gamma Glutamyl Transpeptidase in Transgenic Tobacco Plants. Cellular Localization, Processing, and...

�-Glutamyl Transpeptidase in Transgenic TobaccoPlants. Cellular Localization, Processing, andBiochemical Properties1

Sergei Storozhenko, Enric Belles-Boix2, Elena Babiychuk, Didier Herouart, Mark W. Davey3, Luit Slooten,Marc Van Montagu, Dirk Inze*, and Sergei Kushnir

Vakgroep Moleculaire Genetica, Departement Plantengenetica, Vlaams Interuniversitair Instituut voorBiotechnologie, Universiteit Gent, K.L. Ledeganckstraat 35, B–9000 Gent, Belgium (S.S., E.B.-B., E.B., M.W.D.,M.V.M., D.I., S.K.); Laboratoire de Biologie Vegetale et Microbiologie, Universite de Nice, F–06108 Nicecedex 2, France (D.H.); and Laboratorium voor Biofysica, Vrije Universiteit Brussel, B–1050 Brussels,Belgium (L.S.)

�-Glutamyl transpeptidase (�-GT) is a ubiquitous enzyme that catalyzes the first step of glutathione (GSH) degradation inthe �-glutamyl cycle in mammals. A cDNA encoding an Arabidopsis homolog for �-GT was overexpressed in tobacco(Nicotiana tabacum) plants. A high level of the membrane-bound �-GT activity was localized outside the cell in transgenicplants. The overproduced enzyme was characterized by a high affinity to GSH and was cleaved post-translationally in twounequal subunits. Thus, Arabidopsis �-GT is similar to the mammalian enzymes in enzymatic properties, post-translationalprocessing, and cellular localization, suggesting analogous biological functions as a key enzyme in the catabolism of GSH.

Glutathione (GSH) is involved in a number of im-portant cellular functions, particularly in the storageand transport of reduced S, protection of cells againstoxidative stress, detoxification of xenobiotics andheavy metals, redox regulation of gene expression,and progression through the cell cycle (Meister andAnderson, 1983; May et al., 1998; Noctor et al., 1998;den Boer and Murray, 2000). Because GSH partici-pates directly in the cellular protection against oxi-dative stress, modification of the GSH metabolism bychanging the production of the enzymes involved inits regulation can be a useful approach to obtainingstress-tolerant plants (Noctor et al., 1998).

In mammals, the GSH metabolism is mediated bythe so-called �-glutamyl cycle (Meister and Ander-son, 1983) that includes two ATP-dependent GSHsynthesis steps, catalyzed by �-glutamyl Cys syn-thetase and GSH synthetase, and a specific GSH deg-radation pathway, which allows the reutilization ofthe amino acids for further GSH resynthesis. BecauseGSH is considered to be the main storage compoundof reduced S, GSH degradation is an important pro-

cess to make S available for an organism in the formof Cys (Lieberman et al., 1996).

The first enzyme of the GSH breakdown pathwayis �-glutamyl transpeptidase (�-GT; EC 2.3.2.2). �-GTcatalyzes transfer of the �-glutamyl moiety from GSHto amino acids, peptides (including GSH itself), orwater (Meister et al., 1981; Taniguchi and Ikeda,1998). In contrast to GSH synthesis, GSH breakdownoccurs on the outer surface of the plasma membraneand includes the �-GT reaction followed by the actionof a dipeptidase:

�-Glu-Cys-Gly � AA—�–GTO3 �-Glu-AA � Cys-Gly

Cys-Gly � 2H2OOdipeptidaseO3 Cys � Gly

�-Glutamyl amino acids formed as a result of the�-GT action are then transported back into cellswhere they become substrates for a �-glutamyl cy-clotransferase, an enzyme converting them into5-oxo-Pro and the corresponding amino acids. Fur-thermore, a 5-oxo-prolinase converts 5-oxo-Pro tol-Glu acid, completing the �-glutamyl cycle:

�-Glu-AAO�-glutamyl cyclotransferaseO3 5-oxo-Pro � AA

5-oxo-Pro � 2H2O � ATPO5-oxo-ProO3 l-Glu

The mammalian �-GT is highly active in organswith a secretory or absorptive function. These organsare characterized by a high rate of GSH export out-side the cells (Meister and Anderson, 1983). �-GT isconsidered to be the main enzyme for Cys reabsorp-tion and transport. Knock-out mice lacking a func-tional �-GT lose Cys in the form of GSH because they

1 This work was supported by the Fund for Scientific ResearchFlanders (grant no. G004796).

2 Present address: Laboratoire de Biologie Cellulaire, InstitutNational de la Recherche Agronomique, Route de St-Cyr, F–78026Versailles cedex, France.

3 Present address: Fruitteeltcentrum, Willem de Croylaan 442,B–3001 Heverlee, Belgium.

* Corresponding author; e-mail [email protected]; fax32–9 –264 –5349.

Article, publication date, and citation information can be foundat www.plantphysiol.org/cgi/doi/10.1104/pp.010887.

