Simultaneous Activity Assay of Two Transglutaminase Isozymes

ARTICLE pubs.acs.org/ac

Simultaneous Activity Assay of Two Transglutaminase Isozymes, Blood Coagulation Factor XIII and Transglutaminase 2, by Use of Fibrinogen Arrays Mi-Hye Kwon, Se-Hui Jung, Young-Myeong Kim, and Kwon-Soo Ha* Department of Molecular and Cellular Biochemistry and Institute of Medical Science, Kangwon National University School of Medicine, Chuncheon, Kangwon-Do 200-701, South Korea

bS Supporting Information ABSTRACT: We developed an on-chip activity assay system to simultaneously determine the transamidating activities of blood coagulation factor XIII (FXIII) and transglutaminase 2 (TG2) by use of fibrinogen arrays. FXIII and TG2 are transglutaminase family members that are involved in various physiological functions, including vascular pathophysiology, bone development, and cancer progression. However, investigation of their differential functions is limited by the lack of high-throughput and isozyme-specific activity assays. For the on-chip activity assay, we fabricated protein arrays by immobilizing fibrinogen onto the 3-aminopropyltrimethoxysilane surface of well-type arrays, and we determined transamidating activity by probing biotinylated fibrinogen with Cy3-conjugated streptavidin on arrays. We optimized assay conditions, such as buffer pH, concentrations of dithiothreitol and 5-(biotinamido)pentylamine, and incubation time, and we created equations to determine specific FXIII and TG2 activities in samples. We successfully applied this assay system to monitor changes in FXIII and TG2 activities in THP-1 monocytic cells differentiated with phorbol 12-myristate13-acetate and interleukin-4. This activity assay is sensitive and suitable for highthroughput determination of FXIII and TG2 activities and thus has a strong potential for investigating the differential functions of these isozymes in cell signaling and cardiovascular pathophysiology research.

T

ransglutaminases (TGs) are a family of enzymes consisting of nine isozymes, including TG1
TG7, which are involved in Ca2+- and thiol-dependent post-translational modifications of proteins, and blood coagulation factor XIII (FXIII).1 TGs catalyze the formation of an ε-(γ-glutamyl)lysine bond between the γ-carboxamide group of a peptide-bound glutamine and the ε-group of a peptide-bound lysine, and they also catalyze the formation of a (γ-glutamyl)polyamine bond between a peptidebound glutamine and a polyamine.2 Among the isozymes, TG2 and FXIII are two major members.3 TG2 is involved in celiac disease, cataractogenesis, cardiovascular disease, diabetes,1,4 and neurodegenerative disorders including Huntington’s disease, Alzheimer’s disease, Parkinson’s disease, and nervous system injuries.5,6 Additionally, TG2 plays important roles in cellular responses such as cytoskeletal reorganization, stabilization of the extracellular matrix, cell migration, and apoptosis.2,7
9 Plasma FXIII (pFXIII) is present in blood as a heterotetramer composed of two potentially active A subunits (FXIII-A) and two protective/carrier B subunits (FXIII-B). pFXIII is converted to an active TG (FXIII-A) through the concerted action of thrombin and Ca2+ in the terminal phase of the blood clotting cascade.10,11 FXIII is involved in hemostasis, angiogenesis, wound healing, and maintaining pregnancy.11 Cellular FXIII (cFXIII) is a homodimer of potentially active A subunits (FXIII-A2). cFXIII is activated by nonproteolytic mechanisms r 2011 American Chemical Society

including increased intracellular Ca2+ concentrations in platelets and monocytes.11,12 FXIII is expressed primarily in bone marrow-derived cells, including platelets, megakaryocytes, monocytes, and monocytederived macrophages.1,13 TG2 is widely expressed in cardiac and vascular cells, including endothelial and smooth muscle cells, and cells involved in immunity and inflammation, including lymphocytes, neutrophils, monocytes, and macrophages.1,13,14 Expression of TG2 is also reported in fibroblastic cells and platelets.14,15 The cellular localization of FXIII is often coincident with TG2, but the roles and regulations of the two isozymes are often different. The proangiogenic activity of FXIII, mediated by extracellular cross-linking of VEGFR-2 to β-integrin subunits, is involved in tissue repair and remodeling. On the other hand, TG2 inhibits angiogenesis by accumulation of the extracellular matrix.10,14,16 The dimerization of AT1 receptors by intracellular FXIII-mediated cross-linking contributes to the development of atherosclerosis, whereas TG2 does not effectively form AT1 dimers.17 FXIII contributes to the pathogenesis of atherosclerosis by cross-linking lipoprotein(a).18 However, macrophageexpressed TG2 appears to function as an endogenous apoptotic Received: August 20, 2011 Accepted: September 30, 2011 Published: September 30, 2011 8718

