ARTICLE |
Correspondence to: Shigehisa Hirose, Dept. of Biological Sciences, Tokyo Inst. of Technology, 4259 Nagatsuta-cho, Midoriku, Yokohama 226-8501, Japan. E-mail: shirose@bio.titech.ac.jp
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Summary |
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Histidine-tagged green fluorescent protein (His6-Xpress-GFP), a widely used fluorescent probe, was found to be a good substrate for transglutaminase, an enzyme that catalyzes covalent crosslinking of proteins. GFP alone did not serve as a substrate but its derivative His6-Xpress-GFP was readily crosslinked through the Gln and Lys residues present in the short N-terminal extension (His6-Xpress). His6-Xpress-GFP was sensitive enough to detect the transglutaminase activity in guinea pig liver homogenates. The fluorescent substrate could also be used for activity staining of transglutaminase on histological tissue sections, and such applications revealed a surprisingly wide distribution of transglutaminase in the body, especially in the extracellular matrices of various tissues, suggesting an important role for transglutaminase in maintaining the integrity of the extracellular matrix and connective tissues by crosslinking its constituent proteins.
(J Histochem Cytochem 49:247258, 2001)
Key Words: transglutaminase, green fluorescent protein, extracellular matrix, connective tissue, fluorescent substrate, crosslinking, activity staining, histochemistry
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Introduction |
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TRANSGLUTAMINASES are a family of enzymes that catalyze the transfer of acyl groups between acyl donors and acceptors ( 80110 kD) and subunit structure. They undergo a number of different post-translational modifications, such as proteolytic cleavage, phosphorylation, and fatty acylation, regulating their enzymatic activity and subcellular localization (
-adrenergic receptor signaling (
We report here a simple method for detection and localization of transglutaminase activities in tissue homogenates and histological sections. The method uses, as a substrate, a derivative of green fluorescent protein (GFP), His6-Xpress-GFP, which can be easily obtained by recombinant DNA technology, and monitors its size changes or covalent anchoring to tissue sections. On histological examination, a surprisingly wide variety of tissues gave strong positive signals, especially in their extracellular matrices, suggesting an important role of transglutaminase in the maintenance of the integrity of the extracellular matrix (ECM).
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Materials and Methods |
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Materials
Guinea pigs (male, 8 weeks old) were obtained from Tokyo Laboratory Animal Science (Tokyo, Japan). Guinea pig liver transglutaminase (TGase) and restriction enzymes were purchased from Takara (Kyoto, Japan). pGFPuv vector and rGFPuv were from Clontech (Palo Alto, CA). Ni-NTA agarose resin was from Qiagen, (Chatsworth, CA). pTrcHis A was from Invitrogen (San Diego, CA). Lysyl endopeptidase was from Wako (Tokyo, Japan). µ Bondasphere 5 µ C18-100 Å 3.9 x 150 mm was from Waters (Milford, MA). Tissue-Tek OCT compound was from Miles (Elkhart, IN). Immobilon PSQ membrane was from Millipore (Bedford, MA). 5-(biotinamido)pentylamine was from Pierce (Rockford, IL). Vectabond and horseradish peroxidaseavidin D were from Vector Laboratories (Burlingame, CA). Hydrogenated Triton X-100 and 2,2'-azino-bis(3-ethylbenzthiazoline-6-sulfonic acid) diammonium salt (ABTS) were from Sigma (St Louis, MO). Blood coagulation factor XIIIa (FXIIIa) was a gift from Dr. Yuji Saito (
Construction of His6-Xpress-GFP Expression Vector
A 0.75-kb PstI-EcoRI fragment of pGFPuv, which encodes the entire open reading frame of GFPuv (abbreviated here simply as GFP), was inserted into the PstI-EcoRI cloning sites of pTrcHis A vector and named pHis6-Xpress-GFP.
