Báo cáo khoa học: Identification of preferred substrate sequences for transglutaminase 1 – development of a novel peptide that can efficiently detect cross-linking enzyme activity in the skin pot

Identification of preferred substrate sequences for transglutaminase – development of a novel peptide that can efficiently detect cross-linking enzyme activity in the skin Yoshiaki Sugimura1,*, Masayo Hosono1,*, Miyako Kitamura1, Tatsuya Tsuda2, Kiyofumi Yamanishi2, Masatoshi Maki1 and Kiyotaka Hitomi1 Department of Applied Molecular Biosciences, Graduate School of Bioagricultural Sciences, Nagoya University, Japan Department of Dermatology, Hyogo College of Medicine, Nishinomiya, Japan Keywords epidermis; keratinocyte; phage display; skin; transglutaminase Correspondence K Hitomi, Department of Applied Molecular Biosciences, Graduate School of Bioagricultural Sciences, Nagoya University, Chikusa, 464 8601 Nagoya, Japan Fax: +81 52 789 5542 Tel: +81 52 789 5541 E-mail: hitomi@agr.nagoya-u.ac.jp *These authors contributed equally to this work (Received August 2008, revised 10 September 2008, accepted 18 September 2008) doi:10.1111/j.1742-4658.2008.06692.x Transglutaminase (TGase 1) is an essential enzyme for cornified envelope formation in stratified squamous epithelia This enzyme catalyzes the cross-linking of glutamine and lysine residues in structural proteins in differentiating keratinocytes To gain insight into the preferred substrate structure of TGase 1, we used a phage-displayed random peptide library to screen primary amino acid sequences that are preferentially selected by human TGase The peptides selected as glutamine donor substrate exhibited a marked tendency in primary structure, conforming to the sequence: QxK ⁄ RwxxxWP (where x and w represent non-conserved and hydrophobic amino acids, respectively) Using glutathione S-transferase (GST) fusion proteins of the selected peptides, we identified several sequences as preferred substrates and confirmed that they were isozyme-specific We generated GST-fused alanine mutants of the most reactive sequence (K5) to determine the residues that were critical for reactivity Even in peptide form, K5 appeared to have high and specific reactivity as substrate In situ analysis of mouse skin sections using fluorescence-conjugated K5 peptide resulted in detection of TGase activity with high sensitivity, but no signal was detected in a TGase 1-null mouse In conclusion, we were successful in generating a novel substrate peptide for sensitive detection of endogenous TGase activity in the skin Transglutaminase (TGase; EC 2.3.2.13) is a Ca2+dependent enzyme that catalyzes the formation of isopeptide cross-links between the c-carboxyamide group of glutamine residues and the e-amino group of lysine residues [1,2] In addition, TGases catalyze the incorporation of primary amines into glutamine residues (transamidation), or conversion of glutamine to glutamic acid (deamidation) TGases comprise a family of eight isozymes (Factor XIII and TGases 1–7) that are distributed in a variety of tissues and have unique substrate specificities Factor XIII and TGase (tissuetype TGase), two of the major isozymes, are involved in the stabilization of fibrin clots, and in apoptosis, extracellular matrix stabilization and wound healing, respectively [3–6] TGase is expressed primarily in epithelial cells and plays an essential role in formation of the skin epidermis [7–9] In the outermost layer of the epidermis, the assembly of a highly specialized structure, termed the cornified envelope (CE), is formed as a result of cross-linking of Abbreviations Bio-Cd, 5-(biotinamido)pentylamine; CE, cornified envelope; Dansyl-Cd, monodansylpentylamine; FITC, fluorescein isothiocyanate; GST, glutathione S-transferase; TGase, transglutaminase FEBS Journal 275 (2008) 5667–5677 ª 2008 The Authors Journal compilation ª 2008 FEBS 5667 Preferred substrate sequences for TGase Y Sugimura et al several structural proteins in keratinocytes [10–12] The CE is located underneath the plasma membrane of differentiated keratinocytes and is essential for barrier function of the epidermis The CE comprises several proteins, including involucrin, loricrin, trichohyalin and small proline rich-proteins, that are preferred substrates for TGases in the skin Of the eight TGase isozymes, four (TGases 1, 2, and 5) are expressed in keratinocytes [13–18], and are involved in cross-linking of the structural proteins to form the CE at distinct intracellular sites [8,18] In the current model of TGase function, cytoplasmic TGase catalyzes the cross-linking of several proteins, and these products are then stabilized with a proteinaceous platform beneath the plasma membrane, which is formed by membrane-anchored TGase [19] However, the precise mechanism by which these enzymes coordinate the cross-linking of various substrates is poorly understood Mice that