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Plant Physiol. (1998) 117: 1115-1123
Detection of Ca2+-Dependent Transglutaminase Activity
in Root and Leaf Tissue of Monocotyledonous and Dicotyledonous Plants
Graham R. Lilley,
James Skill,
Martin Griffin, and
Philip L.R. Bonner*
Department of Life Sciences, The Nottingham Trent University,
Clifton Lane, Nottingham NG11 8NS, United Kingdom
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ABSTRACT |
Protein extracted from root and leaf
tissue of the dicotyledonous plants pea (Pisum sativum)
and broad bean (Vicia faba) and the monocotyledonous
plants wheat (Triticum aestivum) and barley (Hordeum vulgare) were shown to catalyze the
incorporation of biotin-labeled cadaverine into microtiter-plate-bound
N ,N-dimethylcasein and the
cross-linking of biotin-labeled casein to microtiter-plate-bound casein
in a Ca2+-dependent manner. The cross-linking of
biotinylated casein and the incorporation of biotin-labeled cadaverine
into N,N-dimethylcasein were
time-dependent reactions with a pH optimum of 7.9. Transglutaminase activity was shown to increase over a 2-week growth period in both the
roots and leaves of pea. The product of transglutaminase's protein-cross-linking activity,  -( -glutamyl)-lysine isodipeptide, was detected in root and shoot protein from pea, broad bean, wheat, and
barley by cation-exchange chromatography. The presence of the
isodipeptide was confirmed by reversed-phase chromatography. Hydrolysis
of the isodipeptide after cation-exchange chromatography confirmed the
presence of glutamate and lysine.
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INTRODUCTION |
Transglutaminases are Ca2+-dependent enzymes
that catalyze an acyl-transfer reaction between primary amino groups
and protein-bound Gln residues. The -carboxamide group of
protein-bound Gln is the exclusive acyl-donor substrate of
transglutaminases, but a variety of primary amino groups may act as
amine donors. These include the -amino group of protein-bound Lys
residues, which result in the formation of inter- or intramolecular
protein cross-links via -(-glutamyl)-Lys isodipeptide bonds. The
covalent cross-links of -(-glutamyl)-Lys are stable and resist
chemical, enzymic, and mechanical disruption (Folk and Finlayson,
1977 ). Alternatively, the primary amino groups of polyamines may be
incorporated into Gln, resulting in covalent posttranslational
modification of proteins through the formation of
N-(-glutamyl)-polyamine bonds. The occurrence of both
types of reaction products has been widely reported in animal tissues,
and the function of several mammalian transglutaminases has been
studied extensively (Folk, 1980; Griffin and Smethurst, 1994).
Factor XIII is the heterotetrameric form of transglutaminase present in
human plasma. Proteolytic cleavage by thrombin to the dimeric form
activates the enzyme, which then stabilizes the fibrin clot during the
final stage of the blood-clotting sequence (Sobel and Gawinowicz,
1996). Keratinocyte transglutaminase present in skin is involved in the
terminal differentiation of keratinocytes via cross-linking of a number
of structural proteins (Thacher and Rice, 1985). Prostate
transglutaminase is responsible for the clotting of rodent seminal
plasma (Folk, 1980). Tissue transglutaminase is the most widespread
member of the transglutaminase family. Proposed roles for the enzyme
include an involvement in programmed cell death (apoptosis) (Fesus et
al., 1987; Knight et al., 1991), in exocytosis (Bungay et al., 1986),
in stabilization of the extracellular matrix (Aeschlimann et al.,
1995), and in transmembrane signal mediation, acting as a G-protein
(G h) (Im et al., 1997).
Transglutaminase activity has been reported in higher plants, although
no clear role for the enzyme in plant tissue has been defined.
