Analytical Biochemistry 394 (2009) 217–222

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Analytical Biochemistry

journal homepage: www.elsevier .com/locate /yabio

A novel array-based assay of in situ tissue transglutaminase activity in humanumbilical vein endothelial cells

Jin-Young Park, Se-Hui Jung, Jae-Wan Jung, Mi-Hye Kwon, Je-Ok Yoo, Young-Myeong Kim, Kwon-Soo Ha *

Department of Molecular and Cellular Biochemistry and Vascular System Research Center, Kangwon National University School of Medicine, Chuncheon,Kangwon-Do 200-701, South Korea

a r t i c l e i n f o

Article history:Received 18 April 2009Available online 30 July 2009

Keywords:In situ transglutaminaseProtein arrayReproducibilityMaitotoxinHuman umbilical vein endothelial cells

0003-2697/$ - see front matter � 2009 Elsevier Inc. Adoi:10.1016/j.ab.2009.07.040

* Corresponding author. Fax: +82 33 250 8807.E-mail address: ksha@kangwon.ac.kr (K.-S. Ha).

1 Abbreviations used: TG, transglutaminase; ROS, rbovine serum albumin; NAC, N-acetyl-L-cysteine; H2Dorescin diacetate; bFGF, basic fibroblast growth facphosphate-buffered saline; BCA, bisinchoninic acid;coefficient of variation; TG2, transglutaminase 2.

a b s t r a c t

Transglutaminases (TGs), a family of calcium-dependent transamidating enzymes, are involved in func-tions such as apoptosis and inflammation and play a role in autoimmune diseases and neurodegenerativedisorders. In this study, we describe a novel array-based approach to rapidly determine in situ TG activityin human umbilical vein endothelial cells and J82 human bladder carcinoma cells. Amine arrays were fab-ricated by immobilizing 3-aminopropyltrimethoxysilane on glass slides. The assay was specific andhighly reproducible. The average coefficient of variation betweens spots was 2.6% (n = 3 arrays), andthe average correlation coefficients between arrays and between arrays/reactions were 0.998 and0.976, respectively (n = 3 arrays). The assay was successfully applied to detect changes in TG activityinduced by maitotoxin and to analyze inhibition of the TG activation with cystamine and monodansylcadaverine. In addition, the assay demonstrated that intracellular reactive oxygen species regulate themaitotoxin-induced activation of TG. Thus, the array-based in situ TG activity assay constitutes a rapidand high-throughput approach to investigating the roles of TGs in cell signaling.

� 2009 Elsevier Inc. All rights reserved.

Transglutaminases (TGs)1 belong to a family of calcium-depen-dent transamidating enzymes that catalyze a calcium-dependentacyl transfer reaction between the c-carboxamide group of a poly-peptide-bound glutamine and the e-amino group of a polypeptide-bound lysine [1,2]. Transamidating activity of TGs (isopeptide link-age by acyl transfer) in a number of proteins has been observed invarious cellular events such as cell growth [3], cell differentiation[4], cell adhesion [5], extracellular matrix crosslinking [6], andapoptosis induction [7,8]. The transamidating activity of TG is reg-ulated by diverse factors such as Ca2+, GTP, nitric oxide, sphingo-syl-phosphocholine, and intracellular reactive oxygen species(ROS) [9–15]. However, dysregulation of TG activity is associatedwith major disruptions in cellular homeostatic mechanisms andcontributes to a number of human disorders such as neurodegen-erative diseases, neoplastic diseases, and autoimmune diseases[16].