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Figure 1. Schematic diagram of simultaneous on-chip activity assay for TG2 and FXIII.

cell clearance and anti-inflammatory factor that limits the expansion of atherosclerotic plaques.19 Further dissection of the differential roles of FXIII and TG2 would be facilitated by the development of TG isozyme-specific activity assay(s). TG activity has been measured by two approaches: probing amine incorporation and monitoring ammonia released during the TG reaction.20
23 Photometric assays based on the continuous monitoring of ammonia release have been used to determine FXIII concentrations in plasma samples.23 Various assays based on the incorporation of exogenous amine substrates have been used to determine FXIII and TG2 activities.15,21,22 Recent studies have reported on-chip activity assays that use fluorescence-based protein arrays to monitor TG2 activities in various cell types, including fibroblasts and neuroblastoma cells, and to determine FXIII activities in human plasma.15,24 Substrate peptides for determining individual activities of FXIII and TG2 have been reported.3,25 However, no assay is available to distinguish the activities of FXIII and TG2 in the same sample. In this study, we developed an on-chip activity assay to simultaneously determine FXIII and TG2 activities by use of fibrinogen arrays. Protein arrays were fabricated by immobilizing fibrinogen onto well-type amine arrays, and transamidation activity assays were performed by probing biotinylated fibrinogen using Cy3-labeled streptavidin. We optimized assay conditions and

created equations to determine specific FXIII and TG2 activities in samples including differentiating THP-1 monocytic cells.

’ EXPERIMENTAL SECTION Chemicals and Reagents. 3-Aminopropyltrimethoxysilane, fibrinogen, Gly-Pro-Arg-Pro amide (GPRP), thrombin, Cy3conjugated streptavidin, and monoclonal anti-mouse β-actin antibody were obtained from Sigma (St. Louis, MO). 5-(Biotinamido)pentylamine (BAPA) was purchased from Pierce (Rockford, IL). Purified human plasma FXIII and TG2 from guinea pig liver were purchased from Innovative Research (Novi, MI) and Oriental Yeast (Tokyo, Japan), respectively. Interleukin-4 (IL-4) was obtained from R&D Systems (Minneapolis, MN). Polyclonal anti-FXIIIA antibody (ab97636) and monoclonal anti-TG2 antibody (CUB7402) were obtained from Abcam (Cambridge, U.K.) and Neomarker (Fremont, CA), respectively. Cell Culture. Human acute monocytic leukemia cell line, THP-1 cells (American Type Culture Collection, Manassas, VA), were cultured in RPMI 1640 medium containing 10% fetal bovine serum, 100 units/mL penicillin, and 100 μg/mL streptomycin at 37 °C under humidified 5% CO2. THP-1 cells were differentiated to an adherent phenotype by treatment with 500 nM 8719

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Figure 2. Standard assay for determination of FXIII activity in various mixtures of human plasma FXIII and guinea pig TG2. (A) Reaction mixtures for selective FXIII activity assay, containing indicated concentrations of human plasma FXIII (0
5 Loewy units/mL) and guinea pig TG2 (0
10 milliunits/ mL), were loaded onto fibrinogen arrays. Arrays were incubated at 37 °C for 15 min and analyzed with a fluorescence scanner. (B) Sigmoidal plots of fluorescence intensities against FXIII concentration in the presence of indicated concentrations of TG2. (C) Correlation coefficients between the sigmoidal plots in panel B. Correlation coefficients were calculated between the plot at 0 milliunits/mL TG2 and the plots at 1 (R0&1), 2 (R0&2), 5 (R0&5), and 10 milliunits/mL TG2 (R0&10). (D) Standard curve for determining FXIII activity. The sigmoidal plot at 0 milliunits/mL TG2 was chosen to create the standard curve, and the equation for calculating the FXIII activity of samples is displayed. Results are expressed as means ( SD from three separate experiments.

