Research article

Transglutaminase-2: a new endostatin partner in the extracellular matrix of endothelial cells

Clément Faye, Antonio Inforzato, Marine Bignon, Daniel J. Hartmann, Laurent Muller, Lionel Ballut, Bjorn R. Olsen, Anthony J. Day, Sylvie Ricard-Blum

Abstract

Endostatin, a C-terminal fragment of collagen XVIII, binds to TG-2 (transglutaminase-2) in a cation-dependent manner. Recombinant human endostatin binds to TG-2 with an affinity in the nanomolar range (Kd=6.8 nM). Enzymatic assays indicated that, in contrast with other extracellular matrix proteins, endostatin is not a glutaminyl substrate of TG-2 and is not cross-linked to itself by the enzyme. Two arginine residues of endostatin, Arg27 and Arg139, are crucial for its binding to TG-2. They are also involved in the binding to heparin [Sasaki, Larsson, Kreuger, Salmivirta, Claesson-Welsh, Lindahl, Hohenester and Timpl (1999) EMBO J. 18, 6240–6248], and to α5β1 and αvβ3 integrins [Faye, Moreau, Chautard, Jetne, Fukai, Ruggiero, Humphries, Olsen and Ricard-Blum (2009) J. Biol. Chem. 284, 22029–22040], suggesting that endostatin is not able to interact simultaneously with TG-2 and heparan sulfate, or with TG-2 and integrins. Inhibition experiments support the hypothesis that the GTP-binding site of TG-2 is a potential binding site for endostatin. Endostatin and TG-2 are co-localized in the extracellular matrix secreted by endothelial cells under hypoxia, which stimulates angiogenesis. This interaction, occurring in a cellular context, might participate in the concerted regulation of angiogenesis and tumorigenesis by the two proteins.

  • endostatin (ES)
  • extracellular matrix
  • protein–protein interaction
  • surface plasmon resonance (SPR) binding assays
  • transglutaminase-2 (TG-2)

INTRODUCTION

Endostatin is a C-terminal fragment of the α1 chain of collagen XVIII, which inhibits angiogenesis and tumour growth [1,2]. It binds to the α5β1 and αvβ3 integrins [3,4], glypicans 1 and 4 [5], and to VEGFR-2 [VEGF (vascular endothelial growth factor) receptor-2; also known as Flk1 or KDR (kinase insert domain receptor)] [6] on the surface of endothelial cells. The binding of human endostatin to the α5β1 integrin leads to the inhibition of the FAK (focal adhesion kinase)/c-Raf/MEK1/2 [MAPK (mitogen-activated protein kinase)/ERK (extracellular-signal-regulated kinase) kinase 1/2]/p38/ERK1 pathway [7]. Endostatin also inhibited the binding of VEGF165 to both endothelial cells and to the purified extracellular domain of VEGFR-2. Furthermore, endostatin blocks VEGF-induced tyrosine phosphorylation of VEGFR-2 and activation of ERK, p38 MAPK and p125(FAK) in HUVECs (human umbilical vein endothelial cells) [6].

Endothelial cells are a rich source of TG-2 (transglutaminase-2) [8] and this enzyme plays an important role at the surface of those cells. For example, evidence has been reported for the protein's externalization and its co-localization with the β1 integrin [9]. Cell surface TG-2 interacts with integrins of the β1 and β3 subfamilies in focal adhesion sites [10] and the formation of a complex between TG-2 and VEGFR-2 has been proposed to modulate the endothelial cell response to VEGF [11]. Conflicting in vivo studies have proposed that the enzyme both stimulates and inhibits angiogenesis [12,13]. However, in an in vitro angiogenesis assay, application of exogenous TG-2 blocks angiogenesis in a dose-dependent manner without causing cell death via a mechanism that involves increased accumulation of extracellular matrix proteins [14]. TG-2 is a calcium-dependent enzyme that catalyses post-translational modification of proteins by forming γ-glutamyl-ε-lysine bonds between glutamine and lysine residues [1517]; it is a multifunctional enzyme that undergoes a GTP-binding/GTPase cycle, with guanine nucleotide and calcium binding reciprocally regulating its transamidation activity [18].