Incorporation of radiolabeled polyamines into the animal transglutaminase substrate N,N-dimethylcasein
by crude cell extracts derived from pea (Pisum sativum) and
Jerusalem artichoke has been demonstrated (Icekson and Apelbaum, 1987;
Serafini-Fracassini et al., 1988). Radiolabeled polyamines covalently
linked to endogenous proteins were detected by autoradiography in
extracts prepared from explants of Jerusalem artichoke tubers (Dinella
et al., 1992; Grandi et al., 1992). Chloroplasts prepared from
Jerusalem artichoke leaf tissue were shown to contain
N-(-glutamyl)-putrescine,
N1,N4-bis-(-glutamyl)-putrescine,
and
N1,N8-bis-(-glutamyl)-spermidine.
The isolation of these polyamine conjugates provides unequivocal proof
of a catalytically active transglutaminase present in Jerusalem
artichoke chloroplasts (Del Duca et al., 1995). The large subunit of
Rubisco has been demonstrated as a substrate in Jerusalem artichoke and
alfalfa, suggesting a possible role for transglutaminase in
photosynthesis (Margosiak et al., 1990; Del Duca et al., 1994). Actin
and tubulin have been identified as substrates in apple pollen (Del
Duca et al., 1997). More recently, a
Ca2+-independent enzyme with
transglutaminase-like activity has been purified from leaves of soybean
using a [14C]putrescine-incorporation assay
(Kang and Cho, 1996).
Transglutaminases in mammalian systems are
Ca2+-dependent enzymes (Folk, 1980). However, in
the intracellular environment this Ca2+
activation is regulated by the binding of GTP and ATP (Smethurst and
Griffin, 1996). Recent findings suggest that Ca2+
ions stimulate the activity of plant transglutaminase but are not
an absolute requirement (for review, see Serafini-Fracassini et al.,
1995). Other investigators have shown Ca2+ ions
to have an inhibitory effect at concentrations greater than 2 mM (Aribaud et al., 1995; Kang and Cho, 1996). The
assays used to demonstrate Ca2+-independent amine
incorporation involve the incorporation of radiolabeled polyamines into
protein substrates such as N,N-dimethylcasein and Rubisco. Transglutaminase assays of this type may not be
appropriate for screening crude plant extracts because of the
possibility of interference by enzymes such as diamine oxidases.
Diamine oxidases are able to incorporate
[14C]putrescine into
N,N-dimethylcasein in a
Ca2+-independent reaction via Schiff base
formation (Siepaio and Meunier, 1995).
During this study the root and leaf tissue of wheat (Triticum
aestivum), barley (Hordeum vulgare), pea, and broad
bean (Vicia faba) were screened for transglutaminase
activity using the conventional [14C]putrescine-incorporation assay (Lorand et
al., 1972) and two microtiter-plate-based assays (Slaughter et al.,
1992; Lilley et al., 1997). In agreement with the findings of other
studies, no absolute Ca2+-ion dependence was
demonstrated using the
[14C]putrescine-incorporation assay. However,
Ca2+-ion-dependent transglutaminase activity was
observed in all tissues using a biotin-labeled casein-cross-linking
assay (Lilley et al., 1997) and a biotin-labeled
polyamine-incorporation assay (Slaughter et al., 1992). Polyamine
incorporation was found to be activated by nanomolar concentrations of
Ca2+, whereas millimolar concentrations were
required to activate protein cross-linking. Transglutaminase activity
was shown to increase over a 2-week growth period in both the root and
leaves of pea.
The definitive evidence for the presence of transglutaminase within a
biological system is the presence of the -(-glutamyl)-Lys isodipeptide (Folk and Finlayson, 1977). We present the first evidence
to our knowledge of -(-glutamyl)-Lys isodipeptide in plant tissue
and show that the isodipeptide is present in the root and shoot tissue
of the dicotyledonous plants pea and broad bean and the
monocotyledonous plants wheat and barley.
