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First published online August 6, 2004; 10.1104/pp.104.042549 Plant Physiology 135:2046-2054 (2004) © 2004 American Society of Plant Biologists AtPng1p. The First Plant Transglutaminase1,[w]Dipartimento di Biologia Evoluzionistica Sperimentale, Università di Bologna, 40126 Bologna, Italy (M.D.M., D.S.-F.); and Consorci CSIC-IRTA Laboratori de Genètica Molecular Vegetal, Jordi Girona 18-26, 08034 Barcelona, Spain (D.C.-R., I.C., J.R.)
Studies have revealed in plant chloroplasts, mitochondria, cell walls, and cytoplasm the existence of transglutaminase (TGase) activities, similar to those known in animals and prokaryotes having mainly structural roles, but no protein has been associated to this type of activity in plants. A recent computational analysis has shown in Arabidopsis the presence of a gene, AtPng1p, which encodes a putative N-glycanase. AtPng1p contains the Cys-His-Asp triad present in the TGase catalytic domain. AtPng1p is a single gene expressed ubiquitously in the plant but at low levels in all light-assayed conditions. The recombinant AtPng1p protein could be immuno-detected using animal TGase antibodies. Furthermore, western-blot analysis using antibodies raised against the recombinant AtPng1p protein have lead to its detection in microsomal fraction. The purified protein links polyamines—spermine (Spm) > spermidine (Spd) > putrescine (Put)—and biotin-cadaverine to dimethylcasein in a calcium-dependent manner. Analyses of the  -glutamyl-derivatives revealed that the formation of covalent linkages between proteins and polyamines occurs via the transamidation of -glutamyl residues of the substrate, confirming that the AtPng1p gene product acts as a TGase. The Ca2+- and GTP-dependent cross-linking activity of the AtPng1p protein can be visualized by the polymerization of bovine serum albumine, obtained, like the commercial TGase, at basic pH and in the presence of dithiotreitol. To our knowledge, this is the first reported plant protein, characterized at molecular level, showing TGase activity, as all its parameters analyzed so far agree with those typically exhibited by the animal TGases.
Transglutaminases (TGases; E.C. 2.3.2.13) catalyze protein cross-linking by the interaction of an acyl acceptor glutamyl residue and an amine donor, a lysyl residue, of the same or another protein, or the formation of a link between a protein and a free primary amine, like a polyamine (PA). The terminal amino-groups of PAs conjugate one or two glutamyl residues giving rise either to mono-( -glutamyl)-polyamines (mono-PAs) or bis-(-glutamyl)-polyamines (bis-PAs; Folk, 1980 ). The posttranslational cross-linking of proteins is one of the essential physiological processes involved in the stabilization of tissue and cellular matrices. High molecular protein networks can be generated by TGase catalysis when cross-links are formed between Gln and either Lys residues or PAs (bis-PA derivatives) thus forming bridges between different proteins. At present, both in eukaryotes and prokaryotes several TGases have been described exhibiting a large number of functions (Aeschlimann et al., 1998; Griffin et al., 2002; Lorand and Graham, 2003). Eight distinct TGase isoenzymes have been identified in different animal tissues, and some of these have been purified and characterized at molecular level (Aeschlimann et al., 1998; Griffin et al., 2002; Lorand and Graham, 2003).