Endostatin and TG-2 share several extracellular partners such as nidogen, SPARC (secreted protein acidic and rich in cysteine), collagen VI and the amyloid β-peptide [15,1922]. Both proteins are also able to bind to heparin [19,23,24], α5β1 and αvβ3 integrins [3,4,10], and VEGFR-2 [6,11]. They are involved in angiogenesis and are increased in brain after trauma [25,26]. Furthermore, they may play a role in Alzheimer's disease. Endostatin is released by neurons and accumulates in amyloid plaques [27] and TG-2 co-localizes with the pathological lesions in the Alzheimer's disease brain [28,29]. All these common properties prompted us to investigate the possible existence of an interaction between these two proteins and we have shown recently that endostatin interacts with TG-2 [22]. In the present paper we report that endostatin binds to TG-2 with an affinity in the nanomolar range in a calcium-dependent manner, that endostatin is not a glutaminyl substrate of TG-2 in vitro and that it is not cross-linked to itself by the enzyme. As endostatin and TG-2 are both involved in the regulation of angiogenesis and have been implicated in Alzheimer's disease, it is likely that their interaction is of major importance for the modulation of endothelial cell migration and/or proliferation, and for the formation and/or the stabilization of amyloid plaques in neurodegenerative diseases.

EXPERIMENTAL

Proteins and antibodies

Recombinant human TG-2 was purchased from Immundiagnostik and Covalab. Guinea-pig TG-2, extracted from the liver, was from Sigma–Aldrich. Recombinant endostatin, the NC1 domain (the NC1 domain is the entire C-terminal globular domain of collagen XVIII of which endostatin is a proteolytic fragment) and NC1 mutants (NC1-D104N and NC1-R27A/R139A) were produced in HEK (human embryonic kidney)-293 cells expressing the EBNA (Epstein–Barr virus nuclear antigen) [4,22,23]. Transfected HEK-293 cells expressing wild-type or mutant endostatin and the NC1 domain were a gift from Naomi Fukai and Reidunn Jetne (Harvard Medical School, Boston, MA, U.S.A.). To avoid the confusion arising from the two different numberings reported for endostatin in the literature, amino acids residues are numbered starting from the first amino acid residue of endostatin (i.e. His1, which corresponds to His132 when numbering starts from the first amino acid of the entire NC1 domain). Both proteins were tagged with the FLAG peptide at the N- or the C-terminus for endostatin and at the C-terminus for the NC1 domain. The conditioned culture media were filtered through 0.22 μm filters and applied on to a 5-ml heparin HiTrap™ column (GE Healthcare) for wild-type and mutant endostatin, and on to an anti-FLAG M2 affinity column (Sigma–Aldrich) for the wild-type and mutant NC1 domain. Endostatin was further purified by gel filtration on a 60 cm×2.6 cm Superdex S75 column (GE Healthcare) and the NC1 domain was further purified on a 60 cm×2.6 cm Sephacryl S200 column (GE Healthcare). The purity of proteins was assessed by SDS/PAGE and immunoblotting (results not shown). The purified proteins were concentrated by ultrafiltration under nitrogen (350 kPa) in a stirred cell (model 8050; Millipore) with regenerated cellulose membranes (YM10, molecular-mass cut-off 10 kDa; Millipore). The concentration process was followed by measuring the absorbance of the protein solution at 280 nm. Proteins were stored at −80 °C. Recombinant human endostatin expressed in Pichia pastoris was from Calbiochem. Anti-TG-2 monoclonal antibody (CUB7402) was from NeoMarker. Bovine collagen XI was a gift from Dr Marie-Claire Ronzière (UMR 5086, CNRS, University Lyon 1, Lyon, France).

Preparation of a polyclonal antibody against human endostatin

Recombinant human endostatin with a FLAG peptide at the C-terminus was used for the immunization of New Zealand white rabbits. All experiments were conducted in the animal facility of the ENVL (Ecole Nationale Vétérinaire de Lyon) (agreement A691270401) with full compliance to the ENVL Ethical Committee guidelines and the current French and European legislation for animal protection. Endostatin diluted in PBS emulsified with an equal volume of Freund's complete adjuvant (Sigma–Aldrich) was injected intradermally. Two booster injections with Freund's incomplete adjuvant (Sigma–Aldrich) were performed 14 and 28 days after the first injection. The IgG fraction was purified from the immunoserum by affinity chromatography on Protein A Ceramic Hyper D®F (Pall Life Sciences). IgGs were eluted with 0.1 M citric acid, pH 3, and neutralized with 1 M potassium phosphate buffer, pH 9. The polyclonal antibody was assayed by immunoblotting and solid-phase assays. The antibody reacted with wild-type and mutant endostatin, and with the wild-type and mutant NC1 domain (see Supplementary Figure S1 available at http://www.BiochemJ.org/bj/427/bj4270467add.htm).

