Molecular cloning and characterization of a maize transglutaminase complementary DNA

www.elsevier.com/locate/gene

Gene 336 (2004) 93–104

Molecular cloning and characterization of a maize

transglutaminase complementary DNA$

E. Villalobos, M. Santos, D. Talavera, M. Rodrıguez-Falcon, J.M. Torne*

Departament de Genetica Molecular, Institut de Biologıa Molecular de Barcelona, (CSIC), Jordi Girona 18-26, 08034 Barcelona, Spain

Received 11 December 2003; received in revised form 2 March 2004; accepted 18 March 2004

Available online 7 June 2004

Received
by B. Hohn
Abstract

Two related complementary DNA clones, TGZ15 and TGZ21, encoding active maize transglutaminase (TGase) have been isolated for the

first time in plants by molecular cloning (Patent Pending PCT/ES03/00247). Southern and northern blot analyses indicate that the two

cDNAs probably corresponded to two different single-copy genes in the maize genome. Northern blot analyses revealed that the transcript is

expressed preferentially in young leaves and differentiated embryogenic maize callus. This expression is dependent on light exposure time.

TGase activity of the proteins encoded by clones TGZ15 and TGZ21 was detected in bacterial extracts overexpressing them, using two

enzymatic assays. TGase activity was significantly higher than that of the empty-phagemid bacterial extracts. As in other TGases, this activity

was inhibited by monodansyl cadaverine (MDC), GTP and the absence of exogenous Ca+ +. Likewise, light-stimulated Ca+ +-dependent

TGase activity was detected in thylakoids and grana of maize chloroplast, which was inhibited by MDC, GTP, DIECA and Diuron.

D 2004 Elsevier B.V. All rights reserved.

Keywords: Plant; Chloroplast; Light dependence; Activity; Protein expression

1. Introduction

Transglutaminases (TGases, EC 2.3.2.13) are a family of

intracellular and extracellular enzymes that catalyse calci-

um-dependent posttranslational modification of proteins by

establishing q-(g-glutamyl) links and covalent conjugation

of polyamines into proteins. TGases are widely distributed

in bacteria, animals and plants.

These enzymes were detected for the first time in

animals, where they modify structural proteins (Folk,

1980). The primary structure of TGase was first established

in 1986 for coagulation Factor XIIIA. In 1988, the sequence

of guinea pig tissue TGase was determined and the homol-

ogy between XIIIA and tissue TGase was recognized for the

0378-1119/$ - see front matter D 2004 Elsevier B.V. All rights reserved.

doi:10.1016/j.gene.2004.03.025

Abbreviations: TGase, transglutaminase; TGZ, maize transglutaminase;

LHCPII, light-harvesting complex of PSII; PSII, photosystem II antenna

complex; PSI, photosystem I antenna complex; MDC, monodansyl

cadaverine; Diuron, (3-(3,4 dichlorophenyl)-1,1 dimethylurea); DIECA,

sodium diethyldithiocarbamic acid); DAO, diamino oxidase.B EMBL Data Library accession numbers: AJ421525 (TGZ15) and

AJ488103 (TGZ21).

* Corresponding author. Tel.: +34-93-4006123; fax: +34-93-2045904.

E-mail address: [email protected] (J.M. Torne).

first time (Ichinose et al., 1990). Animal transglutaminases

possess a catalytic triad composed by a cysteine, a histidine

and an aspartate, and the reaction proceeds via an interme-

diate linked to the cysteine (Hettasch and Greenberg, 1994).

In recent years, many studies of TGase have been

performed in humans, lower vertebrates, bacteria, algae

and yeast (Bergamini et al., 1999; Makarova et al.,

1999). Although some TGases from these organisms have

moderate to significant homology to the known mamma-

lian TGases, others have no structural or functional

similarities.

Of all the reactions that are catalysed by TGases, protein

cross-linking has probably attracted the greatest interest.

Nevertheless, the significance of TGasemediated posttransla-

tional modifications of proteins by deamidation and amine

incorporation is also well recognized. (Lorand and Graham,

2003).

Research on plant TGase is less developed than that in

mammalian systems. Furthermore, TGase-like activity in

plants was first observed in pea seedlings (Icekson and

Apelbaum, 1987) and in sprout apices of Helianthus

tuberosus (Serafini-Fracassini et al., 1988). TGases have

been found in several organs in lower and higher plants.

E. Villalobos et al. / Gene 336 (2004) 93–10494

Other characteristics of various plant TGases, such as Ca2+

dependence and substrate-specificity, were reviewed by

Serafini-Fracassini et al. (1995). Rubisco is a major sub-

strate of TGase in unopened Medicago sativa flower buds

(Margosiak et al., 1990). Del Duca et al. (1994) using

polyclonal antibodies, identified apoproteins of the antenna

complex [such as light-harvesting complex of PSII

(LHCPII), CP24, CP26 and CP29] as endogenous sub-

strates of TGase in chloroplasts of H. tuberosus leaves.

Furthermore, recent studies on H. tuberosus chloroplasts

also indicate that light affects TGase activity in chloroplast

and thylakoid extracts, (Dondini et al., 2003).

Our group, detected a unique 58 kDa band in maize

meristematic calli and their chloroplasts. This putative

TGase of maize calluses was light sensitive, affected by

hormone deprivation and showed a daily rhythm (Bernet,

1997; Bernet et al., 1999).

Subsequently, a polyclonal antibody obtained from a

purified chloroplast TGase, was used, in a comparative

study of the subcellular localization of this plant TGase in

various maize cell types (Villalobos et al., 2001). This study

showed that, in adult leaves, the enzyme was preferentially

present in the grana-appressed thylakoids of mesophyll

light-exposed cell chloroplasts and also dispersed in bun-

dle–sheath cell chloroplasts. The abundance depended on

the degree of grana development, and the enzyme activity

was light dependent. Likewise, the enzyme was not found in

other organelles such as mitochondria.

In light of these results, we attempted to clone TGase

DNA in maize, using the chloroplast TGase antibody

detailed earlier, and a maize-leaf cDNA library. TGase

activity was also examined in subcellular fractions of maize

chloroplasts.

