Báo cáo khoa học: Mammalian transglutaminases Identification of substrates as a key to physiological function and physiopathological relevance pot

REVIEW ARTICLE
Mammalian transglutaminases
Identification of substrates as a key to physiological function and
physiopathological relevance
Carla Esposito and Ivana Caputo
Department of Chemistry, University of Salerno, Italy
Mammalian transglutaminases and
their catalytic activity
Transglutaminases (TGs; EC 2.3.2.13) are encoded by
a family of structurally and functionally related genes.
Nine TG genes have been identified, eight of which
encode active enzymes [1]. Only six TG enzymes have
been isolated and characterized at the protein level.
The TG enzyme family (Table 1) comprises: (a) the
intracellular TG1, TG3 and TG5 isoforms, which are
expressed mostly in epithelial tissue; (b) TG2, which is
expressed in various tissue types and occurs in an
intracellular and an extracellular form; (c) TG4, which
is expressed in prostate gland; (d) factor XIII (FXIII),
which is expressed in blood; (e) TG6 and TG7, whose
tissue distribution is unknown; and (f) band 4.2, which
is a component protein of the membrane that has
lost its enzymatic activity, and serves to maintain
erythrocyte membrane integrity [2]. In addition to
diversity at the genetic level, TGs undergo a number
of post-translational modifications, i.e. phosphoryla-
tion, nitrosylation, fatty acylation and proteolytic clea-
vage [2,3].
In most instances, TGs catalyse the post-transla-
tional modification of proteins, a process that results
in the formation of polymerized cross-linked proteins

[3]. TGs catalyse the formation of isopeptide linkages
between the c-carboxamide group of the protein-
bound glutamine residue and the e-amino group of the
protein-bound lysine residue, so that the reaction prod-
uct results in stable, insoluble macromolecular com-
plexes. In addition, TGs catalyse a number of distinct
Keywords
post-translational modification; protein
substrates; proteomics; transglutaminase
Correspondence
C. Esposito Department of Chemistry,
University of Salerno Via S. Allende, 84081
Baronissi, Salerno, Italy
Fax: +39 089 965296
Tel: +39 089 965298
E-mail:
(Received 27 July 2004, revised 3 November
2004, accepted 10 November 2004)
doi:10.1111/j.1742-4658.2004.04476.x
Transglutaminases form a large family of intracellular and extracellular
enzymes that catalyse the Ca
2+
-dependent post-translational modification
of proteins. Despite significant advances in our understanding of the biolo-
gical role of most mammalian transglutaminase isoforms, recent findings
suggest new scenarios, most notably for the ubiquitous tissue transglutami-
nase. It is becoming apparent that some transglutaminases, normally
expressed at low levels in many tissue types, are activated and⁄ or over-
expressed in a variety of diseases, thereby resulting in enhanced concentra-
tions of cross-linked proteins. As applies to all enzymes that exert their

metabolic function by modifying the properties of target proteins, the iden-
tification and characterization of the modified proteins will cast light on
the functions of transglutaminases and their involvement in human dis-
eases. In this paper we review data on the properties of mammalian trans-
glutaminases, particularly as regards their protein substrates and the
relevance of transglutaminase-catalysed reactions in physiological and dis-
ease conditions.
Abbreviations
CE, cell envelope; ECM, extracellular matrix; FXIII, factor XIII; GAPDH, glyceraldehyde-3-phosphate dehydrogenase; GTP, guanosine
triphoshate; PAI-2, plasminogen activator inhibitor 2; SPR, small proline-rich protein; SV, seminal vesicle; TG, transglutaminase.
FEBS Journal 272 (2005) 615–631 ª 2004 FEBS 615
reactions that lead to post-translational modification
of a specific glutamine residue in the substrate [4]. The
TG-catalysed reaction adds new properties to the pro-
tein substrates, thereby enhancing substrate function,
or more generally, altering it.
The biochemical mechanism underlying the enzyme
action involves a ‘ping-pong’ kinetics. The first, rate-
limiting step is transamidation of the c-carboxamide
group of a glutamine residue to form a thiol ester with
an active site cysteine (resulting in the release of
ammonia) followed by transfer of the acyl intermediate
to a nucleophilic substrate, usually the e-amino group
of a peptide-bound lysine residue (Fig. 1). This process
results in the formation of an intermolecular isopeptide
e-(c-glutamyl)lysine cross-link. However, the monomeric
protein units themselves may become cross-linked
internally [5]. Low molecular mass amines, especially
polyamines, can replace lysines in transamidating
reactions and result in the formation of N-mono(c-

glutamyl)polyamine. In the presence of a second react-
ive glutamine residue, the reaction may proceed to
covalent cross-linking between two polypeptide chains
via a N,N-bis(c-glutamyl)polyamine bridge. In the
absence of suitable amines, water can act as a nucleo-
phile and so cause deamidation of protein-bound
glutamine residues [4].
The various TG gene products share a high degree
of sequence similarity. The sequences around the active
site are the most highly conserved (Fig. 2). Elucidation
of the three-dimensional structure of FXIIIA and TG2
[6,7] revealed a cysteine proteinase-like active site
comprising the catalytic triad cysteine, histidine and
aspartic acid that is required for transamidation. A
four-sequential domain arrangement is highly con-
served in TG isoforms [2]. It consists of an N-terminal
b-sandwich, a core (which contains a transamidation
site and a Ca
2+
-binding site, and has a helices and
b sheets in equal amounts), and two C-terminal b-bar-
rel domains. It has been suggested that glutamyl sub-
strates approach the enzymes from the direction of
two b barrels, whereas lysyl substrates might approach
the enzymes from the direction of the active site [2].
Although the relative positions of residues in the sub-
strate-binding site region are highly conserved in TGs,
the charge distribution differs among the various iso-
enzymes. This difference may account for the different
substrate specificities and hence the specialized func-

