www.elsevier.com/locate/intimp

International Immunopharmac

Preliminary Report

Effects of antiflammins on transglutaminase and phospholipase A2

activation by transglutaminase

Juan Jose Moreno *

Department of Physiology, Faculty of Pharmacy, Barcelona University, Barcelona, Spain

Received 1 July 2005; received in revised form 20 July 2005; accepted 4 August 2005

Abstract

Two anti-inflammatory peptides, named antiflammins (AFs), corresponding to a region with high amino acid similarity

between lipocortin-1 and uteroglobin were tested for their ability to inhibit transglutaminase (TG) and low-molecular-mass

phospholipase A2 (PLA2). Porcine pancreatic PLA2 activity and guinea pig hepatic TG activity were determined by arachidonyl

release from arachidonyl-phosphatidylcholine and by the incorporation of putrescine into succinylated casein, respectively. AFs

inhibited TG activity but did not affect PLA2 activity. Moreover, porcine pancreatic PLA2 was activated by TG and AFs

decreased porcine pancreatic PLA2 activation induced by TG. Taken together, our results support the hypothesis that the anti-

inflammatory effects of AFs are, at least in part, due to the action of AFs on TG activity.

D 2005 Elsevier B.V. All rights reserved.

Keywords: Anti-inflammatory drugs; Lipocortin; Uteroglobin; Arachidonic acid; Eicosanoids

1. Introduction

Transglutaminase (TG, E.C. 2.3.2.13.) is a cal-

cium-dependent enzyme that catalyses the covalent

cross-linking of the g-carboxamide groups of pep-

tide-bound glutamine residues with the q-amino

groups of peptide-bound lysine residues [1]. These

enzymes have been detected both intra- and extracel-

lularly in higher animals including man. Following

blood coagulation, a plasma TG cross-links fibrin

molecules via the formation of interchain q-(g-gluta-

1567-5769/$ - see front matter D 2005 Elsevier B.V. All rights reserved.

doi:10.1016/j.intimp.2005.08.001

* Tel.: +34 93 4024505; fax: +34 93 4035901.

E-mail address: jjmoreno@ub.edu.

myl)-lysine isopeptide bonds and stabilizes the clot by

preventing its hydrolysis by proteases [2]. This parti-

cular form of TG is known as fibrin-stabilizing factor

or coagulation factor XIII. Besides factor XIII, several

extracellular and intracellular TGs have been

described [1]. One of the most thoroughly studied

TGs is derived from guinea pig liver [3]. Intracellular

TGs with apparently identical properties to the liver

enzyme are present in many tissues and organs of

mammals and are generally referred to as tissue TG

or TG-2. Increased TG-2 expression has been reported

in many inflammatory diseases [4–7].

Phospholipase A2 (PLA2, E.C. 3.1.1.4.) are a

family of lipolytic enzymes that play a key role in

ology 6 (2006) 300–303

J.J. Moreno / International Immunopharmacology 6 (2006) 300–303 301

the pathogenesis of inflammation through the hydro-

lysis of arachidonic acid (AA) from the sn-2 position

of phospholipids. In almost every cell studied, low-

molecular-mass PLA2 (14 kDa) and high-molecular-

mass PLA2 (60–115 kDa) are expressed and appear to

be crucial enzymes involved in AA release. Then, free

AA can thus be converted to pro-inflammatory med-

iators like prostaglandins and leukotrienes [8].

TG-2 increased PLA2 activity in vitro. The TG-2-

catalysed conformation of PLA2 can be brought about

by the formation of an intramolecular q-(g-glutamyl)-

lysine cross-link [9] or by the incorporation of poly-

amines [10]. TG-2-mediated modification may thus

activate PLA2 in vivo, which may explain the patho-

logical roles of TG-2 in inflammation.

Glucocorticoids are the most effective drugs for

the treatment of inflammatory diseases. These effects

are mediated, at least in part, by the induction of

regulatory proteins like lipocortins [11] and uteroglo-

bins [12]. Moreover, uteroglobin is an excellent sub-

strate for TGs [13]. Based on computer analysis,

Miele et al. [14] designed several synthetic peptides

corresponding to the region of highest similarity

between uteroglobin and lipocortin-1, which modu-

late inflammation. These peptides, named antiflam-

mins (AFs), inhibited inflammation development in

several experimental models [15,16]. The nonapep-

tide MQMKKVLDS (AF-1) is equivalent to the nine

amino acid C-terminal portion of a-helix 3 in uter-

oglobin, whereas AF-2 (HDMNKVLDL) corre-

sponds to the 246–254 sequence of lipocortin-1.

