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In Situ Detection of Active Transglutaminases for Keratinocyte Type (TGase 1) and Tissue Type (TGase
2) Using Fluorescence-Labeled Highly Reactive Substrate Peptides
Miho Itoh, Tadafumi Kawamoto, Hideki Tatsukawa, Soichi Kojima, Kiyofumi Yamanishi and Kiyotaka Hitomi
J Histochem Cytochem 2011 59: 180
DOI: 10.1369/jhc.2010.957225
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JHC59210.1369/jhc.2010.957225Itoh et

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ArticleJournal of Histochemistry & Cytochemistry 59(2) 180­–187© The Author(s) 2011
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DOI: 10.1369/jhc.2010.957225
http://jhc.sagepub.com

In Situ Detection of Active Transglutaminases for Keratinocyte
Type (TGase 1) and Tissue Type (TGase 2) Using
Fluorescence-Labeled Highly Reactive Substrate Peptides
Miho Itoh,Tadafumi Kawamoto, Hideki Tatsukawa, Soichi Kojima, Kiyofumi Yamanishi
and Kiyotaka Hitomi

Department of Applied Molecular Biosciences, Graduate School of Bioagricultural Sciences, Nagoya University, Chikusa, Nagoya, Japan (MI, KH);
Radioisotope Research Institute, School of Dental Medicine, Tsurumi University, Tsurumi,Yokohama, Japan (TK); Molecular Ligand Biology Research
Team, Genomics Research Group, Chemical Biology Department, Riken ASI, Wako, Saitama, Japan (HT, SK); and Department of Dermatology, Hyogo
College of Medicine, Nishinomiya, Hyogo, Japan (KY)

Summary
Transglutaminase is a calcium-dependent enzyme that posttranslationally modifies proteins by cross-linking between
glutamine and lysine residues or attachment of a primary amine to specific polypeptide-bound glutamine residues. Eight
isozymes play essential roles in various mammalian biological processes.The authors have recently identified 12–amino acidpreferred substrate peptide sequences that are highly reactive and act in an isozyme-specific manner. In this study, a rapid,
isozyme-specific, and sensitive detection of active keratinocyte type (TGase 1) and tissue type (TGase 2) was successful using
fluorescence-labeled peptides. This procedure involved using whole-body sections of a mouse to extensively analyze thetissue distribution of both enzymes that revealed clearly distinct patterns. Strong active TGase 1 was observed in epithelial
tissues such as tongue, developing teeth, forestomach, and skin epidermis. Significantly active TGase 2 was observed invarious types of tissues as predicted and at particularly higher levels in the intestinal mucosa, muscle membrane, and wholeveins in the liver.This manuscript contains online supplemental material at http://www.jhc.org. Please visit this article online
to view these materials. (J Histochem Cytochem 59:180–187, 2011)
Keywords
transglutaminase, calcium, epithelium, connective tissue
Transglutaminase (TGase, EC 2. 3. 2.13) is a calciumdependent enzyme that catalyzes the formation of isopeptide cross-links between glutamine and lysine residues in avariety of proteins (Griffin et al. 2002; Lorand and Graham
2003). In addition, TGase catalyzes incorporation of primary amines into protein-bound glutamine residue or conversion into glutamic acid by deamidation. In mammals,
eight TGase isozymes have been identified (TGases 1-7,
factor XIII), comprising a protein family with unique tissue
distribution and physiological roles.
TGase 1, TGase 3, and TGase 5 are involved in skin formation possibly by cooperatively cross-linking structuralproteins in keratinocytes (Aeschlimann et al. 1998; Eckert
et al. 2005; Hitomi 2005). Among the three isozymes expressing in the epidermis, TGase 1 (keratinocyte type) has beencharacterized as an essential isozyme because its knockout
mice exhibit impaired epidermis (Matsuki et al. 1998). In differentiating keratinocytes, TGase 1 cross-links structural proteins such as involucrin and loricirn to form a highly

specialized protein structure, called a cornified envelop,
which contributes to the barrier function of the outermost layers (Kalinin et al. 2002; Candi et al. 2005).
TGase 2 (tissue-type TGase) is widely expressed andinvolved in multiple events such as cell growth, differentiation, and apoptosis via enzymatic modification of extracellular matrix proteins, transcription factors, and signaling
molecules (Fesus and Piacentini 2002; Beninati andPiacentini 2004; Jeon and Kim 2006; Mehta et al. 2006;
Tatsukawa et al. 2009). The phenotype of TGase 2 knockout