TGases are classified in three structural families: (1) the papain-like TGases, (2) the protein disulfide isomerase-like TGases, and (3) the bacterial toxin TGases. TGases catalyze a variety of posttranslational protein modifications such as transamidation, Lys acylation, esterification, deamidation, and isopeptide cleavage (Lorand and Graham, 2003
One typical feature of TGases is the fact that they exhibit a catalytic triad (Cys, His, and Asp), analogous to that of thiol-proteinases, and a critical Trp located upstream from the active center Cys, which is not involved in disulfide bridges (Weiss et al., 1998
With few exceptions, the activity of TGases and in particular the correspondence to tissueTGase, is activated by calcium and can be inhibited by GTP. These ligands induce opposite structural protein modifications that result in the tightening or relaxing of the four tissueTGase domains (Griffin et al., 2002
At present, several TGase activities have been detected both in higher and lower plants, supporting the presence of this type of enzyme. Indeed, this type of activity has been found in various cell compartments such as chloroplasts, mitochondria, cytoplasm, and cell walls (for review, see Serafini-Fracassini et al., 1995 However, the research on plant TGases has been greatly delayed so far because, although several purified plant protein extracts could establish a close association between their TGase activities with some purified proteins detected by SDS-PAGE, at present the identity of these proteins remains unknown. In addition, in the Arabidopsis, Zea mays, Oryza sativa, and Solanum tuberosum databases no DNA sequence has been found to share homology with animal TGases. This makes it difficult or even excludes the possibility to identify plant TGases by sequence comparison with other well-known animal TGases.
Recently, computational analysis has identified a gene in Arabidopsis, named AtPng1p, coding for a putative peptide N-glycanase (Suzuki et al., 2001 Based on these results, the AtPng1p gene product could therefore be considered as a TGase. Although the existence of some TGase activities have been demonstrated so far in purified plant protein extracts, this is the first characterization to our knowledge of a known protein, AtPng1p, having TGase activity in plants.
AtPng1p Protein Contains the TGase Tripartite Domain AtPng1p gene product is a 721-amino acid residue protein containing the tripartite domain typical of TGases. Alignment of the AtPng1p TGase domain with other well-characterized TGases is shown in Figure 1A . Thus, in common with other TGases and PNGases, AtPng1p contains two cysteinyl residues within the first domain, a histidyl residue within the second domain, and an aspartyl residue within the third TGase domain.
In addition to the tripartite domain, the ScanProsite program predicts in the N-terminal region of AtPng1p (Fig. 1B) a signal peptide which is typically associated with proteins that transit through (or are located in) the Golgi apparatus.
Based on database information, AtPng1p is located in chromosome 5 and contains 17 exons. When searching in Arabidopsis databases, no homologs of AtPng1p were found, indicating that it is a single gene. In addition, only a few expressed sequence tags arising from AtPng1p could be identified in databases so far, suggesting that it is a low-expressed gene. In fact, no mRNA accumulation could be detected by northern-blot analysis (results not shown). Therefore, we performed nested reverse transcription (RT)-PCR for identifying the AtPng1p mRNA accumulation (Yang and Marchand, 2002
Recombinant AtPng1p Purification and Immunodetection To elucidate whether AtPng1p encodes a TGase protein, we have overexpressed its coding sequence in E. coli and purified the recombinant protein by Ni2+-affinity chromatography. The 86-kD band obtained is shown in Figure 3B .
As no plant protein was known as TGase until now, no specific plant antibodies were available, and therefore animal TGase antibodies have been used for the detection of TGases in plant extracts exhibiting TGases activities (Del Duca et al., 1994 ; Waffenschmidt et al., 1999; Dondini et al., 2001; Serafini Fracassini et al., 2002). Therefore, we have also tested whether the recombinant AtPng1p is detected by these animal antibodies (Fig. 3, D–F). As expected, our results indicate that AtPng1p protein is immuno-recognized also by the following animal TGase antibodies, TGase II-Ab 3 (Neomarker, Fremont, CA), anti-Caenorhabditis elegans TGase, anti-rat prostatic gland TGase, which is in agreement with previous results obtained with purified plant protein extracts having TGase activity.
To elucidate more precisely how AtPng1p gene product is expressed in Arabidopsis and which is its subcellular localization, we have raised specific antibodies against the purified recombinant AtPng1p and used them to immuno-localize the protein (Fig. 4M ). Western-blot analyses have detected AtPng1p protein (a single band of 86 kD) in the microsomal-enriched fraction only. This result is therefore in agreement with the ScanProsite program, which predicts AtPng1p gene product as a membrane-associated protein.