phorbol 12-myristate13-acetate (PMA) in the culture medium for 3 h. Adherent cells were incubated with the culture medium overnight and treated with 20 ng/mL IL-4 in the culture medium for 6 days with replacement of the culture medium containing IL-4 every 2 days.26 Cell extracts were prepared by sonication in a lysis buffer (10 μg/mL leupeptin, 10 μg/mL aprotinin, 0.1 mM phenylmethanesulfonyl fluoride followed by centrifugation at 13 000 rpm for 15 min and then subjected to the on-chip assay for FXIII and TG2 activity. Preparation of Well-Type Fibrinogen Arrays. Amine arrays were prepared according to the procedures of Kong et al.27 Briefly, glass slides (75  25 mm) were cleaned with H2O2/ NH4OH/H2O (1:1:5 v/v/v) at 70 °C for 10 min and incubated with 1.5% (v/v) 3-aminopropyltrimethoxysilane solution in 95% ethanol for 2 h. Teflon tapes (75  25 mm) with 200 holes (25  8), 1.5 mm in diameter, were attached on the amine-modified slides. Fibrinogen arrays were fabricated by incubating these well-type amine arrays with 1 μL/well of 50 μg/mL fibrinogen solution in 9.3 mM phosphate buffer (pH 7.4) for 1 h at 37 °C. Simultaneous On-Chip Activity Assay for FXIII and TG2 by Use of Fibrinogen Arrays. Fibrinogen arrays were blocked with 3% bovine serum albumin containing 0.1% Tween 20 in phosphate-buffered saline (PBS; 8.1 mM Na 2 HPO4 , 1.2 mM KH2PO4, pH 7.4, 2.7 mM KCl, and 138 mM NaCl) for 30 min at 37 °C and washed with 0.1% Tween 20 in PBS and Milli-Q water. For measuring FXIII specific activities, samples of cell extracts or various standard mixtures of human plasma FXIII and guinea pig TG2 were prepared in a 30-μL volume in buffer containing 40 mM Tris-HCl, pH 9.5, 2 mM CaCl2, 7 units/mL

thrombin, 1 mM dithiothreitol (DTT), 2 mM BAPA, 0.3 mM GPRP, and 140 mM NaCl. For determining TG2-specific activities, samples of cell extracts or standard mixtures were similarly prepared in a 30-μL volume in buffer containing 40 mM Bis-Tris-HCl, pH 6.0, 2 mM CaCl2, 10 mM DTT, 5 mM BAPA, 0.01% Triton X-100, and 140 mM NaCl. For the simultaneous activity assay, 0.9 μL from each set of reaction mixtures described above for determining FXIII and TG2 activities was applied to the same fibrinogen arrays and incubated at 37 °C for 15 min (Figure 1). Transamidating activity-catalyzed incorporation of BAPA into fibrinogen was probed by incubating the arrays with 10 μg/mL Cy3-conjugated streptavidin at 37 °C for 30 min. Following washing, the arrays were scanned with a fluorescence scanner by use of a 543-nm laser (ScanArray Express GM; Perkin-Elmer, Waltham, MA). The fluorescence intensities of array spots were measured with the ScanArray Express program (Perkin-Elmer, Waltham, MA). Calculation of FXIII and TG2 Activities. FXIII and TG2 activities of samples were calculated from the fluorescence intensities obtained from on-chip activity assays. FXIII activity was initially calculated as follows:     A2
A1
1 =P ð1Þ xFXIII ¼ log x0
log yFXIII
A1 where xFXIII is the FXIII activity, x0 is the FXIII activity at halfmaximal fluorescence intensity, A1 and A2 are the top and bottom asymptotes, respectively, yFXIII is the fluorescence intensity of the samples, and p is the power (Hill slope). FXIII activity was expressed as Loewy units per milligram of protein. 8720

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Table 1. Summary of Assay Conditions for Determining FXIII and TG2 Activities FXIII activity assay