The results implicate this enzyme in photosynthesis-

related processes, such us protection of photosystem anten-

na proteins, and a possible role in the regulation of the ratio

of stacked to unstacked thylakoids is discussed.

2. Materials and methods

2.1. Plant material

Biochemical characterization of maize (Zea mays)

TGase was performed using leaves of 10-day-old dark-

grown plants (germinated at 26–28 jC) or 20-day-old

greenhouse plants from the commercial maize hybrid G-

5054 (Novartis). For PCR, western, Southern and northern

blot analyses of the leaves and roots of 10-day-old seed-

lings of the inbred line B73 (from which the cDNA library

was obtained) were used.

2.2. cDNA library immunoscreening

The E-Zap IIR cDNA library was a generous gift from

Dr. A. Barkan, (University of Oregon, USA). The cDNA

was reverse transcribed from mRNA obtained from 2-

week-old seedlings of the maize cultivar B73. This library

was immunoscreened using a polyclonal antibody raised

against a purified 58 kDa chloroplast TGase of H. tuber-

osus leaves. This antibody inhibits TGase activity in a total

leaf protein extract (Dondini, 1999; Villalobos et al.,

2001). TGase specificity of the plant antibody was checked

previously by dot–blot analysis using commercial guinea

pig TGase (Sigma). Plates with top-agar were incubated at

42 jC, overlaid with a nitrocellulose filter saturated with

10 mM IPTG and placed at 37 jC. Filters were incubated

with a 1:1000 dilution of the plant TGase antibody and

with a peroxidase-conjugated donkey antichicken immu-

noglobulin (IgY) as secondary antibody (Jackson Immu-

noresearch Laboratories, West Grove, PA, USA). Clones

were detected using the electrochemiluminiscence western

blotting detection reagents (Amersham Pharmacia Bio-

tech). Positive phagemids were excised by the ExAssistkInterference-Resistant Helper Phage (Stratagenen) system.

The cDNA sequences were cloned into pBluesript SK�

using the EcoRI and XhoI restriction sites in frame with

the lacZ gene, such that the h-galactosidase-maize cDNA

fusion protein was expressed under the control of the lac

promoter. The plasmids obtained were analyzed by

EcoRI–XhoI digestion.

2.3. Oligonucleotides

To analyse the cDNA sequences, the following oligonu-

cleotides were synthesized :

P1: 5V-oligo1: 3V–5V:GATTCTCCCTGATAAGP2: 5V-oligo2: 3V–5V: GTTCTCCAGCATCTCCAGP3: 5V-CCCTGAGCACCCTGC-3VP4: 5V-GCAAGTTGAGCAGATGCGG-3VP5: 5V-GCAGCTGGTGCACAGGTGAC-3VP6: 5V-GCGCTGCATTTGCAGTGCCACTG-3VP7: 5V-CCCTGAGCACCCTGC-3V

2.4. 5V- end RACE amplification

Total RNA was extracted from maize leaves by the

phenol/SDS extraction method (Takahashi et al., 1992)

and mRNA was purified using the polyATtract mRNA

Isolation System (Promega). For 5V-end amplification, two

nested oligonucleotides (oligo1 and oligo2; Torne et al.,

2002) were synthesized, which were complementary to

a region located about 200 nt from the 5V-end of the

partial cDNA clone. For 5V-end amplification the 5V-RACE system from Life Technologies (GIBCO-BRLR)was used. First-strand cDNA was synthesized from 0.5 AgpolyA+ mRNA, using oligo1 as primer for the reverse

transcriptase; the cDNA was used as a template in a PCR

reaction using the polydC-specific Abridged Anchor Prim-

er (GIBCO-BRLR) and oligo2 as amplification primer.

The PCR product was subcloned in the pGEM-T easy

E. Villalobos et al. / Gene 336 (2004) 93–104 95

vector (Promega) and sequenced to confirm specific

amplification.

2.5. Sequence analysis

DNA was completely sequenced in the IBMB Sequenc-

ing Service, using an ABI Prism 377 DNA Sequencer

(Applied Biosystems, Foster City, CA, USA) The M13

forward and reverse primers, and the oligonucleotides P3

to P7 mentioned above were used. DNA sequencing data

were analysed with GCG software (Genetics Computer

Group, Madison, WI, USA).

2.6. Escherichia coli expression of the TGase proteins

E. coli cells infected with the isolated positive phagemids,

containing packaged TGase cDNA, were induced to over-

express the protein for 2 h at 37 jC, with 1 mM IPTG.

Proteins were extracted in the same buffer as used for plant

extracts, quantified by the Lowry et al. (1951) method and

used to measure the TGase activity or in western blot

analyses. Protein extracts from cells infected with an empty

phagemid were used as negative control. Furthermore, the E.

coli cells were transformed separately with the TGZ15 and

TGZ21 inserts cloned in a pBluescript SK� expression vector

and were induced to overexpress the protein for 2 and 4 h at

37 jC, and 0.5mM IPTG. The TGase activity was analysed in

pellets and supernatants obtained by culture lysis.

2.7. Electrophoresis and western immunoblotting

Western immunoblotting was performed according to the

method of Towbin et al. (1979). Protein was transferred

using a Trans Blot SD transfer system (Bio Rad) . Nitro-

cellulose filters containing the proteins were treated as

described for the cDNA library immunoscreening.

2.8. DNA extraction and Southern blot hybridisation

Genomic DNA was isolated from leaves of 20-day-old

B73 maize plantlets according to the method described by

Dellaporta et al. (1983). For Southern blot hybridisation,

genomic DNAwas digested with EcoRI, HindIII, BamHI or

XhoI and separated in a 0.8% (w/v) agarose gel. DNA was

transferred to a Hybond-N (Amersham) nylon membrane as

described by Maniatis et al. (1982) and hybridised with the32P-labeled complete TGase cDNA coding region. Hybrid-

isation and washing conditions were as described by Ama-

sino (1986).

2.9. Northern blot hybridisation

RNAwas prepared from various tissue samples from 10-

day-old plants and embryogenic callus cultures (Villalobos

et al., 2001) following the method of Chang et al. (1993).