tions of each isoenzyme.
Intriguingly, TG2 and TG3 possess a site that binds
and hydrolyses GTP even though the site lacks any
obvious sequence similarity with canonical GTP-bind-
ing proteins [7,8]. The primary sequence of TG5
contains a similar GTP-binding pocket, and TG5
transamidating activity is also inhibited by GTP
in vitro [9]. It is noteworthy that TG2 intracellular
GTPase activity, which is involved in the transduction
of extracellular a
1
-adrenergic signals [10], occurs inde-
pendently of cross-linking activity, but both activities
are regulated by binding to GTP and Ca
2+
([11] and
references cited therein). GTP-hydrolyzing and tran-
samidating activities are also regulated by enzyme
translocation from the cytosol to the cell membrane.
In fact, TG2 from the cytosolic compartment has
higher cross-linking activity than membrane TG2,
whereas the GTPase function of TG2 predominates
when the enzyme is associated to cell membranes [12].
Substrate requirements for
transglutaminases
Although the mechanism governing the recognition of
the target amino acids within the TG protein sub-
strates is not known, some indications emerge from
in vitro data. As regards glutamine specificity, two
adjacent glutamine residues act as amine acceptors in a

consecutive reaction, e.g. bA
3
-crystallin [13], sub-
stance P [14], osteonectin [15] and insulin-like growth
factor-binding protein 1 [16]. The spacing between the
targeted glutamine and neighbouring residues is a
crucial factor in the specificity of TGs. Positively
charged residues flanking the glutamine residue dis-
courage the TG reaction, at least in unfolded protein
regions. In contrast, positively charged residues at two
or four residues from the glutamine promote the reac-
tion. Glycines and asparagines adjacent to the target
Table 1. The mammalian transglutaminase family.
Names Synonyms kDa Tissue Location
TG1 TG
K
, keratinocyte TG,
type 1 TG
90 Epithelia Cytosolic,
membrane
TG2 TG
C
, tissue TG,
type 2 TG
80 Ubiquitous Cytosolic,
nuclear,
extracellular
TG3 TG
E
, epidermal TG,

type 3 TG
77 Epithelia Cytosolic
TG4 TG
P
, prostate TG,
type 4 TG
77 Prostate Extracellular
TG5 TG
X
, type 5 TG 81 Epithelia Cytosolic
TG6 TG
Y
, type 6 TG Unknown Unknown Unknown
TG7 TG
Z
, type 7 TG 80 Ubiquitous Unknown
FXIII Factor XIIIA,
plasma TG,
fibrin stabilizing
factor
83 Blood
plasma,
platelets
Extracellular
Band
4.2
Erythrocyte
protein band 4.2
77 Erythrocytes Membrane
TGs and their substrates C. Esposito and I. Caputo

616 FEBS Journal 272 (2005) 615–631 ª 2004 FEBS
glutamine may favour substrate accessibility [17,18].
Proline residues seem to be important in the recogni-
tion of a given glutamine residue by the enzyme. In
fact, a glutamine residue is not recognized as a sub-
strate by the enzyme if it occurs between two proline
residues [19].
Arentz-Hansen et al. examined the selectivity of
human TG2 for glutamine residues, in gliadin peptides,
in the generation of epitopes recognized by coeliac
lesion CD4+ lymphocytes [20]. This was a challenging
study because gliadin is an excellent TG2 substrate
being comprised of  30–50 mol% of glutamine (Q),
15 mol% of proline (P) and 19 mol% of hydrophobic
amino acids [21]. TG2 specifically deamidated Q65
(underlined) in the 57–68 peptide (QLQPFPQP
QLPY)
of A-gliadin. Therefore, in most cases the enzyme
recognized QxP (where x represents a variable amino
acid, and indicates the distance between glutamine and
Fig. 1. TG-catalysed acyl transfer reactions.
The c-carboxamide group of a glutamine
residue (Q-donor) forms a thiol ester with
the active site cysteine, and ammonia is
released. (A) e-(c-Glutamyl)lysine cross-link
formation; (B) N-mono(c-glutamyl)polyamine
formation; (C) deamidation of protein-bound
glutamine residue.
Fig. 2. Comparison of the amino acid sequences of human TGs around the active site (black box). Dashes indicate gaps inserted to optimize
sequence alignment. Boxed regions are regions in which amino acids are conserved in at least four gene products. Grey columns indicate

the presence of conserved amino acids in all TGs.
C. Esposito and I. Caputo TGs and their substrates
FEBS Journal 272 (2005) 615–631 ª 2004 FEBS 617
proline), rather than QP or QxxP [22]. Moreover, to
act as TG substrates, glutamine residues must be
exposed at the surface of the protein or, more gener-
ally, located in terminal extensions protruding from
the compactly folded domains, where they can be
accessible to covalent modification; N- and C-terminal
glutamine residues are not recognized by the enzyme
[19]. Therefore, it appears likely that the secondary
and ⁄ or tertiary structure of the protein, rather than
the location of the glutamine within the primary struc-
ture itself, determines where cross-linking occurs [18].
This is supported by evidence that distinct TGs recog-
nize distinct glutamine residues in the same protein;
for instance, several typical FXIIIA substrates may
also serve as substrates for TG2, albeit with a much
lower affinity [23].
TGs are much less selective toward amine donor
lysine residues than toward glutamine residues. For
example, Lys148, 176, 183, 230, 413 and 457 in the
Aa chain of fibrinogen cross-linked to only glutam-
ines 83 and 86 in plasminogen activator inhibitor 2
(PAI-2) during cross-linking by TG2 and FXIIIA
[24]. As in the case of TG recognition of glutamine
residues, the nature of the amino acids directly pre-
ceding the lysine may influence the latter’s reactivity
[25]. Indeed, uncharged, basic polar and small ali-
phatic residues enhance reactivity, whereas aspartic