TG2 activity assay

buffer pH

9.5

6.0

DTT, mM BAPA, mM

1 2

10 5

Ca2+, mM

2

2

Triton X-100, %

0.01

GPRP, mM

0.3

thrombin, units/mL

7

TG2 activity was then determined by use of the FXIII activity from eq 1, as follows: xTG2 ¼

yTG2
ðcxFXIII þ dÞ axFXIII þ b

ð2Þ

where xTG2 is the TG2 activity of the samples, yTG2 is the fluorescence intensity of the samples in the TG2 activity assay, xFXIII is the FXIII activity of the samples, a and c are the slopes for linear regression, and b and d are the y-intercepts (Figure 2C,D). TG2 activity was expressed as milliunits per milligram of protein (nanomoles per minute per milligram of protein). Western Blot. Cell extracts were resolved by sodium dodecyl sulfate
polyacrylamide gel electrophoresis (SDS
PAGE) and transferred to a poly(vinylidene fluoride) membrane. Membranes were blocked with 5% skim milk (Becton Dickinson, Franklin Lakes, NJ) containing 20 mM Tris-HCl, pH 7.6, 150 mM NaCl, and 0.1% Tween 20 and then probed with antirabbit FXIII-A antibody (1:1000 v/v), anti-mouse TG2 antibody (1:4000 v/v), or anti-mouse β-actin antibody (1:2000 v/v). Subsequently, membranes were incubated with horseradish peroxidase-conjugated anti-rabbit or anti-mouse IgG (1:1000 v/v). The protein bands of FXIII-A, TG2, and β-actin were detected by the electrochemical luminescence (ECL) system (Pierce, Rockford, IL). FXIII and TG2 expression were quantitatively analyzed by use of Labworks v4.5 software (UVP Inc., Upland, CA). Statistical Analysis. Calculation of correlation coefficients and fitting of plots were performed by the Origin 6.1 software package (Origin Lab, Northampton, MA).

’ RESULTS AND DISCUSSION Optimization of Conditions for FXIII-Specific Activity Assay. FXIII and TG2 are differentially expressed and regulated,

and their functions are complementary or opposing.1,26 Various activity assays have been reported to determine TG2 or FXIII activities in cells and plasma;15,23,24 however, no assay is available to distinguish the activities of two isozymes in samples. Thus, for simultaneous determination of FXIII and TG2 activities in samples, we developed an on-chip activity assay that uses fibrinogen arrays (Figure 1). In this approach, we used fibrinogen arrays and optimized specific assay conditions, such as buffer pH, BAPA and DTT concentrations, and incubation time, to preserve the activity of one isozyme while minimizing the activity of the other. We performed standard assays with the fibrinogen arrays and created eqs 1 and 2 for determining FXIII and TG2 activities of samples. FXIII activities were initially determined by eq 1, and then TG2 activities were calculated by use of the FXIII activities from eq 2.