Total RNA samples (20 Ag per lane) were separated on a

1.2% agarose/formaldehyde gel and transferred onto

Hybond-N (Amersham) nylon membranes. Blots were

hybridised with the 32P-labeled complete TGase cDNA

coding sequence. Hybridisation was carried out in Church

buffer at 65 jC overnight. To ensure the equal loading of

RNA in the lanes, gels were stained with ethidium bromide

for comparison of the band intensities.

2.10. Chloroplasts, thylakoids and grana isolation

Membranes of chloroplasts, thylakoids and grana were

isolated from leaves as indicated in Berthold et al. (1981),

using 50 mM HEPES instead of morpholinoethanesulfonic

acid (MES) in the fractions, to preserve the TGase activity.

The pH was adjusted to 8.0 in all the extraction methods.

These extracts were used to measure the TGase activity after

the treatments with light and inhibitory compounds.

2.11. Transglutaminase assays

Two types of enzymatic assay were used:

2.11.1. Measure of TGase activity by the radiolabeled

putrescine method

This assay was used with the protein extracts from the

bacteria and from the plant extracts, using labeled putrescine

(PU) as substrate. The light conditions during the assays for

plant extracts were the same as those during sample collec-

tion. In both cases, the pH of the incubation mixture was

adjusted to 8.0. The enzymatic mixture was as described

previously (Bernet et al., 1999). After 30 min of incubation

at 30 jC, the reaction was blocked by adding 300 Al 10%trichloracetic acid (TCA) containing 2 mM PU. Samples

were repeatedly precipitated and the radioactivity was

measured as described by Del Duca et al. (1994).

2.11.2. Measure of the TGase activity by the c-glutamylbiotin cadaverine method

This method is a TGase colorimetric microassay. Covalent

coupled CBZ-GlN-Gly is used as the first substrate, which

solves the problem of leaching out of absorbed substrate, and

biotin cadaverine is used as the second. In the presence of

TGase, biotinylated cadaverine is incorporated on the glu-

tamyl residue of the peptide to form g-glutamyl cadaverine

biotin. The method was developed by Covalab (TG-Covtest

TCMA, ref. 991EL-TG. Covalab, Faculte de Medecine Lyon

Sud, Oullins Cedex, France).

2.12. Diamine oxidase (DAO) activity assay

DAO activity was assayed by the radiochemical method

of Torrigiani et al. (1989), which measures the [C14] pyrro-

line formation from [C14] PU, using cold PU and catalase as

substrates. Aliquots (0.5 ml) of the supernatant or resus-

pended pellet of the leaf homogenate product were used for

the assay.


3. Results

3.1. Isolation of two cDNAs encoding maize chloroplast

transglutaminase

A total of 16 positive clones were isolated and sequenced

starting from several screenings. Finally, two related cDNA

clones (TGZ15 and TGZ21) encoding maize chloroplast

TGase were identified. Both cDNA clones were identical

with the exception of a repeat region near the putative

TGase catalytic domain (Fig. 1A).

A 1748 bp cDNA was obtained for TGZ15 clone com-

prising 15 repeat units in tandem, situated between nucleo-

tides 823 to 1228. The sequence included 126 nucleotides of

the 3V-noncoding region (nucleotides 1603 to 1729). Fur-

thermore, a 1910 bp cDNA was obtained for TGZ21 clone

comprising 21 repeat units in tandem, situated between

nucleotides 823 to 1389. The sequence included 126 nucleo-

tides of the 3V-noncoding region (nucleotides 1765 to 1891).To ensure that this tandem repeat structure was not a

Fig. 1. (A) Primary structure of the maize TGase sequences TGZ15 and TGZ21. (B

DNA obtained by PCR amplification using the oligonucleotides indicated in the

codon.

sequencing artefact, genomic DNA (from the same cultivar

as the cDNA library) was analysed by PCR using primers

situated 5V–3V from this repeat region. The same repeat

structures and sequences were obtained in these PCR reac-

tions, confirming the cDNA sequencing results.

Two 5V noncoding regions (Fig. 1B), including the

first nucleotides of the two 5V cDNA coding regions,

were also obtained by 5V-RACE amplification reverse

transcriptase–PCR. However, due to the sequence simi-

larity between TGZ15 and TGZ21, their respective 5V-noncoding regions could not be assigned to either the

partial cDNA clones.

3.2. Protein sequence analysis of TGZ15 and TGZ21:

chloroplast targeting and catalytic domain homology with

other nonplant sequenced TGases

The deduced TGZ15 protein consists of 534 amino acid

residues with a calculated molecular mass of 60.9 kDa. The

deduced TGZ21 protein has 588 residues and a calculated

) Nucleotide sequence of 5V-end noncoding sequences from maize genomic

text. Small letters: deduced nucleotides of the 5V-coding regions. ***: start


molecular mass of 67 kDa. Outside the repeat region, the

identity between the two proteins is 100% (see Fig. 2).

The two deduced maize TGase protein sequences pos-

sessed a predicted chloroplast import peptide comprised by

the first 47 amino acids (ChloroP, CBS, Denmark). In this

region, a protein kinase C and a casein kinase II phosphor-

ylation sites were also predicted; three myristoylation sites

(only two are presented in Fig. 2) were also observed in this

region. In the deduced protein region C-terminal to the

repeats, a deduced N-glycosylation site, six casein kinase

II, two protein kinase C phosphorylation sites, and two N-

myristoylation sites were observed. The N-glycosylation site

is situated in a glutamic acid-rich region. Furthermore, a

conserved ATP/GTP binding domain appears in the 418 to

425 tandem repeat region. In the repeat region, two more N-

myristoylation sites were also observed using the databases

of NPS@, IBPC, PBIL Lyon, France. The putative catalytic

domain, including the conserved catalytic triad (Cys, His

and Asp), is situated 31 amino acids downstream of the

repeat region.

Hydropathy analysis of maize TGases, revealed that the

deduced active site of these enzymes is located at the

transition region between a hydrophobic region comprised

by the repeat domain and a hydrophilic area that corre-

Fig. 2. Deduced amino acid sequence of the cloned maize TGZ15 (A) and TGZ2

overlap are not shown.

sponds to the C-terminal domain. Due to the similarity of

both sequences, only TGZ15 is shown in Fig. 3.