acid, glycine, proline and histidine residues reduce
reactivity [26]. An exception to this rule is Lys191 in
glyceraldehyde-3-phosphate dehydrogenase (GAPDH)
which is preceded by a glycine that adversely affects
the TG reaction [27]. Moreover, other GAPDH
lysine residues are not amine donors even though
they are located in regions with sequences that
should enhance their reactivity. These observations
suggest that the steric hindrance between enzyme
and substrate prevents TG recognition of specific
lysine residues. As a result, only a limited number of
lysine residues in lysine-rich peptides ⁄ proteins are
able to act as an amine donor for TG, e.g. one of
five lysyl residues in b-endorphin [28], six of nine in
seminal vesicle (SV) protein IV [5], and one of ten
in both aB-crystallin [29,30] and S100A11 [31].
However, conformational changes in the native pro-
tein induced by protein–protein interactions may
affect the ability of some lysine residues to serve as
TG substrates. Lys191, 268 and 331 of the 26 lysine
residues in GAPDH are reactive amine donor sites
that form cross-links with substance P, which bears
the simplest Q
n
domain (n ¼ 2). Other GAPDH
lysine residues (Lys248, 251, 256, 257 and 260) were
recognized by TG2 in the presence of the polyQ
17
and polyQ
43

peptides, thus indicating that the
polyQ
n
–GAPDH interaction makes GAPDH a better
TG2 substrate in vitro [32].
Techniques for identifying
transglutaminase substrates
The intrinsic cross-linking activity of TGs tends to
convert target proteins into massive, probably disor-
dered, insoluble aggregates of multiple proteins. Con-
sequently, it is difficult to identify individual protein
substrates and to investigate alterations in their prop-
erties. Nevertheless, biochemical and functional prote-
omic studies in both in vitro and cellular systems have
furthered our understanding of TG-modified proteins.
However, although numerous TG substrates (both glu-
tamine and lysine donors) have been identified in vitro,
fewer have proved to be substrates in vivo.
The detection of polymer formation by SDS ⁄ PAGE
and ⁄ or western blot, and protein-to-protein cross-link-
ing inhibition by amine- or glutamine-rich peptide
incorporation is the most widely used indirect method
of identifying TG protein substrates. Various proce-
dures are used to identify TG substrates and the
protein domains that function as acceptors in the
cross-linking process, i.e. TG-catalysed labelling of iso-
lated peptides ⁄ proteins with radioactive amines [33],
monodansylcadaverine, fluoresceincadaverine [34] and
5-biotinamidopentylamine [35], or with dansylated or
biotinylated glutamine-containing peptides such as

dansyl-e-aminocaproyl-QQIV, -TVQQEL [29] and dan-
syl-substance P [27].
The reactivity of TG to protein substrates in vitro
does not necessarily mean that the proteins are sub-
strates in vivo. Cross-linking in vivo can be evaluated by
conducting in situ assays with whole cells ⁄ tissue. With
an in situ assay it is possible not only to determine the
amine acceptor ⁄ donor substrates in vivo, but also to
assess the affinity of a TG for the interaction with the
protein substrate in the presence of physiologically
occurring alternative substrates. This procedure also
yields information about the specific functions of a TG
isoform, and about the physiological consequences of
TG-catalysed post-translational modification of the
protein substrate. It entails use of cell-penetrating syn-
thetic TG substrates that do not interfere with normal
cell processes. The donor-carrying reporter groups used
are dansylated or biotinylated amines (e.g. 5-biotin-
amidopentylamine, 3-[N
a
[N
e
-[-2¢,4¢-dinitrophenyl]-
amino-n-hexanoyl-l-lysyl-amido]propane-1-ol) [36] or
glutamine-containing peptides (e.g. penetratin-1-linked
peptide) [37]. The advantage of this strategy is protein
separation via affinity chromatography followed by
identification of the labelled TG-reactive protein.
TGs and their substrates C. Esposito and I. Caputo
618 FEBS Journal 272 (2005) 615–631 ª 2004 FEBS

Depending on the probe used, the labelled substrates
can be visualized by direct fluorescence microscopy,
fluorography and western blot analysis, and identified
by N-terminal sequencing or by MS. FAB ⁄ MS has
yielded data on TG-mediated cross-links in the small
purified monomeric proteins substance P [14], b-endo-
rphin [28] and SV-IV [5]. Currently, TG protein
substrates are identified using a procedure that combines
gel electrophoresis separation with MS-based analyses.
Tandem MS based on data-dependent analyses [38] has
led to functional proteomic strategies in which TG
protein substrates and the enzyme-sensitive amino acid
site are identified in mixtures that have not undergone
gel electrophoretic separation.
Identification of protein substrates
for transglutaminase-catalysed
cross-linkage
The recently created TRANSIT database (http://
crisceb.unina2.it/ASC/) lists  150 protein sequences
that function as TG substrates [39]. The TRANSIT
database also lists protein substrates from food, yeast
and viruses. Our review focuses on mammalian TG pro-
tein substrates.
TG1, TG3 and TG5
Mammalian epidermis harbours at least four TG iso-
forms (TG1, TG2, TG3 and TG5). These play con-
secutive and complementary roles in the formation of
a specialized structure known as the cornified cell
envelope (CE) [40] on the intracellular surface of the
plasma membrane of keratinocytes undergoing ter-

minal differentiation. These TGs induce cross-linking
of the various proteins that constitute the CE. TG2 is
expressed only in the basal layer, whereas TG1, TG3
and TG5 are expressed in the upper layers [41]. Mem-
brane-bound TG1 is the most abundant TG isoenzyme
and is predominantly involved in epithelial differenti-
ation [42]. Moreover, TG1 catalyses the ester linkage
of specialized ceramides to CE proteins [43]. Numerous
CE proteins are substrates cross-linked by TGs: invo-
lucrin [41,44], loricrin [41,45], small proline-rich pro-
teins (SPR) [46], cystatin a [47], trichohyalin [48],
keratins [49], cornifin [50], sciellin [51], S100A11 [31],
filaggrin [45], elafin [45,52], desmoplakin [45], envopla-
kin [53], periplakin [48] and suprabasin intermediate
filaments [45,54] (Table 2). In vitro, loricrin, SPR 1, -2
and -3, and trichohyalin functioned as complete sub-
strates for TG1 and TG3 [48]. In addition, each
Table 2. TG1, TG3 and TG5 protein substrates. IF, Intermediate filaments; SPRs, small proline-rich proteins. Protein substrates were identi-
fied by functional proteomics. RL, radiolabelling; CL, cross-linking; P, proteolysis; L, labelling; S, sequencing; WB, western blot.
Protein substrate Reactive Q
a
Reactive K
a
Method of identification Reference
Cornifin ? ? Epidermal extract; WB [50]
Cystatin a
b
—?L⁄ WB [47]
Desmoplakin
b