Solid-phase binding assays

TG-2 was diluted in TBS (Tris-buffered saline; 25 mM Tris/HCl, pH 7.4, and 150 mM NaCl) for coating. Endostatin and the NC1 domain were diluted either in 10 mM PBS, pH 7.4, containing 138 mM NaCl and 27 mM KCl, or in TBS. Aliquots (100 μl) were added to the wells of a 96-well microplate (MaxiSorp). Plates were incubated overnight at 4 °C and wells were blocked for 2 h with 5% (w/v) BSA in TBS. The plates were incubated for 2 h at room temperature (20 °C) with TG-2 diluted in TBS containing 1 mM EDTA with or without cations (8 mM CaCl2, MgCl2 or MnCl2). The wells were washed three times with TBS containing 0.1% Tween 20. Bound TG-2 was detected with the monoclonal anti-TG-2 antibody, diluted 1:1000 in TBS with 0.1% Tween 20, for 1 h at room temperature. Bound endostatin was detected with the polyclonal anti-endostatin antibody diluted 1:1000. The wells were washed three times with TBS containing 0.1% Tween 20 and were then incubated with either alkaline phosphatase-conjugated or peroxidase-conjugated secondary antibodies. The immunological reaction was detected by adding p-nitrophenyl phosphate (absorbance measured at 405 nm), or 3,3′,5,5′-tetramethylbenzidine (absorbance was measured at 450 nm). Non-specific binding was measured in BSA-coated wells and was subtracted from the values measured in endostatin- or transglutaminase-coated wells. For inhibition experiments with GTP, endostatin was diluted in TBS (0.1 μg/well) and incubated overnight at 4 °C in a 96-well microplate (MaxiSorp). Wells were blocked for 2 h with 5% (w/v) BSA in TBS. TG-2 was pre-incubated in TBS containing 8 mM CaCl2 with various concentrations of GTP (0–100 μM) for 1 h at room temperature, before incubation for 2 h at room temperature. The binding of TG-2 to endostatin was performed as described above. All assays were performed in triplicate.

SPR (surface plasmon resonance) array assay

The SPR array assay was performed using a Biacore Flexchip system (GE Healthcare) as described previously [22]; the available high-density array platform is able to follow the binding of a single analyte to 400 target spots at a time. Proteins were spotted in triplicate at two concentrations (50 and 200 μg/ml) on to the surface of a Gold Affinity chip (GE Healthcare) using a non-contact PiezoArray spotter (PerkinElmer). Six drops of 330 pl each were delivered to the surface of the chip (spot diameter of 250–300 μm and spotted amount of 100–400 pg/spot). The chips were then dried at room temperature and stored under vacuum at 4 °C until their insertion into the Biacore Flexchip instrument. The chip was blocked with Superblock buffer (Pierce) five times for 5 min each time. The blocked chip was then equilibrated with PBS containing 0.05% Tween 20 at a flow rate of 500 μl/min for 90 min. TG-2 (500 nM) was flowed at 25 °C over the chip surface for 25 min at the same flow rate. The dissociation of the endostatin–TG-2 complex was monitored in the buffer flow (PBS containing 0.05% Tween 20) for 40 min. Data collected from reference spots (gold surface) and from buffer spots were subtracted from those collected on the spotted proteins to obtain specific binding curves.

SPR binding assays

The SPR binding assays were performed in a Biacore T100 instrument (GE Healthcare). Recombinant human endostatin (50 μg/ml in 10 mM maleate buffer, pH 6.2) was covalently immobilized to the dextran matrix of a CM5 sensor chip via its primary amine groups according to the manufacturer's instructions (using the Amine Coupling Kit; GE Healthcare) at a flow rate of 5 μl/min using Hepes-buffered saline (GE Healthcare) as a running buffer. An immobilization level ranging between 1800 and 2000 RUs (resonance units) was obtained and a control flow cell was prepared with 10 mM maleate buffer, pH 6.2. ZnCl2 (1 mM) was added to the running buffer for binding experiments. Sensorgrams collected on the control flow cell were automatically subtracted from the sensorgrams obtained on immobilized endostatin to yield specific binding responses. Binding assays were performed at 25 °C, but the sample compartment of the Biacore T100 was kept at 4 °C to maintain TG-2 in its native state. Kinetic and affinity constants were calculated by injecting various concentrations of TG-2 (8–130 nM), in the presence of 2 mM CaCl2, over immobilized endostatin at 60 μl/min. The complexes were dissociated by a 60 s pulse of 1.5 M NaCl containing 2 M guanidinium chloride. The kinetic rates, kass and kdiss (association and dissociation rate constants respectively), were calculated using the Biacore T100 evaluation software 2.0.1. Apparent equilibrium dissociation constants (Kd) were calculated as the ratio of kdiss/kass for a 1:1 (Langmuir) interaction model. Inhibition experiments were carried out by pre-incubating 130 nM TG-2 with 1 mM GTP for 15 min at room temperature before injecting the mixture over endostatin immobilized on the sensor chip.