Due to the importance of TGs in cell regulation and human dis-eases, a number of assays have been developed to determine in situ

ll rights reserved.

eactive oxygen species; BSA,CFDA, 2,7-dichlorodihydroflu-tor; DTT, dithiothreitol; PBS,DCF, dichlorofluorescein; CV,

TG activity. Various strategies used for determination of in situ TGactivity commonly detect incorporation of amine donors into glut-amyl substrates. In immunochemical or enzyme-linked colorimet-ric assays, in situ TG activity is determined by detecting the aminedonor-incorporating proteins in cells using 96-well microtiterplates [17]. In assays using a confocal microscope, in situ TG activ-ity is determined at the single-cell level by measuring relative fluo-rescence intensities of images obtained by confocal microscopy[9,15]. Immunoblotting has also been used to determine in situTG activity using whole-cell extracts [18]. However, these assaysrequire large sample volumes and can be time-consuming. More-over, most are not well suited to high-throughput determinationof in situ TG activity.

Recently, protein arrays have emerged as a powerful technologyfor enzyme activity assays, analysis of protein interactions, serodiag-nosis, and protein profiling because protein arrays allow analysis of avariety of biomolecular interactions using a small sample volume ina single experiment [19]. Protein arrays are analyzed with a numberof detection methods, including surface plasmon resonance, massspectrometry, atomic force microscopy, and fluorescence detection[20–22]. Fluorescence detection is the most favored method becauseit is very simple and quite sensitive. Fluorescence-based protein ar-ray systems have been successfully applied to the serodiagnosis ofhuman diseases, expression profiling of serum proteins, and identi-fication of protein biomarkers of various cancers [23–26]. In addi-tion, these systems have been used for analysis of protein kinase

218 Assay of in situ tissue TG activity in HUVECs / J.-Y. Park et al. / Anal. Biochem. 394 (2009) 217–222

activity and for profiling of phosphorylated proteins [27,28]. How-ever, few studies have reported enzyme activity assays using proteinarrays other than for protein kinases. To our knowledge, there hasbeen no report of protein array-based determination of in situ TGactivity using a high-throughput method.

Here we present a new assay of in situ TG activity using well-type protein arrays in human umbilical vein endothelial cells (HU-VECs) and J82 human bladder carcinoma cells. We fabricated ami-no arrays on glass slides and determined in situ TG activity directlyby analyzing the arrays with a fluorescence scanner. This assay iseasy to perform, simple, and sensitive, and it allows determinationof in situ TG activity in a very small sample volume using a high-throughput format. Thus, as a rapid method for analysis of intracel-lular TG activity, this assay system has great potential for investi-gating the roles of TGs in cell signaling.

Materials and methods

Materials

3-Aminopropyltrimethoxysilane, ammonium hydroxide, hydro-gen peroxide, bovine serum albumin (BSA), Cy3-conjugated strep-tavidin, N,N0-dimethyl casein, cystamine, monodansyl cadaverine,N-acetyl-L-cysteine (NAC), and Trolox were obtained from Sigma(St. Louis, MO, USA). Fetal bovine serum, penicillin/streptomycinsolution, and M199 medium were acquired from Gibco (Gaithers-burg, MD, USA). 5-(Biotinamido)pentylamine was obtained fromPierce (Rockford, IL, USA). TG from guinea pig liver was purchasedfrom Oriental Yeast (Tokyo, Japan). Maitotoxin and 2,7-dichlorodi-hydrofluorescin diacetate (H2DCFDA) were obtained from WakoPure Chemical (Osaka, Japan) and Molecular Probes (Eugene, OR,USA), respectively.

Cell culture

HUVECs were isolated from human umbilical cord veins bytreatment with collagenase as described previously [18]. The cells

Fig. 1. Schematic diagram of in situ TG a

were maintained in M199 medium supplemented with 20% fetalbovine serum, 100 U/ml streptomycin, 100 U/ml penicillin, 3 ng/ml basic fibroblast growth factor (bFGF), and 5 U/ml heparin (cul-ture medium) in a humidified 5% CO2 incubator at 37 �C. For exper-iments, cells between passages 2 and 6 were cultured on gelatin-coated 24-well plates for 2 days and incubated for 6 h with M199medium supplemented with 1% fetal bovine serum and 100 U/mlpenicillin (starvation medium). J82 cells (American Type CultureCollection, Manassas, VA, USA) were maintained at 37 �C in RPMI1640 medium supplemented with 10% fetal bovine serum, 100 U/ml penicillin, and 100 lg/ml streptomycin (culture medium) in a5% CO2 atmosphere.

