Báo cáo khoa học: Functional significance of five noncanonical Ca2+-binding sites of human transglutaminase 2 characterized by site-directed mutagenesis potx

Functional significance of five noncanonical Ca
2+
-binding
sites of human transglutaminase 2 characterized by
site-directed mutagenesis
Ro
´
bert Kira
´
ly
1,2
,E
´
va Cs
}
osz
2
, Tibor Kurta
´
n
3
,Sa
´
ndor Antus
3
, Krisztia
´
n Szigeti
4
, Zso
´

fia Simon-Vecsei
2
,
Ilma Rita Korponay-Szabo
´
5,6
, Zsolt Keresztessy
2
and La
´
szlo
´
Fe
´
su
¨
s
1,2
1 Apoptosis and Genomics Research Group of Hungarian Academy of Sciences, Debrecen, Hungary
2 Department of Biochemistry and Molecular Biology, Medical and Health Science Center, University of Debrecen, Hungary
3 Department of Organic Chemistry, University of Debrecen, Hungary
4 Research Group for Membrane Biology, Hungarian Academy of Sciences, Semmelweis University, Budapest, Hungary
5 Department of Pediatrics, Medical and Health Science Center, University of Debrecen, Hungary
6 Heim Pal Children Hospital, Budapest, Hungary
Introduction
Transglutaminase 2 (TG2), also known as tissue trans-
glutaminase or Gh protein (EC 2.3.2.13), is a unique
multifunctional protein with diverse biological func-
tions. It is present in various cell compartments,
including the cytosol, the nucleus, and the plasma

membrane. TG2 has been implicated in the regulation
Keywords
calcium binding; celiac epitope; GTPase
activity; transglutaminase activity;
transglutaminase 2 (tissue transglutaminase)
Correspondence
R. Kira
´
ly and L. Fe
´
su
¨
s, Department of
Biochemistry and Molecular Biology,
Medical and Health Science Center,
University of Debrecen, Nagyerdei krt. 98,
POB 6, Debrecen, Hungary H-4012
Fax: +36 52 314-989
Tel: +36 52 416-432
E-mail: ;
(Received 28 May 2009, revised 16
September 2009, accepted 1 October 2009)
doi:10.1111/j.1742-4658.2009.07420.x
The multifunctional tissue transglutaminase 2 (TG2) has a four-domain
structure with several Ca
2+
-regulated biochemical activities, including
transglutamylation and GTP hydrolysis. The structure of the Ca
2+
-binding

form of the human enzyme is not known, and its Ca
2+
-binding sites have
not been fully characterized. By mutagenesis, we have targeted its active
site Cys, three sites based on homology to Ca
2+
-binding residues of epider-
mal transglutaminase and factor XIIIa (S1–S3), and two regions with nega-
tive surface potentials (S4 and S5). CD spectroscopy, antibody-binding
assay and GTPase activity measurements indicated that the amino acid
substitutions did not cause major structural alterations. Calcium-45 equili-
brium dialysis and isothermal calorimetric titration showed that both wild-
type and active site-deleted enzymes (C277S) bind six Ca
2+
. Each of the
S1–S5 mutants binds fewer than six Ca
2+
, S1 is a strong Ca
2+
-binding site,
and mutation of one site resulted in the loss of more than one bound
Ca
2+
, suggesting cooperativity among sites. All mutants were deficient in
transglutaminase activity, and GTP inhibited remnant activities. Like those
of the wild-type enzyme, the GTPase activities of the mutants were inhib-
ited by Ca
2+
, except in the case of the S4 and S5 mutants, which exhibited
increased activity. TG2 is the major autoantigen in celiac disease, and test-

ing the reactivity of mutants with autoantibodies from celiac disease
patients revealed that S4 strongly determines antigenicity. It can be con-
cluded that five of the Ca
2+
-binding sites of TG2 influence its transgluta-
minase activity, two sites are involved in the regulation of GTPase activity,
and one determines antigenicity for autoantibodies in celiac patients.
Abbreviations
FXIIIa, coagulation factor XIIIa; GSE, gluten-sensitive enteropathy (celiac disease); GST, glutathione-S-transferase; ICP-OES, inductively
coupled plasma–optical emission spectrometry; ITC, isothermal titration calorimetry; TG2, transglutaminase 2; TG3, epidermal
transglutaminase; TG4, prostate transglutaminase; WT, wild type.
FEBS Journal 276 (2009) 7083–7096 ª 2009 The Authors Journal compilation ª 2009 FEBS 7083
of cell differentiation, apoptosis, phagocytosis, cell
adhesion, wound healing, and the pathophysiology of
various diseases, including celiac disease [gluten-sensi-
tive enteropathy (GSE)], tumor growth, and neurode-
generative disorders [1,2]. In GSE, a chronic
enteropathy with multiple extraintestinal manifesta-
tions and 1–2% prevalence in the general population,
autoantibodies of the IgA (and IgG) class are
produced against TG2 in response to ingestion of
gluten proteins. These antibodies contribute to disease
progression and also have great importance as dia-
gnostic markers [3].
TG2 has several kinds of enzymatic activity. It was
recognized as a Ca
2+
-activated transglutaminase
enzyme performing post-translational protein modifica-
tion via the incorporation of small amines into pro-

teins, forming e-(c-glutamyl)lysine isopeptide bonds
between polypeptide chains, but it can deamidate
glutamine side chains as well [4]. GTP and GDP
inhibit the transamidating activity of the enzyme [5]. It
can bind GTP and ATP [6], and its role in mediating
signal transduction through G-protein-coupled recep-
tors, based on its GTPase activity, has also been
shown [7]. In addition, TG2 demonstrates protein
kinase [8] and protein disulfide isomerase [9] activities.
It also acts as a BH3-only protein, interacting with
proapoptotic factors [10]. Fibronectin-bound TG2
serves as a coreceptor for integrins, contributing to the
adhesive functions of cells [11].
The actual enzymatic activity of TG2 is determined
by its structural state induced by the types and
amounts of bound ligands [12]. The X-ray structure of
TG2 in its GDP-bound and substrate analog-bound
form has been described [13,14]. On comparison of
these two structures, a large difference can be
observed. The beginning of this large conformational
change is induced by calcium ions. However, the exact
sites of bound calcium ions and their functional signifi-
cance have not been determined.
Within the transglutaminase enzyme family, the
calcium-bound X-ray structures of only the human
blood coagulation factor XIIIa (FXIIIa) and epider-
mal transglutaminase (TG3) are known. FXIIIa has
one Ca
2+
-binding pocket, where the main chain oxy-