Received for publication August 10, 2010; accepted September 8, 2010
Corresponding Author:
Kiyotaka Hitomi, Department of Applied Molecular Biosciences,
Graduate School of Bioagricultural Sciences, Nagoya University, Chikusa,
Nagoya 464-8601, Japan.
E-mail: hitomi@agr.nagoya-u.ac.jp

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In Situ Detection of Active Transglutaminases
mice shows abnormal phagocytosis, apoptosis, and woundhealing (Sarang et al. 2009). Despite extensive studies, substrate proteins of physiological relevance acted upon by
TGase 2 and tissue distribution of the enzyme are still underinvestigation.
Factor XIII, which stabilizes fibrin clots, is essential forblood coagulation (Ichinose 2001). TGase 4 is expressed in
the prostate and reported to be involved in the formation ofplug in rodents (Esposito et al. 1996). Little information isavailable for TGase 6 and TGase 7 (Grenard et al. 2001).
In the catalytic reaction of TGase, the glutamine-donor
substrate and the enzyme form the reaction intermediate
and then react to glutamine-acceptor molecules (proteinbound lysine and/or primary amine). Therefore, the recognition of reactive glutamine residue(s) in substrates is
crucial for the enzymatic reaction. Recently, using a random peptide library, we have characterized the preferredglutamine-donor substrate sequences for TGases that showsa unique reaction tendency to each isozyme (Sugimura et al.
2006). In these studies, several 12-mer substrate peptide
sequences for major TGases have been identified: factor
XIII, TGase 1, and TGase 2 (Sugimura et al. 2008; Hitomi,
Kitamura, and Sugimura 2009). The most favorable
sequence for each isozyme appeared to act as a substratewith high reactivities and isozyme specificities, even in the
peptide form. Because the preferred substrate peptides canefficiently react with protein-bound lysine residue and primary amines, these peptides have been used in several
applications, including sensitive in vitro assays (Sugimura
et al. 2007; Perez Alea et al. 2009; Hitomi, Kitamura, Perez
Alea, et al. 2009).
Furthermore, using fluorescence-labeled preferred substrate peptide (pepK5), we were also successful in detecting
active TGase 1 in the human and mouse skin epidermis(Sugimura et al. 2008; Akiyama et al. 2010). The location of
the active TGase 1 was observed by the analysis of the outmost layer of the epidermis. Because this detection method
was simple, sensitive, and isozyme specific, we attempted
to expand the analysis to the whole-tissue sections and to
the detection of active TGase 2.
In this study, frozen sections of the whole mouse wereused to perform rapid and sensitive enzymatic reactions forthe analysis of tissue distribution in mice, similar to thatobserved in mouse skin. Using the sections, both active
enzymes for TGase 1 and TGase 2 could be detected for theextensive evaluation of tissue distribution. Moreover, areas
where both isozymes were enzymatically active have beencharacterized in several tissues, showing distinct patterns
between the two enzymes. This study first describes
the procedure for in situ detection of active TGases in an
isozyme-specific manner with high sensitivity and provides
novel information regarding tissue distribution of TGase 1
and TGase 2.

Materials and Methods
Materials
Fluorescein isothiocyanate (FITC)–labeled oligopeptides
were synthesized by Biologica Co. (Nagoya, Japan). Eachpeptide was dissolved in dimethylsulfoxide at 50 mM as astock solution. Embedding medium for the tissue sections
and whole mouse body was Tissue medium from Chiba
Medical (Chiba, Japan) and SCEM (Leica Microsystems;
Tokyo, Japan), respectively. Other chemical reagents wereobtained from Sigma-Aldrich (St Louis, MO) or WAKO
Chemicals (Osaka, Japan).

