In vitro effect of metal ions on the activity of two amphibian glyceraldehyde-3-phosphate dehydrogenases: Potential metal binding sites 

1096-4959/03/$- see front matter䊚2003 Elsevier Science Inc. All rights reserved.

doi:10.1016/S1096-4959Ž03.00051-4

In vitro effect of metal ions on the activity of two amphibian glyceraldehyde-3-phosphate dehydrogenases: potential metal binding

sites

Khadija Mounaji , Metaxia Vlassi , Nour-Eddine Erraiss , Maurice Wegnez , Aurelio Serrano ,

Abdelaziz Soukri *

Laboratoire de Biologie et Physiologie de la Reproduction et du Developpement, Faculte des Sciences I, BP. 5366, Maarif,

a ´ ´

Casablanca, Morocco

Protein Crystallography Laboratory, Institute of Biology, National Centre for Scientific Research ‘Demokritos’, P.O. Box 60228,

b

15310 Ag. Paraskevi, Athens, Greece

Laboratoire d’Embryologie Moleculaire et Experimentale, UMR 8080 du CNRS, Universite Paris XI, Batiment 445, 91405 Orsay,

c ´ ´ ´ ˆ

France

Instituto de Bioquimica Vegetal y Fotosintesis(CSIC-Universidad de Sevilla),

d

Centro de Investigaciones Cientificas Isla de la Cartuja, Americo Vespucio syn, 41092 Seville, Spain´ Laboratoire de Biochimie, Biologie Cellulaire et Moleculaire, Unite de Genie Enzymatique et Biologie Moleculaire,

e ´ ´ ´ ´

Faculte des Sciences I, BP. 5366, Maarif, Casablanca, Morocco´

Received 28 November 2002; received in revised form 5 February 2003; accepted 10 February 2003

Abstract

Glyceraldehyde-3-phosphate dehydrogenase

GAPDH, EC 1.2.1.12

was purified from two amphibian species,

and

pI values and V8 digested peptide maps. The effect of zinc, cadmium and copper ions on GAPDH enzymatic activity has been examined in vitro. A time, metal concentration and metal type dependent inhibition was observed for both enzymes.

and

GAPDHs exhibit a much greater sensitivity to copper than to cadmium or zinc ions.

Different half-lives and differential sensitivity to various metals was observed between the two enzymes with

GAPDH being remarkably tolerant to cadmium ions compared to the

enzyme. In order to understand the differential sensitivity of the two enzymes to metals, we produced 3D models of both

and

GAPDH structures based upon known 3D structures of GAPDHs from other species. This necessitated, in a first step, to clone a 900 bp cDNA fragment encoding the nearly full-length

GAPDH. Spatial motif searches on the homology models indicated potential metal binding sites involving cysteine and histidine residues outside the catalytic sites, existing only in either the

or the

GAPDH sequences.

䊚

2003 Elsevier Science Inc. All rights reserved.

Keywords: Glyceraldehyde-3-phosphate dehydrogenase; GapC; cDNA; Heavy metals; Homology modelling; Amphibia;Pleurodeles waltl;Xenopus laevis

Abbreviations: D-G3P, D-glyceraldehyde-3-phosphate; DTT, dithiothreitol; GAPDH, glyceraldehyde-3-phosphate dehydrogenase

*Corresponding author. Departement de Biologie, Faculte des Sciences I, BP.5366, Maarif, Casablanca, Morocco. Tel.:´ ´ q212-22- 230672; fax:q212-22-230674.

E-mail address:[email protected](A. Soukri).

1. Introduction

For a variety of organisms, trace metals such as copper and zinc are enzymatic cofactors and their homeostatic control is essential for metabolism.

Copper and zinc are essential but are needed in very low amounts and potentially toxic when present in excess. For example, neuronal death induced by high concentrations of extracellular Zn

has been attributed to energy failure caused by inhibition of glycolysis ( Sheline et al., 2000 ) . In contrast to these metals, cadmium is non- essential and not required in any biological func- tion. When cadmium is present in living tissues, it is mostly bound to metallothioneins ( for review, see Suzuki et al., 1993 ) . The impact of cadmium on metabolism is due to the fact that it is a very toxic transition metal possessing chemical proper- ties close to those of zinc and copper. Its cellular toxicity is thought to be based, at least partially, on its ability to displace these essential heavy metals. Recent studies have proposed that cadmi- um-induced alterations of some metabolic enzymes could be responsible for cell metabolic dysfunction in pathologic conditions ( Casalino et al., 2000 ) . Here, we questioned how the presence of excess zinc, copper and cadmium could affect carbohy- drate metabolism in Amphibia. This is presently not known for this taxonomic group, although Amphibia could be useful study organisms as they are intimately associated with terrestrial and aquat- ic sources of metal pollution. Moreover, because of their phylogenetic position, amphibians offer an opportunity for studying evolutionary trends.