gen of Ala457 mediates Ca
2+
binding, and the other
direct coordinators are five water molecules; Asn436,
Asp438, Glu485 and Glu490 are also involved in the
formation of this negatively charged site [15]. TG3 has
three Ca
2+
-binding sites (S1–S3): S1 is formed by
Asn221–Asn229 and a water molecule – at this site,
TG3 permanently binds a sole Ca
2+
with high affinity
that could derive from the actual expression system
[16]; S2 is similar to the heptacoordinated Ca
2+
-binding
site of FXIIIa, involving Asn393, Ser415, Glu443,
Glu448 and two directly coordinated water molecules;
and at S3, Ca
2+
is coordinated by Asp301, Asp303,
Asn305, Ser307, Asp324, and a water molecule [17].
These three sites are also represented as conserved
sequences in TG2. It has been shown [18] that human
red blood cell TG2 can bind six Ca
2+
, suggesting that
further binding sites exist. Indeed, TG2 has several
negatively charged amino acids with high surface

potential that might serve as Ca
2+
-binding sites [19].
The aim of the present study was to identify the
exact Ca
2+
-binding sites of human TG2, by using site-
directed mutagenesis and targeting sites homologous to
FXIIIa and TG3, and two other sites with highly nega-
tive surface potential. We examined changes in Ca
2+
binding characteristics of the generated mutants, and
investigated the role of these sites in the regulation of
TG2 enzymatic activities. Our results show that each
of these sites contributes to Ca
2+
binding, and that
transglutaminase activities were significantly decreased
or totally lost when any of these sites were mutated.
Two mutants demonstrate higher GTPase activity than
the wild type (WT), and one of them shows very low
affinity for celiac autoantibodies.
Results
Ca
2+
binding of human recombinant TG2
Even after exhaustive dialysis in EDTA-containing
buffer, the bacterially expressed wild-type human TG2
contains 0.45 ± 0.03 mol Ca
2+

per mol WT, as
detected by inductively coupled plasma–optical emis-
sion spectrometry (ICP-OES). This finding suggests
that the recombinant TG2 has a tightly bound Ca
2+
,
which could be derived from the expression system,
similarly to the case with recombinant TG3 [16]. This
tightly bound Ca
2+
has an affinity for TG2 that is
comparable to its affinity for EDTA. To determine the
Ca
2+
-binding properties of the recombinant WT, equi-
librium dialysis measurements were performed. The
results showed that the WT can bind about six Ca
2+
(Fig. 1A), similarly to the native erythrocyte TG2 [18].
The calculated affinity constant of the hyperbolic
saturation curve was 560 lm.
Isothermal titration calorimetry (ITC) measurements
confirmed our equilibrium dialysis and ICP-OES data
(Fig. 1B). The curve of integrated heats shows 0.5 mol
Ca
2+
binding to TG2 per mol protein, with high affin-
ity (K
d
= 0.1 lm). The next five Ca

2+
bind with very
low and comparable affinity to the enzyme. The
observed difference between the Ca
2+
-bound active
form and the inactive form of TG2 suggests a large
Ca
2+
-binding sites of TG2 R. Kira
´
ly et al.
7084 FEBS Journal 276 (2009) 7083–7096 ª 2009 The Authors Journal compilation ª 2009 FEBS
conformational change during the Ca
2+
activation
process, which could be accompanied by a significant
entropy change, explaining the small enthalpy change.
In the presence of Ca
2+
, the WT may work as a
transamidase, even during the equilibrium dialysis and
ITC experiments, and could crosslink itself to a differ-
ent degree, even in the absence of any other substrates
(Fig. S1). This self-crosslinking occurred to a much
smaller extent during the 3–4 h of ITC measurements
at 25 °C than during the 2 days of equilibrium dialysis
at 4 °C. We also wished to clarify whether this process
altered Ca
2+

-binding properties. Therefore, we exam-
ined the C277S active site mutant, which lacks any
transglutaminase activity and does not have the ability
to crosslink itself [20]. On the basis of our equilibrium
dialysis data, the C277S mutant also binds approxi-
mately six Ca
2+
, although the binding is weaker (the
affinity constant is 720 lm) than in the case of the WT
(Fig. 1A). The active site mutant also showed the same
ITC response as the WT (data not shown). These
results demonstrate that self-crosslinking and eventual
polymerization does not have any significant influence
on Ca
2+
binding of the recombinant WT, and that the
possible heat changes related to crosslinking were
probably masked by other concurrent mechanisms.
Design and preparation of site-directed mutants
of TG2
On the basis of the high sequence homology shared by
transglutaminases and the available X-ray structures of
FXIIIa, TG3, and their identified Ca
2+
-binding sites
[15–17], we used homology modeling and comparative
molecular modeling to design seven TG2 mutants. In
these, five different surface sites were altered by intro-
ducing single or multiple point mutations (Fig. 2;
primers are listed in Table S1). The S1 and S3 mutants

were chosen on the basis of homology with TG3
Ca
2+
-binding sites (S1 and S3, respectively). The S2
mutants were planned on the basis of homology to the
0 1
2 3 4
0
1
2
3
4
5
6
7
8
C277S
WT
[Ca
2+
] free (mM)
Bound Ca
2+
(mol/mol)
A
024
–0.5
–0.4
–0.3
–0.2

–0.1
0.0
Molar ratio Molar ratio Molar ratio
kcal·mol
–1
of injectant
–0.10
–0.05
0.00
kcal·mol
–1
of injectant
024681012
0123456
–0.8
–0.7
–0.6
–0.5
–0.4
–0.3
–0.2
–0.1
0.0
kcal·mol
–1
of injectant
B
Strong binding site of wild typeTG2
Weak binding sites of wild type TG2 Weak binding sites of S1 TG2
n = 0.52 ± 0.63

K
d
= 0.1 ± 0.03 µM
DH = –421.5 ± 83.7
Kcal·mol
–1
DS = 30.5 Kcal·mol
–1
K
d
= 4.6 ± 1.3 µM
DH = –58.02 ± 23
Kcal·mol
–1
DS = 24.2 Kcal·mol
–1
n = 5.46 ± 0.08
K
d
= 6.7 ± 3.2 µM
DH = –784.5 ± 38
Kcal·mol
–1
DS = 21.0 Kcal·mol
–1
n = 1.7 ± 0.06
Fig. 1. Ca
2+
binding of recombinant wild-type, C277S mutant and S1 mutant TG2. (A) Ca
2+