Some others bands (showed as doublets) of lower molecular mass (73, 63, 54, and 48 kD, respectively) were identified in the cytosolic fraction (Fig. 4S), opening the question of whether these bands represent soluble fragments of AtPng1p protein produced by proteolytic degradation.
Recombinant AtPng1p Protein Covalently Links PAs to Dimethylcasein
Recombinant AtPng1p Protein Conjugates Spermine to Glutamyl Residues of DMC To confirm that AtPng1p protein conjugates the primary amine of [3H]Spm to Gln residues of DMC, -glutamyl-derivatives were investigated (Fig. 6
). HPLC analysis shows the formation mainly of bis--gln-Spm derivatives in the presence of 2 mM calcium. In addition, neither DMC alone nor AtPng1 protein alone or denatured with 10% (w/v) trichloroacetic acid (TCA) in the presence of substrate are able to generate -glutamyl-derivatives. Furthermore, the biotin-cadaverine assay using DMC as substrate also confirms the amine incorporation due to the action of AtPng1p protein only in the presence of calcium (results not shown).
Recombinant AtPng1p Protein Polymerizes BSA in a pH-Dependent Manner The effect of AtPng1p protein on BSA polymerization was assayed in the absence of PAs to avoid the competition with their primary amino-groups. To avoid an unspecific BSA aggregation, due to the thermal treatment, samples were incubated with 100 mM dithiotreitol (DTT) in the sample buffer prior to and after boiling. Then, samples were run in 10% (w/v) SDS-PAGE, and the effect of polymerization was visualized by Coomassie Blue staining (Fig. 7 ). These results show that polymerization increases starting at pH 7.5 with increasing values of pH, the maximum being reached at pH 8.5, demonstrating that AtPng1p protein polymerizes the substrate in a pH-dependent way.
Recombinant AtPng1p Protein Polymerizes BSA in a Ca2+- and DTT-Dependent Manner We have performed TGase assays using BSA and DMC as substrates to reach a better understanding of the polymerization phenomenon. After these assays, the samples were run in 10% (w/v) SDS-PAGE and the proteins were visualized by Coomassie Blue staining (Fig. 8 ).
Although BSA rarely acts as a TGase substrate, our results show the formation of BSA polymers (>200 kD, indicated by arrow in the figure) through the action of the AtPng1p protein when calcium and DTT are simultaneously present in the reaction mixture (Fig. 8, left, lane 3, and compare with lane 2). In addition, this polymerization event is strongly inhibited when EGTA chelates the calcium in the absence or presence of DTT in the assayed reaction (lane 4 and 5, and compare with lane 3). Similar to the inhibition produced by EGTA, the addition of GTP strongly inhibits polymer formation (lanes 6 and 7, and compare with lane 3). However, the addition of Mg2+ to the reaction mixture does not interfere with the degree of polymerization (lanes 8 and 9, and compare with lane 3). Like magnesium, neither sodium nor potassium affects the polymerization of BSA (results not shown). BSA can also be polymerized using commercial guinea pig liver transglutaminase (GPL-TGase) instead of AtPng1p protein. In this case, the polymerization is also Ca2+-dependent (Fig. 8, right, lane 11), in fact it is strongly inhibited when EGTA is added to the reaction mixture (lane 12). In addition to the polymerization product, 5 to 6 bands of lower molecular mass, with respect to BSA, appear in the presence of the AtPng1p protein, calcium, and DTT (lane 3, and compare with lane 2, indicated with asterisk in the figure). Depletion of Ca by EGTA does not affect the formation of these bands (Fig. 8, right, lane 5, and compare with lane 3), suggesting that this event is DTT-dependent but not Ca2+-dependent. GTP shows a partial inhibition of this phenomenon only at a concentration of 5 mM (lane 6, and compare with lane 3). At variance with polymerization, the generation of these slower bands appears to be specifically related to AtPng1p protein as no such bands are observed when the commercial GPL-TGase is used in the assays (Fig. 8, right, lane 11). In addition to BSA, we have also tested whether DMC is a polymerization substrate for the AtPng1p protein and the commercial GPL-TGase. When the assays were performed in the presence of Ca2+ and DTT, DMC polymerized, although to a lower extent than BSA (results not shown). This phenomenon could indeed be interpreted by the fact that a high percentage of DMC Lys is blocked by methylation.