Cross-linking experiments

Guinea-pig TG-2 (5 m-units/ml, Sigma–Aldrich) was incubated with recombinant human endostatin (10 μg/ml), produced in mammalian cells. The biotinylated peptide TVQQEL was used as an acyl donor [30] (Assay mix A5731; Sigma–Aldrich) and 1 mM dithiothreitol. The reaction was carried out for 2, 5, 10, 20, 30 and 60 min at room temperature and was stopped by the addition of 20 mM EDTA. Control experiments were performed with TG-2 and endostatin alone. The reaction products were analysed by SDS/PAGE (with a 4% stacking gel and a 12% concentration gel) and by immunoblotting with either streptavidin–peroxidase or the polyclonal anti-endostatin antibody, followed by ECL (enhanced chemiluminescence) detection (GE Healthcare). In another series of experiments, 10 m-units/ml TG-2 was incubated with 10 μg/ml endostatin, 2 mM biotin-X-cadaverin (Sigma–Aldrich), 8 mM CaCl2 and 1 mM dithiothreitol. The reaction was carried out for 120 min at room temperature and was stopped by the addition of 20 mM EDTA. Control experiments were performed with either TG-2 or endostatin alone. The reaction products were analysed as described above. The inhibition experiments for the reaction between 476 nM endostatin and the biotinylated peptide TVQQEL, mediated by 5 m-units/ml guinea-pig TG-2, were carried out for 60 min at room temperature in the absence of GTP or in the presence of various GTP concentrations (10–500 µM). The reaction products were analysed by immunoblotting using extravidin–peroxidase and the polyclonal antibodies against endostatin.

Immunocytochemistry

Primary cultures of HUVECs were grown in endothelial cell growth medium 2 (PromoCell). The cells were used for immunofluorescence experiments between passages 2 and 5. HUVECs were seeded on to glass coverslips and cultivated in complete medium under normoxic or hypoxic conditions (1% O2). The cells were then fixed with cold methanol for 5 min before blocking with 10% (v/v) normal goat serum in PBS. They were then incubated either with the anti-endostatin antibody or with the antibody against TG-2, diluted in PBS containing 1% (v/v) normal goat serum. Secondary goat antibodies directed against rabbit or mouse IgG coupled to Alexa Fluor® 488 or 555 were from Invitrogen. Nuclei were stained with TO-PRO®-3 (Invitrogen). Coverslips were mounted with Mowiol and observed with a TCS SP2 confocal microscope (Leica Microsystems).

RESULTS

TG-2 binds to monomeric endostatin and to the C-terminal domain of collagen XVIII

The binding of human TG-2 to endostatin and to the trimeric NC1 domain (of collagen XVIII) was demonstrated by using SPR arrays (Figure 1A). Under the same experimental conditions, TG-2 was found previously to bind to known ligands heparin and heparan sulfate [31], collagen XI [32] (see Supplementary Figure S2 available at http://www.BiochemJ.org/bj/427/bj4270467add.htm), the α5β1 and αvβ3 integrins [10], and amyloid β-peptide [26]. The interaction was confirmed by solid-phase assays (Figure 1B) and was also observed with guinea-pig TG-2 (Figure 2B). Several sources of TG-2 and endostatin were used to support the existence of the interaction between these two molecules.

Figure 1 TG-2 binds to endostatin and NC1 domain

(A) Overlay of sensorgrams resulting from the injection of 500 nM human TG-2 over 154 pg of immobilized wild-type NC1 (continuous line) and 300 pg of wild-type endostatin (broken line). The flow rate was 500 μl/min. (B) Solid-phase binding assay. Binding of 350 nM human TG-2 to immobilized wild-type and mutant endostatin (ES) and the NC1 domain (0.5 μg/well).

The binding properties of the endostatin- and NC1-D104N mutants were investigated because individuals with the homozygous D104N polymorphism in the COL18A1 (collagen XVIII, α1) gene have a high risk of occurrence of cancer [33,34]. The D104N mutation did not alter the folding of endostatin or the NC1 domain as assessed by intrinsic fluorescence (see Supplementary Figure S3 available at http://www.BiochemJ.org/bj/427/bj4270467add.htm), nor did it significantly affect the ability of endostatin or the NC1 domain, to bind to TG-2 (Figure 1B, see also Figure 3B). In contrast, the double NC1-R27A/R139A mutant, which had been previously shown to have lost its capacity to bind heparin [19], but is properly folded as assessed by fluorescence spectroscopy (Supplementary Figure S3), bound to TG-2 to a lesser extent (at 31% of the wild-type NC1 domain) (Figure 1B).