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cannot reuse the GSH that has been exported outsidethe cells. This leads to the development of glutathio-nemia and glutathionuria, and animals eventuallydie from Cys starvation. This phenotype can be par-tially rescued by Cys administration (Lieberman etal., 1996).

Because GSH conjugates are also substrates for�-GT, the enzyme plays a role both in detoxificationof poisonous compounds (Meister, 1988; Ishikawa,1992) and the normal metabolism of biologically ac-tive compounds such as leukotrienes and prostaglan-dins (Cagen et al., 1976; Anderson et al., 1982; Meis-ter, 1988; Ishikawa, 1992; Carter et al., 1997).

�-GT is also an important enzyme that probablymodulates the redox status of thiols in the plasmamembrane proteins (Del Bello et al., 1999; Dominici etal., 1999), because one of the products of the �-GTreaction, Cys-Gly, contains a highly reactive thiolcapable of producing active oxygen species by par-ticipating in the Fenton reaction with iron ions (Hal-liwell and Gutteridge, 1989).

In plants, enzymes with �-GT activity are believedto be involved in secondary metabolism and to cata-lyze the synthesis of a range of �-glutamyl dipep-tides, which are formed during fruit ripening andaccumulate in storage tissues such as seeds or bulbsin certain plants. Such peptides include hypoglycin Bin the ackee plant (Blighia sapida; Kean and Hare,1980); �-Glu linked to Asp, Phe, and Tyr in soybean(Glycine max; Ishikawa et al., 1967); Asp, Glu, and Tyrin asparagus (Asparagus officinalis; Kasai et al., 1982);d-Ala and homo-Ser in pea (Pisum sativum) seedlings(Kawasaki et al., 1982); and some others. In onion,�-GT is supposed to catalyze the last step in theformation of volatile compound precursors by cleav-ing the �-glutamyl moiety off �-glutamyl alk(en) yl-Cys sulfoxides (Lancaster and Shaw, 1994).

Whether plant �-GT participates in GSH metabo-lism is still an open question. It is not clear whetherGSH is a true substrate in vivo for the plant �-GT,because, in some cases, GSH has been reportedto be a poor in vitro substrate for the plant�-GT (Steinkamp and Rennenberg, 1984; Lancasterand Shaw, 1994). Moreover, the existence of the�-glutamyl cycle in plants is not evident. Althoughthe GSH biosynthetic pathway is relatively well stud-ied (Noctor et al., 1998), very little is known about theGSH degradation mechanism(s). Results obtained inexperiments with tobacco (Nicotiana tabacum) suspen-sion cultures indicated that GSH degradation is ini-tiated by a carboxypeptidase (Steinkamp and Ren-nenberg, 1985; Rennenberg and Lamoureux, 1990).The �-Glu-Cys dipeptide produced as a result of thisreaction is further degraded to Glu and Cys by theconsecutive actions of �-glutamylcyclotransferaseand 5-oxo-prolinase (Rennenberg and Lamoureux,1990). On the other hand, Cys-Gly found in soybean(Bergmann and Rennenberg, 1993) and heterotrophictobacco cultures (Schneider and Rennenberg, 1992)

suggests that the first step in GSH degradation can becatalyzed by �-GT, followed by a dipeptidaseactivity.

�-GT activities and the purified enzymes have beencharacterized from a number of plant species(Thompson et al., 1964; Goore and Thompson, 1967;Kasai et al., 1982; Kawasaki et al., 1982; Martin andSlovin, 2000). However, the reported molecularmasses and composition, cellular localization, andbiochemical properties vary considerably.

A cDNA (D22) from Arabidopsis has been clonedthat confers resistance to oxidative stress when ex-pressed in yeast (Saccharomyces cerevisiae; Kushnir etal., 1995). The cDNA encoded a protein with exten-sive homology to the mammalian �-GT. This findingprompted us to overexpress the cDNA in tobaccoplants to characterize further the encoded proteinand to study the possible effects of its overproduc-tion on the GSH metabolism.

RESULTS

D22 cDNA Is Homologous to the Mammalian �-GT

Alignment of the deduced amino acid sequence ofthe protein encoded by the D22 cDNA (Kushnir et al.,1995) with the mammalian �-GT revealed that theputative Arabidopsis �-GT (AtGGT) is very similar tothe animal enzymes (Fig. 1). The AtGGT shares 41%identical and 48% similar amino acids with the hu-man (Homo sapiens) �-GT. A putative hydrophobicmembrane-anchoring domain was found at the Nterminus of the plant protein but was shorter thanthat of the mammalian enzyme. All amino acid resi-dues, which are necessary for the catalytic activity ofthe mammalian �-GT, are conserved in the plantenzyme: Arg-107, Asp-422, and Asp-423 (positionscorrespond to the human enzyme), which are be-lieved to be important for binding of the donor sub-strate, and Ser-451 and Ser-452, which are of criticalimportance in both enzyme catalysis and in the reac-tion with the specific �-GT inhibitor acivicin (Tanigu-chi and Ikeda, 1998). These data suggest that the D22cDNA probably codes for an AtGGT that might havecharacteristics similar to those of the mammalianenzyme.