Preparation of Tissue Sections and Staining
by Hematoxylin and Eosin
All animal experiments were carried out according to theguidelines of each institute (Nagoya University, Hyogo
College of Medicine, RIKEN Institute, and Tsurumi
University). TGase 1 knockout mouse was established previously (Matsuki et al. 1998), and TGase 2 knockout mouse
was provided by Dr. Robert Graham (Nanda et al. 2001).
Frozen tissue sections of skin and liver in TGasedeficient mice were prepared without fixation at a 4- to
6-µm thickness according to the standard method. The sections were stored at −80C until use.
In the case of whole mouse body, 9-day-old mouse(male) was used. According to the method described by
Kawamoto (Kawamoto and Shimizu 2000; Kawamoto2003), the whole-body sections were prepared using a multipurpose cryosection preparation kit (Section Lab Co. Ltd.;
Hiroshima, Japan). First, the mouse was frozen in cold hexane (−93C) and then freeze-embedded with SCEM. Ten-µm
thick sections were cut with a cryomicrotome (Leica
CM3050S; Leica Co. Ltd., Wetzlar, Germany) from the frozen specimen block and collected with cryofilm. After drying the section, the sections were used for in situ enzymatic
reaction without fixation. For hematoxylin and eosin (H&E)
staining, tissue sections were fixed by 4% paraformaldehyde and then mounted with SCEM.

In Situ Detection of Active TGase
Sections were dried and then blocked with 1% bovine
serum albumin (Sigma-Aldrich) in PBS (10 mM
Na-phosphate, pH 8.0, 150 mM NaCl) at room temperaturefor 30 min. The sections were incubated for 90 min at 37C
in a solution containing 100 mM Tris/HCl (pH 8.0), 1 mM
dithiothreitol, and 5 mM CaCl2 in the presence of FITClabeled peptide at the final concentration of 1 µM. In thereaction mixture, calcium ion was contained at higher thanthe physiological level to enhance the products. After enzymatic reaction, PBS containing 25 mM EDTA was added to

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Figure 1. In situ detection of active TGase 1 and TGase 2 in mouse tissue sections. (A) Skin sections from the wild-type (+/+) and TGase
1−null (−/−) mice were reacted with 1 µM FITC-pepK5 in the presence of 5 mM CaCl2. The differential interference images (DIC), thefluorescence images, and their merged images were obtained. As negative controls, reaction samples using 1 µM FITC-pepK5QN or withaddition of 5 mM EDTA were carried out. (B) Liver sections from the wild-type (+/+) and the TGase 2−null (−/−) mice were subjected to insitu reaction using 1 µM FITC-pepT26 or 1 µM FITC-pepT26QN as described in (A). The bars indicate 50 µm.

stop the reaction via chelating calcium ion. Then, after
washing with PBS three times at room temperature for 5min, antifading solution was added to the sections forobservation.

Microscopic Observation and Analyses
Samples were mainly observed under a Keyence fluorescence microscope (BZ-9000; Osaka, Japan) for analyses ofwhole-mouse section using a ×4 lens (NA 0.20). Samples
for H&E staining were also observed with the same lens.
For the skin and liver sections in TGase-deficient mice
(Fig. 1), the samples were observed under a LSM5PASCAL confocal laser-scanning microscope, using a
combination of ×20 (NA 0.50) or ×40 water immersion lens(NA 1.2w) and differential interference contrast (Zeiss,
Göttingen, Germany).
The free software ImageJ (version 1.43u; image processing and analyzing java; http://rsbweb.nih.gov/ij/) wasused for linear adjustment of fluorescent images. This
software was also used for the image mapping using
pseudo-colors from the fluorescent images as a standard
procedure.

Table 1. Isozyme-Specific Substrate Peptides for TGase 1(pepK5) and TGase 2 (pepT26)
PeptideTarget Enzyme

pepK5
pepK5QN
pepT26TGase 1

pepT26QN

TGase 2

SequenceYEQHKLPSSWPFYENHKLPSSWPF
HQSYVDPWNLDHHNSYVDPWNLDH

pepK5QN and pepT26QN are the mutant peptides in which the reactive
glutamine residue was substituted with asparagine. These peptides werelabeled with FITC at the N-terminus.