1646 ? Epidermal extract; P ⁄ S[45]
Elafin 2, 59 6, 58, 60 Epidermal extract; P ⁄ S [45,52]
Envoplakin ? ? Epidermal extract; WB [53]
Epiplakin ? ? Hair follicle; P ⁄ S[48]
Filaggrin 246, 247 ? Epidermal extract; P ⁄ S[45]
IF
b
? ? Epidermal extract; P ⁄ S[45]
Involucrin 465, 496 468 RL ⁄ P ⁄ S [41,44]
Keratins ? 9, 32, 71, 73, Epidermal extract; L ⁄ P ⁄ S[49]
Loricrin 3, 6, 10, 153, 156
215, 216, 219,
225, 305, 306
4, 5, 88,
307, 315
Epidermal extract, L ⁄ P ⁄ S[41,45]
Repetin ? ? Hair follicle; P ⁄ S[48]
S100A7
b
5 ? Epidermal extract ⁄ WB [31]
S100A10
b
4?L⁄ P ⁄ S
S100A11
b
102 3
Sciellin ? ? Epidermal extract; WB [51]
SPRs 4, 5, 16, 17, 18,
19, 87, 167
6, 21, 71, 164,

166, 168
Epidermal extract; P ⁄ S[46]
Suprabasin ? ? CL ⁄ WB [54]
Trichohyalin ? ? L ⁄ WB [48]
a
Q? and K? indicate that reactive glutamine and ⁄ or lysine are present but that the specific residue is not known; – indicates a lack of
evidence for the presence of reactive glutamine.
b
Also in vitro TG2 substrate.
C. Esposito and I. Caputo TGs and their substrates
FEBS Journal 272 (2005) 615–631 ª 2004 FEBS 619
isoenzyme preferred selected reactive glutamine and
lysine residues on the same substrate in vivo. However,
like S100 proteins, which are a family of calcium-
dependent signal transduction mediators, both TG1
and TG2 modify the same sites on S100A11 (i.e.
Q102) and the rank order of reactivity of the three
S100 proteins (A7, A10 and A11) is the same regard-
less of which TG is involved [31]. Key substrates such
as loricrin, involucrin and SPR3 are cross-linked by
TG5 in the initial stage of epidermal differentiation.
The small oligomers formed are cross-linked to the CE
structure by the cytosolic TG3 isoenzyme and subse-
quently by the membrane-bound TG1 enzyme [41].
Derangement of the mechanisms that lead to ter-
minal keratinocyte differentiation might be involved in
lamellar ichthyosis, in hyperkeratinization conditions
such as psoriasis, and in some dermatitis disorders
(e.g. herpetiform disorders through autoimmunity
against TG3). Research is underway to develop drugs

based on natural retinoids and synthetic retinoid-like
agents that will regulate expression of TGs in the skin
[55].
TG2
A large body of data is available for TG2. The results
obtained in structural and functional proteomic studies
are summarized in Tables 3 and 4, respectively. Both
intracellular and extracellular proteins are recognized
and post-translationally modified by TG2. Despite the
lack of a leader sequence, TG2 is externalized from
cells into the extracellular space where it has been
implicated in the stabilization of the extracellular mat-
rix (ECM) and in cell–ECM interactions by cross-link-
ing matrix proteins [56]. Under ‘normal conditions’
TG2 externalized from cells becomes tightly bound to
fibronectin and forms ternary complexes with collagens
that function as a cementing substance in the ECM.
This mechanism probably serves to clear TG2 from
the circulation to prevent it inducing adverse effects.
Fibronectin, a protein abundant in the extracellular
space, is a major TG2 substrate in vitro and in vivo
[57,58]. The other proteins involved in the assembly,
remodelling and stabilization of the ECM are fibrino-
gen ⁄ fibrin [24], von Willebrand factor [59], vitronectin
[60], lipoprotein(a) [61], laminin and nidogen [17]. All
have been identified as TG2 substrates in vitro
(Table 3). The reversible interactions between mole-
cules that form heteromeric complexes in the ECM of
specific tissues, e.g. laminin–nidogen [17], fibronectin–
collagen [62–64] and osteonectin–vitronectin [65], are

stabilized by TG2 [42]. Perturbation of ECM forma-
tion has been implicated in such diseases as liver, renal
and pulmonary fibrosis, as well as atherosclerosis [66].
It is noteworthy that TG2 activity is increased and the
number of e-(c-glutamyl)lysine cross-links is enhanced
in all fibrotic disorders characterized by excessive scar
tissue. Furthermore, TG2 contributes to the organiza-
tion of the ECM by stabilizing the dermo-epidermal
junction via cross-linking of the basement membrane
components fibrillin-1, the major protein of micro-
fibrils, microfibril-associated glycoprotein-1 and latent
transforming growth factor binding protein [67,68].
Latent transforming growth factor binding protein-1 is
particularly interesting because only after its TG2-cata-
lysed linkage to the matrix does it release the active
transforming growth factor b. Consequently, TG2 is
presumed to be involved in the pathogenesis of chronic
inflammatory diseases such as rheumatoid arthritis and
osteoarthritis via regulation of the availability of this
cytokine in the matrix [69]. In addition, extracellular
TG2 might play a role in tissue mineralization by cata-
lyzing the formation of the cross-linked clusters of the
Ca
2+
-binding proteins osteonectin and osteopontin at
the cell surface [70–72].
More intracellular proteins have been identified as
TG2 acyl-donor and ⁄ or acyl-acceptor substrates in
in vitro studies (Table 3) than in functional proteomic
studies (Table 4). However, functional proteomics is a