lack TGase exhibit impaired skin barrier function due to aberrant cornification [20] In humans, several types of TGase mutations have been identified in congenital ichthyoses, such as non-bullous congenital ichthyosiform erythroderma and lamellar ichthyosis [21–25] Thus, TGase appears to be an essential and non-redundant enzyme for CE formation in the epidermis under functional regulation [26] Generally, TGases display strict substrate specificity towards peptide-bound glutamine residues and relatively weak specificity with respect to lysine residues or amine groups Hence, information about the primary and ⁄ or secondary structure of sequences surrounding the reactive glutamine residue in substrates is important for understanding substrate specificity in TGasemediated catalysis [27] Partial amino acid sequences of substrates for TGase and Factor XIII have been identified [28] Although several protein substrates for TGase have been characterized, a consensus sequence for the preferred substrate has not been identified [29–32] Recently, we established a system for the identification of preferred substrate sequences of TGases by screening a phage-displayed random peptide library [33,34] Using this system, we identified the primary sequences of the various preferred, isozyme-specific substrates of TGase and Factor XIII These results provide new insight into the substrate specificity of TGases, and expand the range of application using the enzyme reactions [35] In the current study, we used this system to identify preferred TGase 1-specific peptide sequences We identified several sequences that were preferentially selected by human TGase and exhibited a significant tendency to serve as glutamine donor substrates One 5668 peptide, K5, exhibited strong activity in both recombinant fusion protein and peptide forms Using this peptide, we were able to detect in situ TGase activity in mouse skin sections with a high level of sensitivity and specificity Results Screening of candidate substrate sequences from a random peptide library In the presence of recombinant human TGase 1, phage clones were incubated with biotin-labeled primary amine (Bio-Cd) Phage clones that were covalently bound to Bio-Cd were selected by avidin affinity purification The selected clones were amplified and then subjected to four additional cycles of catalysis and panning as described previously [33] Sequence analysis of the peptides encoded by the final clones revealed that 32 of the 56 clones contained glutamine residues (Fig 1) When the peptides were aligned based on the putative reactive glutamine residue, there were several significant similarities among the primary sequences of more than half of the clones: (1) tyrosine and glutamine were located at the N-terminus of the peptide, (2) there was a lysine or an arginine at position +2 (relative to glutamine), (3) a hydrophobic amino acid was commonly observed at position +3, and (4) tryptophan and proline were frequently located at positions +7 and +8, respectively Among the clones, there was one frequently observed sequence (YEQHKLSSWPF) that was encoded by identical DNA sequences, but treated as an individual clone Evaluation of the selected sequences as recombinant peptide-fused GST proteins To evaluate the ability of the selected peptide sequences to serve as glutamine donor substrates, we measured the amount of monodansyl cadaverine (Dansyl-Cd) incorporated into recombinant peptide–GST(QN) fusion proteins (in which all the glutamine residues in glutathione S-transferase (GST) had been changed to asparagine) in the presence of TGase The sequences marked with an asterisk in Fig were selected for analysis The reaction products were separated by SDS–PAGE and then visualized by UV illumination (Fig 2) GST(QN) fusion proteins of the K1, K2, K5, K13, K26, K51, K57 and K60 sequences exhibited significant levels of incorporation of Dansyl-Cd compared to the other sequences examined All of the highly FEBS Journal 275 (2008) 5667–5677 ª 2008 The Authors Journal compilation ª 2008 FEBS Y Sugimura et al Preferred substrate sequences for TGase Fig Alignment of candidate substrate peptide sequences Amino acid sequences of the peptides contained within the selected phage clones were aligned based on the putative reactive glutamine residues The glutamine residues, the hydrophobic amino acids at position +3 (relative to glutamine), and the most commonly observed amino acids are shaded Clones marked with an asterisk were evaluated as GST(QN) fusion proteins using the Dansyl-Cd incorporation assay reactive sequences had the similarities described above, which suggested that our procedure was successful in identifying the preferred sequences Of these highly reactive sequences, we selected K5, K26, K51 and K60 for further analysis based on their levels of reactivity Isozyme specificity of the peptide sequences The four