To approach these questions, we used as a model system the cytosolic enzyme glyceraldehyde-3- phosphate dehydrogenase ( GAPDH, EC 1.2.1.12 ) . This enzyme plays a key role in the glycolytic pathway by catalysing the oxidative phosphoryla- tion of

-glyceraldehyde-3-phosphate to form 1,3- bisphosphoglycerate in the presence of NAD

and inorganic phosphate ( Harris and Waters, 1976 ) . GAPDH has been well characterised not only because of its central role in the intermediary metabolism but also because of its abundance and ease of preparation. Recent studies have reported that mammalian GAPDHs display a number of diverse activities unrelated to the glycolytic func- tion, whereas a change in GAPDH activity has been reported to be associated with Alzheimer’s and Huntington’s diseases and other cell patholo-

gies ( for review, see Sirover, 1999 ) . An increased expression of GAPDH has been observed in bovine endothelial cells exposed to a variety of transition metals ( Graven et al., 1998 ) and in cultured rat astrocytes following exposure to manganese ( Hazell et al., 1999 ) . In contrast, several in vivo and in vitro studies have clearly demonstrated that physiological dysfunction, stress and exposure to metals result in GAPDH inhibition ( Casalino et al., 2000; Krotkiewska and Banas, 1992; Vieira et al., 1983; Soukri et al., 1995; Nakagawa and Nagayama, 1989; Morgan et al., 2002 ) .

In this work, we report an in vitro analysis of GAPDH activity in the presence of divalent heavy metals. We provide evidence that GAPDHs puri- fied to homogeneity from two different amphibian species Xenopus laevis ( anura ) and Pleurodeles waltl ( urodela ) have distinct biochemical proper- ties and show differential sensitivity to inhibition by zinc, copper and cadmium.

In order to investigate the differential effect of the three tested metals on the two amphibian GAPDHs we first cloned a cDNA fragment encod- ing the nearly full-length P. waltl GAPDH. In a previous work we had partially cloned the GapC gene encoding the P. waltl GAPDH ( Mounaji et al., 2002 ) . The 3D structures of X. laevis and P.

waltl GAPDHs are unknown, although the struc- tures of several GAPDHs have been reported up to date. As GAPDHs are among the most con- served proteins, sharing a high degree of similarity ( Fothergill-Gilmore and Michels, 1993 ) , we sub- sequently produced homology models of both X.

laevis and P. waltl GAPDH structures, based upon known 3D GAPDH structures. The models of both enzymes were subjected to spatial motif searches with the aim of identifying potential metal binding sites that could explain their observed differential sensitivity to the metals tested.

2. Materials and methods

2.1. Animals

Iberian ribbed newts, P. waltl ( Amphibia, Uro-

dela ) , originated from Morocco. Animals used in

this study were from our Casablanca breeding

stock. The frogs X. laevis ( Amphibia, Anura ) were

from the Orsay breeding stock.

2.2. Enzyme purification

GAPDH was purified to electrophoretic homo- geneity from crude cell extracts by a procedure described previously ( Mounaji et al., 2002; Soukri et al., 1996 ) . All steps were performed at 4 8C.

Centrifugations were carried out at 20 000 = g for 45 min.

Adult newts and frogs were anaesthetised by immersion in 0.1% MS 222 ( ethyl m-aminoben- zoate methane sulfonate ) . Skeletal muscle tissue ( f 8 g, fresh weight ) was ground and homogeni- sed using an Ultra-Turrax homogeniser in 25 mM Tris–HCl buffer, pH 7.5, containing 2 mM EDTA, 10 mM 2-mercaptoethanol and protease inhibitors ( 2 mM phenylmethylsulfonyl fluoride, 2 mM ben- zamidine and 5 mM ´ -amino-n-caproic acid ) at a ratio of 3 mlyg of fresh tissue. The supernatant ( soluble protein fraction ) obtained after centrifu- gation was considered as the crude extract.

The crude extract was subjected to protein precipitation in the 66–88% ( wyv ) saturation range of ammonium sulfate. The final pellet was dissolved in a minimal volume of 25 mM Tris–

HCl, pH 7.5, containing 2 mM EDTA and 10 mM 2-mercaptoethanol ( buffer A ) . The protein solution was dialysed twice against 1 l of the same buffer.

The dialysed enzyme preparation was applied to a Blue Sepharose CL-6B column ( 1 = 6 cm ) equili- brated with 2 bed volumes of buffer A. The column was washed with 3 bed volumes of buffer A and 2 bed volumes of the same buffer adjusted to pH 8.5 ( buffer B ) . The enzyme was finally eluted with buffer B containing 10 mM NAD

at a flow rate of 6 mlyh. Active fractions were collected and concentrated by ultrafiltration on a Diaflo PM10-Amicon membrane.

Each step of the purification procedure was monitored with activity assays and SDS-polyacryl- amide gel electrophoresis ( SDS-PAGE ) .

SDS-PAGE was carried out as described by Laemmli ( 1970 ) on 1D 12% polyacrylamide gels containing 0.1% SDS. Gels were run on a minia- ture vertical slab gel unit ( Hoefer Scientific Instru- ments ) . After electrophoresis, gels were stained with Coomassie Brilliant Blue R-250 at 0.2% ( wy v ) in methanolyacetic acidywater ( 4:1:5, vyvyv ) for 30 min at room temperature. The apparent subunit molecular mass was determined by meas- uring relative mobilities and comparing with SDS- PAGE molecular weight standards ( Broad Range MW, Bio-Rad ) .