The binding of recombinant endostatin (i.e. expressed in yeast or in mammalian cells) to immobilized TG-2 was concentration-dependent (Figure 2A). Similar results were obtained in the reverse orientation when TG-2 was incubated on endostatin-coated wells (Figure 2B).

Figure 2 Soluble and immobilized endostatin binds to TG-2

Solid-phase binding assays. (A) Binding of 500 nM recombinant human endostatin, produced in mammalian (black bars) or yeast (white bars) expression systems, to immobilized TG-2. (B) Binding of 350 nM human (white bars) or guinea-pig TG-2 (black bars) to immobilized mammalian human endostatin. Results are means±S.D. for three experiments.

The binding of endostatin to TG-2 is dependent upon cations

Binding assays were performed with human recombinant TG-2 in the presence of 1 mM EDTA, to remove residual cations from the sample and to analyse the effects of different cations added at 8 mM. In the absence of any cation, TG-2 did not bind significantly to endostatin (Figure 3A). The addition of cations increased the binding ~2-fold for MgCl2, ~10-fold for MnCl2 and ~38-fold for CaCl2 (Figure 3A). Similar increases in binding were obtained with guinea-pig TG-2 in the presence of cations (see Supplementary Figure S4 available at http://www.BiochemJ.org/bj/427/bj4270467add.htm). Calcium was used for further experiments because it was the most efficient in promoting binding of endostatin. In the presence of 8 mM CaCl2, the binding of TG-2 to wild-type endostatin and to the D104N endostatin mutant was saturable (Figure 3B).

Figure 3 Binding of endostatin to TG-2 is cation-dependent

(A) Effect of cations in solid-phase assays. Binding of 64 nM human TG-2 to wells coated in endostatin produced in HEK-293 cells (0.1 μg/well) in the presence of 8 mM cations. (B) Binding of human TG-2 (0, 1, 2, 4, 8, 16, 32, 64, 128 and 256 nM) to immobilized human wild-type endostatin (continous line) or mutant endostatin-D104N (broken line) (at 0.1 μg/well) in the presence of 8 mM CaCl2. Results are means±S.D. for three experiments.

Endostatin binds to TG-2 with high affinity

Kinetic analysis was performed by injecting guinea-pig TG-2 at several concentrations over immobilized endostatin produced either in mammalian cells (see Supplementary Figure S5A available at http://www.BiochemJ.org/bj/427/bj4270467add.htm) or in yeast (Supplementary Figure S5B). The sensorgrams were fitted to a 1:1 Langmuir model using the Biacore T100 evaluation software 2.0.1 and kinetics and affinity constants were calculated (Table 1). The affinity constants were 6.8 nM and 5.6 nM for recombinant endostatin produced from mammalian cells and yeast respectively (Table 1).

View this table:
Table 1 Kinetic and equilibrium dissociation constants of the binding of TG-2 to endostatin

Rate and affinity constants for the binding of soluble TG-2 to immobilized endostatin, from human and yeast cells, calculated from SPR binding assays in the presence of 2 mM CaCl2 using the Biacore T100 evaluation software 2.0.1.

TG-2 is regulated by calcium and GTP. The transamidating activity of the enzyme is inhibited by GTP in an allosteric fashion, whereas CaCl2 partially reverses GTP inhibition [35]. We performed inhibition experiments with GTP, a reversible inhibitor of the cross-linking activity of TG-2, to determine whether it was able to interfere with the binding of TG-2 to endostatin. Recombinant TG-2 in TBS containing 8 mM CaCl2 was pre-incubated with increasing concentrations of GTP (0–100 μM) for 1 h at room temperature. It was then added to a 96-well microplate coated with recombinant human endostatin produced from mammalian cells and incubated for 2 h at room temperature. We observed a decrease in the binding level of TG-2 to endostatin upon the addition of increasing concentrations of GTP (Figure 4A). This inhibition was confirmed by SPR experiments where the binding of guinea-pig TG-2 to recombinant human endostatin produced from mammalian cells was nearly abolished (96.7% inhibition) by 1 mM GTP (Figure 4B). A similar effect was observed with human endostatin produced in yeast (100% inhibition, results not shown). The above results suggest that either endostatin binds to TG-2 at the same site as GTP or that the interaction with endostatin requires TG-2 to be in an open conformation, fully activated by calcium.

Figure 4 GTP inhibits the binding of TG-2 to endostatin

(A) Solid-phase binding assays. Effect of GTP (0–100 μM) on the binding of human TG-2 (64 nM) to endostatin (produced in HEK-293 cells; 0.1 μg/well) in the presence of 8 mM CaCl2. Results are means±S.D. for three experiments. (B) SPR binding assays. Injection of 130 nM guinea-pig TG-2 with (continuous line) or without (broken line) 1 mM GTP over immobilized human endostatin.

