

mutation of two aspartate residues (D351 and D356)

resulted in very poor nucleotidase and phosphotransferase

activities and a significantly reduced affinity towards Mg2+

Conservative mutations (mutant D351E and D356E) had a

less pronounced effect than nonconservative mutations

Furthermore, D356N showed a 10-fold increase in the Km

value for inosine, suggesting a possible role for this amino

acid in the interaction with the second substrate All these

data confirmed the nature of the residues present in motif

III, as deduced by sequence alignment

A general comparison of all the kinetically characterized

mutants showed that the nucleotidase vs

phosphotrans-ferase activity ratio was significantly altered in two cases:

mutant S251T and mutant M53I In the first case, this

phenomenon might be caused by a decrease of

phospho-transfer efficiency, as alterations of the Kmvalue measured

for both substrates were very slight In the latter case, the

value observed for this parameter was in line with a 10-fold

increase of the Kmfor IMP

Sensitivity to oxidizing conditions

It has been reported that freshly purified calf thymus cN-II

displays full activity only in the presence of dithiothreitol

[5,11,12], suggesting that oxidation may modulate enzyme

properties To confirm this observation, recombinant

wild-type cN-II was incubated with different concentrations of

CuCl2, and its remaining activity was measured before and

after the addition of dithiothreitol to the reaction mixture

The results reported in Fig 2A demonstrate that CuCl2

treatment inhibited the enzyme in a

concentration-depend-ent manner The loss of activity was reverted by the presence

of the reducing agent Recombinant wild-type cN-II was

also treated with a different oxidizing agent,

5,5¢-dithiobis-(2-nitro-benzoic acid), and its remaining activity was

measured The results shown in Fig 2B clearly demonstrate

that cN-II was strongly sensitive to this reagent After

80 min of incubation, dithiothreitol was added to the

reaction mixture, resulting in a complete recovery of

activity, thus demonstrating that

5,5¢-dithiobis-(2-nitro-benzoic acid) oxidation can be reverted by reducing agents

Proteolytic generation of two cN-II forms

In a previous work, we observed the simultaneous presence

of two cN-II forms in preparations from calf thymus,

distinguishable for electrophoretic, chromatographic and

regulatory properties [35] In fact, the cN-II species (form B)

with faster electrophoretic mobility (54 kDa) was activated

by ADP and BPG, and a synergistic stimulatory effect of

these compounds was also observed On the other hand, the

slower migrating species (form A) (59 kDa), was activated

to a greater extent by ADP and BPG, and the synergistic

effect was absent [35] To ascertain whether form B was

arising from an intracellular proteolytic event or from a

degradative process during preparation, freshly isolated

tissues were solubilized directly in hot sample buffer for

SDS/PAGE, and the extracted proteins were analysed by

Western blotting following SDS/PAGE (Fig 3A) Two

major immunoreactive polypeptides migrating at 54 and

59 kDa were dentified, thus demonstrating that the 54 kDa species is not generated by a preparation artefact

We also noted that highly purified recombinant cN-II preparations, although stable for activity, degraded very slowly to enzyme forms with a lower apparent molecular mass (results not shown) Furthermore, when a freshly prepared recombinant product (60 kDa) was incubated with thrombin to remove the His-tag-containing sequence, different polypeptide species were obtained, depending on the experimental conditions In addition to the expected

59 kDa polypeptide, overnight incubation with thrombin at

25C induced production of a cN-II form with an apparent molecular mass of 54 kDa (Fig 3B), while overnight incubation with thrombin at 4C only gave rise to the expected 59 kDa protein Parallel experiments with native cN-II from calf thymus, containing both form A and form