promising tool with which to identify differently
labelled cellular proteins in relation to physiology and
disease. Indeed, this technique allows one to explore
the cross-linking pattern in such conditions as normal
vs. neoplastic or metastatic cells, and normal vs. prolif-
erating or necrotic ⁄ apoptotic cells, as well as to screen
for differences in TG substrates between quiescent and
differently stimulated cells. A large number of TG2
substrates are proteins involved in the organization of
the cytoskeleton. In the cytoskeleton, the TG2 isoform
colocalizes with stress fibres and, by virtue of its auto-
catalytic activity, it cross-links to myosin. Upon activa-
tion by Ca
2+
, TG2 contributes to the organization of
the cytoskeleton by cross-linking various cytoskeletal
proteins, i.e. microtubule protein tau [73–75], b-tubulin
[76], actin [36,77], myosin [78], spectrin [78], thymo-
sin b [77,79], troponin T [80,81] and vimentin [82].
This extensive polymerization, which occurs during the
final steps of apoptosis, stabilizes the structure of the
dying cells thereby preventing release of cell compo-
nents that might give rise to inflammatory or auto-
immune responses [83]. Interestingly, actin is a TG2
substrate during apoptosis in vivo [74]. Also the retino-
blastoma gene product is a TG2 substrate during
apoptosis in vivo and its polymerization has been indi-
cated as a key signal for the initiation of apoptosis
[84]. Moreover, nuclear proteins such as core histones
TGs and their substrates C. Esposito and I. Caputo

620 FEBS Journal 272 (2005) 615–631 ª 2004 FEBS
Table 3. TG2 protein substrates identified by structural proteomics. BHMT, betaine-homocysteine S-methyltransferase; EMP b-3, erythrocyte
membrane protein band 3; ERM, ezrin–radixin–moesin binding phosphoprotein 50; KGDHC, a-ketoglutarate dehydrogenase; IGFBP-1, insulin-
like growth factor-binding protein 1; MAGP-1, microfibril associated glycoprotein-1; MBP, myelin basic protein; NSB, nuclease sensitive ele-
ment binding protein-1; PGD, phosphoglycerate dehydrogenase; PLA
2
, phospholipase A
2
; Pro-CpU, procarboxypeptidase U; PSA, prostate-
specific antigen; RAP, receptor-associated protein; ROCK-2, Rho-associated coiled-coil-containing protein kinase 2; UV RAD23, UV excision
repair protein RAD23; VIP, vasoactive intestinal peptide. RL, radiolabelling; CL, cross-linking; P, proteolysis; MS, mass spectrometry; L, label-
ling; S, sequencing; WB, western blot; M, mutagenesis.
Protein substrate Reactive Q
a
Reactive K
a
Method of
identification
b
Reference
Actin 41, 167 ? CL ⁄ P ⁄ MS [77]
Aldolase ? — L ⁄ P ⁄ S[35]
Amyloid bA4 15 16, 28 L ⁄ P ⁄ S [86,87]
BHMT ? ? RL [104]
BiP protein ? — L ⁄ in gel P ⁄ MS [78]
C1 inhibitor 453 — L ⁄ P ⁄ S [105]
C-CAM ? ? CL ⁄ WB [106]
Cementoin ? ? CL ⁄ WB [52]
Chaperonin subunit 3 — ? L ⁄ in gel P ⁄ MS [78]
Clathrin heavy chain ? — L ⁄ in gel P ⁄ MS [78]

Collagen III, V, XI 14, ?, ? ? L ⁄ P ⁄ S [63,64]
Crystallin bA3 23, 24 17 L ⁄ P ⁄ S ⁄ M [13,30]
Crystallin bB2 9 —
Crystallin bB3 21 —
Crystallin aB? 175
Cythocrome c 42 — L ⁄ P ⁄ S[33]
Dihydropyrimidinase-2 — ? L ⁄ in gel P ⁄ MS [78]
DNase c ?— L⁄ MS [78]
Elongation factor 1a —? L⁄ in gel P ⁄ MS [78]
Elongation factor 1c —? L⁄ in gel P ⁄ MS [78]
EMP b3 30 ? L ⁄ P ⁄ S [107]
b-Endorphin 11 29 L ⁄ P ⁄ MS [28]
ERM — ? L ⁄ in gel P ⁄ MS [78]
Fatty acid synthase ? — L ⁄ in gel P ⁄ MS [78]
F-box only protein — ? L ⁄ in gel P ⁄ MS [78]
Fibrinogen A 366, 398, 399 148,176, 183, 457 CL ⁄ P ⁄ S[24]
Galectin-3 ? ? L ⁄ WB [101]
GAPDH ? 191, 248, 251, 256,
257, 260, 268, 331
L ⁄ P ⁄ MS [32]
Glucagon 3, 20 — L ⁄ P ⁄ S [108]
a
2
HS-glycoprotein ? ? L ⁄ P ⁄ S[72]
Heat shock 60 kDa — ? L ⁄ in gel P ⁄ MS [78]
Heat shock 70 kDa ? ? L ⁄ in gel P ⁄ MS [78]
Heat shock 70 ⁄ 90 ? ? L ⁄ in gel P ⁄ MS [78]
Heat shock 90 kDa ? ? L ⁄ in gel P ⁄ MS [78]
Huntingtin ? — L ⁄ WB [90]
Histone H1 ? ? L ⁄ P ⁄ S [85,92]