3.3. The TGase genes in the maize genome

Southern blot analysis performed using the TGZ15

cDNA as probe, under stringent hybridisation conditions,

against B73 maize genomic DNA, revealed two hybrid-

isation bands when digested with XhoI, and a thick high-

weight band when digested with BamH1 and EcoRI. Di-

gestion with HindIII revealed four hybridisation bands as

expected (Fig. 4).

Northern blot analyses indicated that this transcript is

expressed in roots (total extract), young leaf and differenti-

ating embryogenic maize callus (Fig. 5A). The expression

was higher in light, with a maximum at 2 h (Fig. 5B).

Moreover, two different bands in the RNA blots were

obtained in all cases.

3.4. Sequence homology of the maize TGases in rice and

maize databases

A FASTA search in the NCBI USA databases revealed

identity of up to 33% with other plant proteins. However, in

1 (B) cDNAs showing the different deduced domains. Note: domains that

Fig. 3. Hydropathy profile of TGZ15 protein-deduced sequence. Cysteine residue is indicated.


a rice (Oryza sativa) database (Gramene Databases Blast

Search), a high identity (87%) with the DNA sequence

AL606595 was obtained including the region with the

putative catalytic domain (see Fig. 6).

With respect to the maize databases (ZmDB, USDA),

94% identity of 613 bp was obtained between our maize

cDNAs and a partial cDNA sequence (EST: A1881274;

from maize immature ear, sequence n. 5555323). This

homology included complete identity with one of our maize

5V noncoding sequence (see Fig. 1B, A). This 5V RACEsequence is 34 bp longer than the 5V- noncoding sequence of

this EST. Moreover, other EST sequence homologies in the

3V-end region were obtained from the same maize data-

bases, i.e., sequence n. 24763828 from maize endosperm

(96% identity of 363 bp), sequence n. 5456439 from maize

ear tissue (89% identity of 383 bp), etc.

Fig. 4. Southern blot analysis of maize genomic DNA digested with XhoI

(X), HindIII (H), BamHI (B) and EcoRI (E). Only HindIII cuts the TGase

cDNA. The membrane was hybridised to the 32P-labeled entire TGZ15

cDNA clone used as probe (see Section 2).

3.5. Relation of the two maize TGases with the TGase gene

family

Based on the Swiss-Prot Release 42.10 database, a

dendrogram was plotted to compare the maize TGases to

the TGase sequences published (Fig. 7). The dendrogram

also included the N-glycanase sequences from O. sativa and

Arabidopsis thaliana, which have been mentioned in other

databases (SMART-EMBL, Heidelberg) as the only putative

plant transglutaminases. It was built from the Clustal W

Service (European Bioinformatics Institute; Higgins et al.,

1994), using the Blosum 30 matrix system (Henikoff and

Henikoff, 1992). As can be seen, the maize TGases (TGZ15

and TGZ21) form part of a subcluster where the other

putative plant TGases and two Streptomyces TGases are

included. Furthermore, in the nearest subcluster, Factor XIII

B chain precursor from human and mouse and four Bacillus

TGases are included.

3.6. Biochemical characterization and TGase activity of

proteins encoded by clones and in plant extracts

TGase activity of the proteins encoded by clones TGZ15

and TGZ21 was detected in bacterial extracts expressing the

proteins (Fig. 8). Likewise, these activities were measured in

different subcellular fractions of maize leaves (Fig. 9).

3.6.1. Both maize cDNA clones encode active TGases

3.6.1.1. Radiolabeled putrescine method. The lysis

extracts from E. coli cells transfected with phagemids

containing inserts of the two maize cDNAs encode active

TGases that incorporate labeled putrescine to proteins (Fig.

8A). Moreover, as shown for other TGases, in both cases,

this activity was also inhibited in the absence of calcium and

in the presence of GTP, monodansyl cadaverine (MDC) and

Fig. 5. Northern blot hybridisation of maize RNA. (A) Different maize tissues. 1: roots of 10-day-old light-growth plantlets; 2: leaves of 10-day-old light-

growth plantlets; 3: leaf of a mature plant; 4: light-grown callus; 5: dark-grown callus; 6: light-differentiating callus. (B) Leaves of 10-day-old plantlets grown

in different light conditions. Down: optical density diagram of the original membrane presented in (B). The membrane was hybridised to the 32P-labeled entire

TGZ15 cDNA clone used as probe (see Section 2). (C) Western blot analyses of protein extracts from leaves of plantlets cultured in the same conditions as in

(B).


EGTA (Fig. 8A). In contrast, TGase activity was the lowest

in the lysis extracts of E. coli cells transfected with a

phagemid that did not contain the cDNA insert.

Furthermore, the supernatant fractions of overexpressed

protein originating from TGZ15- and TGZ21-transformed

bacteria showed significantly higher activity than the pellet

fraction, the noninduced transformed bacteria and the in-

duced bacteria transformed with the empty expression

vector (Fig. 8C).

3.6.1.2. Biotin cadaverine method (TG-Covtest

TCMA). The same protein lysis extracts, assayed at three

concentrations, demonstrated TGase activity by incorpora-

tion of biotinylated cadaverine on the glutaminyl residue

Fig. 6. Clustal W homology between the putative maize TGase domain (TGZ) and

*—identity; :—strongly similar; .—weakly similar.

of the peptide to form g-glutamyl biotin cadaverine (Fig.

8B). In contrast, the presence of TGase in the bacteria

infected with an empty phagemid was scarce. The final

product of TGase activity (g-glutamyl biotin cadaverine)

was formed. Likewise, TGase activity of the protein lysis

extracts was inhibited in the EDTA-negative control

extracts (data not shown).