-binding curve of wild-type and C277S mutant
TG2 measured by equilibrium dialysis. (B) Net heat change of ITC of Ca
2+
binding to wild-type and S1 mutant TG2. The net heat change
curve of wild-type TG2 was divided into two parts to improve the quality of curve regression. For the injection scheme, see Experimental
procedures.
R. Kira
´
ly et al. Ca
2+
-binding sites of TG2
FEBS Journal 276 (2009) 7083–7096 ª 2009 The Authors Journal compilation ª 2009 FEBS 7085
Ca
2+
-binding site of FXIIIa, which has strong similar-
ities to one of the TG3 Ca
2+
-binding sequences. In the
case of S2 and S3, we generated two separate mutants
(S2A and S2B mutants, and S3A and S3B mutants), as
here the suspected Ca
2+
-binding sites are formed by
two opposing loops. We have assumed that mutations
of these sites as a whole may cause significant confor-
mational changes by themselves, and should be
avoided. S4 and S5 were selected on the basis of sur-
face patches characterized by higher local density of
negatively charged amino acids on TG2 [19,21]. Mostly
conservative amino acid replacements were performed

to target Ca
2+
binding specifically and to prevent
significant conformational changes or structural
disruptions. In most cases, only negative charges were
removed (e.g. Glu to Gln, or Asp to Asn) or the
432 G R N Q R Q N I T432 G R D E R E D I TS5
149 Y L N
S Q Q Q R Q Q Y149 Y L D S E E E R Q E YS4
326 D K S Q
M I W N326 D K S E M I W NS3B
305 H N
Q S S S L305 H D Q N S N LS3A
445 Y P Q
G S S Q Q R Q A445 Y P E G S S E E R E AS2B
395 A Q
V S A N V395 A E V N A D VS2A
228 V S
C S N N Q G V228 V N C N D D Q G VS1
Mutant sequenceOriginal sequenceMutant
Fig. 2. Mutagenized sites on the surface of
TG2 (upper part) and detailed location of S4
and S5 Ca
2+
-binding sites in relation to GTP
binding (bottom part). On the Ca backbone
of TG2, the N-terminal domain is blue, the
core domain is red, the first b-barrel is cyan,
and the second b-barrel is green. The red
spheres show the transglutaminase active

site amino acids and the purple ones indi-
cate the bound GTP. The yellow balls and
sticks indicate Lys173 and Phe174, and the
gray spheres show the proposed location of
bound calcium ions.
Ca
2+
-binding sites of TG2 R. Kira
´
ly et al.
7086 FEBS Journal 276 (2009) 7083–7096 ª 2009 The Authors Journal compilation ª 2009 FEBS
potential for Ca
2+
complexation was decreased (e.g.
Asn to Ser). According to previous results [22], this
type of amino acid replacement does not alter the
expression and stability of mutant proteins.
For normalization of protein expression and purity,
the binding of a monoclonal antibody to each mutant
was examined by ELISA, as antibodies are more sensi-
tive to conformational changes [23]. Although most
mutants showed similar antibody binding, the S2B
mutant had lower affinity, because this mutation is at
the recognition site of the antibody used in the experi-
ments (Fig. S2).
Study of CD spectra of mutants
The native states of the purified proteins were tested by
CD spectroscopy (Fig. 3). The CD spectra of the
mutants did not show substantial deviations from that
of the WT, which suggested that their secondary struc-

tures were not altered significantly by the mutations.
The CD deconvolution, performed with the analysis
programs continll, cdsstr, and selcon3 [24,25],
showed that unordered and turn structural segments
contributed about 50% to the secondary structure, and
that their values were very similar for all of the studied
structures (data not shown). The WT and the S2A
mutant had almost identical CD curves and thus very
close secondary segment contributions as well. How-
ever, some minor changes could be observed with the
other mutant proteins: the S4 and S5 mutants had
nearly identical CD curves, but they differed from the
WT in their larger helix and smaller strand contribu-
tions, resulting in somewhat larger negative ellipticities
in the range 200–240 nm. In contrast, the S1, S2B, S3A
and S3B mutants had smaller helix and larger strand
contributions than the WT, resulting in smaller elliptici-
ties in the range 200–240 nm. Subtle differences could
be observed among the members of this group as well;
the S3A and S3B mutants had near-identical CDs and
hence secondary structures, whereas the S1 and S2B
mutants were slightly different from them, with slightly
larger helix and smaller strand segment contributions.
Ca
2+
binding of mutant TG2 proteins
To compare the Ca
2+
binding of wild-type and mutant
enzymes in equilibrium dialysis, a free Ca

2+
concentra-
tion of 1.7 mm was used. In case of the WT, the expo-
nential part of the binding curve reaches the maximum
at this concentration. If the mutants showed lower
Ca
2+
binding than the WT, we would see larger
changes in the exponential part of the binding curve
than in other parts of the curve.
All mutant proteins bound less Ca
2+
than the WT
at 1.7 mm free Ca
2+
concentration (Fig. 4A), and the
mean values were significantly different (P < 0.0001),
as calculated using ANOVA. The experimental Ca
2+
-
binding values confirmed that each of the five mutage-
nized sites contributes to Ca
2+
binding of TG2. It was
also observed that disruption of one site by mutation
leads to weaker ⁄ loss of binding to other sites, and this
suggests cooperative Ca
2+
-binding properties. For
instance, in the S1 mutant, the number of bound Ca

2+
dropped from six to two.
Using ICP-OES, we tested whether TG2 mutated at
the site homologous to the high-affinity Ca
2+
-binding
site of TG3 (S1) still binds Ca
2+
after purification.
The result clearly showed that the S1 mutant cannot
bind Ca
2+
after dialysis with EDTA (< 0.03 mol
Ca
2+
per mol TG2), whereas the WT binds 0.5 mol
Ca
2+
per mol TG2 under the same conditions. This
result means that TG2 also has a Ca
2+
-binding site
with high affinity and that this is S1. The ITC
measurements of the S1 mutant show a stoichiometry
of 1.7, which is the same value as obtained by equilib-
rium dialysis (Figs 1B and 4A).
Ca
2+
-dependent transglutaminase activity of
mutant TG2 proteins