�-GT in Arabidopsis Is Encoded by a SmallGene Family

Purification of enzymes with �-GT activity fromdifferent plant species resulted in contradictions re-garding their molecular masses, subunit composi-tion, cellular localization, and biochemical character-istics. The availability of the nearly completegenomic sequence of Arabidopsis allowed us to as-sess to what extent these discrepancies might be dueto either artifacts of enzyme purification proceduresor natural gene diversity.

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Database searches revealed that the AtGGT gene(accession no. CAB80627) is localized on chromo-some IV. The sequence of the gene is immediatelyfollowed by that of a putative gene (accession no.CAB80628), of which the deduced amino acid se-quence of the encoded protein shares 83% identicaland 87% similar residues with AtGGT. These data arein agreement with the previously obtained DNA gel-blot hybridization data (Kushnir et al., 1995) thatshowed the presence of at least two highly homolo-gous genes for AtGGT. Furthermore, one additionalputative gene (accession no. CAB79679) was foundon the same chromosome with a deduced proteinsequence with 50% identical and 59% similar aminoacids to AtGGT and all of the hallmarks required forcatalytic activity. This putative protein of 69.2 kD hasa long N-terminal extension, which is predicted tocontain a trans-membrane domain. A potential gene(accession no. AAF07391) encoding a small protein of191 amino acids (20.9 kD) was also found on chro-mosome I. The sequence of the encoded proteinmatched the 311- to 501-amino acid fragment ofAtGGT, which contains the active site of the enzyme(Taniguchi and Ikeda, 1998). The putative proteinshares 85% identical and 87% similar amino acidswith AtGGT in this region. Thus, in Arabidopsis,�-GT is probably represented by at least four differ-ent isoforms that might have different cellular local-izations and serve different functions.

Transgenic Tobacco Plants Producing AtGGT PossessHigh �-GT Activity

To further characterize AtGGT, transgenic tobaccoplants were transformed with the D22 cDNA underthe control of the 35S promoter. Integration and ex-pression of the transgenes were checked by DNA andRNA gel-blot hybridizations, respectively (data notshown). Transgenic lines that demonstrated highesttransgene expression at the level of mRNA wereselected for further analysis.

Total protein extracts from the leaves of transgenicplants have been shown to catalyze the release ofp-nitroaniline (PNA) from �-glutamyl-p-nitroanilide(GPNA), a typical reaction used to assay �-GT activ-ity (Orlowski and Meister, 1963; Fig. 2). No activitywas detected in protein extracts isolated from leaves

Figure 1. Amino acid sequences of AtGGT (accession no. S58286)and other putative �-GTs from Arabidopsis (corresponding accessionnos. put as names) aligned with the human (accession no. P19440) andmouse (accession no. Q60928) enzymes. Identical amino acids in allsequences are boxed. Amino acids homologous to AtGGT are shad-owed. Membrane-anchoring domains and putative membrane-anchoring domains of the mammalian and plant enzymes are under-lined. The open arrow points to the protease cleavage site. Aminoacids important for the substrate binding and those essential for thecatalytic activity are marked with asterisks and black circles, respec-tively. Putative glycosylated amino acids in the sequence of AtGGT areindicated by black rectangles.

�-Glutamyl Transpeptidase in Transgenic Tobacco Plants

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of control plants transformed with the “empty” vec-tor under the assay conditions. This observation sug-gests that the activity detected is attributed to theoverproduced protein. This result allowed us to con-clude that AtGGT is a plant �-GT.

AtGGT Is Localized on the Plasma MembraneOutside the Cell

Because the animal �-GT is a membrane-boundprotein, anchored by its short N-terminal hydropho-bic domain to the plasma membrane outside the cell,we addressed the question of where AtGGT is local-ized. Analysis of the AtGGT protein sequence withappropriate software predicted a similar leader se-quence in the N terminus and suggested with equallikelihood that the enzyme was localized either in theplasma or in the vacuolar membranes. To find outwhether AtGGT is a membrane-bound protein, weanalyzed the solubilization of the enzymatic activityas a function of the ionic strength of the extractionbuffer, because it had been shown previously thatmembrane-bound plant �-GTs could be solubilizedin a high-salt buffer (Martin and Slovin, 2000).

No �-GT activity was detected in the soluble frac-tion of the leaf tissue of transgenic plants underlow-ionic-strength conditions, indicating that theoverproduced enzyme was exclusively membranebound. The activity could only be solubilized in ahigh-salt solution, containing 0.5 to 1 m NaCl.

The mammalian �-GT is localized on the plasmamembrane outside the cell. To check whether this isthe case for the plant enzyme, we incubated intactmesophyll protoplasts from the transgenic and con-trol plants in a solution containing GPNA. Whereasno �-GT activity was observed with protoplasts fromthe control plants, incubation of protoplasts fromtransgenic plants resulted in the release of PNA,

suggesting that the overproduced enzyme is local-ized outside the cell. However, this result still leavesopen the possibility that the substrate has been takenup by the protoplasts with further secretion of thereaction product. Furthermore, it cannot be excludedthat a fraction of the enzyme is localized in the in-tracellular membranes, such as the vacuolar mem-brane or the endoplasmic reticulum.