B, demonstrated that thrombin treatment induced increased

Fig 2 Effect of CuCl 2 and 5,5¢-dithiobis-(2-nitro-benzoic acid) treat-ment on the activity of cytosolic nucleotidase-II (cN-II) Purified wild-type recombinant cN-II (1 l M ) was incubated with different concentrations of CuCl 2 , for 10 min, at room temperature (A) The rate of inosine monophosphate (IMP) hydrolysis was measured, as described in the Experimental procedures, before (square) and after (circle) the addition of 5 m M dithiothreitol to the incubation mixture Similarly, wild-type recombinant cytosolic nucleotidase-II (cN-II) (1 l M ) was incubated with (square) or without (circle) 20 l M

5,5¢-dithiobis-(2-nitro-benzoic acid), at room temperature, and cN-II activity was measured as described in the Experimental procedures (B) After 80 min, the Ellman’s reagent treated-enzyme was added with

5 m M dithiothreitol and IMP hydrolysis was measured.

amounts of form B, thus yielding a polypeptide with an

apparent molecular mass of 54 kDa co-migrating with that

obtained from the recombinant enzyme (Fig 3B) These

results suggest that an unpredicted site of protease cleavage

is present in the primary structure of cN-II, in addition to

the predicted site present at the N terminus of the

recombinant product To demonstrate definitively that the

59 kDa and 54 kDa species obtained from the recombinant

enzyme were similar to those occurring in calf thymus cN-II,

their sensitivity towards different regulatory ligands was

investigated As already reported for form B from calf

thymus, the 54 kDa form purified from thrombin-treated

recombinant cN-II was activated by ADP and BPG

(Fig 4A) Also in this case, the reported synergistic

stimulatory effect of these compounds was observed; in

fact, a K50value for ADP of 4 mMand 1 mMwas measured

in the absence and in the presence of 200 lM BPG,

respectively Similarly to form A from calf thymus, the

slower migrating recombinant species (59 kDa) was

activa-ted to a greater extent by ADP and BPG, and the reporactiva-ted

synergistic effect was absent (Fig 4B) In fact, a K50value

for ADP of 2.5 mMwas measured either in the presence or

absence of 200 lMBPG Sequencing analysis of all species

reported in Fig 3B yielded the same N-terminal sequence,

thus indicating that this proteolytic event occurred at the

protein C terminus Hence, a still-unknown intracellular

proteolytic process, which removed a C-terminal

polypep-tide, would seem to control the relative abundance of these

two cN-II forms, thus modulating the different response to

enzyme ligands

Interestingly, in addition to the above-mentioned

differ-ences in regulatory properties, the two cN-II forms

gener-ated by thrombin treatment presented significant differences

also in their activity as a function of dithiothreitol

concen-trations (results not shown) In fact, the 59 kDa species

obtained from recombinant wild-type cN-II, similarly to the

unprocessed product, was highly sensitive to dithiothreitol

On the contrary, the 54 kDa species was fully active, even in

the absence of reducing agents

Structural characterization of 5¢-nucleotidase forms

In order to determine the unexpected site for thrombin

hydrolysis and to identify possible residues involved in

cN-II redox regulation, various protein samples were

subjected or not subjected to the reductive conditions used

in the enzymatic assay, and digested or not digested with thrombin, and then alkylated with iodoacetamide under denaturing nonreducing conditions Following the reac-tion, the samples were desalted and analysed by SDS/ PAGE, and in each case yielded a single component The excised bands were digested either with trypsin or 2% (v/v) formic acid and the corresponding peptide mixtures were analysed by MALDI-TOF-MS or resolved by RP-HPLC and further characterized Table 3 summarizes the results obtained; in all cases, mass spectrometry experiments allowed a complete structural characterization

of the analysed species

As expected, in the sample prepared under reductive conditions and in the absence of thrombin, signals at m/z

Fig 3 Immunoblotting characterization of different cytosolic nucleotidase-II (cN-II) forms (A) Immunoblotting analysis of fresh bovine calf thymus tissues directly homogenized and extracted in a hot sample buffer for SDS/PAGE The analysis was performed on 10 lg of total proteins (B) Immunoblotting analysis of partially purified calf thymus and purified wild-type recombinant cN-II samples following incubation with thrombin Lanes 1–3: 3 lg of calf thymus cN-II treated as follows: lane 1, kept at 4 C; lane 2, incubated overnight at 25 C; lane 3, incubated overnight at

25 C in the presence of thrombin Lanes 4–6: 3 lg of wild-type recombinant enzyme treated as follows: lane 4, kept at 4 C; lane 5, incubated overnight at 25 C; lane 6, incubated overnight at 25 C in the presence of thrombin.