As the transglutaminase activity of TG2 is Ca
2+
-
dependent, decreased activity of the mutants could be
expected (Fig. 4B). In accordance with this, the trans-
glutaminase activity of each mutant decreased to vari-
ous extents, and the S3, S4 and S5 mutants lost their
activity completely in the microtiter plate as well as in
the filter paper assay (Fig. 4C). Interestingly, in the
case of the S2A mutant and, mainly, the S2B mutant,
a substantial difference was observed between the
results of the two methods, which differ in the use of
amine substrate and the availability of Gln substrate
190 200 210 220 230 240 250
–10
–5
0
5
10
15
[θ] x 10
–3
deg cm
2
decimol
–1
Wavelength (nm)
S5
S4
W

S2A
S1, S2B
S3A,S3B
Fig. 3. CD spectra of recombinant TG2 proteins.
R. Kira
´
ly et al. Ca
2+
-binding sites of TG2
FEBS Journal 276 (2009) 7083–7096 ª 2009 The Authors Journal compilation ª 2009 FEBS 7087
in solution versus bound to the surface. At higher
Ca
2+
concentrations, there was no significant increase
in the activities, which means that increased Ca
2+
con-
centrations cannot compensate for the loss of specific
Ca
2+
-binding side chains.
Transglutaminase activity is inhibited by GTP and
GDP. As a proof of GTP sensitivity, we wished to see
a decrease in this remaining, lower transglutaminase
activity of mutant enzymes as compared with the WT.
To obtain a sufficient starting transglutaminase activ-
ity, a relatively high Ca
2+
concentration (5 mm) was
used (Fig. 4D): under these conditions, the presence of

100 lm GTP decreased the transglutaminase activity of
both the WT and the S1, S2A and S2B mutants by
 40%. These results suggested that GTP can effec-
tively bind to the mutants.
GTPase activity of mutant TG2 proteins
Ca
2+
binding also influences both GTP binding and
the GTPase activity of TG2 [5]. Initially, we per-
formed photoaffinity GTP-labeling experiments. As
shown by autoradiography in Fig. 5A, the S2B, S3A
and S3B mutants had similar GTP incorporation to
the WT, whereas the S1 and S2A mutants had lower
GTP incorporation than the WT after UV light
exposure.
The GTPase activity of these mutants correlated well
with photoaffinity GTP labeling. As expected, we also
could see a slight decrease in GTPase activity at
increasing Ca
2+
concentrations for most of the
mutants, similarly to what was seen with the WT
(Fig. 5B).
A
P = 0.0023
P = 0.0016
P < 0.0001
P < 0.0001
P < 0.0001
P < 0.0001

P < 0.0001
P values
3.0
±
1.4
3.2
±
1.1
2.5
±
0.5
2.3
±
0.9
1.6
±
0.3
4.1
±
0.7
1.7
±
0.4
5.6
±
0.7
Bound Ca
2+
at 1.67 mM
[Ca

2+
]
free
mol/mol
S5S4S3BS3AS2BS2AS1WT
Mutants
0
20
40
60
80
100
120
WT S1 S2A S2B S3A S3B S4 S5
Relative activity (%)
Filter paper method
Microtiter plate method
C
D
0
1
2
3
4
5
6
7
8
9
0 10203040

[Ca
2+
] (mM)
Specific activity (DAbs·min·mg
–1
)
WT
S1
S2A
S2B
S3A
S3B
S4
S5
B
0
20
40
60
80
100
120
WT S1 S2A S2B
Relative activity (%)
GTP 0 µM/Ca
2+
5 mM
GTP 100 µM/Ca
2+
5 mM

P = 0.0135
P = 0.0140
P = 0.0005
P < 0.0001
Fig. 4. Ca
2+
-binding and Ca
2+
-dependent transglutaminase activities of wild-type and mutant TG2s. (A) Ca
2+
binding by wild-type and mutant
TG2s at 1.67 m
M free Ca
2+
concentration as measured by equilibrium dialysis. Data are presented as means from four separate experiments
performed in duplicate. The mean values are significantly different (P < 0.0001), as calculated using ANOVA. Unpaired t-tests were per-
formed to compare the Ca
2+
binding of the mutants with Ca
2+
binding of the wild-type enzyme, and these show that each difference is
highly significant. (B) Ca
2+
-dependent transglutaminase activity of recombinant TG2s as determined by using a microtiter plate method. Varia-
tion between experimental values was less than 10%. (C) Transglutaminase activity is shown as a percentage of the activity of wild-type
TG2. One hundred per cent specific activity of wild-type TG2 was 8.4 DA
405
(minÆmg)
)1
protein in the case of the microtiter plate method,

and 77.4 pmol putrescine (minÆmg)
)1
protein in the case of the filter paper method in the presence of 5 mM Ca
2+
. (D) Inhibition of residual
transglutaminase activity of recombinant TG2s by GTP, using the microtiter plate method. Activity is shown as a percentage of the activity
of wild-type TG2; the Ca
2+
concentration was 5 mM.
Ca
2+
-binding sites of TG2 R. Kira
´
ly et al.
7088 FEBS Journal 276 (2009) 7083–7096 ª 2009 The Authors Journal compilation ª 2009 FEBS
Interestingly, the S4 and S5 mutants showed 1.5-fold
to two-fold increased specific GTPase activity despite
an apparent lack of stable UV-induced GTP incorpora-
tion as determined by photoaffinity labeling, and their
GTPase activity was not inhibited by increasing Ca
2+
concentrations. Neither longer UV irradiation nor a
higher amount of the protein led to photolabeling of
these proteins. To confirm increased GTPase activity of
the S4 and S5 mutants, we also expressed them in gluta-
thione-S-transferase (GST)-fused forms with high purity.
The transglutaminase activities of the GST–S4 and
GST–S5 mutants were measured using the two methods
described above, and both failed to detect any
crosslinking activity (data not shown). We found