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MATERIALS AND METHODS |
Treatment of Plant Material
Seeds of broad bean (Vicia faba var
Aquadulce), pea (Pisum sativum var Feltham First), barley
(Hordeum vulgare var Pipkin), and wheat (Triticum
aestivum var Apollo) were soaked overnight in running water and
then germinated in damp vermiculite in a greenhouse at 20°C. PPFD of
300 to 400 µmol m 2 s1
was provided by natural daylight supplemented with high-pressure sodium
lamps and a 16-h photoperiod. Root and leaf tissue were harvested after
14 d unless stated otherwise. Fifty grams of tissue was
homogenized in a Waring blender in ice-cold 50 mM Tris-HCl, pH 7.4, containing 250 mM Suc, 10 mM EDTA, 10 mM 2-mercaptoethanol, 5 µM leupeptin, 1 µM pepstatin, 1 mM PMSF, and 5% (w/v)
polyvinylpolypyrrolidone. The homogenate was filtered through two
layers of muslin, and the pH was readjusted to 7.4 using solid Tris.
The extract was centrifuged at 13,000g for 20 min at 4°C.
The supernatant was clarified further by centrifugation at
80,000g for 45 min at 4°C to sediment the membrane
fraction. The supernatant protein was precipitated by the addition of
solid
(NH4)2SO4
to 90% saturation at 4°C. Precipitated protein was collected by
centrifugation at 13,000g for 20 min at 4°C, redissolved
in 50 mM Tris-HCl, pH 7.4, containing 1 mM
2-mercaptoethanol, and dialyzed against 2.5 L of the same buffer at
4°C. Aliquots of dialyzed protein were stored at  20°C.
Proteolytic Digestion of the Plant Proteins
Twenty-five milligrams of plant proteins extracted from leaf or
root tissue was precipitated from solution by the addition of TCA to a
final concentration of 10% (w/v). The precipitate was collected by
centrifugation in a microcentrifuge for 5 min at 4°C (approximately
10,000g) and then washed twice in 10% (w/v) TCA, twice in
diethyl ether:ethanol (1:1, v/v), and twice in diethyl ether. The
pellet was dried and resuspended in 1.0 mL of 0.1 M (NH4)2CO3,
pH 8.0 (a crystal of thymol was included to inhibit bacterial growth).
The digestion of plant proteins was carried out by the sequential
addition of proteolytic enzymes (subtilisin, pronase, prolidase, Leu
aminopeptidase, and carboxypeptidase Y) according to the method of
Griffin et al. (1982). The incubation time with each of the proteolytic
enzymes was 24 h at 37°C. The digests were mixed with 3.6 mL of
chloroform:methanol:HCl (200:100:2, v/v) and centrifuged (model GPKR,
Beckman) at 2,500g (approximately 3,000 rpm) for 5 min. The
aqueous phase was separated from the organic phase and both were dried
using a centrifugal evaporator (model RC 10.22, Jouan, Winchester, VA).
The aqueous phase containing amino acids and the isodipeptide was
resuspended in 1.0 mL of distilled water. The material in the
dried organic layer was assayed for protein to determine the percentage
of hydrolysis.
Purification of the Isodipeptide Using Anion-Exchange
Chromatography
Three-hundred microliters of the protein digests (pH adjusted to
12.6) was applied to a Dowex (2 × 8-200; Cl form) anion-exchange column (1.5 × 1.1 cm) equilibrated with ultrapure water (Milli-Q system, Millipore) (>18 M ), pH 12.6, and washed with 25 mM NH4HCO3, pH
7.6, at a flow rate of 4.0 mL min1. After 70.0 mL had passed through the column to elute the majority of the Leu, the
column was washed with 10.0 mL of ultrapure water, pH 12.6. The
isodipeptide was eluted with 0.1 N HCl. Five-milliliter fractions were collected and their pH monitored. When the pH decreased to less than 7.0, the next 20.0 mL of eluate was pooled, the pH was
adjusted to 7.0, and the eluate was freeze-dried. After freeze-drying the samples were redissolved in 0.3 mL of ultrapure water and stored at
20°C. After anion-exchange chromatography approximately 70% of
the isodipeptide was recovered and 95% of the Leu was removed.