3.6.2. TGase activity in plant extracts: isolated maize

chloroplasts, thylakoids and grana

EGTA inhibited the TGase activity of chloroplast extracts

assayed without exogenous calcium (Fig. 9A). The addition

of exogenous calcium to the enzymatic assay seems to restore

activity with a bimodal curve, with a decrease at 1 mM, but an

the proposed for the AL606595 rice sequence. The catalytic triad is framed;

Fig. 7. Phylogenetic relationship between the maize TGase sequences and other sequenced TGases (see Results), using the Clustal W program (European

Bioinformatics Institute) on the basis of the aligment of protein sequences.


activation at higher concentrations, indicating that perhaps

two different calcium-dependent processes, might be respon-

sible for TGase activity regulation. TGase activity was

significantly higher when the enzymatic assay was performed

Fig. 8. TGase activity in TGZ15 and TGZ21 transfected (A and B) or transforme

incorporated h� 1 mg protein� 1) of protein extracts of E. coli cells transfected se

(TGZ15 and TGZ21) and incubated in the presence of 600 AM unlabeled PU and

absence of 0.6 mM of CaCl2. 1: TGZ15+Ca2 +; 2: TGZ21+Ca2 +; 3: TGZ15�Ca

mM EGTA; 7: protein extracts of E. coli cells infected with an empty phagemid. T

similar and it is not presented. DataF S.E. are the mean of three replicates. (B) TG

protein extracts from E. coli cells transfected separately with positive phages c

infection; f3: infection with an empty phagemid. a: 100 Ag total protein. b: 150 AgTGase activity (measured in pmol Put incorporated h� 1 mg prot� 1) of protein

overexpressing TGZ15. The TGase activity of TGZ21 overexpression gave simila

IPTG induction of extracts transformed with the empty vector; 2: 2 h 1 mM IPTG

TGZ15 transformed extracts; S: supernatant; P: pellet.

in the light (both in thylakoids and in grana extracts; Fig. 9B).

Similar results were obtained with thylakoids prepared from

dark-grown plants, when the assay was performed in the light

conditions (data not shown).

d (C) bacterial lysis extracts. (A) TGase activity (expressed in pmol of PU

parately with the positive phagemids containing the maize TGase cDNAs

110 Bq [H3-Put] (1.48 TBq/mol). Activity was measured in the presence or2 +; 4*: TGZ15+Ca2 + + 1 mM GTP; 5: TGZ15+ 1 mM MDC; 6: TGZ15+ 5

he activity of the TGZ21 phagemid infection in presence of inhibitors was

ase activity (expressed in mU of enzyme incorporating biotincadaverine) of

ontaining the two maize TGase cDNAs. f1: TGZ15 infection; f2: TGZ21

total protein. c: 200 Ag total protein. Data are the mean of two replicates. (C)

extracts from E. coli cells transformed with plasmids (pBluescript SK�)

r results and it is not presented. Control: noninduced extracts; V: 2 h 1 mM

induction of TGZ15 transformed extracts; 4: 4 h 1 mM IPTG induction of

400

a

b

c

Fig. 9. TGase activity in membrane–chloroplast maize extracts. (A) Effect

of EGTA and Ca2+ addition on TGase activity (percentage of three separate

experiments) of isolated chloroplasts from greenhouse-grown plants. (B)

Effect of light on TGase activity (expressed in pmol of Put incorporated h�1

mg protein�1) of thylakoids (denoted by a) and grana (denoted by b) leaf

extracts. White bars: light enzymatic assay. Black bars: dark enzymatic

assay. GH: greenhouse-grown plants. DataFS.E. are the mean of three

replicates. (C) Percentage of TGase activity inhibition (with respect to

nontreated controls of three separate experiments) induced by different

reagents on chloroplast, thylakoid and grana leaf extracts of greenhouse-

grown plants. 1: Control; 2: 1 mM DIECA; 3: 1 mM DTT; 4: 5 mM DTT;

5: 1 mM GTP; 6: 1 mM nonhydrolizable GTP analog; 7: 6.5 AM Diuron; 8:

13 AM Diuron.

Fig. 10. Western blot analyses. The antibody cross� reaction was

performed using the plant anti-TGase antibody cited in Section 2 as

primary antibody in a 1:10000 dilution. (A) Protein extracts of E. coli cells

infected separately with the cDNA-containing phages. C: E. coli cells

transfected with an empty plasmid; 1: TGZ21 transfected cells; 2: TGZ15

transfected cells. (B) Proteins from different chloroplast fractions of maize

plants cultured under different illumination conditions. I: Greenhouse-

grown plants; II: greenhouse-grown plants cultured for 3 days in dark

conditions; III: dark-grown plants; IV: dark-grown plants grown for 3 days

in greenhouse conditions; V: dark-grown plant extract incubated during 30

min under a white-light lamp in the same conditions as the enzymatic assay.

T: thylakoid protein extract. G: grana protein extract.


Furthermore, we assayed activity inhibition by different

blocking compounds on maize chloroplast, thylakoids and

grana extracts (Fig. 9C). Although the percentage of inhi-

bition was variable, each blocking compound, (DIECA,

DTT, GTP and Diuron) caused inhibition in the enzymatic

assay. In the case of DIECA, inhibition was not due to DAO

activity (data not shown).

3.7. Western blot analysis

To compare the molecular weight of the proteins

expressed in vitro and those detected in plant extracts, we

performed a western blot using the anti-TGase chloroplast

antibody. This antibody recognized bands of 61 and 67 kDa

in the protein lysis extract of bacteria infected with the

cDNA-containing phagemids TGZ15 or TGZ21, respective-

ly. The presence of another band with lower molecular

weight was attributed to protein degradation or postransla-

tional modification . In the protein lysis extracts of bacterial

cells transfected with an empty phagemid, no bands were

recognized by the antibody (Fig. 10A).

Furthermore, leaf protein extracts from young plantlets of

the same age and light treatments as in the northern experi-

ments were analysed (Fig. 5C). In dark-grown plantlets, three

bands of 150, 77 and 58 kDa, respectively, were obtained.

From these bands, the 58 kDa band showed a strong signal.


Nevertheless, after 2 h of illumination and at the same protein

concentration, only 77 and 58 kDa slight bands were

obtained. The same bands appeared after 4, 8 and 48 h of

light treatment, although the concentration of the 58 kDa

band was increased with respect to that of 2 h illumination

(Fig. 5C).