Preparation of amine arrays

Amine arrays were prepared according to the procedure of Jungand coworkers [23]. Briefly, glass slides (75 � 25 mm) werecleaned with NH4OH/H2O2/H2O (1:1:5, v/v) at 70 �C for 10 min,incubated with 1.5% (v/v) 3-aminopropyltrimethoxysilane solutionin 95% ethanol for 2 h, and sequentially washed with ethanol andwater. The amine-modified slides were baked at 110 �C for 1 hand mounted with Teflon tapes (75 � 25 mm) with 108 (18 � 6)2.0-mm diameter holes to fabricate well-type amine arrays. Aminearrays were stored under vacuum until use.

Evaluation of amine arrays for in situ TG activity assays

Reaction mixtures containing 1 mg/ml N,N0-dimethylcasein inreaction buffer (200 mM Tris [pH 7.5], 10 mM CaCl2, 10 mMdithiothreitol [DTT], 1% Triton X-100, and 1 mM 5-(biotinami-do)pentylamine were incubated with various concentrations ofguinea pig TG at 37 �C for 30 min. Each 2 ll of reaction mixtureswas applied to array wells for 1 h, and arrays were washed with0.1% Tween 20 in phosphate-buffered saline (PBS: 8.1 mM Na2H-PO4, 1.2 mM KH2PO4 [pH 7.4], 2.7 mM KCl, and 138 mM NaCl)and Milli-Q filtered water. The arrays were then incubated withblocking solution (5% BSA and 0.1% Tween 20 in PBS) and

ctivity assay using well-type arrays.

Assay of in situ tissue TG activity in HUVECs / J.-Y. Park et al. / Anal. Biochem. 394 (2009) 217–222 219

sequentially washed with 0.1% Tween 20 in PBS and Milli-Q fil-tered water. After incubation at 37 �C for 1 h with 20 lg/ml Cy3-conjugated streptavidin in blocking solution, the arrays werewashed with 0.1% Tween 20 in PBS and Milli-Q filtered waterand scanned in a fluorescence scanner (ScanArray Express, Perk-inElmer, Boston, MA, USA).

In situ TG activity assay

In situ TG activity assays were performed on amine arrays asdescribed in Fig. 1. For in situ TG activity measurements, serum-starved HUVECs or J82 cells were loaded with 1 mM 5-(biotinam-

Fig. 2. Characterization of in situ TG activity assay using well-type arrays. (A,B)Reaction mixtures containing N,N0-dimethylcasein were incubated with variousconcentrations of guinea pig TG. (C) Reaction mixtures containing N,N0-dimethylca-sein were incubated with 10 lg/ml guinea pig TG in the presence of indicatedconcentrations of cystamine or monodansyl cadaverine (MDC). Here 2 ll of reactionmixture was applied to each well of the slides, and TG activity was determined asdescribed in Materials and Methods. Data are means ± standard deviations fromthree separate determinations. BAPA, 5-(biotinamido)pentylamine.

ido)pentylamine for 1 h and treated with maitotoxin at 37 �C. Cellswere incubated with a lysis buffer (50 mM Tris–HCl [pH 7.5], 1%Triton X-100, and protease inhibitor cocktail) for 10 min on ice,sonicated for 60 s, and centrifuged for 10 min at 14,000 rpm. Pro-tein concentrations were determined by the bisinchoninic acid(BCA) method, and lysates were diluted to a final concentrationof 0.2 mg/ml with the lysis buffer. For array assays, 0.8 ll of cell ex-tracts was applied to amine arrays for 1 h, and arrays were washedand incubated with blocking solution. After washing, the arrayswere incubated with 20 lg/ml Cy3-conjugated streptavidin at37 �C for 1 h and scanned using a fluorescence scanner. Fluores-cence intensities of array spots, representing in situ TG activity(fold), were quantified by the fixed circle method using ScanArrayExpress software (version 4.0, PerkinElmer).