-(-Glutamyl)-Lys Isodipeptide Analysis
A sample of either crude plant protein digest or
anion-exchange-purified material was applied to an amino acid analyzer
(Alpha Plus 4151, LKB, Bromma, Sweden) with a 5 × 250 mm
ion-exchange column (Ultrapac 8, LKB; particle size 8 ± 0.5 µm;
lithium form). The amino acids and isodipeptide were eluted with
lithium-citrate buffers and detected using postcolumn
orthophthalaldehyde-2-mercaptoethanol derivatization as described
previously (Griffin et al., 1988) but with the following modifications:
the column temperature was 21°C and the pH of the elution buffer (c)
was adjusted to 3.15. A 2.0-mL postcolumn reaction loop was
incorporated between the mixing loop and the fluorescence detector
(model LS1, Perkin-Elmer; 360 nm excitation, 450 nm emission). The data
were recorded on a computer (model SL1, Viglen, UK) using a series
interface (Analytical 900, Nelson, Cupertino, CA) and chromatography
software (model 2600 V5, Nelson).
Hydrolysis of Ion-Exchange-Purified Sample
Fifty microliters of anion-exchange-purified wheat root sample was
applied to the amino acid analyzer and eluted (profile described above)
without postcolumn derivatization. A 5.0-mL fraction was collected at
the elution point of the isodipeptide, the pH was adjusted to 7.0, and
the sample was dried on a centrifugal evaporator (Jouan) and
redissolved in 0.3 mL of ultrapure water, pH 8.0. One-hundred
microliters of sample in 6.0 N HCl was sealed in a glass
tube under N2 and incubated overnight at 120°C.
The samples were dried using the centrifugal evaporator and redissolved in 0.1 mL of ultrapure water. The pH was adjusted to 8.0 with 8.4 N KOH and the volume was adjusted to 0.2 mL. Pre- and
posthydrolysis reversed-phase analyses were conducted with the
appropriate standard additions.
HPLC Analysis
An autosampler (model 507, Beckman) mixed 25 µL of sample with
an equal volume of orthophthalaldehyde-2-mercaptoethanol derivatizing reagent (Sigma) and injected a fixed 50 µL onto a reversed-phase column (4.6 × 15.0 cm, particle size 5.0 µm; Ultrasphere ODS, Beckman) using an HPLC system (Beckman Gold) and a fluorescence detector (340 nm excitation, 450 nm emission; model 167, Beckman). The
isodipeptide was eluted at a flow rate of 2.0 mL
min1 with a gradient of 60 mM
CH3CO2K, pH 5.9, and
methanol. The presence of the isodipeptide was confirmed by coelution
with an authentic standard.
Protein Assay
The protein content of crude plant extracts was determined using a
modified bicinchoninic acid method (Brown et al., 1989). BSA was used
as the standard protein.
14C-Labeled Putrescine-Incorporation Assay
The method used was a modification of that described by Lorand et
al. (1972). The assay was carried out at 37°C in 100 µL of 77.5 mM Tris-HCl containing 5 mM
CaCl2, 10 mM DTT, 5 mg
mL1 N,N-dimethylcasein,
1.2 mM [1,4-14C]putrescine
(specific activity 3.97 µCi µmol1), and 40 to 770 µg of plant protein. The reaction pH was 7.8 at 37°C.
Putrescine incorporation was terminated after 60 min by pipetting
10-µL aliquots of the reaction mixture onto
1-cm2 Whatman no. 1 filter paper squares
presoaked in 1% (w/v) methylamine and 100 mM EDTA. Protein
was precipitated by washing the filter papers once in ice-cold 10%
(w/v) TCA, three times in ice-cold 5% (w/v) TCA, once in
acetone:ethanol (1:1, v/v), and once in acetone. The filter papers
were then dried and placed into 2.0 mL of liquid scintillant and
counted for 5 min in a liquid scintillation counter (model 300C,
Packard Instruments, Meriden, CT). One unit of transglutaminase
activity was defined as 1 nmol of putrescine incorporated into
N,N-dimethylcasein per hour.