To optimize the FXIII-specific assay, we applied reaction mixtures for FXIII activity assays containing 5 Loewy units/mL human plasma FXIII or 5 milliunits/mL guinea pig TG2 to fibrinogen arrays and determined their transamidating activities. We examined assay conditions that maximally inhibit the transamidating activity of guinea pig TG2 with a minimal effect on human plasma FXIII (Figure S1, Supporting Information). We determined the optimal buffer pH by measuring the transamidating activity of reaction mixtures at pH 6.0
10.0. As shown in Figure S1A (Supporting Information), the transamidating activity of human plasma FXIII was dependent on buffer pH, with a maximal activation at pH 8.3. However, the transamidating activity of guinea pig TG2 was minimal at pH 9.5, demonstrating that pH 9.5 is optimal for the FXIII-specific activity assay. Recently, an on-chip activity assay was reported to analyze FXIII activities by use of fibrinogen arrays in normal human plasma samples and those from patients with liver disease.24 In this report, FXIII activities were determined for reaction mixtures at pH 8.3 and FXIII was found to be a candidate biomarker for hepatocellular carcinoma. In this report, however, we demonstrated that transamidating activity of guinea pig TG2 was not significantly inhibited at pH 8.3 (Figure S1A, Supporting Information). The transamidating activity of guinea pig TG2 was maximally prevented at pH 9.5, with a partial inhibition of transamidating activity of human plasma FXIII. These results demonstrate that buffer pH is a key factor for FXIII-specific activity assay to minimize the transamidating activity of guinea pig TG2. BAPA induced a dose-dependent elevation of the transamidating activity of human plasma FXIII, with saturation at 2 mM (Figure S1B, Supporting Information). However, transamidating activity of guinea pig TG2 was not detectable at the concentrations tested. We also determined the optimal DTT concentration for FXIII activity. As shown in Figure S1C (Supporting Information), DTT caused a dose-dependent increase in the transamidating activity of human plasma FXIII with a minimal stimulation effect on guinea pig TG2 at 1 mM. As expected, thrombin (7 units/mL) increased the transamidating activity of human plasma FXIII but had no effect on guinea pig TG2 (Figure S1D, Supporting Information). Using these optimized conditions of buffer pH and BAPA and DTT concentrations, we investigated changes in transamidating activity as a function of incubation time. The transamidating activity of human plasma FXIII showed a time-dependent increase, with a linear elevation for the initial 15 min (R = 0.988). However, no transamidating activity was detectable with guinea TG2 for 60 min under these assay conditions (data not shown), demonstrating that the FXIIIspecific activity assay can distinguish FXIII from TG2 in samples. Thus, we optimized assay conditions for the FXIII-specific activity by determining buffer pH, concentrations of BAPA and DTT, and incubation time (Table 1). Optimization of Conditions for TG2-Specific Activity Assay. For the TG2 activity assay, we used reaction mixtures containing 5 Loewy units/mL human plasma FXIII or 5 milliunits/mL guinea pig TG2, and we examined assay conditions to distinguish TG2 from FXIII (Figure S2, Supporting Information). Since buffer pH was a critical condition for the FXIII-specific activity assay, we determined the optimal pH for the TG2-specific activity assay. The transamidating activities of human plasma FXIII were higher than those of guinea pig TG2 at all pH values within the tested range except pH 6.0. Therefore, pH 6.0 was chosen as the optimal pH for the TG2-specific 8721

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Figure 3. Standard assays for determination of TG2 activity in various mixtures of human plasma FXIII and guinea pig TG2. (A) Reaction mixtures for selective TG2 activity assay, containing indicated concentrations of human plasma FXIII (0
5 Loewy units/mL) and guinea pig TG2 (0
10 milliunits/mL), were loaded onto fibrinogen arrays. Arrays were incubated at 37 °C for 15 min and analyzed with a fluorescence scanner. (B) Fluorescence intensities were plotted against TG2 concentration in the presence of indicated concentrations of FXIII, and line graphs were obtained from the linear fit function of the Origin program. The results are expressed as mean TG2 activity (FI  103) ( SD from three separate experiments. From the line graphs in panel B, slopes (in panel C) and y-intercepts (in panel D) were calculated and plotted against FXIII concentration.

activity assay. BAPA increased the transamidating activity of guinea pig TG2 in a dose-dependent manner, with saturation at 5 mM (Figure S2B, Supporting Information). We also investigated changes in transamidating activity as a function of DTT concentration and incubation time. DTT induced a dose-dependent increase in the transamidating activities of guinea pig TG2 and human plasma FXIII, and a maximal difference in transamidating activity between the two proteins was observed at 10 mM (Figure S2C, Supporting Information). Transamidating activity increased in an incubation time-dependent manner, with a linear increase up to 15 min (R = 0.986; Figure S2D, Supporting Information). The specific assay conditions for determining TG2 activity are summarized in Table 1. Standard Assay for FXIII Activity Determination. To create equations to determine the activities of FXIII and TG2 in samples, we simultaneously performed FXIII- and TG2-specific activity assays using two sets of standard reaction mixtures (Figure 1), one for the FXIII-specific activity assay and another for the TG2-specific activity assay, consisting of various concentrations of human plasma FXIII (0
5 Loewy units/mL) and guinea pig TG2 (0
10 milliunits/mL). The resulting arrays were analyzed with a fluorescence scanner, and Figure 2A shows a representative image for the FXIII-specific activity assay. The fluorescence intensity, which represents the transamidating activity, increased in a plasma FXIII concentration-dependent manner. However, guinea pig TG2 had no significant effect on FXIII-specific activity assay at the concentrations tested, supporting the selectivity of the FXIII activity assay, which was previously shown in Figure S1 (Supporting Information). To confirm the selectivity of the FXIII-specific assay, we plotted fluorescence intensities against FXIII concentrations in

