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

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

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, nitrosylaphosphoryla-tion, 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: cesposito@unisa.it

(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.

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 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 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.

TG1 TG K , 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 TG P , prostate TG,

type 4 TG

77 Prostate Extracellular TG5 TGX, type 5 TG 81 Epithelia Cytosolic

TG6 TG Y , type 6 TG Unknown Unknown Unknown

TG7 TGZ, type 7 TG 80 Ubiquitous Unknown

FXIII Factor XIIIA,

plasma TG,

fibrin stabilizing

factor

plasma, platelets

Extracellular

Band

4.2

Erythrocyte

protein band 4.2

77 Erythrocytes Membrane

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.

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 propro-tein–propro-tein 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 (n¼ 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 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-[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 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], periplaenvopla-kin [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.

215, 216, 219,

225, 305, 306

4, 5, 88,

307, 315

Epidermal extract, L ⁄ P ⁄ S [41, 45]

19, 87, 167

6, 21, 71, 164,

166, 168

Epidermal extract; P ⁄ S [46]

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.bAlso 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.

Method of identification b Reference

257, 260, 268, 331

149

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-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).

Method of identification b Reference

234, 240

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 functransduc-tion 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, radiolabellcross-link-ing; IP, immunoprecipitation; L, labellcross-link-ing; AC, affinity chromatography; S, sequencing.

Protein

Experimental model

Method of identification Reference

membrane

ECM deposition

a Also an in vitro TG2 substrate.

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

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.

224, 230, 413, 418,

427, 429, 448, 508,

539, 556, 580, 601, 606

VonWillebrand factor 313,509,560,

634

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.