Biotin-Labeled Cadaverine-Incorporation Assay
The assay was carried out according to the method of Slaughter et
al. (1992) with the following modifications. Microtiter-plate wells
were blocked with 3% (w/v) BSA in 0.1 M Tris-HCl, pH 8.5. The incubation time for the transglutaminase reaction was 60 min and
the reaction pH was 7.9. Biotin cadaverine was replaced with 5-({[N-(biotinoyl)amino}hexanoyl]amino)pentylamine
(biotin-X-cadaverine). Streptavidin-alkaline phosphatase (0.25 mg
mL1 [1:150]) was replaced with extravidin
peroxidase (2.0 mg mL1 [1:5000]). Phosphatase
substrate was replaced with 100 mM
NaC2H3O2, pH 6.0, containing 0.310 mM 3,3,5,5-tetramethyl benzidine
and 0.004% (v/v) H2O2.
Color development was terminated by the addition of 50 µL per well of
5.0 M H2SO4.
The A450 was read using a multiscan ELISA
spectrophotometer (Titertek, Flow Laboratories, McLean, VA). One unit
of transglutaminase activity was defined as a change in
A450 of 1.0 per hour.
Biotin-Labeled Casein-Cross-Linking Assay
The -(-glutamyl)-Lys-formation assay was carried out
according to the method of Lilley et al. (1997). One unit of
transglutaminase activity was defined as a change in
A450 of 1.0 per hour.
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RESULTS AND DISCUSSION |
There is a mounting body of evidence to support the presence of
transglutaminase in plant tissue. However, this is the first report to
show a Ca2+-dependent transglutaminase and the
presence of -(-glutamyl)-Lys isodipeptide bonds in plant tissue,
the soundest evidence for transglutaminase activity.
The ability of crude plant extracts to incorporate radiolabeled
polyamines into proteins such as
N,N-dimethylcasein has been demonstrated
(Icekson et al., 1987; Serafini-Fracassini et al., 1988; Margosiak et
al., 1990; Aribaud et al., 1995). Table I shows that the
[14C]putrescine-incorporation assay detected
transglutaminase activity in only three of the eight tissues screened.
The Ca2+-chelating agents EDTA and EGTA at 5 mM were unable to effect more than 35% inhibition of
[14C]putrescine-incorporation activity of
extracts. Similar results have been shown by other investigators using
comparable assays, leading to the proposal that plant
transglutaminase has no absolute Ca2+-ion
requirement (Icekson et al., 1987; Serafini-Fracassini et al., 1988,
1995). Recent research (Siepaio and Meunier, 1995) indicated the
presence of a contaminating diamine oxidase in crude plant extracts
that is able to incorporate [14C]putrescine
into N,N-dimethylcasein in a
Ca2+-independent manner, masking the
transglutaminase activity.
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Table I.
Specific activity of transglutaminase in four plant
species screened using three assay systems
Crude extracts were incubated at 37°C for 60 min (n = 4). Extract boiled for 20 min was used as a negative control. Values are means ± SE.
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In contrast, Table I shows that soluble transglutaminase activity was
detected in all of the extracts screened using both the
biotin-cadaverine-incorporation assay (Slaughter et al., 1992) and the
casein-cross-linking assay (Lilley et al., 1997). Contaminating activities do not appear to interfere with the
biotin-cadaverine-incorporation or casein-cross-linking assays, since
chelation of Ca2+ by 1 mM EDTA and 1 mM EGTA resulted in more than 80% inhibition of pea root
and leaf transglutaminase activity and 100% inhibition of all of the
other extracts screened (data not shown). These data suggest that both
plate assays are more suitable for the study of transglutaminase from
crude plant cell extracts than the conventional
[14C]putrescine-incorporation assay and confirm
the Ca2+ dependency of the transglutaminase found
in the plant tissue studied.