the presence of indicated concentrations of TG2 from 0 to 10 milliunits/mL and obtained sigmoidal plots (Figure 2B). We then analyzed the correlation coefficients between the plot at 0 milliunits/mL TG2 and plots at 1, 2, 5, and 10 milliunits/mL TG2. As shown in Figure 2C, the correlation coefficients ranged from 0.987 to 0.995, demonstrating that the plots were very closely correlated with each other. These results confirmed that the FXIII-specific activity assay was selective for human plasma FXIII, but not affected by the presence of TG2 at concentrations up to 10 milliunits/mL. Therefore, the sigmoidal plot at 0 milliunits/mL TG2 was chosen to create a standard curve by use of eq 1 to calculate FXIII activities in samples (Figure 2D). Standard Assay for TG2 Activity Determination. To create an equation to determine the activity of TG2 in samples, we plotted fluorescence intensities obtained from TG2-specific activity assays (Figure 3A) against TG2 concentrations, in the presence of indicated concentrations of FXIII from 0 to 5 Loewy units/mL (Figure 3B). The measured fluorescence intensity, which represents transamidating activity, increased linearly as a function of TG2 concentration (up to 10 milliunits/mL) and was also dependent on the concentration of human plasma FXIII. These results demonstrated that a linear graph could be created as a standard curve for determining TG2 activity in samples, with FXIII activity as the key determinant for creating the standard curve. To generate a standard curve, plots in Figure 3B were fit via linear regression, and the slopes (A) and y-intercepts (B) of the line graphs were determined. The slopes and y-intercepts of the line graphs were plotted against plasma FXIII concentrations and two linear graphs, for the slopes (A) and y-intercepts (B), were obtained (Figure 3C,D). Since the slope and y-intercept for the standard curve can be calculated from FXIII activity by the 8722

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Figure 4. Determination of FXIII and TG2 activities in THP-1 cells differentiated with PMA and IL4. THP-1 cells were incubated with 500 nM PMA for 3 h, and the resulting adherent THP-1 cells were treated with 20 ng/mL IL-4 for the indicated times. Cell extracts were subjected to Western blot analysis and FXIII and TG2 activity assays. (A) Expression levels of FXIII determined by Western blot. Exposure times for FXIII and TG2 bands were 5 and 0.5 min, respectively. (B) Changes in FXIII activity. (C) Changes in TG2 activity. (D) Correlation between expression and activity of FXIII. (E) Correlation between expression and activity of TG2. Expression levels of FXIII and TG2 (arbitrary units, au) were determined by averaging three values of FXIII and TG2, respectively, relative to β-actin.

terms axFXIII + b and cxFXIII + d, respectively, TG2 activity (xTG2) in samples can be determined by eq 2. Determination of FXIII and TG2 Activities in THP-1 cells Differentiated with PMA and IL-4. To evaluate whether the onchip activity assay was suitable for determination of FXIII and TG2 activities in cells, this assay was used to monitor changes in FXIII and TG2 activities in THP-1 cells after treatment with IL-4.26 It is reported that incubation with IL-4 induces expression of FXIII (cFXIII) and TG2 in PMA-differentiated monocytic cells.26 IL-4 treatment also activates expression of FXIII and TG2 in human-derived monocytes in a time-dependent manner.13,28 Initially, we used Western blot analysis to determine the expression levels of FXIII and TG2 as a function of incubation time with IL-4. Consistent with previous reports,13,26,28 IL-4 induced expression of FXIII and TG2 in an incubation timedependent manner, with the expression level of TG2 being higher and increasing more rapidly than that of FXIII-A (Figure 4A). The expression levels of FXIII and TG2 were quantitatively analyzed by Labworks software and normalized to β-actin levels. We used the simultaneous on-chip activity assay to investigate changes in the activity of FXIII and TG2 in THP-1 cells treated with IL-4 (Figure 4B,C). IL-4 caused an increase in the activities of FXIII and TG2 in a time-dependent manner, with higher