Histone 2B 22, 95 —
Histone 3B 5, 19, 125 —
Histone 4B 27, 93 —
Histone 2A 24, 104, 112 —
IGFBP-1 66, 67 ? M ⁄ CL ⁄ WB [16]
Importin b1 subunit — ? L ⁄ in gel P ⁄ MS [78]
Insulin A chain 5, 15 — L ⁄ P ⁄ S [108]
KGDHC — ? L ⁄ WB [93]
Lipocortin I 19, 23 ? L ⁄ M[98]
MAGP-1 20 — RL ⁄ M[68]
MBP 74,122,146,
149
?L⁄ WB [89]
C. Esposito and I. Caputo TGs and their substrates
FEBS Journal 272 (2005) 615–631 ª 2004 FEBS 621
are able to act as acyl-donor TG2 substrates during
cell death [85].
Amyloid b-A4 peptide [86], a synuclein [86,87], the
microtubule-associated tau protein [88] and myelin
basic protein [89], which are all TG2 substrates in vitro,
are major components of protein aggregates in the
cytosol and nuclei, and in extracellular compartments
in the brains of patients affected by degenerative
neurological diseases. Consequently, TG-mediated
cross-linking has been implicated in the pathogenesis of
Alzheimer’s disease, Parkinson’s disease and in progres-
sive suprabulbar palsy in which the abnormal accumu-
lation of insoluble proteinaceous aggregates cause
progressive neuronal death [66]. A body of evidence
implicates TG2 in the aetiology of (CAG)

n
⁄ Q
n
-diseases
such as Huntington’s disease, i.e. elevated TG2 activity
in the affected regions of diseased brains, colocalization
of TG2 and proteinaceous complexes in cells expressing
truncated huntingtin, c-glutaminyl-lysyl cross-links in
nuclear inclusions in brain, and the finding that TG2
in vitro interacts with the polyglutamine domains to
form cross-links with polypeptides containing lysyl
groups [90–92]. Notably, GAPDH and a-ketoglutarate
dehydrogenase, which are involved in energy metabo-
lism, bind tightly to both huntingtin and several pro-
teins involved in polyglutamine expansion disease [93].
This observation suggested that a slow decline in
energy metabolism of neuronal cells may trigger the
degenerative process that leads to cell death.
TG2 is involved in the activation of members of the
Rho-GTPase family [94–97]. In response to retinoic
acid, TG2 causes transamidation of RhoA and
Table 3. (Continued).
Protein substrate Reactive Q
a
Reactive K
a
Method of
identification
b
Reference

Midkine 42, 44, 95 ? RL ⁄ M [100]
Myosin — ? L ⁄ P ⁄ S [78]
Nidogen 726 — L ⁄ P ⁄ S [17]
NSB — ? L ⁄ in gel P ⁄ MS [78]
Osteonectin 3, 4 — L ⁄ P ⁄ S [15]
Osteopontin 34, 36 — L ⁄ P ⁄ S [71]
Periplakin ? ? L ⁄ M [109]
PGD — ? L ⁄ in gel P ⁄ MS [78]
Phosphorylase kinase ? ? CL ⁄ S [110]
PLA
2
410 CL⁄ WB [111]
PSA ? — RL [113]
RAP 26 ? L ⁄ P ⁄ S [117]
RhoA 52, 63, 136 — L ⁄ P ⁄ S ⁄ MS [94]
40S Ribosomal SA — ? L ⁄ in gel P ⁄ MS [78]
ROCK-2 ? — L ⁄ in gel P ⁄ MS [78]
Sialoprotein ? ? L ⁄ P ⁄ S [72]
Spectrin a ?— L⁄ in gel P ⁄ MS [78]
Statherin ? ? CL ⁄ S [119]
Substance P 5,6 — L ⁄ MS [14]
Synapsin ? — RL [114]
a-Synuclein 79 80 CL ⁄ S [87]
Tau 351, 424 163, 174,180,190, 225,
234, 240
L ⁄ P ⁄ S [88]
T-complex protein ? ? L ⁄ in gel P ⁄ MS [78]
Thymosin b
4
23, 36 3, 18, 38 L ⁄ CL ⁄ P ⁄ MS [77,79]

Thyroglobulin ? ? RL ⁄ CL ⁄ WB [118]
b-Tubulin ? — RL ⁄ CL ⁄ WB [76]
Tumour rejection ag-1 ? ? L ⁄ in gel P ⁄ MS [78]
Uteroglobin ? 43 RL ⁄ CL [115]
UV RAD23 — ? L ⁄ in gel P ⁄ MS [78]
Valosin ? — L ⁄ in gel P ⁄ MS [78]
Vigilin ? — L ⁄ in gel P ⁄ MS [78]
Vimentin 453, 460 97, 104, 294, 439 L ⁄ P ⁄ S [82]
VIP 16 21 L ⁄ P ⁄ MS [116]
a
Q? and K? indicate that reactive glutamine and ⁄ or lysine are present but that the specific residue is not known; – indicates that there is no
evidence for the presence of reactive glutamine and ⁄ or lysine.
TGs and their substrates C. Esposito and I. Caputo
622 FEBS Journal 272 (2005) 615–631 ª 2004 FEBS
formation of the RhoA-Rho-associated coiled-coil-con-
taining protein kinase 2, a complex that promotes the
formation of stress fibres and focal adhesion com-
plexes. RhoA-Rho-associated coiled-coil-containing
protein kinase 2, like the ezrin ⁄ radixin ⁄ moesin intracel-
lular signalling proteins and elongation factors that are
critical for the assembly of junctional proteins and
actin-cytoskeleton organization in intestinal epithelia,
was shown to be a TG2 substrate [78]. These findings
support the notion that TG2 acts as a signal transduc-
tion protein by altering the function of signalling
growth ⁄ differentiation factors such as the CD38 trans-
membrane enzyme [96], dual leucine zipper-bearing
kinase [97], insulin-like growth factor-binding protein 1
[16], lipocortin I [98] and the extracellular midkine
[99–101] that are TG2 substrates in vivo (Table 4).

