Mammalian transglutaminases

REVIEW ARTICLE

Mammalian transglutaminases

Identification of substrates as a key to physiological function andphysiopathological relevance

Carla Esposito and Ivana Caputo

Department of Chemistry, University of Salerno, Italy

Mammalian transglutaminases andtheir 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: [email protected]

(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 Ca2+-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 Ca2+-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 a1-adrenergic signals [10], occurs inde-

pendently of cross-linking activity, but both activities

are regulated by binding to GTP and Ca2+ ([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 fortransglutaminases

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. bA3-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 TGK, keratinocyte TG,

type 1 TG

90 Epithelia Cytosolic,

membrane

TG2 TGC, tissue TG,

type 2 TG

80 Ubiquitous Cytosolic,

nuclear,

extracellular

TG3 TGE, epidermal TG,

type 3 TG

77 Epithelia Cytosolic

TG4 TGP, prostate TG,

type 4 TG

77 Prostate Extracellular

TG5 TGX, type 5 TG 81 Epithelia Cytosolic

TG6 TGY, type 6 TG Unknown Unknown Unknown

TG7 TGZ, 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 (QLQPFPQPQLPY)

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


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 Qn domain (n ¼ 2). Other GAPDH

lysine residues (Lys248, 251, 256, 257 and 260) were

recognized by TG2 in the presence of the polyQ17

and polyQ43 peptides, thus indicating that the

polyQn–GAPDH interaction makes GAPDH a better

TG2 substrate in vitro [32].

Techniques for identifyingtransglutaminase 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-[Na[Ne-[-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.



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 substratesfor transglutaminase-catalysedcross-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 Qa Reactive Ka Method of identification Reference

Cornifin ? ? Epidermal extract; WB [50]

Cystatin ab — ? L ⁄WB [47]

Desmoplakinb 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]

IFb ? ? 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]

S100A7b 5 ? Epidermal extract ⁄WB [31]

S100A10b 4 ? L ⁄ P ⁄ SS100A11b 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.



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

Ca2+-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 Ca2+, 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



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; PLA2, phospholipase A2; 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 Qa Reactive Ka

Method of

identificationb 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 2HS-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]



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 ⁄Qn-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-ketoglutaratedehydrogenase, 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 Qa Reactive Ka

Method of

identificationb 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]

PLA2 4 10 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 b4 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.



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 A2 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

AchEa Neurotransmission Membrane ECM Myotubes CL ⁄WB [103]

Actin Cell morphology Cytosol HL-60 cells L ⁄AC ⁄ S [74]

CD38a 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]

GSTa 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 Ta Cell morphology Cytoskeleton Isolated hearts perfused CL ⁄WB [81]

a Also an in vitro TG2 substrate.



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

Qa

Reactive

Ka

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 IVb 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 Qa Reactive Ka Method of identification Reference

a2-Antiplasminb 2 ? CL ⁄P ⁄MS [24]

Collagen XVI ? ? RL ⁄CL ⁄WB [132]

Fibronectinb 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]

Filaminb ? ? CL ⁄WB [134]

Lipoprotein(a)b ? ? RL ⁄CL ⁄WB [61]

a-Macroglobulin 670 — L ⁄ P ⁄ S [131]

PAI-2b 83, 84,86 ? CL ⁄P ⁄MS [133]

Pro-CpUb 2, 5, 292 ? RL ⁄P ⁄MS [112]

S19 ribosomal protein ? ? CL ⁄Coomassie [137]

Thrombospondin ? ? RL ⁄CL [135]

Vinculin ? ? CL ⁄WB [134]

Vitronectinb 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.



(A2B2). FXIII is a proenzyme that is activated by

Ca2+ 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 achain 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 Aachain 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

a2-antiplasmin and a2-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, a2-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 ‘bete 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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