AFs may reduce leukocyte migration and infiltration

in inflamed tissues [17,18]. These effects may be

correlated with the modulation of the expression of

adhesion molecules and the subsequent binding of

leukocytes to endothelial cells [19,20].

Here, we report the effect of AFs on TG-2 activity

and on the modification of PLA2 activity by TG-2.

2. Material and methods

2.1. Materials

AF-1 and AF-2 were purchased from Bachem Feinche-

mikalien AG (Switzerland) with a purity of N98% (HPLC).

They were stored under argon in sealed glass vials and

desiccated at �20 8C until use. AFs were dissolved in

Tris–HCl 10 mM pH 8.0 buffer to prepare stock solution

at 0.1 mM. AFs were never stored in solution. 1-Stearoyl-2-

[1-14C]arachidonyl-phosphatidylcholine (52 mCi/mmol)

and [1,414C]-putrescine (118 Ci/mole) were from NEN

(Boston, MA). Porcine pancreatic PLA2, TG-2 from guinea

pig liver and succinylated casein were obtained from Sigma

Chem Co. (St. Louis, MO). All other reagents were of

analytical grade.

2.2. PLA2 activity assay

PLA2 activity was measured as described by Miele et

al. [14]. Briefly, 1-stearoyl-2-[1-14C]arachidonyl-phosphati-

dylcholine used as substrate was dispersed with 5 mM

sodium deoxycholate. Reaction mixtures in a total volume

of 500 Al consisted of 10 AM of the substrate phospholipid

(20,000 cpm), 100 mM NaCl, 2 mM CaCl2 and 1 mM

sodium deoxycholate in 100 mM Tris pH 8.0. The reaction

was started by addition of 15 mU of porcine pancreatic

PLA2 and stopped after 15 min at 37 8C with 3 ml of

CHCl3 /CH3OH (1 /2 v /v). Products were extracted fol-

lowing Bligh and Dyer [21] and separated by thin layer

chromatography. Radioactivity was quantitated by liquid

scintillation.

2.3. TG activity assay

TG activity was determined by measuring the incor-

poration of [1,414C]-putrescine into succinylated casein

[3]. TG-2 from guinea pig liver (1 mU) was incubated

in 0.1 ml of buffer containing 0.1 M Tris–acetate pH 7.5,

1% succinylated casein, 1 mM EDTA, 10 mM CaCl2,

0.5% lubrol PX, 5 mM dithiothreitol, 0.15 M NaCl and

0.5 mCi 14C-putrescine. Following incubation at 37 8C for

20 min, the reaction was terminated by addition of 4.5 ml

of cold 7.5% (w /v) trichloroacetic acid (TCA). The TCA-

insoluble precipitates were collected onto GF/A glass fibre

filters, washed with cold 5% (w /v) TCA, dried and

counted.

2.4. Statistics

Data are the meanFSEM of three determinations per-

formed in triplicate. Statistical significance was assessed by

one-tailed Student’s t-test for unpaired samples with signif-

icance set at P b0.05.

3. Results and discussion

In 1988, Mukherjee and co-workers suggested that

short peptides derived from uteroglobin and lipocortin-1

have the same spectrum of activity as these proteins.

Fig. 1. Effects of AFs on PLA2 activation by TG-2. Purified porcine

pancreatic PLA2 (15 mU) was pre-incubated with TG-2 from guinea

pig liver (1 mU) in the absence (circle) or presence of AF-1 (10 AM,

square) or AF-2 (10 AM, triangle) for 15 min before the assay. The

assay was started by addition of PLA2 substrate and stopped after 15

min at 37 8C. Data are meanFSEM from three experiments per-

formed in triplicate. *P b0.05 compared with control conditions.

J.J. Moreno / International Immunopharmacology 6 (2006) 300–303302

Thus, they proposed that AFs prevent type I low-molecu-

lar-mass PLA2 activity and they correlated these biochem-

ical action with an anti-inflammatory effect in vivo [14].