Measurement of intracellular ROS

The amount of intracellular ROS was determined using the pro-cedure of Lee and coworkers [15]. Briefly, serum-starved cells onround coverslips were stabilized in serum-free culture mediumwithout phenol red for at least 30 min and then treated with10 lM 2,7-dichlorodihydrofluorescein diacetate for 10 min. Forsome experiments, cells were pretreated with 5 mM Trolox or10 mM NAC for 30 min. Cells were washed and immediately ob-served using a confocal microscope (FV300, Olympus, Tokyo, Ja-

Fig. 3. Reproducibility of in situ TG activity assay on well-type arrays. (A) Interarrayreproducibility, that is, reproducibility between same batches of reaction mixturesanalyzed on different array slides. (B) Interarray/Interreaction reproducibility, thatis, reproducibility between different batches of reaction mixtures analyzed ondifferent array slides. The correlation coefficients between arrays were analyzedusing the Linear Fit of the Origin program. The average correlation coefficient, r2, isgiven (n = 3 arrays). FI, fluorescence intensity; a.u., arbitrary units.

220 Assay of in situ tissue TG activity in HUVECs / J.-Y. Park et al. / Anal. Biochem. 394 (2009) 217–222

pan). Approximately 30 cells were randomly selected from threeseparate experiments, and dichlorofluorescein (DCF) fluorescenceintensities of stimulated cells were compared with those ofunstimulated control cells (fold).

Results

Characterization of array-based in situ TG activity

To answer the need for a simple, sensitive, and high-throughputmethod for analysis of in situ TG activity, we developed a novel as-say system using amine arrays. As illustrated in Fig. 1, we loadedHUVECs with 5-(biotinamido)pentylamine (an amine donor forin situ TG activity assay), lysed the cells, applied the cell extractsto well-type amine arrays, and probed the resulting biotinylatedproteins with Cy3-conjugated streptavidin. We then determinedin situ TG activity using fluorescence intensities obtained by ana-lyzing the arrays with a fluorescence scanner.

To investigate whether the assay system is appropriate fordetermination of in situ TG activity, we incubated reaction mix-tures containing TG substrates (N,N0-dimethylcasein and 5-(bioti-namido)pentylamine with various concentrations of guinea pigTG and analyzed the resulting biotinylated dimethylcasein onamine arrays using a fluorescence scanner. As shown in Figs. 2Aand B, the relative fluorescence intensity, representing the amount

Fig. 4. Analysis of in situ TG activity changes induced by maitotoxin in HUVECs andJ82 cells. Serum-starved HUVECs (A) and J82 cells (B), loaded with 1 mM 5-(biotinamido)pentylamine, were treated with indicated concentrations of maitotox-in (MTX) for 1 h. In situ TG activity was determined by the array-based assay systemas described in Materials and Methods. Data are means ± standard deviations fromthree separate determinations.

of biotinylated dimethylcasein, increased according to the concen-tration of guinea pig TG. However, in the absence of 5-(biotinami-do)pentylamine, fluorescence was not detectable at anyconcentrations of guinea pig TG, indicating that most of the fluo-rescence intensity was contributed by the transamidated dim-ethylcasein. In addition, transamidation of N,N0-dimethylcaseinwith guinea pig TG was inhibited by the TG inhibitors, cystamineand monodansyl cadaverine, in a dose-dependent manner(Fig. 2C). Thus, the assay system we have developed is quite suit-able for in situ TG activity assays.