Another interesting aspect of TG2 function is its
involvement in receptor-mediated endocytosis in various
cellular systems [102]. In vitro, valosin and clathrin,
which are implicated in transport processes, are gluta-
mine-donor substrates, whereas importin is a lysine
donor [78]. Phosphoglycerate dehydrogenase and fatty
acid synthase, which are involved in different metabolic
processes, are TG2 substrates in vitro [78,103–119]
(Table 3).
Finally, the presence of autoantibodies against
TG2 and its protein substrates in autoimmune
diseases such as coeliac disease suggests that TG2
may cross-link potential autoantigens to itself and to
other protein substrates so triggering the humoral
response in autoimmune diseases [66,120]. In this
scenario, TG2–protein complexes formed in vivo may
function as hapten–carrier complexes [120]. An
immune reaction was observed against the well-
known TG2 substrates actin, myosin, tubulin, lipo-
cortin I and histone H2B in patients with systemic
lupus erythematosus, and against collagen and myelin
basic protein in bullous pemphigoid and multiple
sclerosis, respectively [66].
Besides its involvement in protein cross-linking,
within the intracellular compartment, TG2 is more
likely to catalyse the incorporation of polyamines into
specific acyl-donor substrates especially when the con-
centration of polyamines in the cell ⁄ tissue is in the
millimolar range. Numerous proteins are covalently
modified by polyamination in intact cells, and poly-

amines can modulate the function and metabolism of
the protein substrate. For example, TG2-catalysed
polyamination of phospholipases A
2
increased activity
of the enzyme in vitro [111], polyamination of micro-
tubule-associated protein tau inhibits calpain-mediated
proteolysis [73], and modification of substance P by
spermine and spermidine incorporation protects the
peptide against proteolysis [121].
Table 4. TG2 protein substrates identified by functional proteomics. AChE, acetylcholine esterase; GST, glutathione S-transferase; IGFBP-1,
insulin-like growth factor-binding protein 1; LTBP-1, latent transforming growth factor-b binding protein-1; pRB, retinoblastoma; CL, cross-link-
ing; WB, western blot; RL, radiolabelling; IP, immunoprecipitation; L, labelling; AC, affinity chromatography; S, sequencing.
Protein
substrate Function Localization
Experimental
model
Method of
identification Reference
AchE
a
Neurotransmission Membrane ECM Myotubes CL ⁄ WB [103]
Actin Cell morphology Cytosol HL-60 cells L ⁄ AC ⁄ S[74]
CD38
a
Signalling Membrane HL-60 cells RL ⁄ IP ⁄ WB [96]
Collagen V ⁄ XI Fibrillar organization ECM A204 cells CL ⁄ WB [64]
DLK Signalling Cytosol NIH 3T3 cells CL ⁄ WB [97]
GST
a

Cytoprotection Cytosol Neuroblastoma cells IP ⁄ WB ⁄ S[75]
Fibrillin-1 Myofibrils formation ECM Amniotic membranes CL ⁄ S[67]
Fibronectin Matrix assembly Extracellular ECV304 cells L ⁄ WB [58]
IGFBP-1 Signalling Extracellular Fibroblasts CL ⁄ WB [16]
Lipocortin I Signalling Cytosol
membrane
A431 cells CL ⁄ WB [98]
LTBP-1 Signalling
ECM deposition
Extracellular HT 1080 cells IP ⁄ WB ⁄ CL [69]
Midkine Signalling EC Neurons L ⁄ WB [99]
Osteonectin Bone mineralization ECM Organ culture tracheae L ⁄ WB [70]
pRB Cell cycle control Nucleus U937 cells L ⁄ WB [84]
RhoA Signalling Cytosol HeLa cells L ⁄ WB [95]
Tau Cytoskeleton organization Cytoskeleton Brain tissue CL ⁄ WB [73]
Troponin T
a
Cell morphology Cytoskeleton Isolated hearts perfused CL ⁄ WB [81]
a
Also an in vitro TG2 substrate.
C. Esposito and I. Caputo TGs and their substrates
FEBS Journal 272 (2005) 615–631 ª 2004 FEBS 623
TG4
TG4 is the only TG with prostate-specific and andro-
gen-regulated expression. In rodents, TG4 is secreted
by the anterior lobe of the prostate, also called ‘coagu-
lating gland’, and induces the postmating formation of
a vaginal coagulatory plug by cross-linking the major
coagulating proteins, SV proteins I–V, which are secre-
ted by the SV epithelium [122,123]. The SV I–V pro-

teins are TG4 substrates, and SV IV was one of the
first TG substrates in which glutamines and lysine resi-
dues were identified by MS [5] (Table 5). TG4-cata-
lysed polymeric forms of SV IV suppress epididymal
sperm immunogenicity. Although no physiological
function has yet been assigned to human TG4, the
functions identified in the rat enzyme could apply to
the human isoform because TG4 activity occurs both
in human seminal plasma and on the spermatozoon
surface [124]. Moreover, the major gel-forming pro-
teins in human semen, semenogelin I and II, which
correspond to rat SV proteins, are substrates for TG4
[125]. However, even though the rat and human
enzymes are synthesized in the same organ and are
unconventionally secreted, there are several differences
between the rodent enzyme and the human homologue
[126]. Human TG4 is expressed at a much lower level
than the rat enzyme, and the two sequences share an
amino acid identity of no more than 53%. Rat TG4 is
very complex [127]. In fact, it is highly glycosylated
and possesses a lipid anchor that is retained during
enzyme apocrine secretion. It binds GTP, which acts
as a negative modulator [128], and it is positively influ-
enced by phosphatidic acids and SDS [127]. Finally,
rat prostate secretion contains a kinesin-like protein
able to act as an efficient acyl donor substrate for the
enzyme in vitro. This protein substrate may be import-
ant for the correct extrusion of TG4 from the coagula-
ting gland [129].
FXIII

Coagulation FXIII is a plasma TG, and circulates in
blood as a heterotetramer consisting of two catalytic A
(XIIIA) and two noncatalytic B (XIIIB) subunits
Table 5. Protein substrates of TG4. SV IV, seminal vesicle I–V; CL,
cross-linking; P, proteolysis; MS, mass spectrometry; L, labelling;
WB, western blot.
Substrate
protein
Reactive
Q
a
Reactive
K
a
Method of
identification Reference
Kinesin-like ? ? L ⁄ in gel P ⁄ MS [129]
Semenogelin I–II ? ? CL ⁄ WB [125]
SV II–IV–V ? ? L ⁄ CL [123]
SV IV
b
9, 86 2, 4, 59,
78, 79, 80
L ⁄ P ⁄ MS [5]
a
Q? and K? indicate that reactive glutamine and ⁄ or lysine are
present but that the specific residue is not known.
b
Also an in vitro
TG2 substrate.