To localize the AtGGT activity in a more directway, leaf discs from the transgenic and control plantswere infiltrated with a fluorescent �-GT substrate,�-glutamyl-7-amido-4-methylcoumarin (Smith et al.,1979). The fluorescence was monitored in vivo usinglaser confocal microscopy. As is seen in Figure 3,intense fluorescence is associated with the plasmamembrane/cell wall of the AtGGT-overproducingplants. No membrane-associated fluorescence wasdetected in control plants. Thus, the AtGGT is aplasma membrane-bound protein and localized out-side the cell.

AtGGT Consists of Two Unequal Subunits

The mammalian �-GT is composed of two subunitsderived from the processing by a protease of a singlepolypeptide chain precursor. When high-salt proteinextracts from the transgenic and control plants wereanalyzed by protein gel blots with a polyclonal anti-body raised against AtGGT (see “Materials andMethods”), two bands with calculated molecularmasses of 29 and 41 kD in a 1:1 ratio were found toreact with the antibody (Fig. 4). The sum of their

Figure 2. �-GT activities detected in the transgenic plants expressingD22 cDNA determined by the rate of PNA release in the standardtranspeptidation reaction with GPNA as donor and Gly-Gly as ac-ceptor substrates. D22/*, Protein extracts from different transgeniclines incubated with the substrates; control, protein extract from thecontrol plant incubated with the reaction substrates.

Figure 3. Confocal images of tobacco leaf discs infiltrated with�-glutamyl-7-amido-4-methylcoumarin. A and B, AtGGT-overproducing plants. C and D, Control plants. Bars � 60 �m (A andC) and 25 �m (B and D).

Storozhenko et al.




calculated molecular masses, 70 kD, was roughlyequal to the expected molecular mass of AtGGT (61.1kD). The AtGGT proteolysis did probably not occurduring the protein isolation, because the same bandswere obtained in the protein gel blots of total proteinextracts isolated under denaturing conditions (datanot shown). From these data, the bands can be as-sumed to be products of the single-chain precursor

cleavage. The discrepancy between expected and de-termined molecular masses can be explained by pos-sible glycosylation of the plant �-GT, because animal�-GTs are known to be heavily glycosylated (Tanigu-chi and Ikeda, 1998). Two potential glycosylationsites were found in the large subunit sequence andone in the sequence of the small subunit (Fig. 1).

AtGGT Is Similar to the Mammalian Enzyme in ItsBiochemical Characteristics

Given the similarity between the mammalian �-GTand AtGGT sequences, processing, and extracellularlocalization, the question arises as to whether theplant enzyme also possesses similar biochemicalcharacteristics. The plant enzyme efficiently hydro-lyzes GPNA in a relatively wide pH range (Fig. 5),and by adding the “classical” �-glutamyl group ac-ceptor, dipeptide Gly-Gly, the reaction is stimulatedby approximately 1.5-fold (Vmax of the hydrolysisand transpeptidation reactions, 5 and 8 �m min�1,respectively). The transpeptidation was stimulatedalso to a different extent by addition of differentamino acid acceptors, each having a different pHoptimum (data not shown). The Km value of AtGGTfor GPNA as determined in the transpeptidation re-action with GPNA as donor and Gly-Gly as acceptorwas in a range similar to those of the enzymes fromother organisms (Table I).

Because one of the supposed functions of AtGGT isconnected to GSH catabolism, it was important todetermine whether AtGGT could readily use GSH asdonor substrate. Incubation of a purified bovine�-GT with GSH as the donor and [35S]Met as theacceptor of the �-glutamyl group led to the formationof the 35S-labeled �-Glu-Met, which was visualizedby thin-layer chromatography (Fig. 6). Essentially thesame reaction was catalyzed by the protein extractfrom transgenic plants but not by that from the con-trol plants. These data prove unequivocally that GSHcan be used in vitro by AtGGT as donor of the�-glutamyl group.

The ability of AtGGT to use GSH as donor sub-strate in vitro raises another important question as towhether GSH is a high-affinity substrate that can beused by the enzyme in vivo. When GSH was addedto the standard activity reaction, the release of PNAfrom GPNA was inhibited competitively (Fig. 7). In

Figure 5. pH dependence of the AtGGT transpeptidation and hydro-lysis. Two buffers, 1,4-piperazinediethanesulfonic acid (PIPES) andTris, were used to cover the pH range from 6.0 to 9.5. Both activitieswere 1.8-fold lower in PIPES buffer. The corresponding curves wereshifted to the level of the activities in Tris buffer to make a continuouscurve.

Figure 4. Protein gel blots of protein extracts solubilized in 0.5 M

NaCl from transgenic plants with polyclonal anti-AtGGT antibody.D22, Protein extract from the transgenic plant overproducing AtGGT;C, protein extract from the control plant transformed with an “empty”vector.