In the case of thylakoids and grana extracts, bands of

different molecular weight were obtained depending on the

light conditions and on the type of extract, reinforcing the

results obtained in the activity assays. While a single 58 kDa

band was obtained in the grana extracts, a 150 and a 77 kDa

band were also obtained in the case of thylakoid extracts,

depending on the light treatment (Fig. 10B).

4. Discussion

4.1. TGase gene and sequence characteristics

Southern and northern blot analyses of the two sequences

TGZ15 and TGZ21, indicate that the two cloned cDNAs

probably correspond to two single-copy TGase genes in the

maize genome. The two 5V-end noncoding regions obtained

reinforce this hypothesis, although, to confirm it, more

experiments on genomic maize DNA must be done.

The level of similarity observed between maize protein

and nonplant TGases may indicate convergent evolution.

Maize protein appears to form a distinct family of proteins

with TGase activity (Lorand and Graham, 2003).

Furthermore, the observed proximity between maize

TGases and the A. thaliana and O. sativa N-glycanase

sequences, which are the only plant putative TGases

referenced, suggests the appearance of a new group in

which some microbial TGases are included. The similarity

found between our deduced protein sequences of maize

TGases with the human Factor XIII (B chain precursor) is

localized in the tandem repeats region. These repeats seem

to stabilise the Factor XIII A subunits and regulate the rate

of transglutaminase formation activated by thrombin (Ichi-

nose et al., 1990, 1996). The presence of a repeat region in

the maize TGases and many myristoylation sites might be

related to a similar function of this protein in stabilization of

photosystems I and or II, located in the thylakoids.

Likewise, when a rice DNA database is consulted, a

hypothetic protein that shares a highly significant percentage

of identity with the deduced maize protein is revealed. This

homology includes the putative maize TGase domain. With

respect to maize database, the high identity (94%) observed

between our maize cDNAs and a published partial cDNA

maize sequence confirms our results on the full-length cDNA

TGase sequencing. This is also reinforced by the high

homology obtained with other 5V–3V ESTs in the maize

database. In conclusion, all these data confirmed the results

obtained from the maize TGase cDNAs sequences.

With respect to the putative catalytic domain, the pres-

ence of the conserved catalytic triade (Cys, His and Asp), as

described in many other TGases (Lorand and Graham,

2003), is a new evidence that confirms the identification

of these proteins as authentic TGases. Likewise, the cysteine

location in a transition region between a hydrophobic and a

hydrophilic area is consistent with previous reports on liver

tissue TGase (Ikura et al., 1988; Ichinose et al., 1990) and

on A subunit of Factor XIII (Takahashi et al., 1986).

4.2. The TGase protein and its possible function

Considering the published results about different factors

that regulate TGase activity in other plant and nonplant

species, our assays demonstrate that maize TGase exhibit a

similar pattern of regulation.

With respect to calcium dependence, glutamic acid-rich

regions, which are potentially involved in Ca2 + binding,

were identified in nonplant TGases: Factor XIIIb A subunit

(Takahashi et al., 1986), guinea pig liver TGase (Ikura et al.,

1988), rat prostate TGase (Ho et al., 1991), etc. The deduced

maize TGase proteins were found to exhibit a putative

Ca2 +-binding site. This is reinforced by the observed Ca

dependence of TGase activity in the cDNA-transfected

bacterial lysis product and in the chloroplast maize leaf

protein extracts. Ca dependence has also been observed in

extracts of other monocotyledoneae and dicotyledoneae

plants (Lilley et al., 1998).

Some TGases may have two distinct catalytic functions,

one related to their cross-linking capacity (calcium-depen-

dent) and the other related to GTP and ATP hydrolysis

(calcium-independent; Lorand and Graham, 2003). GTP

hydrolysis was detected in purified guinea pig TGase

(Nakaoka et al., 1994; Zhang et al., 1998). A conserved

ATP/GTP binding domain was localized in the deduced

maize TGase protein sequences. The enzymatic activity

decrease observed in our protein extracts after GTP addi-

tion, reinforces these functional similarities with some non-

plant TGases.

The dose-dependent inhibitory effect of the sulfhydryl

modifying agent DTT, observed on maize TGase activity

of chloroplast, thylakoids and grana, has been reported by

other authors in chloroplasts of other plant species (Ice-

kson and Apelbaum, 1987; Del Duca et al., 2000). This

effect has been explained as a possible block of the

enzyme activity by a disulfide exchange reaction involving

the cysteinyl thiol group of the TGase active site (Lorand

and Conrad, 1984). The putative active site deduced in the

maize TGase sequences (including cysteine) supports the

involvement of SH groups in the TGase-mediated covalent

modification of the chloroplast, thylakoids and grana

proteins.

With respect to the possible functionality of the maize

TGase, certain aspects have been reinforced in the present

study. We previously reported that maize TGase is specif-

ically associated with the membrane of the thylakoid-

appressed grana in chloroplasts of adult plants (Villalobos

et al., 2001), so it might be implicated in the photosyn-


thesis related processes. A putative chloroplast signal

peptide, including the protein kinase phosphorylation sites,

has been identified in the maize TGase sequences as a

‘‘binding-protein-dependent transport system inner mem-

brane complementary signal’’ and described by many

authors (Waegemann and Soll, 1996; Peltier et al., 2000).

Although a predicted mitochondrial targeting peptide is

also obtained from the TargetP database, this possibility is

not supported by the immunolocalization results reported

previously nor by the molecular and biochemical data

presented here.

The predicted molecular weight (60.9 kDa) of the

protein corresponding to the TGZ15 cDNA clone is in

agreement with that of the protein recognized by the

antibody raised against plant TGase in transfected bacterial

lysis extracts and probably corresponds to the 58 kDa band

detected by the antibody in maize grana extracts. Likewise,

the presence of the 58 kDa band in dark-grown plant

extracts might also indicate that this polypeptide band

corresponds to a storage form of the protein, which needs

to be further modified for higher TGase activity. Further-

more, the immunorecognition of a 150 kDa band indicates

that the 73–77 kDa band might derive from a high

molecular weight agglomerate which is disrupted by light

conditions (in the case of 10-day-old plants) or by other

processes (in the case of oldest plants) in which active

TGase is required.