This paper reports the first known protein, AtPng1p, showing TGase activity in plants. Furthermore, as the protein catalyzes the formation of covalent linkages between primary amino-groups of PAs and glutamyl-residues in a Ca2+-dependent way (Figs. 5 and 6), we suggest that the tripartite Cys-His-Asp TGase domain of the AtPng1p protein (Fig. 1) may be implicated in this enzymatic process. However, whether one or more of these residues are critical for enzymatic activity is still unknown and directed mutagenesis should be performed to answer this question. In addition, AtPng1p protein recognizes as substrates and polymerizes BSA and DMC (Figs. 5, 6, and 8) in a Ca2+-, DTT-, GTP-, and pH-dependent way (Figs. 7 and 8), like GPL-TGase and other well-known animal TGases reported so far (Aeschlimann et al., 1998 ; Griffin et al., 2002; Lorand and Graham, 2003). The biochemical features exhibited by the AtPng1p gene product agree with previous results obtained with partially purified plant protein extracts showing TGase activity. In these different plants, the calcium-dependent TGase activity appears to be regulated by particular physiological conditions such as senescence (Serafini-Fracassini et al., 2002), light (Dondini et al., 2003), or salt stress (Dondini et al., 2001). However, the precise binding sites for calcium and GTP in the AtPng1p protein remain to be determined. In addition, this protein is immuno-recognized by three animal TGase antibodies (Fig. 3), a feature that is in agreement with those previously reported for plant extracts exhibiting TGase activity (Del Duca et al., 1994, 2000; Waffenschmidt et al., 1999; Dondini et al., 2001).
The fact that very few expressed sequence tags are found in databases arising from AtPng1p agrees with our difficulty detecting AtPng1p mRNA accumulation. This situation was solved by RT-PCR assays (Fig. 2), which demonstrated that this gene is ubiquitously expressed. The presence of the AtPng1p mRNA, even though low expressed, in all tissues, growth stages, and light conditions suggests that AtPng1p gene product could be a constitutive enzyme. Lilley et al. (1998)
Western-blot analysis also revealed that the amount of AtPng1p protein is low in the cell, in agreement with the low AtPng1p mRNA levels detected by RT-PCR. In fact, these low amounts of AtPng1p protein in the cell could explain why several laboratories have failed in isolating plant TGases over the years. To our knowledge, only Kang and Cho (1996)
Interestingly, an 86-kD band corresponding to the AtPng1p gene product has been detected mainly in the microsomal protein fraction. This result is in agreement with the ScanProsite program, which predicts an N-terminal region typically associated to proteins that transits through (or are located in) the Golgi apparatus. However, a few other smaller bands were detected in the soluble protein fraction (Fig. 4). Therefore, although AtPng1p appears to be a single gene, these results could indicate that its product may undergo posttranslational modifications that could modulate its subcellular location. This possibility is in keeping with results obtained from TGases of animal origin, in which both soluble and plasma membrane-bound TGases have been identified. For example, tissue TGase is found at several intracellular sites including mitochondria, the nuclear membrane, and the nucleoplasm (Griffin et al., 2002
In conclusion, although the Arabidopsis AtPng1p protein only shares a TGase domain (Suzuki et al., 2001 Although the existence of some TGase activities have been demonstrated so far in plant protein extracts, this is the first report in which a known protein, AtPng1p, is characterized as a TGase enzyme in plants.