2.3. Analytical procedures

2.3.1. GAPDH activity determination

GAPDH activity in the oxidative phosphoryla- tion was determined spectrophotometrically at 25 8C by monitoring NADH generation at 340 nm ( Serrano et al., 1993 ) . The reaction mixture of 1 ml contained 50 mM Tricine–NaOH buffer pH 8.5, 10 mM sodium arsenate, 1 mM NAD

and 2 mM D-G3P. A coupled assay in which aldolase ( 1 Uyml ) produced the stoichiometric breakage of

- fructose 1-6 biphosphate ( 2 mM ) to D-G3P and dihydroxyacetone-phosphate, the first product being the actual substrate of the oxidative reaction, was usually used during enzyme purification. One unit of enzyme is defined as the amount which catalyses the formation of 1 mmol of NADH per min under the conditions used. Concentrations of the enzymes were estimated by the Bradford meth- od ( Bradford, 1976 ) using bovine serum albumin as a standard. Activity levels in cell-free extracts were expressed as specific activity ( Uymg of protein ) .

2.3.2. Metal tests

The reaction mixture ( 200 ml of 50 mM Tris–

HCl buffer pH 7.5 ) contained 0.4 mM of purified GAPDH ( Holoenzyme ) and various amounts of heavy metal salts ( ZnSO , CuSO , CdCl

) . All incubations were carried out at 4 8C. Samples were removed in defined time intervals for GAPDH activity determination as described above.

2.3.3. Isoelectric focusing

The isoelectric point of GAPDH was determined by the method of Robertson et al. ( 1987 ) , using electrophoretic system in 5% ( wyv ) acrylamide slab gels, holding ampholyte-generated pH gradi- ents ( pH range, 3.5–10; Pharmalyte 3.5–10, Phar- macia Biotech, Uppsala, Sweden ) . Twenty-five millimolar NaOH and 20 mM CH COOH were

used as cathode and anode solutions, respectively.

The marker kit was Sigma 3.6–9.3 IEF-Mix isoe- lectric focusing protein. The proteins were visual- ised by Coomassie Brilliant Blue R250 staining.

2.3.4. Peptide mapping

GAPDH peptide mapping by endoproteinase Glu-C from Staphylococcus aureus strain V8 ( Sig- ma ) was performed according to Cleveland et al.

( 1977 ) . Briefly, pure GAPDH was digested by V8

protease for 30 min at 37 8C at a ratio GAPDH y

V8 of 4y1 ( wyw ) in 125 mM Tris–HCl pH 6.8 buffer containing 0.5% SDS, 10% glycerol and 0.0001% Bromophenol Blue. Proteolysis was stopped by addition of 2-mercaptoethanol and SDS at final concentrations of 10 and 2%, respectively.

After boiling, the samples were loaded on a 12%

SDS-PAGE gel.

2.4. Nucleic acid techniques

2.4.1. RNA isolation and reverse transcriptase- polymerase chain reaction

We used the procedure described by Mounaji et al. ( 2002 ) . Total RNA was isolated from skeletal muscle using the method of Chomczynski and Sacchi ( 1987 ) . First-strand cDNA was produced by reverse transcription ( RT ) using MMLV reverse transcriptase ( Promega ) in conjunction with 2 mg total RNA and the reverse primer named Gap2;

59-CCC ( G ) CAC ( T ) TCG ( A ) TTG ( A ) TCG ( A ) TA- CCA-39 for 1 h at 42 8C. An aliquot from this template ( 1y10 of the reaction ) was used in a subsequent polymerase chain reaction ( PCR ) using Taq DNA polymerase ( Promega ) , Gap2 and for- ward primer named Gap4; 59AAT(C)GGG (ATC)TTC(T)GG(ACT)A(C)GA(G)AT(ACT)GG G(ACT)A(C)G-39. PCR conditions included 30 cycles of 92 8C for 1 min, 45 8C for 1 min and 72 8C for 1 min. Gap4 and Gap2 are degenerated oligonucleotides constructed from conserved regions ( NGFGRIGR and WYDNEW ( C ) G, respectively ) present in all GAPDHs studied so far ( Fothergill-Gilmore and Michels, 1993 ) . Aga- rose gel electrophoresis of the PCR-amplified cDNA from four independent amplification exper- iments showed a single fragment of approximately 0.9 kb—the expected size for the GapC fragment to be amplified with the above described primers—

comprising the almost complete coding region.

2.4.2. Cloning and sequencing

Two PCR products from independent experi- ments were purified using the Geneclean II Kit ( BIO 101, La Jolla, CA ) . These PCR products were subcloned into the pGEM-T vector system ( Promega ) and the nucleotide sequence was deter- mined on both strands using universal primers T7 and SP6 ( Sequencer ABI PRISM model 377, Eurogentec s.a., DNA Sequencing Department, Belgium ) . This nucleotide sequence data corre- sponding to P. waltl GAPDH has been deposited

within the GenBankyEMBL database under the accession number AF 482996.

2.5. Sequence alignment and modelling of X. laevis and P. waltl GAPDH

A sequence similarity search against the PDB Data Bank ( Berman et al., 2000 ) was first per- formed in order to identify GAPDHs of known 3D structure that share high percent sequence identity with the X. laevis enzyme. The X. laevis GAPDH sequence retrieved from the

databank ( entry P51469 ) was used as a query in the

-

program ( Altschul et al., 1997 ) on the NCBI non-redundant database, for this pur- pose. GAPDH sequences sharing high similarity with the query were subsequently subjected to multiple sequence alignments using the program

CLUSTALX

( Thompson et al., 1997 ) .