The RNA expression and the significant increase ob-

served in plantlets after 2 h of light exposition also reinforce

the western blot data. The significant decrease in the signal

of the 58 kDa protein bands after 2 h of illumination may

indicate that, when light is switched on, TGase is rapidly

utilized and a new synthesis of TGase–mRNA is needed for

enzymatic activity. Moreover, these findings are consistent

with the knowledge that the thylakoid membrane system

starts to be formed after 4–6 h of etioplast illumination.

Here, we demonstrate that TGase activity increases signif-

icantly in maize thylakoids and grana extracts when light is

present in the enzymatic assay, both in light-grown and in

dark-grown plants (Talavera, 2001).

If a TGase function related with the chloroplast devel-

opment is assumed, these results would indicate that the

enzyme is required shortly after light exposure, when

thylakoids and grana start to be formed. Later, when the

protein present in the system is sufficient to support TGase

activity, a gradual decrease in RNA accumulation would be

detected. The low expression observed in mature leaves and

in plants receiving continuous light, would agree with this

hypothesis. This is also in agreement with our previous

work on immunolocalization (Villalobos et al., 2001).

Recent studies of other groups with H. tuberosus chloro-

plasts also indicate that light affects TGase activity in

chloroplast and thylakoid extracts. The authors hypothesise

that light may cause conformational changes on the protein

substrates and on the enzyme itself, although the mechanism

is still unknown (Dondini et al., 2003).

The significant reduction of TGase activity on purified

grana maize proteins treated with an inhibitor of the PSII

activity (Diuron; Talavera, 2001) reinforces the hypothesis

that TGase not only uses proteins of the PSII antenna

complex as substrates but they must also be active. Like-

wise, the inhibition by DIECA (a chelating agent of copper

and another metals) might indicate inhibition of plastocia-

nine or other reducing agents in the photosystem antenna

complex, which proteins are substrates of the TGase activ-

ity, as indicated by other authors working with H. tuberosus

chloroplasts (Del Duca et al., 1994; Dondini et al., 2003).

These observations may reinforce the hypothesis of

TGase implication in the light-dependent thylakoid–mem-

brane system formation. The ratio of stacked to unstacked

thylakoids might be regulated by TGase via polyamine

conjugation to photosystem antenna proteins. Such regula-

tion should contribute to the efficient distribution of light

energy between PSI and PSII complexes. Further investiga-

tions at molecular and cellular levels are in progress to

clarify new aspects of this functionality in the related

photosynthetic processes.

Acknowledgements

We thank Dr. A. Barkan (USA) for her generous gift of

the maize expression library. We particularly thank Dr. Joan

Rigau for his support and Dr. Salome Prat for her assistance

and for critical reading of the manuscript. We thank Dr.

Picorel’s group for fruitful discussions. We thank Merce

Miquel (IBMB. Sequencing Service) for the rigorous

sequencing of the cDNA clones, Angel Sanchez (IBMB.

Imaging Service) for the processing of the figures and Robin

Rycroft for correcting the English text.

This study was supported by a Spanish grant DGICYT

PB-97 1138 and forms part of the PhD thesis of Enrique

Villalobos (AECI grant) and includes postgraduate master

work by David Talavera.

References

Amasino, A., 1986. Acceleration of nucleic acid hybridization rate by

polyethyleneglycol. Anal. Biochem. 152, 304–307.

Bergamini, C.M., Dean, M., Tanfani, F., Ferrari, C., Scatturi, N., 1999.

Conformational stability of human erythrocyte transglutaminase: pat-

terns of thermal unfolding at acid and alkaline pH. Eur. J. Biochem.

266, 575–582.

Bernet, E., 1997. Studies on putrescine metabolism and related enzymes

during the differentiation of Zea mays meristematic callus. PhD thesis.

University of Barcelona, Spain.

Bernet, E., Claparols, I., Dondini, L., Santos, M.A., Serafini-Fracassini, D.,

Torne, J.M., 1999. Changes in polyamine content, arginine and orni-

thine decarboxylases and transglutaminase activities during light/dark

phases (of initial differentiation) in maize calluses and their chloro-

plasts. Plant Physiol. Biochem. 37, 899–909.

Berthold, D.A., Babcock, G.T., Yokum, C.F., 1981. A highly resolved


oxygen evolving PSII preparation from spinach thylakoid membranes.

FEBS Lett. 134, 231–234.

Chang, S., Poryear, J., Cairney, J., 1993. A simple and efficient method for

isolation RNA from pine tree. Plant Mol. Biol. Rep. 11, 113–116.

Del Duca, S., Tidu, V., Bassi, R., Esposito, C., Serafini-Fracassini, D.,

1994. Identification of chlorophyll-a/b proteins as substrates of trans-

glutaminase activity in isolated chloroplasts of Helianthus tuberosus L..

Planta 193, 283–289.

Del Duca, S., Dondini, L., Della Mea, M., Munoz de Rueda, P., Serafini-

Fracassini, D., 2000. Factors affecting transglutaminase activity catalys-

ing polyamine conjugation to endogenous substrates in the entire chlo-

roplast. Plant Physiol. Biochem. 38, 429–439.

Dellaporta, S.L., Wood, J., Hicks, J.B., 1983. A plant DNA miniprepara-

tion, version II. Plant Mol. Biol. Rep. 1, 19–21.

Dondini, L., 1999. Poliammine legate e transglutaminasi nelle plante. PhD

thesis. University of Bologna, Bologna, Italy.

Dondini, L., Del Duca, S., Dall’Agata, L., Bassi, R., Gastaldelli, M., Della

Mea, M., Di Sandro, A., Claparols, I., Serafıni-Fracassini, D., 2003.

Suborganellar localisation and effect of light on Helianthus tuberosus

chloroplast transglutaminases and their subtrates. Planta 17, 84–95.

Folk, J.E., 1980. Transglutaminases. Annu. Rev. Biochem 49, 517–531.

Henikoff, S., Henikoff, J., 1992. Amino acid substitution matrices from

protein blocks. Proc. Natl. Acad. Sci. U. S. A. 89, 10909–10915

(biochemistry).