Plant Material and Vectors Arabidopsis L. cv Columbia was used. All plants were grown in greenhouse conditions (23°C) with 16 h light/8 h dark or only on dark conditions (light intensity, 55 µmol photons m–2 s–1). The coding sequence of AtPng1p (2,163 bp; N96715) was obtained from the Arabidopsis G3B4T7 cDNA clone from TAIR database. At the beginning of this work, this clone was the longest one, but by sequence comparison with genomic DNA, it lacked the start Met and two other amino acid residues of the coding sequence. Therefore, during the course of the cloning step, we added these three amino acid residues prior to cloning this cDNA into the pET-28a (+) expression vector (Novagen, La Jolla, CA). The coding sequence was cloned in frame by adding the NdeI and SacI restriction sites. This coding sequence was confirmed by another longer cDNA clone (GenBank accession no. AI993733) containing these three amino acid residues and the 5' untranslated region that appeared later in databases. All reagents were obtained from Sigma-Aldrich (St. Louis) unless otherwise specified.
The sequence of the upstream and downstream primers is:
Underlined sequences refer to NdeI and SacI restriction sites, respectively, and correspond to the restriction sites used for cloning in-frame into pET-28a (+) expression vector. Heat shock transformed Escherichia coli (BL21 strain) were grown in 250 mL Luria-Bertani medium at 37°C. When the culture reached optical density (O.D., 600 nm) of 0.7, the production of recombinant protein was induced with 1 mM isopropil-
mRNAs deriving from different organs in different growth condition (as reported in Fig. 2) of Arabidopsis were isolated using standard procedures. The RNA was suspended in 50 µL of diethyl pyrocarbonate-treated distilled H2O, analyzed by gel electrophoresis, and quantified spectrophotometrically. Ten micrograms of RNA were used to synthesize cDNAs using oligo(dT) primers according to manufacturer's conditions (Invitrogen, Giuliano Milanese, Italy). First round PCR was performed with 5 µL of cDNA in 30 cycles using specific primers (upstream primer sequence, 5'-GCAGTTTCTTCCCGCACTAGGC-3'; downstream primer sequence, 5'-CTTCTGTACAGATCGATGCTCCC-3'). Four microliters of first round PCR product were added to 46 µL of a second round master mix containing nested primers (upstream primer sequence, 5'-GCATTGCCAGTTGCATTGGACGC-3'; downstream primer sequence, 5'-CCACCATCGTTACTTCCTTCAAG-3'), followed by a further 30 cycles of amplification.
Protein extracts were prepared from 20-d-old Arabidopsis plants, with all steps performed at 4°C. Four grams of whole 20-d-old plants were added to 30 mL ice-cold extraction buffer (0.25 M sorbitol, 50 mM Tris-HCl, pH 7.4, 3 mM EGTA, 1 mM EDTA supplemented immediately prior to use with 1 mM DTT, 10 mg/mL pepstatin, 0.5 mg/mL leupeptin, and 1 mM phenylmethylsulfonyl fluoride) and homogenized. The separation of microsomal and soluble proteins was then performed as already described (Terry and Williams, 2002 In all cases, isolated proteins were frozen with liquid nitrogen and stored at –80°C prior to use.
A quantity of 50 µg x 3 of affinity-purified AtPng1p protein were injected in chicken. Antibodies were recovered from 80 mL egg yolks 30 d after each injection. Each sample was suspended in 2 volumes of phosphate-buffered saline (PBS) 100 mM, pH 7.5, 5.25% (w/v) of polyethyleneglycol-8000, gently mixed for a period of 5 min, then centrifuged at 12,000g for 10 min at 4°C. The supernatant was recovered, filtered through two layers of Miracloth, incubated for 10 min at room temperature with 25 mL of PBS 100 mM, pH 7.5, 37.5% (w/v) of polyethyleneglycol-8000, then centrifuged at 12,000g for 15 min at 4°C. The pellet was recovered and resuspended in 25 mL 100 mM PBS, pH 7.5. Finally, antibodies were aliquoted and stored at –80°C.