A model of the X. laevis GAPDH structure was generated using the program

( Sali and Blundell, 1993 ) . The 2 A crystal structures of ˚ Palinurus versicolor ( Chinese lobster ) ( Song et al., 1998 ) and of Escherichia coli ( Yun et al., 2000 ) GAPDHs and the 1.8 A structure of the ˚ Bacillus stearothermophilus GAPDH ( Skarzynski et al., 1987 ) were used as templates. The high percent sequence identity shared between the target protein and the templates and the high resolution at which the structures were determined, were used as criteria for the selection of the templates. Co- ordinates from the PDB entries 1CRW, 1DC5 and 2GD1 corresponding to the apo form of P. versi- color, E. coli and B. stearothermophilus GAPDH, respectively, were used in order to model the apo form of the X. laevis enzyme. The initial model was optimised using the conjugate gradient algo- rithm combined with the variable target function method ( Braun and Go, 1985 ˜ ) followed by molec- ular dynamics simulations combined with simulat- ed annealing as implemented within the program

MODELLER

( Sali and Blundell, 1993 ) . The above optimisation procedure was repeated three times.

The optimised model was subsequently used to

generate the tetrameric form of the enzyme by

applying transformation matrices given in the PDB

entries corresponding to the templates. The entries

1SJZ, 1DC6 and 1GD1 corresponding to the holo

forms of the templates were used in order to model

the quaternary structure of the X. laevis holo-

enzyme. The quality of the final models was

Fig. 1. Comparison of GAPDH subunit molecular masses from X. laevisandP. waltl skeletal muscle. Purified proteins were run on 12% SDS-PAGE and gels were stained with Coomassie Brilliant Blue. Lanes 2 and 3 show pure GAPDHs(10mg) fromX. laevis andP. waltl, respectively. Lane 1 corresponds to molecular mass standards(Broad Range MW, Bio-Rad).

Fig. 2. Isoelectric focusing ofX. laevisandP. waltlGAPDHs.

Isoelectric focusing was performed on 5%(wyv)acrylamide gel holding ampholyte-generated pH gradients(pH range, 3.5–

10). Lane 1 corresponds to isoelectric focusing protein markers (pIrange, 3.6–9.3). Lanes 2 and 3 correspond to pure GAPDH fromX. laevis(pI7.3)andP. waltl(pI7.6), respectively. Iso- electric points are indicated with arrows.

assessed with the

suite of programs ( Laskowski et al., 1993 ) .

The homology model of the P. waltl GAPDH structure was constructed as described above for the X. laevis enzyme. The same templates were used due to high percent sequence identity shared between X. laevis and P. waltl GAPDHs. Display and handling of the models was performed within the

-

program ( Guex and Peitsch, 1997 ) .

Potential metal binding sites on the two amphib- ian GAPDHs were identified by spatial motif searches using the program

( Kleywegt, 1999 ) and coordinates from the models of the tetramers in the apo form. The program

makes searches against libraries of predefined motifs derived from known protein structures. The library produced by the editors of the program was modified by adding metal binding sites from PDB entries not included in the original library. For comparison, we also used the program

on known GAPDH crystal structures.

3. Results

3.1. Purification of GAPDH and electrophoretic characterization

The GAPDHs from muscle tissues of the Amphibia P. waltl and X. laevis were purified using ammonium sulphate precipitation followed by Blue Sepharose CL-6B chromatography. The purity of the preparations was verified by SDS- PAGE analysis. The gel pattern in Fig. 1 reveals the presence of a single protein band for both purified X. laevis and P. waltl GAPDHs. The corresponding apparent molecular masses are 35 and 37 kDa for X. laevis and P. waltl GAPDH, respectively. These values correspond to the expected size for one subunit indicating that the two native enzymes are probably homotetramers with molecular masses within the range of 140–

150 kDa ( Mounaji et al., 2002; Fothergill-Gilmore

and Michels, 1993 ) . The value obtained for X.

Fig. 3. V8 protease peptide maps of X. laevis and P. waltl GAPDHs. Proteolytic fragments of GAPDHs produced by V8 digestion were separated on 12% SDS-PAGE gels. Lane 1:

Markers with corresponding molecular masses(kDa)indicated on the left.Lane 2:10mg of V8 protease. Lane 3; 10mg of undigested X. laevis GAPDH. Lane 4: 8 mg of X. laevis GAPDH digested with 2mg of V8 protease.Lane 5:4mg of undigestedP. waltlGAPDH.Lane 6:4mg ofP. waltlGAPDH digested with 1mg of V8 protease.

laevis enzyme agrees with that reported for X.

laevis embryonic GAPDH ( Nickells and Browder, 1988 ) .

The X. laevis and P. waltl GAPDH pI values, determined by isoelectric focusing, differ slightly ( 7.3 and 7.6, respectively ) ( Fig. 2 ) . A pI value of 7.57 for P. waltl GAPDH ( Mounaji et al., 2002 ) and 7.28 for X. laevis GAPDH were obtained by chromatofocusing ( data not shown ) . Both tech- niques thus reveal a difference in the protein charge of the two GAPDHs.

The electrophoretic pattern in Fig. 3 shows the resulting peptides obtained after limited V8 prote- olysis of X. laevis and P. waltl GAPDHs. The comparative peptide profiles reveal that the two amphibian GAPDHs have dissimilar peptide patterns.

3.2. Effect of zinc, cadmium and copper on GAPDH activity

To determine the effect of heavy metal ions on the activity of X. laevis and P. waltl GAPDHs, pure enzymes were incubated in vitro in the presence of either ZnSO , CdCl or CuSO . All

experiments were performed at the same enzyme concentration. The effect of individual metals on GAPDH activity is reported in Fig. 4 for both X.

laevis and P. waltl. A decrease in enzymatic activity was obtained for the three tested metals.