Results
Reactions of Fluorescence-Labeled Favorable
Substrate Peptides for TGase 1 (pepK5) andTGase 2 (pepT26)
We previously identified favorable substrate sequences thatwere screened from the M13 phage-displayed peptide
library (Hitomi, Kitamura, and Sugimura 2009). The
respective sequences of pepK5 and pepT26 for TGase 1
and TGase 2 are shown in Table 1. Taking advantage of the

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In Situ Detection of Active Transglutaminases

Figure 2. In situ reaction patterns of whole-mouse body sections using FITC-pepK5 and FITC-pepT26. Whole-body sections wereprepared and stained with hematoxylin and eosin (HE). The sections were reacted with 1 µM FITC-labeled peptides (pepK5, pepK5QN,
pepT26, and pepT26QN) in the presence of CaCl2. (A) Midline and (B) sideline.The bars indicate 1 cm. Nonspecific signals in the pictures
for the QN mutants may be due to excrement in the bowel.

isozyme-specific reactivities of these peptides, in situ
active TGase 1 and TGase 2 were detected using FITClabeled peptides. In this procedure, the peptides reacted tothe enzyme and covalently cross-linked to glutamineacceptor substrate proteins in tissues, which provide fluorescence signals. Mutant peptides, in which each reactive
glutamine residue had been replaced by asparagine, werealso prepared as negative controls (pepK5QN, pepT26QN).

To confirm the efficiency of staining, we initially investigated detection using the sections from knockout mice foreach TGase (Fig. 1). Because highly active TGase 1 andTGase 2 are respectively expressed in the skin and the liver(Lorand and Graham 2003), these tissue sections in both wildtype and knockout mice were subjected to the reactions.
In the skin section from wild-type mice, a significant fluorescence signal resulting from cross-linked FITC-pepK5 was

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Itoh et al.

Figure 3. Color images of fluorescence signals detected in the sections reacted with FITC-pepK5 and FITC-pepT26. Based on the
fluorescence images of Figure 2, pseudo-color images were produced using ImageJ (version 1.43u). To indicate the fluorescence intensity,
we divided level of brightness into six classes (from background to maximum area). The six colors, as shown in the box (left in each
picture), indicate the intensity in five grades (white, yellow, orange, purple, dark blue) and black as background. Tissues are indicated asfollows: Br, brain; Es, esophagus; Fst, forestomach; H, heart; It, intestine; Ki, kidney; Li, liver; Lu, lung; Sp, spleen; Spi, spine; St, stomach; Te,
testis; Th, thymus; Tn, tongue. HE, hematoxylin and eosin. The bars indicate 1 cm.

obtained in cells below the outermost layers, which is consistent with previous results (Fig. 1A; Sugimura et al. 2008). No
apparent signal was observed in the section from the TGase
1–deficient mouse or in the reaction using FITC-pepK5QN.
In addition, no signal was obtained in the presence of EDTA,
suggesting that the reaction is calcium dependent. These
results indicated that active TGase 1 was specifically detectedby the procedure.
In the liver section, a signal for wild-type TGase 2 wasobserved around the intimal and medial area at the central
vein (Fig. 1B). This signal was not observed in the section
from TGase 2 knockout mice. No signal was obtained in thereaction either in the presence of EDTA or after use of themutant peptide, indicating that the detection was specific toactive TGase 2.

Detection of In Situ Active TGases Using
Whole-Mouse Section
To obtain the tissue distribution patterns of the active
enzymes, the same analyses were carried out using sectionsof whole-mouse body. Midline and sideline sections of
9-day-old mice were prepared for the above-mentioned
analyses and were then subjected to in situ detection ofactive TGase as well as H&E staining.

In both midline and sideline sections, apparent signals
were obtained for FITC-pepK5 and FITC-pepT26 reactions(Fig. 2A, midline; Fig 2B, sideline). No significant fluorescence was observed when using the mutant peptide (FITCpepK5QN and FITC-pepT26QN), indicating that the signals
are specifically derived from the enzymatic reaction.
Distinct fluorescence patterns for each cross-linked peptide
indicate that the active forms of each enzyme were detected,
reflecting the different tissue distribution of the two
enzymes. In addition, this reaction is calcium dependent(Suppl. Fig. S1A,B).
To evaluate the localization of active enzymes using
FITC-peptide incorporation, fluorescence intensity was
expressed as pseudo-colors in the region of interest (Fig. 3).
For TGase 1, which is mainly involved in skin formation,much highly active enzyme was observed in the epidermis.
Furthermore, other epithelial tissues such as tongue andforestomach showed the existence of relatively high active
enzyme. Although they had weaker signals than those ofabove tissues, hepatocytes showed a significant level. On
the other hand, wide tissue distribution patterns wereobtained for TGase 2 with various intensities. Digestive tissues such as tongue, esophagus, stomach, and intestine
appeared to contain remarkably high active enzyme.
Significant active TGase 2 was also observed in veins in the

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In Situ Detection of Active Transglutaminases
liver in addition to spine and muscles. The brain membrane
showed significantly active TGase 2, whereas fewer
enzymes were observed in the thymus, spleen, and kidney.

