Each of the S1–S5 mutants binds fewer than six Ca2+, S1 is a strong Ca2+-binding site, and mutation of one site resulted in the loss of more than one bound Ca2+, suggesting cooperativity
Trang 1sites of human transglutaminase 2 characterized by
site-directed mutagenesis
Ro´bert Kira´ly1,2, E´va Cs}osz2, Tibor Kurta´n3, Sa´ndor Antus3, Krisztia´n Szigeti4, Zso´fia Simon-Vecsei2, Ilma Rita Korponay-Szabo´5,6, Zsolt Keresztessy2and La´szlo´ Fe´su¨s1,2
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: kiralyr@dote.hu; fesus@dote.hu
(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 Ca2+-regulated biochemical activities, including transglutamylation and GTP hydrolysis The structure of the Ca2+-binding form of the human enzyme is not known, and its Ca2+-binding sites have not been fully characterized By mutagenesis, we have targeted its active site Cys, three sites based on homology to Ca2+-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 Ca2+ Each of the S1–S5 mutants binds fewer than six Ca2+, S1 is a strong Ca2+-binding site, and mutation of one site resulted in the loss of more than one bound
Ca2+, 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 Ca2+, 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 Ca2+-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.
Trang 2of 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 Ca2+-activated transglutaminase
enzyme performing post-translational protein
modifica-tion via the incorporamodifica-tion 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 Ca2+-binding pocket, where the main chain
oxy-gen of Ala457 mediates Ca2+ 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 Ca2+-binding sites (S1–S3): S1 is formed by
Asn221–Asn229 and a water molecule – at this site,
TG3 permanently binds a sole Ca2+with high affinity
that could derive from the actual expression system
[16]; S2 is similar to the heptacoordinated Ca2+-binding
site of FXIIIa, involving Asn393, Ser415, Glu443, Glu448 and two directly coordinated water molecules; and at S3, Ca2+ 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 Ca2+, suggesting that further binding sites exist Indeed, TG2 has several negatively charged amino acids with high surface potential that might serve as Ca2+-binding sites [19] The aim of the present study was to identify the exact Ca2+-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 Ca2+ 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 Ca2+ 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
Ca2+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 Ca2+ 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 Ca2+, which could be derived from the expression system, similarly to the case with recombinant TG3 [16] This tightly bound Ca2+ has an affinity for TG2 that is comparable to its affinity for EDTA To determine the
Ca2+-binding properties of the recombinant WT, equi-librium dialysis measurements were performed The results showed that the WT can bind about six Ca2+ (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
Ca2+binding to TG2 per mol protein, with high affin-ity (Kd= 0.1 lm) The next five Ca2+ bind with very low and comparable affinity to the enzyme The observed difference between the Ca2+-bound active form and the inactive form of TG2 suggests a large
Trang 3conformational change during the Ca2+ activation
process, which could be accompanied by a significant
entropy change, explaining the small enthalpy change
In the presence of Ca2+, 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 25C than during the 2 days of equilibrium dialysis
at 4C We also wished to clarify whether this process
altered Ca2+-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 Ca2+, 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 Ca2+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 Ca2+-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
Ca2+-binding sites (S1 and S3, respectively) The S2 mutants were planned on the basis of homology to the
0 1 2 3 4 5 6 7 8
C277S WT
[Ca 2+ ] free (m M )
A
–0.5
–0.4
–0.3
–0.2
–0.1
0.0
–0.10 –0.05 0.00
–0.8 –0.7 –0.6 –0.5 –0.4 –0.3 –0.2 –0.1 0.0
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
DH = –421.5 ± 83.7
DS = 30.5 Kcal·mol–1
DH = –58.02 ± 23
DS = 24.2 Kcal·mol–1
n = 5.46 ± 0.08
DH = –784.5 ± 38
DS = 21.0 Kcal·mol–1
n = 1.7 ± 0.06
Fig 1 Ca2+binding of recombinant wild-type, C277S mutant and S1 mutant TG2 (A) Ca2+-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.