3.2.1. Zinc

Addition of 20 mM Zn

decreases the specific activity by 50% for the X. laevis GAPDH and by approximately 70% for the P. waltl enzyme ( Fig.

4a ) . Increasing the incubation time does not sig- nificantly modify the metal effect. The inhibitory effect of Zn

is significantly higher at a 50 mM ZnSO

concentration for the X. laevis enzyme ( Fig. 4b ) . Addition of 1 mM dithiothreitol ( DTT ) prior to incubation with 20 mM ZnSO

has a protective effect since it yields to a 20% recovered activity for P. waltl GAPDH ( Fig. 5 ) .

3.2.2. Cadmium

Cadmium has a less pronounced effect than zinc on P. waltl GAPDH activity ( Fig. 4a and c ) . A 70% remaining activity was observed in the pres- ence of 20 mM CdCl , whereas a 30% activity

was still observed after increasing the concentra- tion of cadmium ions to 100 mM ( Fig. 4c and d ) . Addition of 1 mM DTT prior to incubation of P.

waltl GAPDH with 100 mM cadmium resulted in only a 10% recovery of activity ( Fig. 5 ) . At the same ionic concentration ( 20 mM ) , cadmium has a slightly higher inhibitory effect than zinc on the X. laevis GAPDH activity ( Fig. 4a and c ) . 3.2.3. Copper

In contrast to the results obtained with cadmium and zinc ions, a very high inhibitory effect on GAPDH activity was obtained with copper. At a dose of 1 mM, copper caused a 70% inhibition of the X. laevis enzyme in 2 min of incubation ( Fig.

4e ) . An even stronger effect is observed with the P. waltl enzyme since a nearly 90% inhibition is observed under these conditions ( Fig. 4e ) . A rapid and complete inhibition was obtained for both enzymes in less than 1 min after addition of 10 mM copper. Addition of 1 mM DTT abolished almost completely the inhibition of P. waltl GAPDH caused by 1 mM copper ( Fig. 5 ) .

In order to complete these data, further experi-

ments were carried out using combinations of the

three metals. GAPDH retained significant activity

in the presence of both 10 mM CuSO and 20 mM

Fig. 4. Effect of metal ions onX. laevisandP. waltlGAPDH activity in vitro. Purified GAPDH fromX. laevis(j)andP. waltl(⽧) was incubated in the presence of(a)20 mM ZnSO ,₄ (b) 50 mM ZnSO ,₄ (c) 20 mM CdCl ,₂ (d) 100 mM CdCl ,₂ (e) 0.001 mM CuSO . Each incubation mixture contained 0.44 mM of pure GAPDH in 50 mM Tris–HCl, pH 7.5 and various concentrations of metal ions. At the indicated times, GAPDH activity was monitored following the reduction of NADqto NADH as described in Section 2.

The residual activity is given as the percentage of the total activity measured without metal addition(control). The points correspond to average values from three determinations.

ZnSO or 20 mM CdCl , although both amphibian

enzymes are completely inhibited in the presence of 10 mM copper. Simultaneous incubation with the three metals also diminished the inhibitory effect produced by copper alone ( data not shown ) . 3.3. Cloning of a P. waltl GAPDH cDNA ( GapC gene )

The complete amino acid sequence of X. laevis GAPDH is known (

entry:

G3P_XENLA ) . In a previous work, we have

cloned a fragment ( f 0.5 kb ) of the GapC gene

of P. waltl, i.e. that encoding for its catalytic

domain ( Mounaji et al., 2002 ) . In this study, and

with the aim to further investigate the observed

differential effect of various metals on the two

amphibian GAPDHs, we cloned a cDNA of 900

bp encoding for the almost complete P. waltl

GAPDH ( Section 2 ) . The derived 311 amino acid

sequence was subjected to sequence similarity

searches and alignments and was further used in

order to model the 3D structure of P. waltl

GAPDH.

Fig. 5. Effect of DTT on GAPDH activity in presence of metal ions. Incubation mixture contained 0.4mM of pure GAPDH in 50 mM Tris–HCl pH 7.5 and 20 mM ZnSO , 100 mM₄ CdCl or 0.001 mM CuSO_{2 4} (h). To determine the effect of DTT, 1 mM of DTT was added in the mixture prior to incu- bation with metal ions (j). The measurements of GAPDH activity were taken after 10 min of incubation. The values reported are calculated as percent of control(GAPDH without metal addition) and are the mean of three independent experiments.

3.4. Alignment of P. waltl and X. laevis GAPDH sequences on the sequences of GAPDHs with known 3D structure

The sequence alignment of GAPDHs of known 3D structure sharing high percent identity with both X. laevis and P. waltl GAPDHs is shown in Fig. 6. P. waltl and X. laevis GAPDHs share 80%

sequence identity and 87% similarity. The conser- vation is slightly higher in the catalytic domain ( 86% identity, 93% similarity ) compared to the NAD binding domain ( 80% identity, 85% similar- ity ) . The minor differences between the two sequences include different content and distribu- tion of glutamates and aspartates accounting for the different V8 proteolysis patterns ( Fig. 3 ) . In addition, the X. laevis GAPDH sequence comprises two cysteine residues ( Cys10, Cys315 ) that are not found in other GAPDHs ( Fig. 6 ) . Cysteine10 corresponds to a conserved arginine residue in other GAPDHs that is involved in the binding of NAD

phosphates. It is not known whether a third cysteine residue ( Cys326 ) found in the X. laevis GAPDH is also present in the P. waltl enzyme due to lack of sequence data in this region ( Fig. 6 ) . On the other hand, the P. waltl GAPDH sequence comprises three histidine residues ( His157, His161, His298 ) that are not found in the X. laevis

enzyme, one of which ( His161 ) existing only in the P. waltl protein when comparing all known GAPDH sequences ( Fig. 6 ) .