activities of TG2 and saturation at 5 days. We then studied the relationship between the expression levels of the two TG isozymes and their activities by analyzing correlation with a sigmoidal fit. As shown in Figure 4D,E, the activity of FXIII increased more rapidly than did that of TG2, and the activities of both isozymes showed high correlation coefficients (0.987 and 0.986, respectively) with their expression levels. These results indicate that FXIII-A was mainly activated at the early stage of IL4 incubation, while TG2 stimulation occurred at the late stage. Thus, the on-chip activity assay was sensitive enough to monitor changes in the activities of FXIII and TG2 in monocytic cells differentiated with IL-4. Macrophages are activated by two major pathways: the classical pathway induced by Th1 cytokines, such as interferonγ, and the alternative pathway activated by Th2 cytokines, such as IL-4.12,28,29 It is reported that activation of macrophages with IL-4 induces the expression of FXIII at the mRNA and protein levels and that FXIII expression is an intracellular marker for the alternative pathway of macrophage activation.12,28 In accordance with those reports, we showed here that IL-4 induced the expression and activation of FXIII in PMA-differentiated monocytic cells. Additionally, TG2 activation occurred with its expression at the late stage of IL-4 treatment. Thus, in addition to FXIII, TG2 might be another intracellular marker for the alternative 8723

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Analytical Chemistry pathway of monocyte-derived macrophage activation. However, it is necessary to elucidate the exact role(s) of TG2 in the IL-4 activation of macrophages. FXIII and TG2 are involved in tissue remodeling associated with cancer, wound repair, and bone development.1 The functions of FXIII and TG2 are often complementary, as observed for bone development.14,30 Two isozymes can also have opposing roles, including tumor progression,1,31 angiogenesis,10,14,16 and development of artherosclerosis.17
19 However, the mechanisms responsible for these differential functions remain unknown. In this report, we describe a newly developed on-chip activity assay for simultaneous determination of individual activities of FXIII and TG2 in samples including differentiated monocytes. Therefore, this on-chip activity can assist in understanding pathophysiological mechanisms for osteoarthritis, cancer, artherosclerosis, and vascular disorders. In addition, this assay has a strong potential in investigating the specific functions of FXIII and TG2 in the cardiovascular system and in various TG-related diseases and, furthermore, in investigating the two isozymes as distinct therapeutic targets for treatment of various diseases, including neurodegerative disorders, cataracts, and gluten sensitivity.

’ CONCLUSION We present a new on-chip activity assay for simultaneous determination of FXIII and TG2 activities by use of fibrinogen arrays in samples including differentiated THP-1 monocytic cells. This activity assay is sensitive and suitable for high-throughput determination of FXIII and TG2 activities, and thus it has a strong potential for investigating the specific functions of these TG isozymes in cardiovascular pathophysiology and in TGrelated diseases. ’ ASSOCIATED CONTENT

bS

Supporting Information. Two figures, showing optimization of buffer pH, BAPA and DTT concentrations, and incubation time for on-chip FXIII- and TG2-specific activity assays. This material is available free of charge via the Internet at http://pubs.acs.org.

’ AUTHOR INFORMATION Corresponding Author

*Telephone: +82-33-250-8833. Fax: +82-33-250-8807. E-mail: [email protected].

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’ ACKNOWLEDGMENT This work was supported in part by the Korean Ministry of Health and Welfare through the National R&D Program for Cancer Control (1020420), the Korea Research Foundation through the Basic Research Program (2008-05943), and the Regional Research Universities Program/Medical & Bio-Materials Research Center. ’ REFERENCES (1) Iismaa, S. E.; Mearns, B. M.; Lorand, L.; Graham, R. M. Physiol. Rev. 2009, 89, 991–1023. (2) Esposito, C.; Caputo, I. FEBS J. 2005, 272, 615–631. (3) Sugimura, Y.; Hosono, M.; Wada, F.; Yoshimura, T.; Maki, M.; Hitomi, K. J. Biol. Chem. 2006, 281, 17699–17706. 8724

dx.doi.org/10.1021/ac202178g |Anal. Chem. 2011, 83, 8718–8724