Hettasch, J.M., Greenberg, C.S., 1994. Analysis of the catalytic activity of

human Factor XIIIa by site-directed mutagenesis. J. Biol. Chem. 269,

28309–28313.

Higgins, D., Thompson, J., Gibson, T., Thompson, J.D., Higgins, D.G.,

Gibson, T.J., 1994. Clustal W: improving the sensitivity of progressive

multiple sequence alignment through sequence weighting, position-spe-

cific gap penalties and weight matrix choice. Nucleic Acids Res. 22,

4673–4680.

Ho, K.Ch., Quarmby, V.E., French, F.S., Wilson, E.M., 1991. Molecular

cloning of rat prostate transglutaminase complementary DNA. Am. Soc.

Biochem. Mol., 12660–12667.

Icekson, I., Apelbaum, A., 1987. Evidence for transglutaminase activity in

plant tissue. Plant Physiol. 84, 972–974.

Ichinose, A., Bottenus, R.E., Davis, E., 1990. Structure of transglutami-

nases. J. Am. Chem. 265, 13411–13414.

Ichinose, A., Izumi, T., Hashiguchi, T., 1996. The normal and abnormal

genes of the a and b subunits in coagulation Factor XIII. Semin.

Thromb. Hemost. 22, 385–391.

Ikura, K., Nasu, T., Yokota, H., Tsuchiya, Y., Sasaki, R., Chiva, H., 1988.

Amino acid sequence of guinea pig liver transglutaminase from its

cDNA sequence. Biochemistry 27, 2898–2905.

Lilley, G.R., Skill, J., Griffin, M., Bonner, P.L., 1998. Detection of Ca2 +-

dependent transglutaminase activity in root and leaf tissue on monoco-

tyledoneus and dicotyledoneus plants. Plant Physiol. 117, 1115–1123.

Lorand, L., Conrad, S.M., 1984. Trans Transglutaminases. Mol. Cell. Bio-

chem. 58, 9–35.

Lorand, L., Graham, M.G., 2003. Transglutaminases: crosslinking enzymes

with pleiotropic functions. Nat. Rev. 4, 140–157.

Lowry, O., Rosebrough, N.J., Farr, A.L., Randall, R.J., 1951. Protein mea-

surement with the Folin phenol reagent. J. Biol. Chem. 193, 265–275.

Makarova, K.S., Aravind, L., Koovin, E.V., 1999. A superfamily of archae-

al, bacterial, and eukaryotic proteins homologous to animal transgluta-

minases. Prot. Sci. 8, 1714–1719.

Maniatis, T., Fristch, E.F., Sambrook, J., 1982. Molecular Cloning. A

Laboratory Manual. Cold Spring Harbor Laboratory, Cold Spring Har-

bor, NY, pp. 197–198.

Margosiak, S.A., Dharma, A., Bruce-Carver, M.R., Gonzales, A.P., Louie,

D., Kuehn, G.D., 1990. Identification of the large subunit of ribulose

1,5-bisphosphate carboxylase/oxygenase as a substrate for transgluta-

minase in Medicago sativa L. (alfalfa). Plant Physiol. 92, 88–96.

Nakaoka, H., Perez, D.M., Baek, K.J., Das, T., Husain, A., Misono, K.,

Im, M., Graham, R.M., 1994. Gh: a GTP binding protein with trans-

glutaminase activity and receptor signaling function. Science 264,

1593–1596.

Peltier, J.B., Friso, G., Kalume, D.E., Roepstorff, P., Nilsson, F., Adamaka,

I., van Wijk, K.J., 2000. Proteomics of the chloroplast: systematic iden-

tification ad targeting analysis of lumenal and peripheral thylakoid

proteins. Plant Cell 12, 319–341.

Serafini-Fracassini, D., Del Duca, S., D’Orazi, D., 1988. First evidence for

polyamine conjugation mediated by an enzymatic activity in plants.

Plant Physiol. 87, 757–761.

Serafini-Fracassini, D., Del Duca, S., Beninati, S., 1995. Plant transgluta-

minases. Phytochemistry 40, 355–365.

Takahashi, N., Takahashi, Y., Putnam, F.W., 1986. Primary structure of

blood coagulation Factor XIIIa (fibrinoligase, transglutaminase) from

human placenta. Proc. Natl. Acad. Sci. U. S. A. 83, 8019–8023.

Takahashi, T., Naito, S., Komeda, Y., 1992. Isolation and analysis of the

expression of two genes for the 81 kilodalton heat shock protein from

Arabidopsis. Plant Physiol. 99, 383–390.

Talavera, D., 2001. Maize transglutaminase biochemical characterization

and western blot analysis in different leaf cellular fractions. Postgradu-

ate Master in Biology. University of Barcelona, Spain.

Torne, J.M., Santos, M.A., Talavera, D., Villalobos, E., Rigau, J., 2002.

Sequence of maize nucleotides codifying a protein with transglutami-

nase activity and their use. OEPM, Patent Pending no. 200201253;

PCT/ES03/00247.

Torrigiani, P., Serafini-Fracassini, D., Fara, A., 1989. Diamine oxidase

activity in different physiological stages of Helianthus tuberosus tuber.

Plant Physiol. 89, 69–73.

Towbin, H., Stachelin, T., Gordon, J., 1979. Electrophoretic transfer of

proteins from polyacrylamide gels to nitrocellulose sheets: procedure

and some applications. Proc. Natl. Acad. Sci. U. S. A. 76, 4350–4354.

Villalobos, E., Torne, J.M., Rigau, J., Olles, I., Claparols, I., Santos, M.,

2001. Immunogold localization of a transglutaminase related to grana

development in different maize cell types. Protoplasma 216, 155–163.

Waegemann, K., Soll, J., 1996. Phosphorylation of the transit sequence of

chloroplast precursor proteins. J. Biol. Chem. 271, 6545–6554.

Zhang, J., Lesort, M., Guttmann, R.P., Johnson, V.W., 1998. Modulation of

in situ activity of tissue transglutaminase by calcium and GTP. J. Biol.

Chem. 273, 2288–2295.

Molecular cloning and characterization of a maize transglutaminase complementary DNA

Documents

Transcript of Molecular cloning and characterization of a maize transglutaminase complementary DNA