3.5. Modelling of X. laevis and P. waltl GAPDH structures

X. laevis GAPDH shares a sequence identity of 81% ( 90% similarity ) , 73% ( 82% similarity ) , 67%

( 80% similarity ) and 52% ( 71% similarity ) with the human, P. versicolor, E. coli and B. stearoth- ermophilus GAPDHs, respectively. The crystal structures of P. versicolor GAPDH ( Song et al., 1998 ) , E. coli GAPDH ( Yun et al., 2000 ) and B.

stearothermophilus GAPDH ( Skarzynski et al., 1987 ) , in both the apo ( equivalent PDB entries:

1CRW, 1DC5, 2GD1 ) and the holo forms ( equiv- alent PDB entries 1SZJ, 1DC6, 1GD1 ) have been determined at 2, 2 and 1.8 A, respectively. The ˚ above structures were used as templates for mod- elling the X. laevis GAPDH structure as they fulfil both the criteria of high percent sequence identity and high resolution structure determination. Fur- thermore, the crystal structures of both their apo and holo forms are known. Although the human GAPDH shares a high sequence identity with the X. laevis GAPDH ( 81% ) , its crystal structure ( PDB entry: 3GPD ) has been determined at low resolution ( 3 A ˚ ) and therefore was not included in the templates.

The final models fulfil all the

stere- ochemical criteria: 99.7% of the residues have combinations of w and c main chain conforma- tional angles in sterically allowed regions accord- ing to a diagram called the Ramachandran plot after the biophysicist G.N. Ramachandran who first made calculations of regions of not allowed w , c combinations because of steric collisions between the side chains and main chain of proteins.

All stereochemical parameters were found to be

better than expected, showing a quality of the final

models equivalent to that of structures of other

proteins determined at 2 A resolution. The quality ˚

of the final models reflects the high percent

sequence identity shared between X. laevis

GAPDH and the templates. The final model cor-

responding to one subunit of the holo-enzyme is

shown in Fig. 7. The cofactor was modelled using

the crystal structures corresponding to the holo

forms of the templates. As expected by the high

sequence similarity, the overall folding of X. laevis

GAPDH is quite similar to that of other GAPDHs.

Fig. 6. Sequence alignment ofP. waltlandX. laevisGAPDHs on the sequences of GAPDHs of known 3D structure. Only GAPDH sequences sharing more than 60% similarity with theP. waltlandX. laevisenzymes were included in the alignment. TheE. coli,B.

stearothermophilus,P. versicolor, human andX. laevisGAPDH sequences were retrieved from the equivalent entries(G3P1_ECOLI, G3P_BACST, G3P_HOMAM, G3P1_HUMAN and G3P_XENLA)of theSWISSPROTdatabase(Bairoch and Apweiler, 2000). The 311 amino acid sequence corresponding to the GAPDH encoded by the RT-PCR amplified cDNA fragment of theGapCgene fromP. waltl produced in the present work(accession number AF482996), was used in the alignment. The sequence alignment was performed using theCLUSTALXprogram(Thompson et al., 1997). Conserved residues throughout all the aligned sequences are read shaded, whereas similarities according to physico-chemical properties, are boxed. The conserved amino acid sequences used to design the degenerated oligonucleotides used as primers for the PCR amplification, are depicted as Gap4 and Gap2 for the forward and reverse primer, respectively. Cysteine residues found only in theX. laevisGAPDH sequence are indicated with asterisks, whereas histidine residues of P. waltlGAPDH not found in the X. laevisenzyme, are indicated with an arrowhead( )ⁿ. The upper line corresponds to secondary structure elements(ai;a-helix,bi;b-strand)as deduced by theDSSP(Kabsch and Sander, 1983)program and the modelled structure ofX. laevisGAPDH. The figure was made using theESPRIPTsoftware(Gouet et al., 1999).

The structure of the catalytic site, comprising the residues Cys149 and His176 ( Fig. 7 ) , is highly conserved. The conformation of residues involved in NAD binding is also conserved apart from

Cys10 ( Fig. 7 ) that corresponds to an arginine residue in other GAPDHs.

The homology model of the P. waltl GAPDH

structure was constructed as above based on its

Fig. 7. Stereoview of the structural alignment of the modelled structures ofP. waltlandX. laevisGAPDHs. The backbone ofP. waltl andX. laevisGAPDHs are shown as gold and light blue ribbons, respectively. For clarity, only one subunit and the cofactor(in magenta) from theX. laevisholo-enzyme model are depicted. The catalytic cysteine and the histidine residue involved in the catalysis are coloured in green. Residues ofX. laevisGAPDH potentially involved in metal binding are labelled and coloured in light blue. Histidine residues of theP. waltlGAPDH involved in zinc and cadmium binding are also labelled and coloured in gold. The figure was created with the

SWISS-PDBVIEWERprogram(Guex and Peitsch, 1997).