sequences with the highest reactivity were further analyzed for isozyme specificity with respect to TGases 1, and 3, and Factor XIIIa (activated Factor XIII) GST(QN) fusion proteins of each peptide were incubated with [3H]-putrescine in the presence of each TGase Most of the peptide sequences exhibited high reactivity with TGase 1, consistent with the results of the Dansyl-Cd incorporation assay (Fig 3) We did not observe cross-reactivity for any of the peptides, with the exception of K60, which showed significant incorporation in the presence of Factor XIIIa K5 exhibited the lowest cross-reactivity to TGases and 3, indicating the highest specificity Thus, we selected K5 for further analysis Substitution mutant analysis of the K5 peptide sequence To determine the role of each amino acid of K5 in TGase 1-mediated catalysis, we generated alanine substitution mutants for every residue of the 12-amino-acid peptide Wild-type and mutant peptides were produced as GST(QN) fusion proteins, and evaluated for their reactivity in the presence of TGase As shown in Fig 4, mutation of positions +2 (K), +3 (L), +5 (S), +6 (S), +7 (W), +8 (P) or +9 (F) to alanine decreased the level of incorporation of DansylCd Substitutions at positions )2 (Y), )1 (E), +1 (H) or +4 (P) had a smaller effect on the reactivity of the peptide Assessment of the K5 sequence for reactivity and specificity in the peptide form To assess the reactivity and specificity of K5 in the peptide form, biotinylated K5 (pepK5: YEQHK LPSSWPF), or K5 in which the reactive glutamine residue was replaced by asparagine (pepK5QN: YENHKLPSSWPF), were incubated with casein as a glutamine acceptor substrate in the presence of TGases 1, or For comparison, we also examined the reactivity of the TGase preferred substrate peptide (pepT26: HQSYVDPWMLDH) [33] Incorporation of pepK5 into casein was observed in the presence of TGase in a time-dependent manner, but pepT26 showed no apparent reactivity (Fig 5A) In contrast, in the presence of TGase 2, pepT26 showed apparent incorporation into casein, whereas pepK5 reacted at FEBS Journal 275 (2008) 5667–5677 ª 2008 The Authors Journal compilation ª 2008 FEBS 5669 Preferred substrate sequences for TGase CBB 0.5 Y Sugimura et al 10 15 20 (min) DMC Ctrl K1 K2 K5 Detection of in situ TGase activity in skin K13 K26 K51 K57 K60 K11 K29 K42 K56 K59 K70 Fig Evaluation of the reactivity of selected peptides as peptide GST fusion proteins Incorporation of Dansyl-Cd into peptide– GST(QN) fusion proteins of the selected amino acid sequences in the presence of TGase The numbers prefixed with K (K1, K2, K5, etc.) for each peptide sequence correspond to the ID numbers of the clones (Fig 1) At the indicated times, the reaction products were separated by 12.5% SDS–PAGE and then illuminated by UV light Unreacted (0 min) peptide–GST(QN) fusion proteins were stained with Coomassie brilliant blue and are shown on the left of the fluorograph Ctrl, GST(QN) fusion protein of a peptide that does not contain a glutamine residue (K3, SPLAYVAPWSNL) Dimethylcasein (DMC) was examined in parallel as a positive control much lower level (Fig 5B) However, at a higher concentration of the peptide (above 0.1 mm), pepK5 showed cross-reactivity to TGase (data not shown) pepK5QN was not incorporated into casein, also acting as glutamine-donor substrate, in the presence of either TGase or TGase This result indicates that the lysine residue in the sequence of K5 does not participate in the enzymatic reaction To determine whether K5 cross-reacts with TGase 3, we compared the incorporation rates of pepK5 in the presence of TGases or 3, as a preferred peptide sequence for TGase has not yet been established (Fig 5C) In the presence of TGase 3, the incorporation of pepK5 was similar to control reactions that did 5670 not contain any enzyme, whereas there was a significant increase in incorporation in the presence of TGase pepK5QN also failed to react with casein in the presence of TGase (data not shown) These results indicate that the K5 peptide sequence acts as a glutamine donor substrate in an isozyme-specific manner, even in the peptide form As pepK5 appeared to be a highly preferred and specific substrate for TGase 1, we examined whether this peptide could be used to detect endogenous TGase activity in the skin epidermis (Fig 6A) In the presence of CaCl2 in the reaction mixture, we detected specific incorporation of fluorescein isothiocyanate (FITC)labeled K5 peptide (FITC-pepK5) (1 lm) into substrate proteins in the epidermis, mainly at the cell periphery of the upper spinous and granular layers This staining pattern of reactivity corresponded well with the localization of TGase by immunostaining with monoclonal anti-mouse IgG No signals were detected when we used FITC-pepK5QN mutant peptide, or in the presence of EDTA, which indicated that the cross-linking reaction was catalyzed