Table I shows that 14-d-old root tissue exhibited a higher specific
activity in all species than leaf tissue of the same age. Using both
assays the greatest specific activity was observed in wheat root. In
pea root extract both the biotin-cadaverine-incorporation and
casein-cross-linking reactions were time dependent and linear up to 60 min (Fig. 1); therefore, this time period
was used for all subsequent experiments.
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| Figure 1.
Time-dependence curves for soluble pea root
transglutaminase extracted from 14-d-old tissue.  , Biotinylated
casein cross-linking;  , biotin-cadaverine incorporation. Data points
represent means ± SE of four replicates.
CaCl2 (5 mM) was replaced by 250 µM EDTA in the negative control reaction buffer.
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Resting levels of plant cytosolic Ca2+ have been
found to be in the nanomolar range, with brief increases to micromolar
levels in response to the appropriate stimulation.
Ca2+ levels in the extracellular matrix and in
Ca2+ stores have been detected in the millimolar
range (Bush, 1995). Figure 2a shows that
activation of the biotin-cadaverine-incorporation activity of soluble
pea root transglutaminase by Ca2+ was observed at
levels of 20 nM with an apparent
Km for Ca2+ of 50 nM. Maximum activity was achieved at 94 nM free
Ca2+, suggesting that soluble pea root
transglutaminase is able to incorporate polyamines into proteins at
resting levels of cytosolic Ca2+. Figure 2b shows
that activation of the protein-cross-linking function of soluble pea
root transglutaminase occurs at 250 µM free
Ca2+ and peaks at 3 mM, with an
apparent Km for Ca2+
of 2 mM. This observation may also suggest that to carry
out the protein-cross-linking reaction, soluble pea root
transglutaminase must be in a high-Ca2+
environment, such as the extracellular matrix, or in the intracellular environment when Ca2+ stores are released through
cellular damage.
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| Figure 2.
Ca2+-activation curve for
biotin-cadaverine incorporation (a) and casein cross-linking (b) by
soluble pea root transglutaminase extracted from 14-d-old tissue. ,
Biotinylated casein cross-linking; , biotin-cadaverine
incorporation. Zero free Ca2+ was achieved by the addition
of 1 mM EGTA to the reaction buffer. The amount of
CaCl2 required to give the desired free
Ca2+-ion concentrations at pH 7.9 and 37°C was then
calculated using a computer program (Fuhr et al., 1993). Data points
represent means ± SE of four replicates.
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Figure 3 shows that the optimum pH for
both casein-cross-linking and biotin-cadaverine-incorporation activity
of soluble pea root transglutaminase was 7.9. The profiles of both pH
plots are similar, suggesting that both assays are measuring the same
enzymic activity. Other investigators have demonstrated pH optima
between 7.9 and 8.4 for transglutaminase-like activities in different tissues of Jerusalem artichoke (Falcone et al., 1993).
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| Figure 3.
pH-dependence curve for soluble pea root
transglutaminase extracted from 14-d-old tissue. , Biotinylated
casein cross-linking; , biotin-cadaverine incorporation. The pH
values were measured at 37°C after the addition of pea root extract.
Biotin-cadaverine incorporation was measured in the presence of 100 µM CaCl2. Casein cross-linking was measured
in the presence of 5 mM CaCl2. Data points
represent means ± SE of four replicates. EDTA (250 µM) was used as a negative control.
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Mammalian transglutaminases have a Cys residue at the active site and
are irreversibly inhibited by reagents such as iodoacetamide (Smethurst
and Griffin, 1996). In this study the biotin-cadaverine-incorporation activities of soluble pea root and leaf extracts were inhibited by 32%
and 24%, respectively, by 10 mM iodoacetamide, suggesting that the active site of plant transglutaminase may be similar but not
identical to the active site of mammalian tissue transglutaminase, since a greater inhibition may have been expected at this concentration of iodoacetamide. Furthermore, the activity of mammalian tissue transglutaminase is regulated by GTP at low concentrations of Ca2+ (Smethurst and Griffin, 1996). The
biotin-cadaverine-incorporation activity of pea root transglutaminase
was not inhibited by 1 mM GTP at 1 µM free
Ca2+, indicating that in this respect plant
transglutaminase may be different from the mammalian tissue
transglutaminase (data not shown).