However, the ability of AFs to inhibit low-molecular-mass

PLA2 and their anti-inflammatory action have been ques-

tioned [22–24]. In our experimental conditions, porcine

pancreatic PLA2 activity in the absence of AFs was

assigned a value of 100% (3.56F0.05 pmol arachido-

nyl/15 min) and AFs did not significantly inhibit this

PLA2 activity (Table 1). These data are consistent with

previous results [25].

To determine the inhibitory effect of the AFs on puri-

fied guinea pig liver TG-2, this was pre-incubated with

peptides for 15 min before starting the TG assay. The

activity of TG-2 incubated without AFs was assigned a

value of 100% (2.21F0.06 pmol putrescine/20 min). AFs

significantly inhibited TG-2 activity by about 50% at 10

AM. Higher AF concentrations were not able to increase

the inhibition of guinea pig liver TG-2 activity (Table 1).

Analysis of AF peptide sequences showed that nonapep-

tides contain a common sequence KVLD with lysine. The

inhibitory effect of AFs on TG activity may be due to the

lysine residue in the AFs, which behaves as an acyl

acceptor for TG catalysis thereby competing with TG

substrates.

PLA2 activation may be involved in the development of

inflammatory diseases such as rheumatoid and juvenile

rheumatoid arthritis [26]. Since inflammatory cells are

known to secrete low-molecular-mass PLA2 and this

enzyme may cause synovial tissue damage, the release of

TG and further PLA2 activation may occur as this process

continues. As a result, the increase in TG-2 may trigger

porcine pancreatic PLA2 activity. Indeed this PLA2 activity

Table 1

Effects of AFs on low-molecular-mass PLA2 and TG-2 activities

PLA2 activity (%) TG activity (%)

AF-1 (0.1 AM) 98F1.9 93F1.7

AF-1 (1 AM) 97F2.2 82F1.8*

AF-1 (10 AM) 96F2.1 51F1.5*

AF-1 (50 AM) 92F2.5 49F1.7*

AF-2 (0.1 AM) 96F2.3 91F1.4

AF-2 (1 AM) 97F2.3 86F1.5*

AF-2 (10 AM) 92F1.5 62F1.3*

AF-2 (50 AM) 91F1.8 59F1.6*

Purified porcine pancreatic PLA2 (15 mU) and TG-2 from guinea

pig liver (1 mU) were pre-incubated with AFs for 15 min before the

assay and both enzymatic activities were measured as described in

the Material and methods section. Enzymatic activities were

expressed as percentage with respect to non-treated enzyme. Data

are meanFSEM from three experiments performed in triplicate.

* Pb 0.05 compared with control conditions (absence of AFs).

was increased by incubation with TG-2 (Fig. 1) as reported

elsewhere [10]. Thus, porcine pancreatic PLA2 activity was

doubled when TG-2 in the mixture rose by 1 mU. Interest-

ingly, AFs (10 AM) inhibited the increase in porcine pan-

creatic PLA2 activity by about 80% in this experimental

conditions (Fig. 1).

TG is also involved in the adhesion and migration of

leukocytes. Akimov and Belkin [27] reported that TG serves

as an integrin-associated adhesion receptor that may be

involved in the extravasation and migration of monocytic

cells into tissues containing fibronectin matrices during

inflammation. Recently, Mohan et al. [28] demonstrated

that TG may have a role in mediating T cell migration

across cytokine-activated endothelium and infiltration of

tissues during inflammation. Considering that neutrophil/

monocyte and lymphocyte adhesion to endothelial cells is

a pivotal step in the inflammatory process, TG may affect

cell migration and thus contributes to the development of the

inflammatory lesion. In this regard, the anti-inflammatory

effects of AFs as well as the inhibitory action of AFs on cell

infiltration during inflammation observed previously [19,20]

may be, at least in part, due to the inhibition of TG activity.

Therefore, TG-2 inhibitors like AFs may have dual anti-

inflammatory properties, decreasing cell infiltration and

PLA2 activation by TG as proposed recently by Sohn et

al. [29] who reported that dual inhibitory effect on TG-2 and

PLA2 reverted the inflammation of allergic conjunctivitis to

ragweed in a guinea pig model.

J.J. Moreno / International Immunopharmacology 6 (2006) 300–303 303

Acknowledgments

This study was supported by grants from the

Spanish Ministry of Education (DGESIC PM98-

0191) and Generalitat de Catalunya (Autonomous Gov-

ernment of Catalonia, 2001SGR00134). The author is

very grateful to Robin Rycroft for his valuable assis-

tance in the preparation of the manuscript.

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