Table I. Km values of different �-GTs for GPNA donor substrate

Enzyme Source Km Value Reference

mM

Human 0.9 Visvikis et al. (1991)Rat kidney 2.0 Thompson and Meister (1977)Yeast 1.5 Penninckx and Jaspers (1985)Onion 0.6 Lancaster and Shaw (1994)Tomato I 1.7 Martin and Slovin (2000)Tomato II 2.1 Martin and Slovin (2000)Arabidopsis 0.8 This work





such a reaction in which one substrate inhibits theother substrate, the inhibition constant Ki is actuallyequal to the kinetic constant Km of the inhibitingsubstrate (Cornish-Bowden and Wharton, 1988). Thekinetics of PNA release at different GSH concentra-tions allowed us to determine the Ki as 255 � 4 �m.Therefore, the Km value of AtGGT for GSH is 255 �4 �m, which is low enough to claim that GSH is agood substrate for AtGGT in vitro and can poten-tially be the main in vivo substrate.

Taken together, these results confirm that AtGGThas catalytic characteristics very similar to those ofyeast, mammalian, and other plant �-GTs and, more

importantly, that it has a high in vitro affinity toGSH, suggesting that GSH may be the functionalsubstrate for the plant �-GT in vivo.

Transgenic and Control Plants Have Similar GSHDegradation Rates

By assuming that GSH is a good substrate forAtGGT, does the high �-GT activity affect GSH me-tabolism in transgenic plants? Quantitative determi-nation of GSH and oxidized glutathione (GSSG) dem-onstrated that the steady-state levels of thesecompounds (800 � 100 pM �g�1 protein with a GSH:GSSG ratio of 9:1 [w/w]) are essentially the same inleaves of transgenic and control plants. This obser-vation does not exclude the possibility that the GSHturnover rate can be different in transgenic plants,because the higher GSH degradation rate broughtabout by the AtGGT overproduction might be com-pensated by an increase in the rate of GSH synthesis.To check whether this is the case, leaf discs fromtransgenic and control plants were infiltrated withl-buthionine-[S,R]-sulfoximine, which inhibits GSHsynthesis, and the time course of the GSH degrada-tion rate was followed (Fig. 8). Upon inhibition ofGSH synthesis with l-buthionine-[S,R]-sulfoximine,the level of GSH slowly dropped to approximately10% of the initial value after 32 h of incubation in leafdiscs from both transgenic and control plants. Nosignificant differences were observed between thetransgenic and control plants in the degradation rateof GSH.

DISCUSSION

In transgenic plants overproducing AtGGT, the�-GT activity is high in the leaves, whereas no activ-

Figure 6. Radio-image of thin-layer chromatograph showing the re-action products of AtGGT with GSH as donor and [35S]Met asacceptor of the �-Glu group. AtGGT and BovGGT reactions werecatalyzed by the protein extract from AtGGT-overproducing plantsand by purified bovine �-GT, respectively. Control, Reaction wascarried out in the presence of the protein extract from control plants;�, no GSH added; �, 1 mM GSH added as donor substrate.

Figure 7. Competitive inhibition by GSH of PNA release catalyzedby AtGGT, when added to the standard transpeptidation reaction.GPNA and Gly-Gly were used as donor and acceptor substrates,respectively.

Figure 8. Time course of GSH breakdown in leaf discs of transgenicand control plants infiltrated with 1 mM L-buthionine-[S,R]-sulfoximine. To simplify the picture error bars were not added. SD inthree independent experiments was not more that 15%.

Storozhenko et al.




ity is detected in the leaves of control plants, at leastunder our assay conditions. This observation allowedus to use crude protein extracts from the transgenicplants to study the catalytic properties of AtGGTwithout further purification.

We show that AtGGT is a membrane-bound pro-tein localized on the plasma membrane outside thecell. This result is consistent with most data concern-ing mammalian enzymes, which have been shown tohave the same cellular localization (Meister et al.,1981; Taniguchi and Ikeda, 1998). In contrast, theyeast enzyme is confined not only to the plasmamembrane (Payne and Payne, 1984), but also to thevacuolar membrane (Jaspers and Penninckx, 1984).Existing data on the cellular localization of plant�-GTs are controversial. Two isoforms of �-GT fromtomato (Lycopersicon esculentum) fruit have been re-ported to be exclusively membrane-bound proteins(Martin and Slovin, 2000). Steinkamp and Rennen-berg (1984) attributed only 30% of the total �-GTactivity in a tobacco suspension culture to theplasma membrane-bound fraction and the remain-ing 70% to the cytoplasm. This result was explainedeither by the enzyme solubilization during the tis-sue homogenization step or by the existence of sev-eral �-GT isoforms in tobacco (Steinkamp and Ren-nenberg, 1984). The Arabidopsis database searchrevealed that the plant �-GT is represented by amultigene family, in which the protein productsmay have different cellular localizations and servedifferent functions.