slightly increased GTPase activity in the case of the
GST–S4 mutant (130.7% ± 3.4% as compared with
GST–WT as 100%), and greatly increased GTPase
activity in the case of the GST–S5 mutant (353 ± 38%
as compared with GST–WT). Nonspecific GTP degra-
dation was excluded by the use of appropriate controls.
Antigenicity of mutant TG2 forms
TG2 is recognized as an autoantigen in GSE. The
epitopes are conformational [26], and the presence of
Ca
2+
can increase the binding of celiac autoantibodies
to TG2 [27,28], although there are some contradictory
results [29,30]. In an attempt to resolve this discrep-
ancy, our Ca
2+
-binding site mutants were tested in
ELISA with a large panel (n = 62) of serum samples
obtained from GSE patients (age 1.1–69 years, mean
10.4) prior to treatment (Fig. 6; for statistical analysis,
see Table S2). The S4, S5, S3B and S3A mutants
showed decreased affinity for celiac autoantibodies
(P < 0.001), and the S4 mutant showed the lowest
binding (11.5 ± 8.2% as compared with the WT as
100%).
The binding of celiac autoantibodies to TG2 is influ-
enced by the presence or absence of Ca
2+
in the case
of guinea pig TG2 [27]. Therefore, we also examined

the effect of Ca
2+
and GDP on the binding of celiac
autoantibodies to mutant TG2s. The presence of 2 mm
EDTA, 20 lm GDP or 5 mm Ca
2+
failed to alter the
antigenicity of the enzymes (Fig. 6, insert). It has been
recently reported [31] that mutation of the transgluta-
minase catalytic triad of the active site decreased the
binding of celiac autoantibodies to the enzyme. In our
experiments, celiac autoantibodies could bind to the
C277S mutant and to the WT with similar affinity
(data not shown).
Discussion
In this article, we describe the Ca
2+
-binding properties
of TG2 and its mutants and the role of five Ca
2+
-
binding sites in the regulation of transglutaminase and
GTPase activities, as well as the binding of celiac
0
20
40
60
80
100
120

140
160
180
A
B
WT S1 S2A S2B S3A S3B S4 S5
Relative activity (%)
Without Ca
2+
With 3.5 mM Ca
2+
With 7 mM Ca
2+
TG2
TG2
WT S1 S2A S2B S3A S3B S4 S5
Fig. 5. Photoaffinity GTP labeling and GTPase activity of recombi-
nant TG2s. (A) Photoaffinity labeling of TG2 proteins; 2.2 lg protein
per lane (upper panel). Proteins were visualized with Coomassie
BB staining (lower panel). (B) GTPase activity and effect of Ca
2+
on
the GTPase activity of recombinant TG2s. GTPase activity was
calculated as percentage of activity of the WT [99.1 ± 7.4 pmol
GTP (minÆmg protein)
)1
] in the absence of Ca
2+
. Data are presented
as means with ± standard deviations from three separate

experiments performed in triplicate.
S1 S2A S2B S3A S3B S4 S5 WT
0
25
50
75
100
125
150
175
Relative binding (%)
0.00
20.00
40.00
60.00
80.00
100.00
120.00
WT S4
Relative binding (%)
+Ca
2+
+EDTA
+GDP
Fig. 6. Binding of IgA class celiac antibodies to wild-type and
mutant TG2s. Binding to wild-type TG2 is 100%, serum dilution is
1 : 200, and n = 62 from biopsy-proven untreated celiac disease
patients. The mean values are significantly different (P < 0.0001),
as calculated using ANOVA. Effects of 2 m
M EDTA or 20 lM GDP

are compared with those of 5 m
M Ca
2+
on antibody binding (insert).
Results were similar for IgG class antibodies (data not shown).
R. Kira
´
ly et al. Ca
2+
-binding sites of TG2
FEBS Journal 276 (2009) 7083–7096 ª 2009 The Authors Journal compilation ª 2009 FEBS 7089
autoantibodies to the enzyme. We found five nonca-
nonical Ca
2+
-binding sites of TG2, and determined
that one of these, S1, has a tightly bound Ca
2+
. There
are canonical and noncanonical protein structures that
bind Ca
2+
, and common to all of them is a high nega-
tive surface potential, derived mainly from Asp or Glu
residues. Ca
2+
, as a ‘hard’ metal ion, can coordinate
six or seven ligands with negative character or charge
in a pentagonal bipyramidal arrangement. There are
some well-known canonical Ca
2+

-binding domain
structures: EF-hand domain, C-type lectin-like domain,
Ca
2+
-dependent phosphatidylserine-binding domains
(C2, annexin and Gla domains), and EGF-like
domain, which have been characterized in detail by
X-ray diffraction and NMR spectroscopy. These
known Ca
2+
-binding domains, which are present in a
large number of Ca
2+
-binding proteins, do not share
significant similarities with the Ca
2+
-binding motifs of
the transglutaminase family. Interestingly, TG2 also
has a GTP-binding site and can hydrolyze GTP, but
does not have a typical GTP-binding site [13].
Members of the transglutaminase family have some
highly conserved negatively charged amino acids with
high surface potential. Ikura et al. [22] mutated two
highly conserved anionic sites of the guinea pig TG2
that were earlier proposed, on the basis of sequence
comparison, as putative Ca
2+
-binding sites [21]; how-
ever, their data showed that these sites are not essen-
tial for or directly involved in Ca

2+
binding. The
Ca
2+
-binding sites of human TG2 studied here
partially overlap with these negatively charged surface
patches in the guinea pig enzyme sequence. Every
mutated site investigated by us is located on a loop or
border of a loop, which could allow the appropriate
coordination of Ca
2+
and, in addition, may induce a
change in the structure of the protein. Without a
Ca
2+
-bound X-ray structure, however, it is not possi-
ble to establish the exact participation of the different
side chains in Ca
2+
binding and selected functions.
For TG2, the most important regulatory function
of bound Ca
2+
is the initiation of transglutaminase
activity. The tightly bound Ca
2+
at S1 is not enough
for transglutaminase activity in the case of TG3; addi-
tional Ca
2+

binding to S3 is needed to open the
active site and to form a substrate channel [16].
According to our data, the measurable transglutamin-
ase activity of the S1 mutant suggests that, although
Ca
2+
binding to this site is important for this activity,
binding of Ca
2+
to other sites also contributes to the
effective induction of an active transglutaminase con-
formation. Binding of Ca
2+
to S2 plays only a minor
role in the formation of the active state of TG2,
because mutation of S2 resulted in the highest resid-
ual transglutaminase activity. The loss of S3 Ca
2+
binding leads to an enzyme without transglutaminase
activity, suggesting that the binding of Ca
2+
to S3 in
TG2 plays a significant role in the induction of this
activity, similarly to the case of TG3. It is very likely
that Glu329 (replaced in the S3B mutant) plays a
crucial role in Ca
2+
coordination and regulation of
transglutaminase activity. It is interesting to note that,
by activation of TG2 [14], S3 undergoes significant