Fig 4 Regulatory effect of ADP and 2,3-biphosphoglycerate (BPG) on the different cytosolic nucleotidase-II (cN-II) forms generated following thrombin treatment of the wild-type recombinant enzyme Enzyme assays were performed in the absence of ATP, as described in the Experimental procedures (A) Twenty-five nanograms of the 54 kDa form were assayed with a variable amount of ADP, in the presence (open symbols) or absence (closed symbols) of 200 l M BPG (B) Twenty-five nanograms of the 59 kDa form were assayed with variable amounts of ADP, in the presence (open symbols) or absence (closed symbols) of 200 l M BPG Inserts show the SDS/PAGE ana-lysis of the respective enzyme species.

1093.2, 1731.2, 2052.1, 2066.7, 2178.4, 2180.3, 2968.5,

3457.2, 3502.4, 4023.6 (trypsin) and 2131.4, 2920.2,

3035.4, 3242.8, 3252.6, 3728.5, 3813.3, 3928.7, 3944.8,

4001.1, 4334.9 (2% formic acid), corresponding to the

carboxyamidomethylated peptide species, clearly

demon-strated that all eight cysteine residues occurring in the

polypeptide chain were present in a reduced state On the

other hand, the cN-II sample prepared in the presence of

reducing agents and thrombin treatment, in addition to the

mass signals reported above, showed the occurrence of clear

MH+signals at m/z 7363.4 (trypsin) and 5742.8, 5959.1,

6349.4 (2% formic acid) that were tentatively assigned to

S-S-containing peptides (Table 3) These peaks suggested

the occurrence in this sample of an oxidized cN-II form

containing the disulfide bridge C175-C547, in addition to a

fully reduced enzyme species This hypothesis was

con-firmed by Edman degradation analysis of the purified

disulfide-containing peptides In fact, the tryptic peptide

(MH+at m/z 7363.4) revealed the occurrence of

PTH-Cys-carboxyamidomethylated (PTH-Cys-CAM) at position 167

and PTH-cystine at position 175, together with the absence

of any PTH amino acids at position 547 [36] Similarly,

sequencing analysis of the acid-generated peptides (MH+at

m/z 5742.8, 5959.1, 6349.4) demonstrated the presence of

PTH-Cys-CAM at position 181 and PTH-cystine at

posi-tion 547, together with the absence of any PTH amino acids

at position 175 [36] These results definitively demonstrate

the nature of the oxidized enzyme and the extreme sensitivity of C175 and C547 to changes in redox conditions Different data were generated from the

MALDI-TOF-MS analysis of a cN-II sample obtained following pro-longed thrombin treatment This species migrated with an apparent mass of 54 kDa The occurrence in the spectra of new signals at m/z 1101.8, 1318.5, 1398.7, 1709.2, 2167.8, 3623.7 (2% formic acid), as well as the disappearance of the signals at m/z 1901.3, 4023.6 (trypsin) and 3280.6, 3728.5, 3944.8, 4334.9 (2% formic acid), corresponding to the N- and C-terminal region of the intact protein, clearly demonstrated that, in addition to the expected site (R-6) present at the N terminus, cN-II was hydrolysed by thrombin also at R526 (Table 3) Consequently, in the spectrum there were no mass signals corresponding to peptides containing the disulfide bridge C175–C547 These data were in perfect agreement with the above-mentioned insensitivity of the thrombin-generated 54 kDa species to the oxidative conditions In fact, as thrombin-treated cN-II

is devoid of the C-terminal peptide containing C547, involved in the S-S bridge, it is no longer sensitive to changes in redox conditions