79, 75 and 64% sequence similarity shared with the P. versicolor, E. coli and B. stearothermophilus GAPDHs, respectively. Spatial alignment of the P.

waltl and X. laevis GAPDH modelled structures ( Fig. 7 ) shows an extremely high structural simi- larity: the rms deviation on backbone atoms is 0.6 A. The minor differences in the backbone tracing ˚ concern loop regions and the N-terminal b-strand ( b

) and C-terminal a-helix ( a

) of X. laevis GAPDH. These two secondary structure elements could not be modelled in the P. waltl GAPDH structure due to lack of sequence data for these regions. The high structural similarity reflects the 81% sequence identity and 90% sequence similar- ity shared between the X. laevis and P. waltl GAPDH sequences ( Fig. 6 ) .

3.6. Metal binding sites

Previous kinetic studies have suggested the pres- ence of zinc ions in some mammalian GAPDH preparations ( Krotkiewska and Banas, 1992 ) . In order to identify any potential metal binding sites

in the X. laevis and P. waltl GAPDHs we per- formed spatial motif searches ( Section 2 ) using their modelled structures.

3.6.1. Zinc

For both X. laevis and P. waltl GAPDHs the

spatial motif searches indicated a potential zinc

binding site involving their catalytic cysteine. This

putative site is spatially reminiscent of a zinc

binding site in GTP-specific succinyl-Coa synthe-

tase ( PDB entry: 1EUC ) . The same result was

obtained for other GAPDHs. The spatial motif

searches suggested the existence of two additional

zinc binding sites that involve conserved residues

among GAPDHs ( equivalent to Asp47, His50,

Asp186 and Glu314 of X. laevis GAPDH ) . The

above putative sites ( 3 per subunit s 12 per mole-

cule ) are common in both X. laevis and P. waltl

as well as in other GAPDHs. Our observations are

in agreement with previous studies that have sug-

gested the involvement of the catalytic cysteine of

GAPDHs in the binding of divalent metal ions

( Casalino et al., 2000; Krotkiewska and Banas,

1992; Maret et al., 1999 ) and have shown binding of approximately 10 zinc ions per molecule of two mammalian GAPDHs ( Krotkiewska and Banas, 1992 ) .

In addition, the spatial motif searches have suggested the existence of an extra potential zinc binding site ( 4 per molecule ) in X. laevis and P.

waltl GAPDHs that does not exist in other GAPDHs. This site is, however, different for each enzyme. In the X. laevis GAPDH it involves the unique Cys315 among GAPDHs. According to the spatial motif searches, this putative site also involves His50 ( conserved in all GAPDH sequenc- es ) and is spatially reminiscent of the catalytic zinc binding site found in the Clostridium NADP alcohol dehydrogenase ( PDB entry: 1KEV ) and of a zinc binding site in a chloroplastic malate dehydrogenase ( PDB entry: 7MDH ) . The equiva- lent of Cys315 in the P. waltl GAPDH sequence is a tryptophan and therefore cannot be involved in zinc binding. Instead, the spatial motif searches suggested the existence of a different potential zinc binding site for the P. waltl GAPDH that involves residues His157 and His161 not found in the X. laevis enzyme. This putative site—also involving Glu215 and Asp158—is spatially remi- niscent of a zinc binding site present in the binuclear zinc cluster found in the active site of a phosphotriesterase-like protein ( PDB entry:

1BF6 ) . The implication of groups other than thiols in zinc binding is in accordance with the results of DTT experiments showing that only 20% of the P. waltl GAPDH activity is recovered after addition of DTT prior to incubation with zinc ( Fig. 5 ) . On the 3D models, residues Cys315 and His50 and residues His157 and His161 are located far away from the catalytic site of X. laevis and P. waltl GAPDH, respectively ( Fig. 7 ) . The existence of extra zinc binding sites involving residues located far away from the catalytic site of the enzymes may explain the higher tolerance to zinc ions exhibited by X. laevis and P. waltl GAPDHs compared to other GAPDHs.

3.6.2. Copper

The spatial motif searches suggested that Cys326 of X. laevis GAPDH may be potentially involved in copper binding. This putative site also involves spatially neighbouring histidine residues ( His134 and His327 ) and is reminiscent, in 3D space, of the Cu ( II ) binding site of the blue-green alga plastocyanin ( PDB entry:1FA4 ) . However,

due to lack of sequence data in the equivalent region of P. waltl GAPDH, it is not known whether such a site could also exist in the P. waltl enzyme.

The spatial motif searches detected no potential copper binding sites in P. waltl and other GAPDHs.

However, the strong inhibition of both amphibian GAPDHs by copper suggests the involvement of mainly their catalytic cysteine in the binding of this metal. Involvement of the catalytic cysteine of GAPDHs in copper binding has been also suggested previously ( Krotkiewska and Banas, 1992 ) and it is supported by DTT experiments;

addition of DTT prior to incubation with copper ions abolished almost completely the inhibition of P. waltl GAPDH by this metal ( Fig. 5 ) .