Expression and Purification of His6-Xpress-GFP
Escherichia coli JM109 was transformed with pHis6-Xpress-GFP and grown in 2 x Luria Bertani (LB) medium containing 50 µg/ml ampicillin at 37C with aeration until the cells reached an optical density of 0.6 at 600 nm. The expression of His6-Xpress-GFP was induced by addition of 1 mM isopropyl-ß-D-thiogalactopyranoside (IPTG) and incubation was continued at 37C for 5 hr. The cells were harvested by centrifugation at 4000 x g for 20 min and suspended in binding buffer (50 mM phosphate, pH 8.0, containing 500 mM NaCl). The suspensions were treated with lysozyme (1 mg/ml) for 30 min at 4C, and lysed by sonication for 2 min at 4C. The extracts were centrifuged at 10,000 x g for 20 min and supernatant was applied to a column containing 2 ml of Ni-NTA agarose resin. The column was washed with 30 ml of wash buffer (50 mM phosphate, pH 6.0, containing 500 mM NaCl) and wash buffer containing 50 mM imidazole. His6-Xpress-GFP was eluted stepwise with wash buffer containing 100, 200, 350, and 500 mM imidazole, and then dialyzed against 20 mM Tris-HCl, pH 7.5.
Assay by SDS-PAGE of Transglutaminase Activity Using His6-Xpress-GFP as a Substrate
One microgram of tissue transglutaminase was incubated with 0.1 µg of His6-Xpress-GFP or rGFPuv for 1 hr in 20 µl of transglutaminase assay buffer (50 mM Tris-HCl, pH 7.5, 1 mM dithiothreitol, and 5 mM CaCl2) in the presence and absence of 50 mM EDTA or 10 mM putrescine, a transglutaminase inhibitor. The reaction was stopped by adding 5 µl of 5 x Laemlli sample buffer and samples were then separated by SDS-PAGE on a 12.5% gel. After electrophoresis, fluorescent bands in the gel were directly visualized with a Bio-image Analyzer FLA-2000 (Fuji Film; Tokyo, Japan).
Normalization of Activities of Tissue Transglutaminase and FXIIIa and Determination of Their Affinities for His6-Xpress-GFP
For comparison of the affinities of tissue transglutaminase and FXIIIa for the substrate His6-Xpress-GFP, the enzyme levels should be adjusted using a combination of control substrates. This was done by a modified method of
Isolation, Digestion, and Amino Acid Sequencing of Crosslinked His6-Xpress-GFP
Five hundred pmol of His6-Xpress-GFP and 1 µg of transglutaminase were incubated in 200 µl of transglutaminase assay buffer at 37C for 1 hr. The reaction was stopped by adding 50 µl of 5 x SDS sample buffer. The sample was heat-denatured at 100C for 2 min, electrophoresed in 12.5% SDS-polyacrylamide gels, and then electrotransferred onto an Immobilon-PSQ membrane. After transfer, the membrane was stained with Coomassie Brilliant Blue. The crosslinked band that was seen in the presence of transglutaminase was excised and sequenced either directly for the N-terminal sequence or after digestion with lysyl endopeptidase in 100 mM Tris-HCl, pH 9.5, 10% acetonitrile, and 1% hydrogenated Triton X-100 for C-terminal analysis. The digestion was performed for 24 hr at 37C. Peptide fragments released were purified by reverse-phase HPLC on a Waters C18 column (3.9 x 150 mm) using a 060% gradient of acetonitrile in aqueous trifluoroacetic acid (0.8%). Amino acid sequencing was performed with a peptide sequencer (Shimazu PSQ-1100, Kyoto, Japan) using approximately 100 pmol of peptides.