A relationship exists between transglutaminase activity and the age of
pea root tissue. Figure 4a shows that
transglutaminase activity increased during the first 18 d of
growth, using both the biotin-cadaverine-incorporation and the
casein-cross-linking assays. This was followed by a decrease in
activity in 22- to 32-d-old root tissue, indicating that
transglutaminase may be involved in early root growth and development.
This suggestion is supported by a similar observed increase and
decrease in transglutaminase activity in developing roots of
Chrysanthemum morifolium (Aribaud et al., 1995). In pea leaf
tissue (Fig. 4b) both casein-cross-linking activity and
biotin-cadaverine-incorporation activity increased to a peak at d 25 and 15, respectively. Activity did not decline rapidly, as in pea root,
but remained at a level above that detected at d 8. The profiles for
the casein-cross-linking activity and biotin-cadaverine-incorporation
activity in root and shoot tissue during the 30-d period assayed were
similar, indicating, like the pH profile (Fig. 3), that one enzyme is
capable of the two activities. Transglutaminase has been detected in
both developing and mature leaf tissue, and this observation may
support other reports proposing roles for transglutaminase in
photosynthesis (Margosiak et al., 1990; Del Duca et al., 1994, 1995).
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| Figure 4.
Variation in soluble transglutaminase activity
with age of developing pea root (a) and shoot (b) tissue. ,
Biotinylated casein cross-linking; , biotin-cadaverine
incorporation. EDTA (250 µM) replaced 5 mM
CaCl2 in the negative controls. Data points represent means ± SE of four replicates.
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Despite reports of the presence of transglutaminase in plants
(Serafini-Fracassini et al., 1995; Kang and Cho, 1996; Del Duca et al.,
1997), there have been no previous reports of -(-glutamyl)-Lys in
plant tissue, which is the definitive proof of the presence of the
enzyme (Folk and Finlayson, 1977). This may indicate that the
isodipeptide is not present, suggesting that the reports of transglutaminase activity in plants are indeed reports of other types
of enzymes such as diamine oxidases (Siepaio and Meunier, 1995).
Alternatively, the levels of the cross-link in plant tissue may be low,
making detection difficult.
This work shows that the low levels of -(-glutamyl)-Lys
isodipeptide in plant tissue can be detected if an additional
purification step is undertaken before cation-exchange chromatography.
In addition, application of a concentrated crude digest of plant
protein to the amino acid analyzer results in the Leu and the Tyr peaks
masking the -(-glutamyl)-Lys peak. To overcome this problem, the
resolution between Tyr and Leu was maximized by modification of the pH
of the elution buffer and the temperature of the column. Figure
5a shows that levels of
-(-glutamyl)-Lys isodipeptide in plant tissue were low. Figure 5b
shows that detectable levels of the isodipeptide could be observed
using the amino acid analyzer after the sample had been purified using
anion-exchange chromatography.
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| Figure 5.
Amino acid profile from digested wheat root
protein using cation-exchange chromatography. Protein extracted from
wheat root tissue was digested with proteolytic enzymes. a, Ten
microliters of the digest was applied to an amino acid analyzer. b,
Fifty microliters of anion-exchange-purified material was applied to an
amino acid analyzer. The amino acids and isodipeptide (solid lines)
were eluted by increasing the salt concentration, and the position of
the isodipeptide was confirmed by a run containing 1.0 nmol of
authentic standard plus the wheat root samples (dashed lines).
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To confirm the presence of -(-glutamyl)-Lys, the peak from the
amino acid analyzer was collected and subjected to reversed-phase chromatography. Figure 6a shows the
profile of the isodipeptide peak analyzed by reversed-phase
chromatography (37 pmol). The presence of the isodipeptide was
confirmed by coelution with authentic standard. In addition, the
underivatized sample from the amino acid analyzer was hydrolyzed
overnight in 6.0 N HCl at 120°C. Figure 6b shows the
changes that occurred in the wheat root sample hydrolyzed by HCl; the
isodipeptide peak (18.5 pmol applied to the column) decreased and this
was mirrored by increases in glutamate (17 pmol) and Lys (16.9 pmol).