dislocation, just like the GTP-binding site, which is
also composed of two or three loops. Datta et al. [32]
studied, without determining actual Ca
2+
binding,
how three Ca
2+
-binding site mutants of TG2
influence cell survival; these sites correspond to our
S1, S2, and S3A. They changed two amino acids to
Ala at targeted sites, and this resulted in decreased
transglutaminase activity; similarly to our results,
there was no change in GTPase activity and GTP
binding, except for the N229A ⁄ D233A mutant
(labeled S1 by us).
Each Ca
2+
-binding site is in the core domain of
TG2, and they could influence each other, leading to
an energetically favorable arrangement of the enzyme
structure (Fig. 2). Our finding that mutation of one
site leads to the loss of more than one bound Ca
2+
supports an assumption of positive cooperativity [33]
among the Ca
2+
-binding sites of TG2. Ahvazi et al.
[16] also found indications that S2 and S3 may coop-
erate in TG3. S4 and S5 may have similar roles in
the process of fine tuning cooperativity, as mutation

of these sites also leads to the loss of Ca
2+
-inducible
transglutaminase activity. Our data suggest that the
cooperativity may be strong between S3, S4, and S5,
because loss of any of these results in binding of
about three Ca
2+
and total inactivation, which means
that Ca
2+
binding at these sites is needed for the
active conformation of transglutaminase activity. This
also raises the possibility that a sequential mechanism
of site occupancy may operate in Ca
2+
binding of
TG2.
How can weak Ca
2+
-binding sites play such an
important role in determining transglutaminase activity
when various biophysical measurements did not show
significant changes of TG2 after Ca
2+
binding [34]? In
the case of the canonical C2 domain, it is known that
a third Ca
2+
binds with lower affinity to the domain,

but in the presence of an interaction partner – phos-
pholipid in the case of C2, but it could be any appro-
priate substrate in the case of TG2 – the affinity for
Ca
2+
is higher, owing to completed coordination
spheres [35] of Ca
2+
. Further study is required to clar-
ify whether substrates, other interacting partners or
lipid molecules can regulate Ca
2+
affinity of TG2.
Ca
2+
-binding sites of TG2 R. Kira
´
ly et al.
7090 FEBS Journal 276 (2009) 7083–7096 ª 2009 The Authors Journal compilation ª 2009 FEBS
Interestingly, the S1–S3 mutants and the WT
showed Ca
2+
-sensitive GTPase activity, but this was
not observed in the case of the S4 and S5 mutants. In
the TG3 structure, Asp324, which coordinates the S3
analog Ca
2+
-binding site directly and is located on a
loop forming a part of the S3B analog site, is responsi-
ble for a switch between GTP and Ca

2+
binding by
opening a channel for the acyl Gln donor substrate
[16,17]. As Ca
2+
binding can decrease GTP binding
and GTPase activity in the case of TG2, too, S4 and
S5 could be responsible for regulatinon of GTPase
activity and the proper regulation of the distinct trans-
glutaminase and GTPase activities. When these two
sites could not bind Ca
2+
, the GTPase activity of TG2
was not inhibited by increasing the Ca
2+
concentra-
tion. Moreover, these two mutations resulted in
increased basal GTPase activity and altered
GTP ⁄ GDP binding. The two mutated sites are steri-
cally close to the hydrophobic pocket for GTP ⁄ GDP
binding, which is formed by the side chains from
Phe174, Val479, Met483, Leu582, and Tyr583 [13].
They may conformationally influence GTP binding
and the GTPase activity of TG2 by changing the posi-
tion of Phe174, the docking amino acid, and of
Lys173, which is the nucleophilic attacking group in
GTP hydrolysis (Fig. 2, lower panel). The mutations
can result in a conformational state that speeds up
GDP ⁄ GTP exchange via decreasing the docking time
of GTP and facilitating the release of GDP, which ulti-

mately results in higher GTPase activity and lower
GTP binding. Such a change could also be responsible
for the lack of GTP incorporation signal in our phot-
olabeling experiments. However, it cannot be excluded
that this happened because the altered surface did not
support the UV light-induced artificial trapping of
GDP after hydrolysis via its guanosine group. A simi-
lar finding was described when the core domain of
TG2 was expressed alone and tested for GTP bind-
ing ⁄ hydrolysis with the same methods [36]. Interest-
ingly, two shorter alternatively spliced forms of TG2
that have lower GTP-binding affinity also have higher
GTPase activity [36a].
Mutagenesis of some of the Ca
2+
-binding sites leads
to decreased binding of celiac autoantibodies against
TG2 to the enzyme. GSE is a chronic disorder of the
small intestine in genetically susceptible individuals.
Wheat gliadin and related prolamins in other cereals
can trigger an autoimmune reaction to TG2 [37,38],
and the resulting autoantibodies might play a role in
the pathogenesis of GSE by modifying the enzyme’s
activities or other functions [39]. Previous results sug-
gested that binding of Ca
2+
to TG2 is needed to pro-
mote the binding of celiac antibodies to the enzyme
[27,28]. S1, S2 and S3A do not have a role in antibody
binding, because the S1, S2 and S3A mutants were

recognized equally as well as the WT. Also, the S3B
and S5 mutants retained considerable antigenicity
towards patient serum samples, making a direct role in
antibody binding improbable for the majority of
patients. In contrast, binding of celiac serum samples
to TG2 was greatly affected by changing S4, suggest-
ing that it may be needed to form a main celiac
epitope. Further clarification of potential anchor
points in this region may help us to understand the
role of antibodies in the pathogenesis of GSE and in
designing new therapy for it.
Members of the mammalian transglutaminase family
have evolved through duplication of a single gene and
subsequent redistribution to distinct chromosomes [40].
On the basis of the available and presented data, a
description of the subsequent evolution of the Ca
2+
-
binding sites of the human enzymes can be attempted.
Sequence comparison (Fig. 7) clearly shows that S2 is
conserved in each transglutaminase, and this by itself
can determine the Ca
2+
dependency of transglutamin-
ase activity, as FXIIIa has only the S2-equivalent site.
Similarly, prostate transglutaminase (TG4) seems to
have only this site. It is likely that these two secreted
enzymes are sufficiently activated by Ca
2+
through