Trang 4Ca2+-binding site of FXIIIa, which has strong
similar-ities to one of the TG3 Ca2+-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 Ca2+-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 Ca2+ 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 T
432 G R D E R E D I T
S5
149 Y L N S Q Q Q R Q Q Y
149 Y L D S E E E R Q E Y
S4
326 D K S Q M I W N
326 D K S E M I W N
S3B
305 H N Q S S S L
305 H D Q N S N L
S3A
445 Y P Q G S S Q Q R Q A
445 Y P E G S S E E R E A
S2B
395 A Q V S A N V
395 A E V N A D V
S2A
228 V S C S N N Q G V
228 V N C N D D Q G V
S1
Mutant sequence Original sequence
Mutant
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.
Trang 5potential for Ca2+ 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
Ca2+binding of mutant TG2 proteins
To compare the Ca2+binding of wild-type and mutant enzymes in equilibrium dialysis, a free Ca2+ 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
Ca2+ 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 Ca2+ than the WT
at 1.7 mm free Ca2+ concentration (Fig 4A), and the mean values were significantly different (P < 0.0001),
as calculated using ANOVA The experimental Ca2+ -binding values confirmed that each of the five mutage-nized sites contributes to Ca2+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 Ca2+-binding properties For instance, in the S1 mutant, the number of bound Ca2+ dropped from six to two
Using ICP-OES, we tested whether TG2 mutated at the site homologous to the high-affinity Ca2+-binding site of TG3 (S1) still binds Ca2+ after purification The result clearly showed that the S1 mutant cannot bind Ca2+ after dialysis with EDTA (< 0.03 mol
Ca2+ per mol TG2), whereas the WT binds 0.5 mol
Ca2+ per mol TG2 under the same conditions This result means that TG2 also has a Ca2+-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)
Ca2+-dependent transglutaminase activity of mutant TG2 proteins
As the transglutaminase activity of TG2 is Ca2+ -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
–10
–5
0
5
10
15
2 decimol
Wavelength (nm)
S5 S4 W
S2A
S1, S2B S3A,S3B
Fig 3 CD spectra of recombinant TG2 proteins.
Trang 6in solution versus bound to the surface At higher
Ca2+ concentrations, there was no significant increase
in the activities, which means that increased Ca2+
con-centrations cannot compensate for the loss of specific
Ca2+-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 Ca2+ 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
Ca2+ 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 Ca2+ concentrations for most of the mutants, similarly to what was seen with the WT (Fig 5B)
A
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
mol/mol
S5 S4 S3B S3A S2B S2A S1 WT
Mutants
0
20
40
60
80
100
120
Filter paper method Microtiter plate method
0 1 2 3 4 5 6 7 8 9
[Ca 2+ ] (m M )
WT S1 S2A S2B S3A S3B S4 S5
B
0 20 40 60 80 100 120
GTP 0 µ M /Ca 2+ 5 m M GTP 100 µ M /Ca 2+ 5 m M
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 DA405 (minÆmg))1protein in the case of the microtiter plate method, and 77.4 pmol putrescine (minÆmg))1protein in the case of the filter paper method in the presence of 5 m M 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 Ca2+concentration was 5 m M
Trang 7Interestingly, 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 Ca2+
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
Ca2+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 Ca2+-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 Ca2+ in the case
of guinea pig TG2 [27] Therefore, we also examined the effect of Ca2+ and GDP on the binding of celiac autoantibodies to mutant TG2s The presence of 2 mm EDTA, 20 lm GDP or 5 mm Ca2+ 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 Ca2+-binding properties
of TG2 and its mutants and the role of five Ca2+ -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
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 Ca2+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 Ca2+ Data are presented
as means with ± standard deviations from three separate
experiments performed in triplicate.
0 25 50 75 100 125 150 175
0.00 20.00 40.00 60.00 80.00 100.00 120.00
+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 l M 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).