The protein gel-blot analysis strongly suggests thatAtGGT is a heterodimer that is formed from thecleavage of the single-chain precursor with the largeand small subunits of approximately 41 and 29 kD,respectively, as determined from the protein gel-blothybridization. Moreover, the amino acid sequence ofthe cleavage site is well conserved in all �-GT se-quences available, and the AtGGT sequence is noexception to this rule (Fig. 1). The data are in a goodagreement with other reports showing that all�-GTs from bacteria, yeast, and mammals that havebeen studied to date, to our knowledge, are post-translationally cleaved into two subunits with mo-lecular masses ranging from 38 to 72 kD for the largesubunit and from 21 to 66 kD for the small subunit(Taniguchi and Ikeda, 1998). It is noteworthy thatsuch a high variation in the molecular masses can beexplained by a high glycosylation of the animal(Taniguchi and Ikeda, 1998) and plant enzymes(Lancaster and Shaw, 1994; Martin and Slovin,2000).

Previous studies concerning purified �-GT fromplants reported that the enzymes were singlepolypeptides with highly variable molecular masses:12.5 kD in the ackee plant (Kean and Hare, 1980), 43kD in tomato fruits (Martin and Slovin, 2000), 56.7 kDin onion (Lancaster and Shaw, 1994), and 180 kD inkidney bean (Goore and Thompson, 1967). These

differences can be explained by the existence of dif-ferent enzyme isoforms, by partial proteolysis of thepurified proteins, or by glycosylation, in the case ofthe very high-molecular masses.

The catalytic properties of AtGGT are very similarto those of other �-GTs studied to date. When theartificial substrate GPNA is used, the kinetics con-stants are in the same range as those described for themammalian, yeast, bacterial, and plant enzymes (Ta-ble I). However, a discrepancy in the enzyme affinitytoward GSH is found in different �-GTs of plantorigin. �-GT purified from onion has a Km value of4.97 mm for GSH (Lancaster and Shaw, 1994). Indi-rect evidence presented by Steinkamp and Rennen-berg (1984) also confirmed that GSH is a poor sub-strate for �-GT from a tobacco suspension culture,challenging the assumption that plant �-GT mayfunction in vivo as an enzyme of the GSH catabolism.On the other hand, two �-GTs isolated from tomatofruits (Martin and Slovin, 2000) have a high affinityto GSH (Km values 90 and 110 �m). Like the enzymesfrom tomato fruits, AtGGT has a high affinity to GSH(Km value 255 �m), which strongly suggests that GSHcan be used in vivo by AtGGT as the main donorsubstrate.

If we assume that AtGGT can use GSH, the AtGGT-overproducing transgenic plants may be an interest-ing experimental system to study the role of �-GT inGSH metabolism. We did not find a significant dif-ference in either the steady-state levels of GSH or inthe GSH to GSSG ratio in transgenic plants whencompared with control plants under “normal” con-ditions. Moreover, the GSH degradation rate wasessentially the same in transgenic and control plants.

Although seemingly surprising, this fact makesdirect sense when the extracellular localization ofAtGGT is borne in mind. For example, under “nor-mal conditions” the apoplast of barley (Hordeum vul-gare) does not contain detectable amounts of GSH(Vanacker et al., 1999), which probably explains whythe increase in the �-GT activity brought about by theAtGGT overproduction did not lead to a higher GSHdegradation rate and why the endogenous �-GT ac-tivity was practically undetectable in the controlplants. However, the situation can dramaticallychange when plants are challenged by biotic or abi-otic stresses. In case of barley, the hypersensitiveresponse to the mildew pathogen results in the in-creased GSH synthesis rate with the amount of apo-plastic GSH rising to 2% of the total GSH content.Under such conditions, AtGGT potentially can play arole in GSH salvage and recycling as does the animalenzyme.

In conclusion, AtGGT is very similar to the mam-malian enzyme in its in vitro biochemical properties,molecular structure, and cellular localization. Its pos-sible in vivo role in GSH metabolism requires furtherinvestigation.





MATERIALS AND METHODS

Bacterial Strains

For the molecular cloning procedures, the Escherichia colistrain JM109 (Promega, Madison, WI) was used, and ex-pression of the D22–6x-His fusion was carried out in the E.coli BL21(DE3) strain (Promega). Agrobacterium tumefaciensstrain C58C1RifR (pGV2260; Deblaere et al., 1985) was usedfor the transformation of tobacco plants.

Transformation of Tobacco (Nicotiana tabacum)Plants and Analysis of the TransgeneIntegration and Expression

Tobacco SR1 (Maliga et al., 1975) was used for the gen-eration of stable transformants according to the procedureof leaf disc cocultivation (De Block et al., 1987). Potentialtransgenic lines were analyzed by DNA and RNA gel-blothybridizations. In both procedures, Hybond-N membranes(Amersham Pharmacia Biotech, Little Chalfont, UK) wereused, and hybridizations were done under stringent con-ditions according to the manufacturer’s specifications.Genomic DNA was isolated using DNeasy Kit (Qiagen,Hilden, Germany). Total RNA was isolated with the TRIzolreagent (Invitrogen, Gaithersburg, MD). Probes were la-beled with Redivue [32P]dCTP (3,000 Ci mmol�1) with theRediprime DNA labeling kit (Amersham PharmaciaBiotech).