Table 6. Protein substrates of Factor XIII. Pro-CpU, procarboxypeptidase U. Proteins shown in bold have been identified by functional pro-
teomics. RL, radiolabelling; CL, cross-linking; P, proteolysis; L, labelling; S, sequencing; WB, western blot.
Substrate protein Reactive Q
a
Reactive K
a
Method of identification Reference
a
2
-Antiplasmin
b
2? CL⁄ P ⁄ MS [24]
Collagen XVI ? ? RL ⁄ CL ⁄ WB [132]
Fibronectin
b
3— L⁄ P ⁄ S [57]
Fibrinogen A 398, 399 148, 176, 208, 219,
224, 230, 413, 418,
427, 429, 448, 508,
539, 556, 580, 601,
606
CL ⁄ P ⁄ S [24,130]
Filamin
b
?? CL⁄ WB [134]
Lipoprotein(a)
b
?? RL⁄ CL ⁄ WB [61]
a-Macroglobulin 670 — L ⁄ P ⁄ S [131]
PAI-2

b
83, 84,86 ? CL ⁄ P ⁄ MS [133]
Pro-CpU
b
2, 5, 292 ? RL ⁄ P ⁄ MS [112]
S19 ribosomal protein ? ? CL ⁄ Coomassie [137]
Thrombospondin ? ? RL ⁄ CL [135]
Vinculin ?? CL⁄ WB [134]
Vitronectin
b
73, 84, 86, 93 ? RL ⁄ CL ⁄ MS [60]
VonWillebrand factor 313,509,560,
634
—L⁄ P ⁄ S [59]
a
Q? and K? indicate that reactive glutamine and ⁄ or lysine are present but that the specific residue is not known; indicate that there is no
evidence for the presence of reactive glutamine and ⁄ or lysine.
b
Also an in vitro TG2 substrate.
TGs and their substrates C. Esposito and I. Caputo
624 FEBS Journal 272 (2005) 615–631 ª 2004 FEBS
(A
2
B
2
). FXIII is a proenzyme that is activated by
Ca
2+
and thrombin generated in the final stage of the
blood coagulation cascade. The A subunit of coagula-

tion FXIII (an FXIII–A dimer) has been identified in
the cytoplasm of platelets, megakaryocytes and mono-
cytes–macrophages [2,3]. FXIIIA catalyses the cross-
linking of the fibrin(ogen) c-chains to form c–c dimers
involving glutamine 398 or 399 of one chain and
Lys406 of another fibrin. Successively, the enzyme sta-
bilizes the blood clot by cross-linking lysine and gluta-
mine of the a and c chains of fibrin molecules [24].
Sobel and Gawinowicz [130] found that Lys556 and
Lys580 of fibrinogen accounted for 50% of the total a
chain donor cross-linking activity observed; Lys539,
508, 418 and 448 accounted for 28% and Lys601, 606,
427, 429, 208, 224 and 219 each accounted for between
2 and 5% (Table 6). TG2 can also cross-link the Aa
chain of fibrinogen to PAI-2, but it does so by medi-
ating cross-linking to Lys183 and Lys457 instead of
Lys230 and Lys413 [24].
FXIII plays an important role in haemostasis,
wound healing and the maintenance of pregnancy. It
protects clots from plasminolysis by covalently linking
a
2
-antiplasmin and a
2
-macroglobulin to the a chain of
fibrin [131]. Cross-linking of fibrin with fibronectin and
collagen at the site of injury may facilitate wound heal-
ing by providing a scaffold for fibroblasts to prolifer-
ate and spread [57,132]. Other plasma and tissue
proteins such as vitronectin [60], PAI-2 [133], lipopro-

tein(a), platelet vinculin [134], thrombospondin [135],
von Willebrand factor [136], procarboxypeptidase U
[112] and S19 ribosomal protein [137] are reported to
be substrates of FXIIIA (Table 6), however, the role
of the enzyme-mediated modification is not always
clear. The procoagulant proteins von Willebrand fac-
tor, thrombospondin, fibronectin, a
2
-antiplasmin and
fibrinogen are anchored, via the TG-mediated addition
of serotonin, on the surface of activated platelets,
where both TG2 and FXIII are present, thereby form-
ing ‘COAT-platelets’ [138]. These platelets may be
formed under circumstances of extreme haemostatic
need, but may cause thrombosis if inappropriately
elicited.
Conclusion
In most cases, the cross-linking of proteins mediated
by TG isoforms results in well-defined functions in
various biological processes: cross-linking of fibrin in
haemostasis, of semenogelin in semen coagulation and
of involucrin in keratinocyte CE formation. An excep-
tion is the ubiquitous TG2, designated the ‘be
ˆ
te noire’
of the TG family [2], whose physiological role remains
largely obscure. TG2 appears to be involved in biologi-
cal phenomena ranging from ECM stabilization to
intracellular signalling and apoptosis, as well as in
neurodegenerative and autoimmune diseases. Conse-

quently, its function may depend on its subcellular and
cellular localization and on access to proteins able to
act as substrates. Thus far, many more substrate
proteins have been identified for TG2 than for the
other TG isoforms. However, TG2-mediated changes
to a substrate protein have not always been linked to a
given function. In order to make this correlation, there
is a need to identify as many in vivo TG substrates as
possible. The combined structural and functional pro-
teomic approach is a promising procedure with which
to verify the identification of TG2 protein substrates
in vivo.
Finally, the identification of TG substrates may lead
to novel drug targets and new diagnostic markers for
several TG-related diseases. In this context, peptides or
recombinant proteins that inhibit TGs or their pepti-
domimetic derivatives are potential therapeutic ⁄ pro-
phylactic agents.
Acknowledgements
We would like to acknowledge Professor Carlo Berga-
mini for his comments to the manuscript, Drs Frances-
co Facchiano and Angelo Facchiano for their help in
consulting the TRANSIT database, and Dr Eleonora
Candi for her contribution in the preparation of
Table 2. We are grateful to Jean Gilder for text edit-
ing. This work was supported by grant from MURST
40% grant to CE.
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