this site in the extracellular space, where the Ca
2+
concentration is high. Transglutaminase 1 works in the
terminally differentiating keratinocytes, where Ca
2+
concentration rises; sequence data show that, in addi-
tion to S2, it may have S1 as well. It seems that intra-
cellular transglutaminases need more sophisticated
Ca
2+
regulation. We propose that, for intracellular
transglutaminase activation, S1, which binds Ca
2+
tightly, is essential, as all intracellular forms have
potential S1s. Actually, sequence comparison suggests
that even the red sea bream and invertebrate Drosoph-
ila transglutaminases have S1 and S2. Sequence com-
parisons also explain why FXIIIa does not have S1:
FXIIIa has a positively charged amino acid (Lys) in
this region. A similar sequence difference may preclude
Ca
2+
binding at S1 of TG4. There are some amino
acids with apolar or positive side chains in S3, S4 and
S5 of FXIIIa, transglutaminase 1 and TG4, and
S3 and S5 in TG3, suggesting that they do not bind
Ca
2+
there. S3 is needed to open the substrate channel
in intracellular transglutaminases. Transglutaminase 5

and transglutaminase 7 probably lost this site; these
two enzymes are located on a different arm of the phy-
logenetic tree of transglutaminases than TG2 or TG3
and transglutaminase 6 [40], and may use another site
for this purpose. Transglutaminase 5, transglutaminase
6 and transglutaminase 7 also have S4 and S5, and
R. Kira
´
ly et al. Ca
2+
-binding sites of TG2
FEBS Journal 276 (2009) 7083–7096 ª 2009 The Authors Journal compilation ª 2009 FEBS 7091
therefore may have similar Ca
2+
regulatory mecha-
nisms as TG2. This perhaps explains how these trans-
glutaminases may compensate for the loss of TG2 in
knockout mice [41].
Experimental procedures
Materials
All materials were purchased from Sigma (St Louis, MO,
USA) unless otherwise indicated.
Transglutaminase enzyme preparations
Wild-type recombinant human TG2s were expressed in
N-terminally (His)6-tagged [42] and GST-fused forms [19].
The fusion tags, which were not found to alter the enzymo-
logical properties of TG2 [36], were left on the protein.
Site-directed mutants were constructed using the Quik-
Change Site-Directed Mutagenesis Kit (Stratagene, La
Jolla, CA, USA). Mutant constructs were checked by

restriction analysis and DNA sequencing (ABI PRISM,
Applied Biosystems, Foster City, CA, USA). Rosetta 2
(Novagen, Darmstadt, Germany) strains were transformed
with wild-type or mutant TG2 containing pET-30 Ek ⁄ LIC–
TG2 vectors. The (His)6-tagged proteins were expressed in
a similar way to that described previously [42], using Pro-
Bond Ni
2+
–nitrilotriacetic acid resin (Invitrogen, Carlsbad,
CA, USA), according to the manufacturer’s instructions.
The protein concentration was determined using the Brad-
ford method (Bio-Rad, Mu
¨
nchen, Germany). The purity
and self-crosslinking activity of proteins were checked by
Coomassie BB staining of SDS ⁄ polyacrylamide gels and by
western blotting (Fig. S1).
Equilibrium dialysis
Ca
2+
binding to TG2 was measured by equilibrium dialy-
sis, with modification of a published procedure [18]. For
every Ca
2+
-binding experiment, only EDTA-rinsed plastic-
ware and high-purity water (Millipore, Billerica, MA,
USA) were used, to prevent Ca
2+
contamination. Recom-
binant TG2 ( 1.7 mgÆmL

)1
) was dialyzed for 48 h at 4 °C
in a 96-well equilibrium dialyzer plate (molecular mass
cutoff 10 kDa; Harvard Bioscience, Holliston, MA, USA)
against 150 lL of dialysis buffer (50 mm Tris ⁄ HCl, 5 mm
mercaptoethanol, pH 7.5) supplemented with 8.3 lCi of
45
CaCl
2
per mL (PerkinElmer, Boston, MA, USA) and
containing different concentrations of cold CaCl
2
. After
the equilibration, the radioactivity was measured by liquid
scintillation counting using Tritosol [43]. The results were
normalized for the protein content of the sample deter-
mined by Bradford reagent and protein purity, which was
measured with alpha imager software ( 90%). The free
Ca
2+
concentration was calculated by maxchelator and
Fabiato and Fabiato’s computer program [44,45]. The
Ca
2+
-binding curves and binding parameters were fitted
and calculated using graphpad prism software (GraphPad
Software, Inc., La Jolla, CA, USA). To reduce the possible
incidental errors of the radioactive method, we used Ca
2+
solutions of high specific activity: this was 10 000–

23 000 c.p.m. per nmol Ca
2+
in the case of measurements
at 1.67 mm Ca
2+
, and  3.2 000 000 c.p.m. per nmol
Ca
2+
at smaller Ca
2+
concentrations, to increase the accu-
racy of measurement. The standard errors of parallel coun-
ter measurements and protein determinations were lower
than 6%, mostly less than 3%, in every case. The differ-
ence in radioactivity between the two chambers was always
highly significant (P < 0.01).
Fig. 7. Multiple sequence alignment of Ca
2+
-binding sites of transglutaminases. Sequence alignments of Ca
2+
-binding sites of TG2 com-
pared with the other members of transglutaminase family using
CLUSTALW. The bold characters mark the proven Ca
2+
-binding sites, and the
underlined characters indicate the amino acids that coordinate Ca
2+
in known crystal structures. Characters in bold italics indicate potential
Ca
2+