Discussion

The HAD fold defines a versatile hydrolase/mutase/trans-ferase superfamily which appears to function on the

Table 3 MALDI-TOF-MS analysis of air-exposed, reduced and thrombin-treated cytosolic nucleotidase-II (cN-II) samples Protein samples sub-jected or not subsub-jected to the reductive conditions used in enzymatic assay, and digested or not digested with thrombin, were alkylated with iodoacetamide in denaturing, nonreducing conditions and separated by SDS/PAGE under nonreducing conditions Bands were digested in situ with trypsin or 2% (v/v) formic acid and peptide extracts were analysed by MALDI-TOF-MS For simplicity, the table only reports the carboxy-amidomethylated (CAM), disulfide-linked (S-S) and N-/C-terminal peptides The mass is reported as average values.

Trypsin

Peptide

2% (v/v) Formic acid

Peptide

Native

cN-II

MH + (m/z)

Reduced

cN-II

MH + (m/z)

Thrombin-treated cN-II

MH + (m/z)

Native cN-II

MH + (m/z)

Reduced cN-II

MH + (m/z)

Thrombin-treated cN-II

MH + (m/z) 634.6 634.7 634.2 (35–39) 1101.8 (519–526)

654.8 654.9 654.7 (522–526) 1318.5 (517–526)

675.7 675.4 675.9 (30–34) 1398.7 ( )5–7)

1093.5 1093.2 1093.0 (178–186)CAM 1709.2 (514–526)

1433.3 1433.6 1433.5 (516–526) 1934.3 1934.1 1934.6 (497–513)

1475.4 1475.4 1475.8 (510–521) 2131.2 2131.4 2131.7 (171–187)CAM 2

1554.6 1554.7 1554.1 ( )5–8) 2324.9 2324.2 2324.5 (497–516)

1730.9 1731.2 1730.6 (48–61)CAM 2920.7 2920.2 2920.7 (147–170)CAM

1901.0 1901.3 ( )23–6) 3035.3 3035.4 3035.7 (146–170)CAM

2052.2 2052.1 2052.6 (426–442)CAM 3242.5 3242.8 3242.9 (307–337)CAM

2067.0 2066.7 2066.9 (112–129)CAM 3252.9 3252.6 3252.4 (432–459)CAM

2178.2 2178.4 2178.2 (178–195)CAM 3280.7 3280.6 ( )23–7)

2180.8 2180.3 2180.9 (425–442)CAM 3623.7 (497–526)

2968.3 2968.5 2968.1 (314–342)CAM 3728.3 3728.5 (519–549)CAM

3253.5 3253.6 3253.9 (479–507) 3813.1 3813.3 3813.6 (114–145)CAM

3456.7 3457.2 3457.0 (150–177)CAM 2 3928.5 3928.7 3928.3 (114–146)CAM

3502.1 3502.4 3501.9 (99–129)CAM 3944.5 3944.8 (517–549)CAM

4023.4 4023.6 (527–560)CAM 4000.8 4001.1 4000.5 (21–52)CAM

7363.4 (150–177)CAM +

(527–560)S-S

4335.2 4334.9 (514–549)CAM 5742.8 (171–187)CAM + (519–549)S-S 5959.1 (171–187)CAM + (517–549)S-S 6349.4 (171–187)CAM + (514–549)S-S