Endostatin is not a glutaminyl substrate of TG-2

We next performed enzymatic assays to determine whether endostatin was a substrate of TG-2. Endostatin was incubated with the enzyme in the presence of an acyl donor, i.e. the biotinylated peptide TVQQEL. Analysis of the reaction products by SDS/PAGE and immunoblotting showed that the biotinylated peptide was cross-linked to endostatin, and to a lesser extent to the enzyme (Figure 5A). The amount of peptide incorporated increased as a function of time. In the absence of TG-2, or in the presence of a calcium ion chelator (20 mM EDTA), the biotinylated peptide was not cross-linked with either endostatin or TG-2. Immunodetection with a polyclonal anti-endostatin antibody showed that endostatin was not cross-linked to itself or to TG-2 under those experimental conditions (Figure 5B). When endostatin was reacted with the biotinylated peptide TVQQEL in the presence of TG-2 and GTP, the amount of peptide incorporated into endostatin decreased when the GTP concentration increased (Figure 6A), whereas the amount of endostatin did not change under these conditions (Figure 6B). The incorporation of the peptide was totally inhibited by 500 μM GTP as shown in Figure 6(A). As GTP is an inhibitor of the transamidating activity of TG-2, these results confirm that the cross-linking of endostatin to TVQQEL is mediated by the transamidating activity of TG-2 and that endostatin behaves as an acyl acceptor in the transamidation reaction.

Figure 5 Endostatin is as an acyl acceptor in the transamidation reaction catalysed by TG-2

Cross-linking experiments. Reaction between 476 nM endostatin and the biotinylated peptide TVQQEL (Peptide), mediated by 5 m-units/ml guinea-pig TG-2. The reaction was carried out for 0, 2, 5, 10, 20, 30 and 60 min at room temperature. Results were analysed by immunoblotting using (A) extravidin–peroxidase and (B) the polyclonal antibody against endostatin. The molecular mass in kDa is indicated on the left-hand side.

Figure 6 GTP inhibits the cross-linking between endostatin and an acyl donor mediated by guinea-pig TG-2

The reaction between 476 nM endostatin and the biotinylated peptide TVQQEL (Peptide) mediated by 5 m-units/ml guinea-pig TG-2. Reactions were carried out for 60 min at room temperature in absence of GTP or in presence of increasing GTP concentrations (10–500 μM). Results were analysed by immunoblotting using (A) extravidin–peroxidase and (B) the polyclonal antibody against endostatin. The molecular mass in kDa is indicated on the left-hand side.

When endostatin was incubated with TG-2 and cadaverin, an acyl acceptor, no cross-linking reaction was detected, even when the reaction was performed with an increased amount of enzyme (10 m-units/ml instead of 5 m-units/ml) and for a longer period of time (120 min compared with 60 min) (Figure 7). These results show that endostatin is an acyl acceptor, but not an acyl donor in the transamidation reaction catalysed by TG-2. Preliminary results showed that endostatin did not inhibit the cross-linking activity of TG-2 in vitro (see Supplementary Figure S6 available at http://www.BiochemJ.org/bj/427/bj4270467add.htm), suggesting that endostatin does not bind to the transamidating active site of TG-2.

Figure 7 Endostatin is not a glutaminyl substrate of TG-2

Cross-linking experiments. Reaction between 476 nM endostatin (ES) and biotin-X-cadaverin (Cad) in presence of 10 m-units/ml human or guinea-pig TG-2. Analysis of the reaction products by immunoblotting using (A) extravidin–peroxidase and (B) the polyclonal antibody against endostatin. The molecular mass in kDa is indicated on the left-hand side.

Endostatin and TG-2 are co-localized in the extracellular matrix secreted by endothelial cells and in vessel walls

To determine whether the binding of endostatin with TG-2 occurs within the extracellular matrix secreted by endothelial cells and/or at the cell surface, immunostaining experiments were performed with the anti-endostatin and anti-TG-2 antibodies. As hypoxia induces the expression of the COL18A1 gene in a HIF (hypoxia-inducible factor)-1α-dependent manner in endothelial cells [36], HUVECs were incubated in a hypoxic environment for 6 days to enhance detection of endostatin by immunofluorescence. Indeed an increased level of endostatin was detected in the extracellular matrix synthesized by cultured endothelial cells under hypoxia, which stimulates angiogenesis when compared with normoxia (Figures 8A and 8B). As shown in Figure 8, TG-2 co-localized with endostatin/collagen XVIII in the extracellular matrix secreted by HUVECs cultured under hypoxic conditions (Figures 8C–8E). We have confirmed the co-localization of TG-2 and endostatin in tissue by performing double immunostaining of foreskin sections with antibodies directed against TG-2 and endostatin. TG-2 and endostatin were co-localized in vessel walls (see Supplementary Figure S7 available at http://www.BiochemJ.org/bj/427/bj4270467add.htm).