A total of 100 µg of Arabidopsis enriched microsomal and soluble proteins were boiled in SDS loading buffer, loaded onto a denaturing 10% (w/v) SDS-PAGE (Laemmli, 1970 Studies on cross-reactivity between AtPng1p and animal TGase antibodies was performed as above using: anti-TGase II-Ab 3 (Neomarker), anti-Caenorhabditis elegans TGase (kind gift of Prof. K. Mehta, Houston, TX), and anti-rat prostatic gland TGase (kind gift of Prof. C. Esposito, Salerno, Italy). Anti-mouse or anti-rabbit horse radish peroxidase conjugates (Sigma-Aldrich) were used as secondary antibodies. Proteins were detected using amino-ethilcarbazole tablets (Sigma-Aldrich).
Prior to enzymatic assays, the recombinant purified AtPng1p protein was renatured according to Novagen protocols. Refolding was obtained by performing a multistep dialysis in 100 mM Tris-HCl, pH 8.5 buffer with a progressive depletion of urea under the presence of 5 mM Ca2+, 10 mM DTT, and 10% (w/v) Suc. Finally, with the same technique, samples were deprived of Ca2+ and DTT.
Radiometric Assay
Biotin-Labeled Cadaverine Incorporation Assay
Identification of
Polymerization Assay
The densitometric analysis of stained and labeled hydrated gels was performed with the specific software Total Lab (Raytest, Milan) on the gel scanned by a FLA 3000 Laser System (Fuji) provided with an imaging plate ultrasensitive for tritium (BAS-TR 2025, Fuji).
Quantitative determinations were repeated at least three times. All values are means with indicated SEs. The Student's t test was used to compare means, as reported in the legends.
Upon request, all novel material described in this publication will be made available in a timely manner for noncommercial research purposes, subject to the requisite permission from any third-party owners of all or parts of the material. Obtaining any permission will be the responsibility of the requestor. Sequence data from this article have been deposited with the EMBL/GenBank data libraries under accession number AI993733.
We thank the Arabidopsis TAIR database for providing us with the AtPng1p cDNA clone used in this work. Authors are very indebted to Dr. J. Casas (CSIC) for producing antibodies against AtPng1p, Dr. M. Capellades (IBMB-CSIC) for the technical support in greenhouses for growing Arabidopsis, the DNA sequencing team of IBMB-CSIC of Barcelona, Mr. N. Mele (University of Bologna) for the images elaboration, and Professor A. Serafini-Fracassini, Emeritus of St. Andrews University, for scientific and language suggestions. Received March 12, 2004; returned for revision April 30, 2004; accepted May 11, 2004.
1 This work was supported by Ministero dell'Università e della Ricerca Scientifica e Tecnologica (FIRB 2001), by the ESF Transglutaminases Program and the COST 844 Transglutaminases in Apoptosis (grant to M.D.M.), and by the Spanish Government (Secretaría de Estado de Educación, Universidades e Investigación, support to D.S.-F.). 
2 These authors contributed equally to the paper.
[w] The online version of this article contains Web-only data. Article, publication date, and citation information can be found at www.plantphysiol.org/cgi/doi/10.1104/pp.104.042549. * Corresponding author; e-mail donatella.serafini{at}unibo.it; fax 39–051242576.
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-D-thiogalactopyranoside and the culture incubated at 37°C for 3 h. Then, bacteria were centrifuged (3,000g) at 4°C and the pellet was manipulated according to manufacturer's conditions (Novagen). Finally, as the recombinant AtPng1p protein was present in the bacteria inclusion bodies, it was purified by Ni2+ affinity chromatography in the presence of 8 M urea (Novagen). 