common scheme of an enzyme-aspartate ester formation

followed by the transfer of the ester group to a nucleophile

molecule, i.e water for hydrolases and dehalogenases, a

different chemical moiety on the initial substrate for

mutases, or a second substrate for transferases With the

sole exception of dehalogenases, all family members require

Mg2+ion in the active site to promote covalent intermediate

formation Enzyme functionalities involved in catalysis are

conserved within members They appear to be juxtaposed in

space, as determined on the basis of the crystallographic

structures solved to date [7,9,37,38] This determines the

occurrence of three specific sequence motifs conserved

among all superfamily members [9,23] On the other hand,

the utilization of the HAD fold for diverse functions is

demonstrated by its rearrangement in a variety of

topolo-gical variants [38] In all cases, catalysis always involves

residues present in an a/b Rossmann-fold domain consisting

of a centrally located parallel b-sheet surrounded by

a-helices Depending on the nature of the enzyme, this core

presents large helical insertions which lead to additional

noncatalytic domain(s) Excluding the three conserved

motifs, the level of sequence divergence among proteins

belonging to the HAD superfamily is so elevated (sequence

identity < 10%) that the relationship between the family

members would go unrecognized This means that some

members of this family may not yet have been identified, as

a result of the limitation in the approaches used for sequence

database search and/or various molecular dimensions of the

analysed polypeptides In our opinion, this is true for the six

mammalian 5¢-nucleotidases reported in Fig 1 This

hypo-thesis, originally proposed by our laboratory [6], has been

recently confirmed by the resolution of the crystallographic

structure of mdN [7]

The characterization of the reaction mechanism [5], the

absolute requirement of a Mg2+ion [11,12], the detection of

a phosphorylated intermediate involving the first aspartate

of its DMDYT motif [6] and the identification of the three

conserved motifs reported in this study, all strongly support

the idea that cN-II also belongs to the HAD superfamily

Mutagenesis studies at specific amino acid positions

predicted by the reported alignment allowed us to identify

a series of residues essential for cN-II catalysis In fact, the

modification of measured kinetic parameters caused by

conservative and nonconservative substitutions suggested a

specific role of these amino acids in the cN-II active site Our

results are perfectly in line with those already reported for

phosphoserine phosphatase [8,39] and Ca2+-ATPase [40–

42], thus demonstrating that cN-II presents a catalytic

machinery which very much resembles those of the other

members of the HAD superfamily Table 4 summarizes the

results obtained for these three enzymes with mutants at

equivalent positions

Similarly to phosphoserine phosphatase (PSP) and Ca2+

-ATPase, mutations of the two aspartates in motif I (D52

and D54) totally abolished cN-II activity The first residue is

directly responsible for the formation of the enzyme–

phosphate intermediate [6], and the second would seem to

be involved in adduct stabilization and Mg2+ion

coordi-nation, as already observed for the other two enzymes [9,38]

Conservative mutations at cN-II D54 had a more

pro-nounced effect than those observed at the corresponding

residues in phosphoserine phosphatase and Ca2+-ATPase,

thus emphasizing the important role of this amino acid for effective cN-II catalysis In addition, our experiments demonstrated, for the first time, that mutations at other conserved positions of motif I also affect enzyme function-ing (Fig 1) (Table 2) In fact, substitution at position 56 (mutant T56R) resulted in a protein totally devoid of nucleotidase and phosphotransferase activity Even though this effect might be caused by differences in relative steric hindrance, this conserved threonine residue has been reported to establish a specific hydrogen bond essential for a correct positioning of the active site nucleophile in the structure of 2-haloacid dehalogenases and phosphonoacet-aldehyde hydrolases [43] Similarly, kinetic analyses of M53 mutants demonstrate that replacements in this position can also affect cN-II catalysis, probably by influencing the correct orientation of the two aspartate residues

On the other hand, the clear similarity observed in the effect of conservative and nonconservative mutations at T249, with respect to S109 in phosphoserine phosphatase and T625 in Ca2+-ATPase, identified this amino acid as the cN-II residue of motif II essential for enzyme catalysis (Table 4) These results are in line with the hypothesis that the hydroxyl group of T249 is implicated in stabilization of the covalent intermediate, as already demonstrated for the corresponding residues of motif II in mdN, HAD and PSP [7,37,38] Finally, the effect of mutations at the conserved lysine and the two negatively charged residues putatively present in cN-II motif III paralleled well with those observed for phosphoserine phosphatase and Ca2+-ATPase (Table 4) A comparison of the kinetic parameters observed for these proteins suggests that, in cN-II, K292 is the basic amino acid essential for the stabilization of the negatively