Activity Staining of Transglutaminase in Tissue Sections with His6-Xpress-GFP
Guinea pigs (n = 10) were sacrificed by guillotine or lethal IP injection of sodium pentobarbital. Tissues were immediately removed, frozen in Tissue-Tek OCT compound on dry ice, and stored at -80C. Frozen sections (7 µm) were cut in a cryostat at -16C, mounted on gelatin-coated slides, and air-dried at RT for 30 min. The use of Vectabond for coating slides sometimes gave nonspecific staining (bright dots). Sections were incubated with His6-Xpress-GFP by overlaying 50200 µl of its solution (0.1 mg/ml) in transglutaminase assay buffer (50 mM Tris-HCl, pH 7.5, 1 mM dithiothreitol, and 5 mM CaCl2) with or without transglutaminase inhibitors (e.g., 50 mM EDTA or 10 mM putrescine) at RT for 1 hr in a moist chamber. The sections were washed three times in excess PBS and covered with coverslips. The samples were analyzed using either a normal fluorescent microscope with mercury lamp and filters for fluorescein isothiocyanate (FITC) fluorescence (Fig 5A5C) or a laser scanning confocal microscope (TCS4D, Leica Laser Teknik) based on a Zeiss Axiovert microscope (Carl Zeiss; Oberkochen, Germany) interfaced with a mixed gas argonkrypton laser. Fluorescence acquisitions were performed with the 488-nm laser line to excite GFP (Fig 5D5U). The fluorescence was very stable. The stained specimens could be viewed repeatedly for at least 3 weeks if stored in a humidified chamber at 4C.
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Results |
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Construction and Purification of GFP Derivatives
The E. coli expression vector pTrcHis used in this study is designed for high-level expression of His-tagged recombinant proteins. The proteins produced have a polyhistidine (6 x His) tag on the N-terminus for rapid purification, which is followed by the Xpress epitope and the enterokinase cleavage site for removal of the fused tags. We noted the presence of two Gln and one Lys in the short stretch of the Xpress epitope and enterokinase cleavage site (Fig 1) and considered the possibility that if GFP is produced as a His6-Xpress-GFP fusion protein using the pTrcHis vector, it may serve as a useful fluorescent substrate of transglutaminase. We therefore produced the His6-Xpress-GFP fusion protein in E. coli and purified it by Ni-chelate chromatography as described in Materials and Methods. The yield was about 3 mg per liter of bacterial culture. The purified protein gave a single fluorescent band of 41 kD on SDS-PAGE (Fig 2, Lanes 4 and 10). Like the wild-type GFP, the N-terminally extended GFP was resistant to the reducing agent 2-mercaptoethanol but sensitive to heat denaturation in the presence of SDS (Fig 2, lane 11). The heat-denatured nonfluorescent His6-Xpress-GFP migrated as a band of 34 kD, a value close to the calculated molecular weight (32,776).
Electrophoretic Mobility Shift of Crosslinked GFP Derivatives
When His6-Xpress-GFP was incubated with transglutaminase and analyzed by SDS-PAGE, significant changes were observed in its electrophoretic mobility (Fig 2, Lane 3). The wild-type GFP showed no such changes (Fig 2, Lane 1). EDTA, an inhibitor of transglutaminase, completely inhibited the mobility changes (Fig 2, Lane 4). Similar inhibitory effects were also seen with putrescine, a competitive inhibitor of transglutaminase (data not shown). Depending on the incubation time, the transglutaminase-treated His6-Xpress-GFP behaved as compact molecules at the initial stage (Fig 2, Lane 3, lower band of 36 kD) and as much larger molecules at later stages of the treatment or at higher concentrations of the substrate (Fig 2, Lane 5). Fig 3 shows the time course of the crosslinking reaction monitored by measuring the fluorescence intensity of the 36-kD band (open circle). It appears unlikely that the lower molecular weight species are proteolytic degradation products because (a) the transglutaminase used is a highly purified preparation, (b) the N-terminal amino acid sequence of the lower molecular weight species (RGSHHHHHH; Fig 1, shading) matched perfectly to that of His6-Xpress-GFP, and (c) amino acid sequencing of lysyl endopeptidase-generated peptide fragments of the lower molecular weight species revealed the presence of the C-terminal fragment beginning with the sequence RDHMVLLEFVTA (Fig 1, shading). The initial lowering of the apparent molecular size (from 41 kD to 36 kD) and the later appearance of the higher molecular weight species (>50 kD) may therefore be due to intramolecular and intermolecular crosslinking, respectively, of His6-Xpress-GFP by transglutaminase.