-binding sites as compared with those in TG2. The amino acids in the frames may preclude Ca
2+
binding as compared with the homo-
logous sites in other members of the transglutaminase family in which Ca
2+
-binding sites have been verified. Invertebrate transglutaminase
used in the alignments: Q9VLU2_DROME is the A isoform of Drosophila melanogaster transglutaminase. TGM2_PAGMA is the red sea
bream (Pagrus major) TG2. ‘x’ indicates the amino acids that are conserved in the enzyme family.
Ca
2+
-binding sites of TG2 R. Kira
´
ly et al.
7092 FEBS Journal 276 (2009) 7083–7096 ª 2009 The Authors Journal compilation ª 2009 FEBS
ITC and ICP-OES
The Ca
2+
-binding properties of TG2 were measured using
ITC (VP-ITC MicroCalorimeter; MicroCal, Piscataway,
NJ, USA). High-purity recombinant TG2 was produced
by ion exchange for ITC measurements. After affinity
chromatography, the sample buffer was changed to FPLC
A binding buffer containing 50 mm Tris ⁄ HCl (pH 7.8),
50 mm NaCl, and 1 mm EDTA. After filtration, the pro-
tein was loaded onto a HiTrap Q HP column (AP Bio-
tech, Uppsala, Sweden). TG2 was eluted with a linear
gradient of FPLC B buffer (FPLC A buffer containing
1 m NaCl). The purity of eluted fractions was checked by
SDS ⁄ PAGE and Coomassie staining (Fermentas, Burling-
ton, ON, Canada). The appropriate fractions were concen-

trated using centrifugal concentrators (Millipore), and
dialyzed against ITC buffer (25 mm Tris ⁄ HCl, pH 7.5,
1mm 2-mercaptoethanol) with 0.5 mm EDTA and then
twice against ITC buffer with Chelex 100 at 4 °C for 6 h
each. The ITC experiments were performed at 25 °C; a
40 lm wild-type TG2 sample was placed into the sample
chamber, and then, from a 2 mm CaCl
2
solution (in ITC
buffer), the following volumes were injected into it:
5 · 2 lL, 6 · 5 lL, 5 · 15 lL, 7 · 20 lL, and 2 · 25 lL.
In the case of S1 mutants, a 23 lm TG2 sample was used,
and from a 0.6 mm CaCl
2
solution, 4 · 2 lL, 5 · 4 lL,
6 · 8 lL, 5 · 20 lL and 5 · 30 lL volumes were injected.
ITC data were analyzed using the origin data analysis
package. Data were fitted using the two and the single set
of identical sites models, respectively. All parameters were
allowed to float freely during fitting.
The sample was prepared in the same way for ICP-OES
(Analab Ltd, Debrecen, Hungary) experiments. The effec-
tive concentrations of Ca
2+
stock solution and buffers were
also checked by ICP-OES.
CD analysis
CD spectra were recorded on a Jasco-810 spectropolarime-
ter at room temperature in 50 mm Tris ⁄ HCl (pH 7.5) con-
taining 1 mm EDTA. CD deconvolutions were performed

with the continll, cdsstr and selcon3 analysis programs,
kindly provided by Dichroweb [24,25].
Microtiter plate assay of transglutaminase
activity
The microtiter plate assay, based on the incorporation of
5-(biotinamido)pentylamine (Molecular Probes, Invitrogen,
Carlsbad, CA, USA) into immobilized N,N-dimethylated
casein was used as described previously [39], with the
following modifications. Transglutaminase activity was
measured using 5 mm CaCl
2
and 0.4 lg of TG2. The
reaction was started with 50 lL of enzyme mixture, and
was performed at 37 °C for 30 min.
Filter paper assay of transglutaminase activity
The assay is based on the incorporation of
[1,4(n)-
3
H]putrescine (30 CiÆmmol
)1
; PerkinElmer) into
N,N-dimethylated casein as described previously [39]. The
100 lL reaction mixture contained 5 lg of recombinant
TG2. The reaction was started by the addition of CaCl
2
(5 mm final concentration) and incubated at 37 °C for
5 min.
GTPase activity assay and direct GTP
photolabeling
GTPase activity was determined by the charcoal method as

previously described [39], with minor modifications. The
released [
32
P]P
i
was determined by counting of 150 lLof
the supernatant. GTP photolabeling was performed as
previously described [46].
Molecular modeling and sequence alignment
The X-ray structures of human TG2, TG3 and FXIIIa were
retrieved from the RCSB Protein Data Bank (http://
www.rcsb.org/pdb). Graphical analysis was performed on a
Silicon Graphics Fuel workstation using the grasp and
sybyl program packages (Tripos, St Louis, MO, USA),
rasmol, and vmd [47].The sequence alignments were
performed using clustalw [48].
Statistical analysis
For statistical data analysis, one-way ANOVA (microsoft
excel; Microsoft Inc., Denver, CO, USA), Mann–Whitney
U-test (analyse-it; Analyse-it Software Ltd, Leeds, UK)
and unpaired t-test (graphpad prism) were used. The
Ca
2+
-binding parameters were determined using graphpad
prism and origin software.
Acknowledgements
We thank P. Bagossi for his help with molecular model-
ing, Mrs Z. Darai and Mrs A. Klem for technical assis-
tance, J. Fidy of Semmelweiss University for providing
the possibility of ITC measurement, M. Braun and

Analab Ltd, Debrecen, Hungary for ICP-OES measure-
ments, the Retroviral Biochemistry Laboratory in our
department for suggestions concerning protein purifica-
tion procedures, and M. Hughes for hosting R. Kira
´
ly
at the Institute for Cell and Molecular Biosciences,
University of Newcastle upon Tyne, UK. This work was
supported by the following grants: Hungarian Scientific
Research Funds (OTKA NI 67877, K 61868) and EU
grants MRTN-CT-2006-036032, MRTN-CT-2006-
035624, and LSHB-CT-2007-037730. R. Kira
´
ly was the
R. Kira
´
ly et al. Ca
2+
-binding sites of TG2
FEBS Journal 276 (2009) 7083–7096 ª 2009 The Authors Journal compilation ª 2009 FEBS 7093
recipient of a travel grant from the European Science
Foundation’s Transglutaminases Program and Erasmus
Program and the Dea
´
k Ferenc Fellowship of the
Hungarian Ministry of Education and Culture.
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Supporting information
The following supplementary material is available:
Doc. S1. Supplementary Experimental procedures.
Fig. S1. Self-crosslinking of TG2 variants.
Fig. S2. Purity of and monoclonal antibody binding to
recombinant wild-type and mutant TG2s.
Table S1. Primers used for mutagenesis.
R. Kira
´
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2+
-binding sites of TG2
FEBS Journal 276 (2009) 7083–7096 ª 2009 The Authors Journal compilation ª 2009 FEBS 7095
Table S2. Statistical analysis of celiac antibody binding
in Fig. 6.

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Ca
2+
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´
ly et al.
7096 FEBS Journal 276 (2009) 7083–7096 ª 2009 The Authors Journal compilation ª 2009 FEBS