Table 4 Comparison of the effect of mutations on the activity of three different enzymes of the L-2-haloacid dehalogenase (HAD) superfamily The values reported indicate the relative activity with respect to the wild-type enzyme Residues in equivalent positions in Fig 1 are in the same row Results are taken from the following references: cytosolic nucleotidase-II (cN-II), this article and [6] phosphoserine phosphatase [8], and [29] Ca2+ATPase [30–32] PSP, phosphoserine phosphatase;

WT, wild type; low exp, low expression.

cN-II PSP Ca 2+ -ATPase Mutation Activity Mutation Activity Mutation Ca 2+ transport

WT 100 WT 100 WT 100 Motif I

D52E 0 D20E 0 D351E 0 D52A 0 D20N 0 D351N 0 D54E 0 D22E 50 T353S 20 D54A 0 D22N 0 T353A 0 Motif II

T249S 20 S109T 115 T625S 79 T249V 1.6 S109A 6 T625A low exp Motif III

K292R < 0.1 K158R 1 K292M 0.1 K158A < 0.4 D351E 0.7 D179E 78 D703E 31 D351N < 0.1 D179N 0.6 D703N < 5 D356E 2.3 D183E 63 D707E < 5 D356N 0.6 D183N < 0.4 D707N < 5

charged reaction intermediate On the other hand, D351

and D356 seem to be involved in metal ion coordination, as

substitutions at these positions completely abolished

enzyme activity and caused an increase in the Mg2+K50

values However, if conservative mutations at the

corres-ponding residues in PSP and Ca2+-ATPase generated

enzyme species still presented a certain residual activity,

these mutations in cN-II strongly affected enzyme catalysis

suggesting that, as already observed for motif I, Mg2+

coordination in cN-II strictly requires aspartate residues

Therefore, according to the mutagenesis and chemical

labelling experiments reported above, the active site of cN-II

should be similar to that schematically represented in Fig 5

A careful comparison of the sequence and secondary

structure elements with other 5¢nucleotidases and members

of the HAD superfamily revealed that cN-II, in addition to

the canonical a/b core domain responsible for enzyme

catalytic activity, presents a large noncatalytic extension at

its C terminus A differential processing of this domain

identifies the two naturally occurring cN-II forms that

present a distinct response to the regulatory properties

exerted by various ligands and oxidizing reagents This

phenomenon was originally hypothesized by us [35] and is

now demonstrated in the work presented above by a

biochemical and structural characterization of the 54 and

59 kDa species observed in calf thymus or generated by

thrombin treatment of the wild-type recombinant product

These results would seem to show that the cN-II C-terminal

domain is probably involved in the modulation of enzyme

activity, although the fine structural details associated

with this regulation have not yet been elucidated A

still-unknown protease present in calf thymus and other cells

should cleave intact cN-II closely to R526, thus generating

the two differently regulated enzyme forms Their

simulta-neous presence should make the enzyme less susceptible not

only to physiological variations in cell energy charge, but

also to fluctuations of cellular redox status, as demonstrated

by the results reported in this work

In conclusion, the essential residues involved in the

catalysis and regulation of the cN-II-assisted hydrolysis/

phosphate transfer of purine monophosphates were

ascer-tained by a combined mutagenesis/proteomic investigation

An extended structure-based sequence alignment of 5¢-nucleotidases provided support for a common structural and mechanistic origin of these enzymes, revealing a strong relationship to the HAD superfamily We are currently studying the crystallographic structure of the two naturally occurring forms of cN-II, in the presence or absence of various nucleotide analogs These studies should be able to ascertain the 3D details responsible for the enzyme prop-erties reported in this work They will also be useful for therapeutic developments aimed at improving nucleotide-based drugs against malignancies and neurological dis-orders caused by purine dismetabolisms

Acknowledgements

This work was financially supported by grants from the Italian National Research Council and from the Italian MURST.

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