Tissue Distribution of In Situ Active TGases
In Figure 4, we show enlarged patterns for the nine selected
tissues. Results from five epithelial tissues are depicted in
Figure 4A. In the tongue, both active TGase 1 and TGase 2
are expressed mainly in the filiform papilla, where TGase 1
is not expressed in the outermost cornified layer but highly
expressed in underlying cell layers. TGase 2 is also present
in the muscle layers. Bright signals in developing teeth
were obtained only for active TGase 1 and were mainly
located in odontoblast layers and sterate reticulum.
In the stomach of rodents, the glandular stomach is positioned after the forestomach, in which epithelial cells
undergo an organized progressive differentiation to form amature stratified squamous epithelium. Interestingly, active
TGase 1 was significantly detected beneath the outer cornified layer limited to the forestomach area, whereas TGase 2
was weakly expressed in both the glandular and forestomach muscle layers. In the intestine, strong signals wereobtained only for TGase 2, and they were found in themucosal and muscle layers. Enlarged observation of mucosal layers showed that the extracellular matrix area beneaththe epithelial cells on the surface was stained (data notshown). In the skin, active TGase 1 was expectedly detectedin the epidermal layer and hair follicles. In hair follicles, aweak level of active TGase 2 was detected in the central
region, which is different from the pattern of TGase 1.
Results for the other four tissues, where significant signals and mostly distinct patterns for TGase 1 and TGase 2
were observed, are shown in Figure 4B. In the liver, active
TGase 2 was expressed around the endothelium in theregion of the central and interlobular portal veins. In thespine, diaphragm, and testis, active TGase 2 but not TGase
1 was observed. In the spine, TGase 2 was active at theepiphyseal plate and intervertebral disk. These results suggested that active TGase 1 and TGase 2 were located largelyin the epithelial and connective tissues, respectively.

Discussion
Because posttranslational modifications by TGases are
essential for various biological processes in mammals, it isimportant to evaluate their activities with high sensitivity
and in an isozyme-specific manner. Although many in vitro
assay systems have been established to measure the TGase
activity, there is no method for monitoring isozymespecific activity in situ. We recently obtained highly reactive substrate peptides that act as efficient tools for

Figure 4. Tissue distribution of in situ active area of TGase 1
and TGase 2. Enlarged pictures of hematoxylin and eosin (HE)staining (left), fluorescence imaging of FITC-pepK5 (center), and
FITC-pepT26 (right) are shown for each tissue. (A) Tongue: FP,
filiform papilla; ML, muscle layer; L, lip, TB, taste bud. Teeth: DP,
dental papilla; O, odontoblast; D, dentin; E, enamel; A, ameloblast;Sr, sterate reticulum. Stomach: Fst, forestomach (stratified);
GlS, Glandular stomach (monolayer); CL, cornified layer; GP,
gastric pits; ML, muscle layer. Intestine: McL, mucosal layer; MsL,
muscle layer; IV, intestinal villi; IG, intestinal glands; SM, seronous
membrane. Skin: SC, skin cornified layer; E, epidermis; D, dermis;
ST, subcutaneous tissue; HF, hair follicle. (B) Liver: PV, interlobular
portal vein; Hp, hepatocyte. Spine (bone): M, bone marrow; ID,
intervertebral disk; EP, epiphyseal plate. Diaphragm (D): Li, liver;
Lu, lung. Testis: St, stroma; SeT, semiferous cells. The bars indicate
200 µm.