Higher Affinity for Tissue Transglutaminase
There are several types of transglutaminase that are encoded by separate genes, including keratinocyte transglutaminase, plasma transglutaminase (blood coagulation factor XIIIa, FXIIIa), tissue transglutaminase, and prostate gland transglutaminase. Among these, plasma and tissue transglutaminases are widely distributed in the body. We therefore determined whether the fluorescent substrate His6-Xpress-GFP serves as a substrate for plasma transglutaminase as well as for tissue transglutaminase by first activating the purified preparation of plasma transglutaminase with thrombin and then mixing it with the substrate. His6-Xpress-GFP turned out to be a very poor substrate for plasma transglutaminase (Fig 4). When both enzyme activities were adjusted to the same level by using the synthetic substrate 5-(biotinamido)pentylamine and their crosslinking abilities toward His6-Xpress-GFP were compared, plasma transglutaminase exhibited only weak activity. This weak activity of plasma transglutaminase toward His6-Xpress-GFP is consistent with its narrow substrate specificity; it is highly specific for fibrin.
Detection of Transglutaminase Activity in Tissue Extracts
To evaluate the usefulness of His6-Xpress-GFP as a transglutaminase substrate, we prepared guinea pig liver homogenate, incubated it with His6-Xpress-GFP, and analyzed the reaction products by SDS-PAGE (Fig 2B). Incubation of the fluorescent substrate with the liver homogenate resulted in its intra- and inter-molecular crosslinking (Fig 2, Lanes 6 and 8), indicating that His6-Xpress-GFP is sensitive enough to detect the transglutaminase activity in tissue extracts.
Detection of Transglutaminase Activity on Tissue Sections
Fig 5 shows that His6-Xpress-GFP can also be used for histological purposes, i.e., activity staining of transglutaminase on tissue sections. When incubated with non-fixed frozen sections of a variety of guinea pig tissues shown in Fig 5, His6-Xpress-GFP was crosslinked to certain discrete structures, such as the stratified squamous epithelium of the skin, smooth muscle layers of the blood vessels and ducts, and connective tissues, giving a clear fluorescent pattern of the localization of active transglutaminase. In general, transglutaminase activities appear to be relatively concentrated in the ECM of various tissues.
In the esophagus, very bright fluorescent staining was seen in the endomysium of the muscularis mucosae (Fig 5A). In the skin, fluorescent labeling was seen in the upper spinous and granular layers of epidermis (Fig 5B and Fig 5R), where membrane-bound keratinocyte transglutaminase has been demonstrated by immunohistochemistry (
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Discussion |
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Two types of histological methods have been used for determining cellular localization of transglutaminase: immunohistochemistry and activity staining. In the previous activity staining method, active transglutaminases are detected on tissue sections by incubation with monodansylcadaverine (
Our original intention to devise an in situ transglutaminase assay was to demonstrate the presence of active transglutaminase in the brain and in the nucleus of the cell, the locations currently receiving considerable attention in relation to transglutaminase and neuronal development (
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Acknowledgments |
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Supported by Grants-in-Aid for Scientific Research from the Ministry of Education, Science, Sport and Culture of Japan, by a Research Grant for Cardiovascular Diseases (11C-1) from the Ministry of Health and Welfare of Japan, and by an SRF Grant for Biomedical Research. YF is supported by a Research Fellowship for Young Scientists from the Japan Society for the Promotion of Science.
We thank Dr Yuji Saito and Hiroo Takahashi for discussion and help in the FXIIIa assay, and Setsuko Satoh for secretarial assistance.
Received for publication April 7, 2000; accepted August 23, 2000.
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