Figure 8 Endostatin and TG-2 are co-localized in the extracellular matrix synthesized by HUVECs under hypoxia

Immunostaining of (AC) endostatin/collagen XVIII and (D) TG-2 in the extracellular matrix secreted by cultured HUVECs. (E) The merge of images (C) and (D). Cells were grown to confluence under (A) normoxic or (BE) hypoxic conditions. Nuclei (blue) were stained using TO-PRO®-3. Scale bar, 30 μm.

DISCUSSION

The interaction between endostatin and TG-2 was demonstrated by SPR and by solid-phase assays with two different sources of TG-2 (human recombinant TG-2 and guinea-pig TG-2 extracted from liver) and two different sources of recombinant human endostatin (produced by HEK-293 EBNA-expressing cells or by yeast). These experiments revealed that endostatin binds to TG-2 with an affinity in the nanomolar range and in a cation-dependent manner, with calcium ions being the most potent among those assayed.

TG-2 catalyses post-translational modification of proteins by the formation of γ-glutamyl-ε-lysine bonds between glutamine and lysine residues. Endostatin contains eight glutamine residues that could potentially act as glutaminyl substrates (acyl donors) in the transamidation reaction. However, endostatin is not a glutaminyl substrate of TG-2 in vitro, but rather an acyl acceptor, providing lysine residues in the transamidation reaction. This is in agreement with the fact that none of the glutamine residues of endostatin is found within a Gln-Xaa-Pro sequence, a preferred sequence for glutamine-donor substrates [37] and with the fact that the N-terminus of endostatin contains a Gln7-Pro8 sequence reported to abolish transamidation [37]. The Leu-Gly-Gln-Ser sequence found in the N-propeptide of the α1 chain of procollagen III, a physiological substrate of TG-2 [38], is present in endostatin (residues 158–161) but it is located on the side of endostatin that is not involved in the binding to TG-2. Therefore Gln160 does not appear to be a glutaminyl substrate of TG-2.

GTP inhibits the binding of endostatin to TG-2, suggesting that endostatin could bind to the GTP-binding site on TG-2. However, the binding of endostatin to TG-2 is calcium-dependent and calcium is known to activate TG-2 by switching on its transamidating (i.e. cross-linking) activity [15]. As the activation of TG-2 by calcium can be counteracted by the allosteric inhibitor GTP [35], the inhibition of endostatin binding by GTP might alternatively suggest that endostatin binds to the transamidating active form of the enzyme. Thus we used TG-2 in an extended active conformation [39] to further investigate the location of the endostatin-binding site on TG-2. In present study we have shown that the Arg27 and/or Arg139 residues participate in the binding of endostatin to TG-2. These residues both form part of a large basic patch on endostatin, comprising the heparin-binding site [19], that is likely to interact with negatively charged amino acids on the surface of TG-2. In addition to our experimental results, we have used the Connolly molecular surface of TG-2 to find clues to the possible binding site(s) for endostatin on TG-2. There are three negatively charged areas on the enzyme: one in the N-terminal β-sandwich domain, one close to transamidating catalytic core, and a third one close to the GTP-binding site. We have shown that endostatin does not interfere with TG-2 cross-linking activity in vitro and that GTP blocks the binding of endostatin to TG-2. These results suggest that endostatin may bind to, or close to, the GTP-binding site but not to the transamidating active site of TG-2.

The requirement for calcium ions in order for endostatin to bind to TG-2 suggests, as discussed above, that endostatin binds to the open form of the enzyme. Extracellular TG-2 is predominantly maintained in the catalytically inactive closed conformation under ordinary physiological conditions and is able to promote cell adhesion, spreading, migration or differentiation independently of its catalytic activity [15]. Extracellular TG-2 is transiently activated in response to innate immune signals, such as exposure to polyinosinic-polycytidylic acid, a potent ligand of TLR (Toll-like receptor)-3 [40]. The stimulation of innate immune pattern recognition receptors such as TLR-3 triggers the release of extracellular TG-2 from cell surface integrins and the concomitant enzyme activation [40]. It is thus possible that, in the course of angiogenesis, an up-regulated innate immune receptors triggers the activation of extracellular TG-2 leading in turn to endostatin binding. This hypothesis is supported by the fact that multiple human endothelial cell types express surface TLR-3, which play a key role in disrupting the haemostasis balance on endothelial cells [41], and by the suppression of sequence- and target-independent angiogenesis by TLR-3 siRNA (small interfering RNA)-mediated down-regulation [42].