Molecular Cloning Procedures

For the expression of D22 cDNA in transgenic plants, aplant expression cassette pDH51 (Pietrzak et al., 1986) wasused containing the 35S promoter and the 3�nos transcrip-tion terminator. The vector was digested with the restric-tion enzyme XbaI (Amersham Pharmacia Biotech). The re-sulting 5�-protruding ends were dephosphorylated withcalf intestinal phosphatase (Promega) followed by end-filling with the Klenow fragment (Amersham PharmaciaBiotech). D22 cDNA was cut out from the pFL61 yeast(Saccharomyces cerevisiae) vector with NotI (AmershamPharmacia Biotech) and ligated with the vector after theends were filled in with the Klenow fragment. The result-ing fusion between the promoter cDNA and the terminatorwas excised from the vector with EcoRI (Amersham Phar-macia Biotech) and subcloned into the plant SalI-linearized(Amersham Pharmacia Biotech) transformation vectorpGSC1704 (Aventis CropScience, Gent, Belgium). Beforethe ligation, the cohesive ends were converted to blunt asdescribed above. The resulting construct was used for planttransformation.

For the expression of the D22 cDNA in E. coli as a 6x-Hisfusion, the expression vector pET-19b (Novagen, Madison,WI) was used. The cDNA has been amplified with Pfupolymerase (Stratagene, La Jolla, CA) with a pair of specificprimers containing the NdeI site in frame with the 6x-Histag sequence of the expression vector. The amplified cDNAwas digested with NdeI (Amersham Pharmacia Biotech)

and ligated with the correspondingly digested and dephos-phorylated vector.

Antibody Production

AtGGT was purified from the E. coli-overproducingD22–6x-His fusion on His-affinity resin (Qiagen) underdenatured conditions according to the manufacturer’s in-structions. The produced protein was insoluble and ap-peared in the SDS gel as a single band of the expectedmolecular mass of approximately 60 kD. Because the pro-tein was insoluble, it was suspended in phosphate-bufferedsaline buffer to immunize rabbits according to a standardimmunization protocol (Harlow and Lane, 1988) of subcu-taneous antigen administration with boost injections after14, 28, and 84 d after the first antigen administration at theLaboratory of Hormonology (Marloie, Belgium).

Protein Extraction for the Analysis of �-GTActivity and Protein Gel-Blot Hybridization

Typically, 5 g of tobacco leaf tissue was homogenized inliquid nitrogen and resuspended in 25 mL of cold extrac-tion buffer, containing 50 mm Tris-HCl (pH 8.0), 50 mmNaCl, and one tablet of a protein inhibitor cocktail (RocheDiagnostics, Brussels). All procedures were carried out at4°C. At this point, an aliquot was taken and, after the celldebris had been precipitated by centrifugation at 20,000g,used to determine the activity in the soluble fraction. Toobtain a final NaCl concentration of 0.5 m, 5 m NaClsolution was added to the remainder of the homogenate.The homogenate was incubated for 30 min with periodicalsteering to allow solubilization of membrane-bound pro-teins. The homogenate was cleared by centrifugation at20,000g, and proteins were precipitated by addition of crys-talline ammonium sulfate to 70% saturation. Precipitatedproteins were collected by centrifugation at 20,000g anddiluted in 3 mL of homogenization buffer. The proteinextract was desalted on the Econo-Pac 10DG column (Bio-Rad, Hercules, CA) and used for the �-GT activity assay.As an alternative, for the protein gel blots, total proteinswere isolated by using the phenol extraction method toprevent protein degradation (Hurkman and Tanaka, 1986).Protein concentration in the samples was measured accord-ing to the method of Bradford (1976).

The protein extracts were separated by SDS-PAGE witha Mini-Protean II apparatus (Bio-Rad) and subsequentlyblotted onto nitrocellulose membranes (Hybond C super,Amersham Pharmacia Biotech) with a mini trans-blot elec-trophoretic transfer cell (Bio-Rad) according to the manu-facturer’s specifications. Antisera against AtGGT wereused at a dilution of 1:1,000,000 (v/v). For the primaryantibody detection, the secondary anti-rabbit peroxidase-linked goat antibody (Amersham Pharmacia Biotech) wasused in combination with the Renaissance ECL detectionkit (NEN Life Science Products, Boston).

�-GT Activity Assay

Standard �-GT activity assays were performed withGPNA (Sigma-Aldrich, St. Louis; Orlowski and Mister,

Storozhenko et al.