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Itoh et al.

detecting enzymatic activity of several TGases (Sugimura
et al. 2006; Sugimura et al. 2008). More recently, using afluorescence-labeled peptide (pepK5) specific for TGase 1,
we have successfully completed in situ detection of active
TGases both in human and mouse skin (Sugimura et al.
2008; Akiyama et al. 2010). In the present study, we demonstrated that this system could be adapted to investigate
another major isozyme, TGase 2, and to analyze the wholemouse body.
To date, tissue distribution of the expression pattern of
TGase analysis has been reported by immunochemical
analyses (Hiiragi et al. 1999; Griffin et al. 2002). However,immunoblotting of lysates prepared from tissues does notallow precise localization of active TGase. Furthermore,
TGases detected by antibodies are not necessarily active
because the enzymes may be cross-linked themselves orwith other proteins, resulting in inactivation. The procedure
performed in this study, however, could detect active TGaselocated in tissues.
In situ detection of active TGase has been possible byobserving reaction products that incorporate FITC-labeled
cadaverine or green fluorescent protein (GFP) fused withglutamine-containing proteins (Furutani et al. 2001; Ojiet al. 2006). However, isozyme specificity cannot be evaluated by these methods. Two substrate peptides (pepK5 andpepT26), which react specifically with each isozyme invitro, enable detection of the distribution of active TGase in
an isozyme-specific manner as shown in this study.
TGase 1 and TGase 2 are mainly localized in the membrane and the cytoplasm, respectively (Lorand and Graham
2003). During reaction, there may be some leakage of the
enzymes from the tissue sections, which might decrease thedetection of intracellular active TGase. However, the resultsof the detection can be evaluated because the signals in theliver and skin as well as other tissues are consistent with theprevious results obtained by immunohistochemical analysis.
Although the results of in situ detection of both active
TGases were mostly consistent with previous reports, novel
findings were observed in several tissues. TGase 1, originally identified as a skin keratinocyte-type TGase, wasdetected at significant levels in other epithelial tissues suchas the tongue and forestomach. Thus, TGase 1 is found in an
active form primarily in such stratified epithelial tissues. In
these TGase 1–expressing tissues, the enzyme may contribute to sustaining these tissues against the external environment by cross-linking structural proteins. In addition, novel
findings include having significant expression in the odontoblast and epiphyseal plate in developing teeth and being
weakly active in hepatocytes. Analysis using cultured cells
for these cells may provide information on the physiological significance of TGase 1.
In contrast, active TGase 2 was observed primarily
around single epithelial cells and in connective tissues.
For example, significant signals were obtained beneathepithelial cells in the intestinal mucosa and around veins in
the liver. Furthermore, other connective tissues such asmuscle membrane and chondrocytes (data not shown)showed apparent signals. TGase 2, which plays a role instabilizing the extracellular matrix by cross-linking reactions, may maintain the integrity of these connective tissues. Identification of enzyme substrates and observation of
signals in these tissues will provide valuable information on
the physiological significance of TGases.
In conclusion, in situ sensitive and specific detection of
the active form of two major isozymes, TGase 1 and TGase2, was successful. A number of diseases such as autoimmune disease, neoplastic disease, neuronal degeneration,
and aberrant skin formation result from the aberrant expression of these TGases (Huber et al. 1995; Sollid 2002; Ruan
and Johnson 2007; Mehta 2009). Our procedure may be
applicable for clinical diagnosis of diseases in which there
is abnormal TGase activity. Furthermore, it will be applied
for several investigations such as intracellular activity by
efficiently introducing peptides into cells (Van Nooden2010).
Acknowledgments
We greatly appreciate Dr. Masatoshi Maki and Dr. Hideki Shibata
in our laboratory for providing valuable suggestions. The knockout mouse for TGase 2 was kindly provided by Dr. Robert
Graham (Victor Chang Cardiac Institute, Australia).

Declaration of Conflicting Interests
The author(s) declared no potential conflicts of interest with respect
to the authorship and/or publication of this article.

Funding
The author(s) disclosed receipt of the following financial support for the research and/or authorship of this article: This work
was supported by a Grant-in-Aid for Scientific Research on
Innovative Areas (No. 20200072) (to KH) from the Ministry of
Education, Sports, Science and Technology (MEXT, Japan).
This work was also supported in part by a grant from the
Chemical Genomics Research Program from RIKEN (to SK)
and by a Grant-in-Aid for Scientific Research (C) from JSPS
(No. 20591359) (to KY).

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