Reproducibility of the array-based in situ TG activity assay

We next evaluated the reproducibility of the array system forin situ TG activity assays by analyzing interspot, interarray, andinterarray/interreaction reproducibility. Interspot reproducibilitywas measured by analyzing 10 replicate spots, and the averagecoefficient of variation (CV) was found to be 2.6% (n = 3 arrays)(data not shown). Interarray reproducibility was evaluated by ana-lyzing the same batch of reaction mixtures on different arrays. Theinterarray reproducibility was high, with an average correlationcoefficient of 0.998 (n = 3 arrays) (Fig. 3A). In addition, interar-ray/interreaction reproducibility was evaluated by analyzing dif-ferent batches of reaction mixtures on different arrays. Theinterarray/interreaction reproducibility was also found to be high,

Fig. 5. Inhibition of TG activity with cystamine and monodansyl cadaverine inHUVECs. Serum-starved HUVECs, loaded with 1 mM 5-(biotinamido)pentylamine,were treated with 200 pM maitotoxin in the presence of various concentrations ofcystamine (A) or monodansyl cadaverine (MDC) (B). In situ TG activity wasdetermined by the array-based assay system as described in Materials and Methods.Data are means ± standard deviations from three separate determinations.

Fig. 6. Regulation of TG activity by intracellular ROS in HUVECs. HUVECs grown onround coverslips were incubated with 5 mM Trolox or 10 mM NAC for 30 min andwere treated with 200 pM maitotoxin (MTX) for 1 h. Intracellular ROS (A) andin situ TG activity (B) were determined as described in Materials and Methods. Dataare means ± standard deviations from three independent determinations.

Assay of in situ tissue TG activity in HUVECs / J.-Y. Park et al. / Anal. Biochem. 394 (2009) 217–222 221

with an average correlation coefficient of 0.976 (n = 3 arrays)(Fig. 3B). Taken together, the results demonstrated that the assayresults are highly reproducible and appropriate for high-through-put analysis of in situ TG activity.

Application of array-based assay system for determination of in situTG activity in HUVECs

Maitotoxin has been reported to stimulate transglutaminase2 (TG2) in HUVECs in experiments where proteins incorporating5-(biotinamido)pentylamine were blotted and detected with horse-radish peroxidase-conjugated streptavidin [18]. However, thisapproach for determination of in situ TG activity is time-consuming.Thus, in this study we applied the array-based assay system to thehigh-throughput analysis of in situ TG activity in HUVECs. We loadedHUVECs with 5-(biotinamido)pentylamine, treated the cells withvarious concentrations of maitotoxin, and applied lysates from thetreated cells to amine arrays. As shown in Fig. 4A, maitotoxin stimu-lated in situ TG activity in a dose-dependent manner. At 100 pM, TGactivity increased significantly to approximately 9-fold that of con-trol levels. Maitotoxin-induced stimulation of in situ TG activitywas time-dependent (data not shown). In addition, we applied theassay system to rapid determination of in situ TG activity in J82human bladder carcinoma cells stimulated with maitotoxin. Maito-toxin induced a dose-dependent increase of in situ TG activity, with asignificant stimulation at 800 pM (Fig. 4B).

We next investigated the effects of cystamine and monodansylcadaverine, competitive inhibitors of TGs, on the stimulation ofin situ TG activity by maitotoxin. As shown in Fig. 5A, cystamineshowed dose-dependent inhibition of TG activity. Cystamine inhi-bition was evident at 10 lM, and 500 lM cystamine almost com-pletely abolished maitotoxin-induced stimulation of TG activity.Monodansyl cadaverine demonstrated dose-dependent inhibitionof TG activity with a maximal effect at 100 lM, as shown inFig. 5B. Based on these results, we conclude that the assay systemis appropriate for the high-throughput determination of in situ TGactivity.