specifically by TGase To further evaluate the specificity of the assay for TGase 1, skin sections from a TGase knockout mouse were also examined No signals were detected in TGase 1-null sections in the presence or absence of Ca2+, which indicates that FITC-pepK5 specifically detects endogenous TGase activity We also compared the reactivity of FITC-pepK5 and FITC-cadaverine, which has been used previously for detection of in situ TGase activity, over a range of concentrations (0.1–100 lm) As shown in Fig 6B, endogenous TGase activity was detected at 0.1–1 lm FITC-pepK5, but no apparent signal was observed with FITC-cadaverine at the same concentrations In the presence of higher concentrations of cadaverine, the staining pattern became somewhat ambiguous in mouse skin sections, possibly because of cross-reactivity with other TGases These results suggest that FITC-pepK5 detects endogenous TGase activity with greater sensitivity than FITC-cadaverine Discussion In terminally differentiating stratified epithelial cells, isopeptide cross-linking of keratinocyte proteins into organized protein structures lead to the formation of the CE Although the cooperative action of several TGases in the keratinocytes plays a role in this process, the precise role of each isozyme in cross-linking FEBS Journal 275 (2008) 5667–5677 ª 2008 The Authors Journal compilation ª 2008 FEBS 10 DMC Ctrl K5 K26 K51 K60 DMC Ctrl K5 K26 K51 K60 DMC Ctrl K5 K26 K51 K60 DMC Ctrl K5 K26 K51 K60 Fig Cross-reactivity of the preferred sequences with TGase 2, TGase and Factor XIIIa GST(QN) fusion proteins of peptides K5, K26, K51 and K60, and the control sequence (WGHTIYHLHPTI [33]) (Ctrl), were incubated for 10 with 1.0 mM [3H]-putrescine in the presence of TGase 1, TGase 2, TGase or Factor XIIIa Dimethylcasein (DMC) was used in parallel to adjust the enzymatic activities Data are the means ± SD of triplicate assays Preferred substrate sequences for TGase [3H]-putrescine incorporated (×104 dpm) × Y Sugimura et al TGase TGase TGase Factor XIIIa 1.4 Relative value 1.2 0.8 0.6 0.4 0.2 WT –2A –1A QN +1A +2A +3A +4A +5A +6A +7A +8A +9A Fig Assessment of the contribution of each amino acid residue of K5 to substrate recognition Alanine substitution mutants of K5 were produced as GST(QN) fusion proteins, and then incubated with Dansyl-Cd for 10 in the presence of TGase The reaction products were separated by SDS–PAGE, followed by UV irradiation The fluorescence intensity was analyzed by SCION imaging software for quantification The relative values shown are normalized to the intensity for the reaction of wild-type Data represent the means of duplicate samples Numbers ()2A to +9A) indicate the position of substitution mutations WT, peptide in which there were no amino acid substitutions; QN, peptide in which the glutamine residue was changed to asparagine The mutations that resulted in an apparent decrease in the reactivity are shaded in darker gray of the target substrates remains elusive TGase has unique substrate specificity, which results in a distinct pattern of in vitro cross-linked substrate proteins compared to other TGases [32,36,37] A better understanding of the preferred substrate structure for TGase is essential to evaluate substrate reactivity, and also to develop isozyme-specific regulatory molecules In the current study, preferred substrate sequences for TGase selected through screening of a phage-displayed peptide library exhibited different tendencies compared to those for Factor XIIIa and TGase [33,38] The preferred substrate sequence for TGase conformed to the consensus motif QxR ⁄ KwxxxWP, whereas those for TGase and Factor XIIIa conform to the motifs QxPwDP and QxxwxWP, respectively A hydrophobic amino acid at +3 relative to the glutamine residue is present in the substrate motifs of all three TGases Based on previous studies, the preferred sequence for TGase contains the sequence QxP, which has been shown to be an in vivo transamidation motif [39] In the case of TGase 1, most of the selected substrates contained QxK or QxR This motif is frequently observed in involucrin, trichohyalin and small proline-rich proteins, and has been implicated as a putative cross-linking site in these proteins [32,36,37] Sequences homologous to the motif QxR ⁄ KwxxxWP are present in the C-terminal regions of involucrin (QQKQEVQWP) and loricrin (QQKQAPTWP), which are the main components of the CE In the developing TGase substrate database (TRANSDAB; http:// genomics.dote.hu/wiki), several sequences containing the reactive glutamine residue have been listed for TGase as well as other isozymes When these sequences were compared with our proposed consensus sequence, half of the sequences were found to contain QxK or QxxK, which is partially consistent with the obtained motif Interestingly, the TGase substrate motif was similar to that of Factor XIIIa, QxxwxWP Among TGase