3.6.3. Cadmium

In contrast to the results obtained with copper, the binding of cadmium ions by the catalytic cysteine of P. waltl GAPDH seems to be very weak since the remaining activity after incubation with cadmium is 70%, whereas addition of DTT prior to incubation results in only a 10% recovery of P. waltl GAPDH activity ( Fig. 5 ) . In addition, the spatial motif searches indicated a potential cadmium binding site in only P. waltl GAPDH that involves histidine residues His157 and His161 not found in the X. laevis enzyme. According to the program used for the analysis, this putative site is spatially reminiscent of cadmium binding sites found in thermolysin ( PDB entry: 1LNE ) and ATP sulfurylase ( PDB entry:1G8F ) , where cad- mium has substituted a catalytic and a potential structural zinc, respectively. Interestingly, the above potential cadmium binding site of P. waltl GAPDH coincides with one of its putative zinc binding sites suggested by the spatial motif search- es. Since the equivalent of His161 in other GAPDHs is not a histidine ( Fig. 6 ) , such a site could not exist in the X. laevis GAPDH nor in GAPDHs from other species. The binding of cad- mium ions by residues existing only in P. waltl GAPDH and located far away from its catalytic site may contribute to the extremely high tolerance to cadmium ions exhibited by P. waltl GAPDH compared to X. laevis and other GAPDHs.

Potential cadmium binding sites have been also

identified by the spatial motif searches in the

Leishmania mexicana ( PDB entry: 1A7K ) and

Homarus americanus ( PDB entry: 4GPD )

GAPDH structures. However, the effect of cadmi-

um ions on the activity of the above enzymes has not been reported so far.

4. Discussion

We observed that zinc, cadmium and copper inhibit in vitro the activity of two amphibian X.

laevis and P. waltl GAPDHs. Inhibition of GAPDH activity by these metals has been also reported previously for other species ( Casalino et al., 2000;

Krotkiewska and Banas, 1992; Vieira et al., 1983;

Nakagawa and Nagayama 1989 ) . Both amphibian GAPDHs are highly sensitive to copper whereas they show strong tolerance to high concentrations of zinc and cadmium. The observation that Cu

ions cause a much larger decrease of the GAPDH activity than Zn

is in agreement with the results of a similar work on two mammalian GAPDHs ( Krotkiewska and Banas, 1992 ) .

Furthermore, we observed that X. laevis and P.

waltl GAPDHs exhibit a differential sensitivity to metals with the X. laevis GAPDH being less sensitive to zinc ions compared to the P. waltl enzyme. Both amphibian GAPDHs, however, show higher tolerance to zinc ions from that reported for GAPDHs from other species ( Krotkiewska and Banas, 1992; Vieira et al., 1983 ) . We also found that P. waltl GAPDH activity shows a remarkable high tolerance to cadmium ions compared to the X. laevis GAPDH.

In order to identify any specific metal binding sites in the amphibian enzymes, we also construct- ed 3D models of the X. laevis and P. waltl GAPDH structures. Spatial motif searches on the modelled structures suggest the existence of some potential zinc binding sites in both enzymes spatially remi- niscent of catalytic or structural zinc binding sites found in other proteins. Some of these putative zinc binding sites are shared among all GAPDHs.

An extra potential zinc binding site was identified by the spatial motif searches only in the amphibian GAPDHs and involves cysteine and histidine res- idues found only in X. laevis and P. waltl GAPDH sequence, respectively. Such extra zinc binding sites being located far away from the catalytic site may account for the observed higher tolerance of the amphibian GAPDHs to zinc ions compared to other GAPDHs. Potential zinc binding sites includ- ing residues other than the catalytic ones could bind a portion of zinc ions thus protecting the catalytic site of the enzyme from high concentra- tions of this metal and may thus contribute to

relatively high tolerance to zinc ions exhibited by all GAPDHs studied so far. Our observation is in accordance with kinetic studies that have shown binding of several zinc ions by bovine heart and rabbit muscle GAPDHs ( Krotkiewska and Banas, 1992 ) .

Furthermore, the spatial motif searches suggest the existence of a cadmium binding site only on the P. waltl GAPDH modelled structure. According to the program used for the analysis, this potential site involves spatially neighbouring histidine resi- dues that are found only in the P. waltl GAPDH sequence. On the 3D model, this histidine cluster is located far away from the catalytic site of the enzyme. The involvement of such residues in cadmium binding in conjunction with a somehow weak binding of cadmium by the catalytic cysteine of P. waltl GAPDH may contribute to the observed extremely high tolerance of P. waltl GAPDH to cadmium ions compared to X. laevis and other GAPDHs.

The existence of potential zinc binding sites only in amphibian GAPDHs suggests that GAPDHs in these two species undergo adaptive molecular changes that may allow a better zinc tolerance of these enzymes in metal-rich niches.

In summary, our analysis suggests the existence of potential zinc binding sites in both P. waltl and X. laevis GAPDHs and a cadmium binding site only in the P. waltl enzyme. These putative metal binding sites appear to be different between the two amphibian enzymes and involve residues other than the ones involved in the catalysis.

Our study is far from being exhaustive. More

GAPDHs from other species have to be studied in

regard to their interaction with metals and their

influence on GAPDH activity. Site directed muta-

genesis and structural studies on GAPDHs in the

presence of metal ions are to be performed in

order to confirm the existence of specific metal

binding sites, especially zinc, in GAPDHs. It

would be particularly interesting to co-crystallise

the P. waltl GAPDH with cadmium salts and to

determine its crystal structure in order to test our

data that postulate the existence of a cadmium

binding site for this enzyme. Finally, functional

studies have to be undertaken in order to investi-

gate any physiological role of zinc ions in

GAPDH.

Acknowledgments

This work was supported by grant from CNCPRST, project PARS ( Morocco ) ; PB 97-1135 ( Spain ) ; group PAICVI-261 ( Junta de Andalucia, Spain ) . We thank Dr A. Iddar for his technical help, Prof. M. Loutfi and all the members of laboratoire EMEX ( Universite Paris XI ´ ) .

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