Role of intracellular ROS in the activation of in situ TG activity

The transamidating activity of TG is regulated by intracellularROS [9,15]. We employed the assay system to investigate the roleof intracellular ROS in the stimulation of TG in HUVECs. First, wetreated HUVECs with 200 pM maitotoxin for 1 h in the presenceof the ROS scavenger, Trolox or NAC, and determined the level ofintracellular ROS by confocal microscopy using the cell-permeablefluorophore H2DCFDA. As shown in Fig. 6A, maitotoxin increasedthe level of intracellular ROS to approximately 2-fold that of thecontrol cells, and ROS production was inhibited with Trolox orNAC. We then measured in situ TG activity in maitotoxin-treatedcells treated with the ROS scavengers. As shown in Fig. 6B, maito-toxin induced an increase of in situ TG activity by approximately30-fold compared with the control cells, and TG stimulation wassignificantly suppressed with Trolox or NAC. Thus, we concludethat the array-based in situ TG activity assay system has strong po-tential for investigating the regulation of TG in cell signaling.

Discussion

In this article, we have presented a new approach for a high-throughput analysis of in situ TG activity in HUVECs using well-type arrays. TGs are a family of transamidating enzymes and par-ticipate in numerous cell functions, including apoptosis, cell adhe-sion, inflammation, and wound healing, as well as in diseases suchas neurodegenerative and autoimmune disorders [5,29,30]. A rapid

method of analysis of in situ TG activity is needed because detec-tion of transamidating activity is essential for investigating thefunctions of TGs in cells [31]. A number of assays have been usedto determine in situ TG activity, including enzyme-linked colori-metric assays, confocal microscopy assays, and immunoblots[9,17,18]. However, these conventional methods require large sam-ple volumes and are time-consuming and rather insensitive. Inaddition, these assays are not suited for use in high-throughputformats. To our knowledge, this is the first report of an array-basedassay for high-throughput determination of in situ TG activity.

The in situ TG activity assay system using amine-modified well-type arrays is simple, specific, reproducible, and rapid. We demon-strated that the assay system was highly specific because TG activ-ity was not detectable at any concentrations of TG in the absence of5-(biotinamido)pentylamine. Our results showed that the assaywas highly reproducible, as illustrated by the interspot reproduc-ibility (average CV of 2.6%) and interarray and interarray/interreac-tion reproducibility (correlation coefficients of 0.998 and 0.976,respectively). In addition, the assay allows simple and rapid analy-sis of in situ TG activity because it employs direct application ofcell lysates loaded with 5-(biotinamido)pentylamine onto theamine arrays and sequential probing of biotinylated proteins withCy3-conjugated streptavidin, as shown in Fig. 1. We successfullyapplied well-type amine arrays to high-throughput analysis ofmaitotoxin-induced changes in in situ TG activity in HUVECs and

222 Assay of in situ tissue TG activity in HUVECs / J.-Y. Park et al. / Anal. Biochem. 394 (2009) 217–222

J82 cells. The expression level of TG2 in J82 cells is much lowerthan that in HUVECs (data not shown). Consistent with a previousreport [15] in which stimulation of TG2 was demonstrated usingimmunoblotting with extracts from HUVECs, we showed withthe array-based activity assay that in situ TG activity was stimu-lated by maitotoxin in a dose- and time-dependent manner andthat this stimulation was significantly decreased by cystamineand monodansyl cadaverine, competitive inhibitors of TGs. Fur-thermore, we successfully applied the high-throughput assay sys-tem to investigate the regulation of in situ TG activity byintracellular ROS.

In summary, we have developed a novel assay system usingwell-type amine arrays to determine relative levels of in situ TGactivity in HUVECs. This new approach is rapid and allows use ofvery small sample volumes for determining in situ TG activity.Thus, it has strong potential for use in cell research and activity-based proteomics as well as for diagnosis of human diseases suchas celiac disease.

Acknowledgments

This work was supported in part by grants from the KoreaHealth 21 R&D Project (Ministry of Health, Welfare, and Family Af-fairs, A030003) and the Basic Research Program of the Korean Sci-ence and Engineering Foundation (KOSEF, 2008-05943). We thankthe Korea Basic Science Institute (Chuncheon Center) for help witharray preparation.

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