Trang 8autoantibodies to the enzyme We found five
nonca-nonical Ca2+-binding sites of TG2, and determined
that one of these, S1, has a tightly bound Ca2+ There
are canonical and noncanonical protein structures that
bind Ca2+, and common to all of them is a high
nega-tive surface potential, derived mainly from Asp or Glu
residues Ca2+, 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 Ca2+-binding domain
structures: EF-hand domain, C-type lectin-like domain,
Ca2+-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 Ca2+-binding domains, which are present in a
large number of Ca2+-binding proteins, do not share
significant similarities with the Ca2+-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 Ca2+-binding sites [21];
how-ever, their data showed that these sites are not
essen-tial for or directly involved in Ca2+ binding The
Ca2+-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 Ca2+ and, in addition, may induce a
change in the structure of the protein Without a
Ca2+-bound X-ray structure, however, it is not
possi-ble to establish the exact participation of the different
side chains in Ca2+binding and selected functions
For TG2, the most important regulatory function
of bound Ca2+ is the initiation of transglutaminase
activity The tightly bound Ca2+ at S1 is not enough
for transglutaminase activity in the case of TG3;
addi-tional Ca2+ 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
Ca2+binding to this site is important for this activity,
binding of Ca2+ to other sites also contributes to the
effective induction of an active transglutaminase
con-formation Binding of Ca2+ 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 Ca2+ binding leads to an enzyme without transglutaminase activity, suggesting that the binding of Ca2+ 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 Ca2+ 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 Ca2+ binding, how three Ca2+-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 Ca2+-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 Ca2+ supports an assumption of positive cooperativity [33] among the Ca2+-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 Ca2+-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 Ca2+ and total inactivation, which means that Ca2+ 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 Ca2+ binding of TG2
How can weak Ca2+-binding sites play such an important role in determining transglutaminase activity when various biophysical measurements did not show significant changes of TG2 after Ca2+binding [34]? In the case of the canonical C2 domain, it is known that
a third Ca2+ 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
Ca2+ is higher, owing to completed coordination spheres [35] of Ca2+ Further study is required to clar-ify whether substrates, other interacting partners or lipid molecules can regulate Ca2+affinity of TG2
Trang 9Interestingly, the S1–S3 mutants and the WT
showed Ca2+-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 Ca2+-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 Ca2+ binding by
opening a channel for the acyl Gln donor substrate
[16,17] As Ca2+ 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 Ca2+, the GTPase activity of TG2
was not inhibited by increasing the Ca2+
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 Ca2+-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 Ca2+ 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 Ca2+ -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 Ca2+ 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 Ca2+ through this site in the extracellular space, where the Ca2+ concentration is high Transglutaminase 1 works in the terminally differentiating keratinocytes, where Ca2+ 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
Ca2+ regulation We propose that, for intracellular transglutaminase activation, S1, which binds Ca2+ 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
Ca2+ 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
Ca2+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
Trang 10therefore may have similar Ca2+ 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 Ni2+–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
Ca2+ binding to TG2 was measured by equilibrium dialy-sis, with modification of a published procedure [18] For every Ca2+-binding experiment, only EDTA-rinsed plastic-ware and high-purity water (Millipore, Billerica, MA, USA) were used, to prevent Ca2+ contamination Recom-binant TG2 ( 1.7 mgÆmL)1) was dialyzed for 48 h at 4C
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
CaCl2 per mL (PerkinElmer, Boston, MA, USA) and containing different concentrations of cold CaCl2 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
Ca2+ concentration was calculated by maxchelator and Fabiato and Fabiato’s computer program [44,45] The
Ca2+-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 Ca2+ solutions of high specific activity: this was 10 000–
23 000 c.p.m per nmol Ca2+in the case of measurements
at 1.67 mm Ca2+, and 3.2 000 000 c.p.m per nmol
Ca2+at smaller Ca2+concentrations, to increase the accu-racy of measurement The standard errors of parallel coun-ter measurements and protein decoun-terminations 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 Ca2+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.