1963) as donor substrate and the dipeptide Gly-Gly (Sigma)as an acceptor substrate. The assay was carried out in96-well microtiter plates. The release of PNA was moni-tored with a SpectraMAX 250 microplate spectrophotome-ter system (Amersham Pharmacia Biotech) at 405 nm (� �8,800). A standard reaction was carried out typically in avolume of 300 �L and contained 0.1 m Tris-HCl (pH 7.5), 1mm GPNA, 40 mm Gly-Gly, and 200 �g of the proteinextract. One unit of enzyme is defined as the amount thatreleases 1 �mol PNA min�1 at 20°C. For the determinationof the enzyme activity in a pH range of 6.0 to 7.5,piperazine-N,N�-bis(ethanesulfonic acid) buffer was usedat 0.1 m. All amino acid acceptors were used at 40 mmconcentration.

To determine the kinetics parameters, a time course ofthe reaction at different concentrations of GPNA was re-corded. Km and Vmax were determined from a Lineweaver-Burk plot as described (Wilson and Goulding, 1986). Forthe Ki determination, the reaction was performed at differ-ent concentrations of GSH, and a Lineweaver-Burk plot foreach GSH concentration was constructed. Ki was deter-mined as a negative intercept of a secondary plot of theslopes of the primary plots against the GSH concentrationon the GSH concentration axis (Wilson and Goulding,1986).

Reaction of AtGGT with GSH was done in a total volumeof 50 �L with 0.1 mm Tris-HCl (pH 7.5), 1 mm GSH, 10 mmMet (Sigma-Aldrich), 10 �Ci of Redivue [35S]Met (Amer-sham Pharmacia Biotech), and 200 �g of a protein extract.As a positive control and marker for �-Glu-Met, a reactionwith bovine kidney �-GT (Sigma-Aldrich) was used. Beforethe reaction with GSH, the bovine enzyme was titrated in areaction with GPNA to have approximately the same ac-tivity as the protein extract from a transgenic plant. Thereaction was carried out at 20°C and was stopped after 30min by adding an equal volume of absolute ethanol. Afterincubation on ice for 10 min and removal of the proteinprecipitate, 2 �L of the reaction was analyzed by ascendingthin-layer chromatography on Polygram CEL 300 plates(Macherey-Nagel, Duren, Germany) in a pyrydine:butanol:water (1:1:1, v/v) buffer system. The reaction productswere visualized on a phosphor imager (model 445SI, Am-ersham Pharmacia Biotech).

For the AtGGT activity tests with intact protoplasts,tobacco mesophyll protoplasts were isolated from theleaves of transgenic and control plants according to a stan-dard protocol (Kushnir et al., 1991). Protoplasts (106 mL�1)were incubated in the standard reaction mix with GPNAsupplemented with 0.4 m sorbitol. After 1 h of incubation,protoplasts were floated by centrifugation at 100g, and theunderlying supernatant was used to determine the amountof PNA released.

Determination of the AtGGT Cellular Localization

For the confocal microscopy, leaf discs with a diameterof 8 mm cut from transgenic and control plants were vac-uum infiltrated with a solution containing 0.1 m Tris-HCl(pH 7.5), 1 mm �-glutamyl-7-amido-4-methylcoumarin, 40

mm Gly-Gly, briefly washed in water, and mounted inwater. The leaf discs were examined with a 20�/0.5 Plan-Neo fluor lens on a LSM510 laser scanning confocal micro-scope (Karl Zeiss Inc., Thornwood, NY). Images were ob-tained with the Enterprise UV laser (351 and 364 nm) withthe band-pass emission filter 385 to 470 nm. Optical sec-tions were taken and maximal brightness was projectedusing the LSM510 software package supplied with themicroscope. The images were converted to black and whitefor best contrast.

GSH Content Determination

The GSH content was analyzed according to the HPLCmethod described by Willekens et al. (1997).

Determination of the Rate of GSH Breakdown

To determine the GSH breakdown rate, 0.8-cm-di-ameter leaf discs from transgenic or control plantswere vacuum infiltrated with 1 mm l-buthionine-[S,R]-sulfoximine (Sigma-Aldrich) or water (negative control).At certain time points, four leaf discs from transgenic orcontrol plants were taken to determine the GSH content.

Computer Analysis

For computer analyses, software from the Genetics Com-puter Group (Madison, WI) was used. Multiple sequenceswere aligned with the PILEUP Program and the alignedsequences were edited with the SeqVu program (The Gar-van Institute of Medical Research, Sydney). Pair-wiseamino acid sequence similarities were calculated with theGAP program (Genetics Computer Group). For the homol-ogy searches, the BLAST program was used. The searcheswere done at http://www.bork.embl-heidelberg.de andhttp://Arabidopsis.org. Prediction of the cellular localiza-tion was made by using the PSORT program athttp://psort.nibb.ac.jp/form.html.

ACKNOWLEDGMENTS

We thank Gilbert Engler and Tom Beeckman for �-GTcellular localization determination, Srikrishnan Mani forthin-layer chromatography and fruitful discussions, Mar-tine De Cock for help in preparing the manuscript, andRebecca Verbanck for excellent artwork.

Received September 28, 2001; returned for revision October11, 2001; accepted December 6, 2001.

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