To confirm the presence of glutamate and Lys, Figure 6b shows the
hydrolyzed wheat root sample with additions of authentic glutamate,
Lys, and -(-glutamyl)-Lys isodipeptide.
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| Figure 6.
Reversed-phase chromatography profile from
digested wheat root protein after anion-exchange and cation-exchange
chromatography. Protein extracted from wheat root tissue was digested
with proteolytic enzymes. The digest was purified using anion-exchange
resin and applied to an amino acid analyzer. The underivatized
-(-glutamyl)-Lys isodipeptide peak was collected, concentrated,
and hydrolyzed in 6.0 N HCl. Twenty-five microliters of
isodipeptide before (a) and after (b) hydrolysis was applied to a
reversed-phase column. The isodipeptide (solid lines) was eluted by
increasing the methanol concentration (dotted/dashed lines), and the
position of the isodipeptide (dashed lines) was confirmed by a run
containing 100 pmol of authentic isodipeptide plus the wheat root
isodipeptide sample or 50 pmol of authentic isodipeptide, 25 pmol of
Lys, and 25 pmol of glutamate plus the hydrolyzed wheat root
isodipeptide sample.
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Table II shows the -(-glutamyl)-Lys
isodipeptide present in 14-d-old leaf and root protein of the
dicotyledonous plants pea and broad bean and the monocotyledonous
plants wheat and barley, with more cross-linking in the proteins
extracted from root tissue. The isodipeptide content follows the
pattern of extractable transglutaminase shown in Table I. The level of
-(-glutamyl)-Lys isodipeptide present in plant protein is
approximately 3% of that found in clotted fibrin (Griffin and Wilson,
1984). In pea root protein the levels of -(-glutamyl)-Lys
isodipeptide during a 32-d period varied between 0.15 and 0.55 nmol
mg1 (data not presented), without any pattern
being apparent.
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Table II.
 -(-Glutamyl)-Lys isodipeptide content of
digested plant proteins analyzed using cation-exchange chromatography
(n = 3)
Values are means ± SE.
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This is the first report, to our knowledge, to show the presence of
-(-glutamyl)-Lys isodipeptide and a
Ca2+-dependent transglutaminase in plant tissue.
The protein-cross-linking function of the plant transglutaminase is
activated by millimolar concentrations of Ca2+,
suggesting a possible extracellular protein-cross-linking role or a
role in cell death similar to that of the mammalian tissue transglutaminase when Ca2+ levels are likely to
increase within the cell (Fesus et al., 1987). In contrast, the
polyamine-incorporation function is activated by nanomolar
concentrations of Ca2+, suggesting that this is
an intracellular function of plant transglutaminase. Furthermore,
polyamine incorporation is not inhibited by 1 mM GTP at low
free-Ca2+ concentrations, indicating that in
plants the enzyme may not be coupled to transmembrane signal
transduction and that substrate availability may regulate intracellular
plant transglutaminase activity.
The activity of plant transglutaminase is altered during the growth of
pea root and leaf tissue, suggesting an involvement in developmental
processes, possibly including cell wall development, root-tip
development, organelle development, or cellular differentiation. In
mammalian species the presence of -(-glutamyl)-Lys cross-links in
proteins gives rise to protein stability (Folk, 1980; Smethurst and
Griffin, 1994), so it is not unreasonable to assume that similar cross-links fulfill the same role in plant tissue. Further work will be
required to characterize the enzyme and to identify cellular protein
substrates to help establish a role for transglutaminase in plant
tissue.
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FOOTNOTES |
*
Corresponding author; e-mail p.bonner{at}ntu.ac.uk; fax
44-115-948-6636.
Received January 28, 1998;
accepted April 16, 1998.
| |
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Abstract
Introduction