Endostatin might co-operate with TG-2 in several physio-pathological processes either through direct interactions or through more complex mechanisms involving multimolecular complexes. Direct interactions between endostatin and TG-2 might play a role in organizing the extracellular matrix in skin basement membrane, endostatin being present in this location [43], and in TG-2 stabilizing basement membranes [44]. At the endothelial cell surface, endostatin inhibits both the extracellular activation of proMMP (matrix metalloproteinase)-2 by inhibition of MT1 (membrane-type 1)-MMP as well as the catalytic activity of MMP-2, thus blocking the invasiveness of tumour cells [45]. MMP-2, functioning in concert with MT1-MMP, hydrolyses cell-surface-associated TG-2, and the cleavage eliminates both the adhesion and the enzymatic activity of TG-2 [46]. It should be noted that endostatin and TG-2 bind to the catalytic domain of MMP-2 and both proteins inhibit MMP-2 maturation [46,47].

Besides MMP-2, endostatin and TG-2 share heparan sulfate, VEGFR-2 and integrins as binding partners at the cell surface, and both regulate endothelial cell adhesion [3,6,10,11,48]. As reported in the present paper, two arginine residues implicated in the binding of endostatin (Arg27 and/or Arg139) to TG-2 are also involved in the binding to α5β1 and αvβ3 integrins [4], and to heparin [19]. This suggests that endostatin is not able to bind simultaneously to TG-2 and α5β1/αvβ3 integrins, or to TG-2 and heparan sulfate. The co-localization of endostatin/collagen XVIII and TG-2 in the extracellular matrix secreted by cultured endothelial cells indicates that this interaction may occur in a cellular context and supports its physiological relevance. These proteins are co-localized in the extracellular matrix synthesized under hypoxia, which stimulates angiogenesis. This raises the possibility that endostatin and TG-2 regulate endothelial cell behaviour and control angiogenesis in a concerted fashion. However, their molecular interplay in the extracellular matrix secreted by endothelial cells and/or at the surface of these cells remains to be deciphered.

AUTHOR CONTRIBUTION

Clément Faye, Antonio Inforzato, Marine Bignon, Daniel J. Hartmann, Laurent Muller and Lionel Ballut performed the experiments. Bjorn Olsen provided the HEK-293 cells transfected with plasmids coding for endostatin, mutant endostatins and the NC1 domain, and contributed to the discussion. Antony Day and Sylvie Ricard-Blum designed the research and analysed the results. Sylvie Ricard-Blum wrote the paper.

FUNDING

This work was supported by the Association pour la Recherche contre le Cancer [grant number 3652 (to S.R.B.)]; the GIS-Institut des maladies rares [grant number Inserm A04115SP (to S.R.B.)]; Le Contrat de projets Etat-Région Rhône-Alpes (to C.F. and S.R.B.); the Emergence Research Program Région Rhône-Alpes (to S.R.B.); a Explora'doc grant Cluster 10, Région Rhône-Alpes (to C.F.); a BQR grant from the University Lyon-1 (to S.R.B.); and by the National Institutes of Health [grant number 36820 (to B.O.)].

Acknowledgments

We thank Dr C. Marquette and Professor L. Blum (UMR 5246 Centre National de la Recherche Scientifique, University Lyon 1, Lyon, France) for their help in spotting on to the Gold affinity chips, Sylviane Guerret (Novotec, Lyon, France) for her skilful assistance in the production of the polyclonal anti-endostatin antibody and Naomi Fukai and Reidunn Jetne for their gift of transfected HEK-293 cells expressing wild-type or mutant endostatin and the NC1 domain. The support of GE Healthcare (Dr C. Quétard, P. Linden, T. Salomon, X. Rousselot and A. Sylvan) is gratefully acknowledged. SPR experiments were performed in the facility of L'Institut Fédératif de Recherche 128, Gerland Lyon Sud, Lyon, France.

Abbreviations: COL18A1, collagen XVIII, α1; EBNA, Epstein–Barr virus nuclear antigen; ERK, extracellular-signal-regulated kinase; FAK, focal adhesion kinase; HEK, human embryonic kidney; HIF, hypoxia-inducible factor; HUVEC, human umbilical vein endothelial cell; MAPK, mitogen-activated protein kinase; MMP, matrix metalloproteinase; MT1, membrane-type 1; NC1 domain, entire C-terminal globular domain of collagen XVIII; SPR, surface plasmon resonance; TBS, Tris-buffered-saline; TG-2, transglutaminase-2; TLR, Toll-like receptor; VEGF, vascular endothelial growth factor; VEGFR, VEGF receptor

References

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