family members, the primary structures of Factor XIII and TGase display the most homology, which suggests that the catalytic domains of these two enzymes may recognize similar substrate structures Most of the highly reactive peptide sequences identified in this study (K1, K2, K5, K26, K57 and K60) contained the TGase substrate consensus sequence The peptide sequences K5, K26 and K51 exhibited less FEBS Journal 275 (2008) 5667–5677 ª 2008 The Authors Journal compilation ª 2008 FEBS 5671 Preferred substrate sequences for TGase Absorbance at 492 nm A 1.2 Y Sugimura et al TGase 1 0.8 0.6 0.4 K5 K5QN T26 0.2 0 10 15 20 25 Reaction time (min) Absorbance at 492 nm B 1.2 TGase 0.8 0.6 0.4 K5 K5QN T26 0.2 0 10 15 20 Reaction time (min) 25 C 1.2 Absorbance at 492 nm 0.8 0.6 0.4 TGase TGase -TGase 0.2 0 10 20 Reaction time (min) 30 Fig Analysis of the reactivity of the K5 peptide with casein in the presence of TGases The time-dependent incorporation of biotinylated peptides (pepK5, pepK5QN and pepT26) into casein was examined in the presence of TGase (A) and TGase (B) The amount of reaction product was determined by microtiter assay, and is represented as absorbance at 492 nm The square, circle and triangle represent pepK5, pepK5QN and pepT26, respectively (C) Time-dependent incorporation of pepK5 measured in the presence of TGase (closed circle) or TGase (open circle) Reaction mixtures that did not contain enzyme were analyzed in parallel (triangle) as a negative control Data represent the means ± SD of triplicate samples cross-reactivity with other TGases, and K60 exhibited some reactivity towards Factor XIIIa These results indicate that the consensus motif identified in this 5672 study defines a unique structure of preferred substrates of TGase This information should be useful for predicting substrate reactivity and designing regulatory molecules that can interact with the active site of the enzyme K5 (YEQHKLPSSWPF) showed the highest level of reactivity and specificity as a substrate for TGase Of note, the K5 sequence was the most frequently selected clone during screening, which suggests that (a) this sequence is a highly preferred substrate as compared to other sequences, and ⁄ or (b) the phage clone that contained this sequence was efficiently amplified in bacteria Mutational analysis of each amino acid residue in the K5 sequence demonstrated that the consensus TGase motif, as well as additional C-terminal amino acids, is important in substrate activity This was expected because the other selected peptides had conserved amino acid residues among analyzed sequences It would be interesting to explore whether shorter peptides can function as substrates with similar levels of reactivity The reactivity and specificity of K5 for TGase was maintained even in peptide form Initially, we expected that the lysine residue of K5 would react with the glutamine residue of pepK5 itself or with casein during the catalytic reaction However, pepK5QN, in which the glutamine was mutated but lysine was not, failed to react with casein (Fig 2), which indicates that there is no reactivity of K5 as a glutamine acceptor Although there is far less selectivity of TGase for glutamine acceptor substrates in general, this result indicates that the lysine residue in the pepK5 is not strongly reactive Thus, pepK5 represents a potentially valuable tool for determining the specific partners that are favored substrates during TGase catalysis When we examined the ability of pepK5 to detect endogenous TGase activity in mouse skin sections (Fig 6), Ca2+-dependent incorporation of FITCpepK5 into glutamine acceptor substrates in keratinocytes was seen, and this activity was undetectable in sections from TGase 1-null mice As observed in immunostaining analysis, TGase protein localized to the peripheral regions of the keratinocytes in the granular and upper spinous layers, consistent with previous reports [40,41], and the K5-reactive proteins most likely co-localize with TGase at these sites These results indicate that the K5 peptide acts as a sensitive and precise probe to detect in situ activity of endogenous TGase In the current study, K5 did not exhibit cross-reactivity to TGase 3, skin-specific TGase In the in vitro assay with TGase 2, K5 reacted to a small extent at higher enzyme activity or higher peptide FEBS Journal 275 (2008) 5667–5677 ª 2008 The Authors Journal compilation ª 2008 FEBS Y Sugimura et al Preferred substrate sequences for TGase A Fig Detection of in situ TGase activity in mouse skin sections (A) FITC-labeled peptides (1 lM), pepK5 (upper) or pepK5QN (lower), were incubated with mouse skin sections in the presence of CaCl2 or EDTA TGase activity in skin sections from a TGase knockout mouse was also examined (right) Sections analyzed by immunostaining using monoclonal anti-mouse TGase IgG (TGase1 mAb) and by hematoxylin and eosin staining are also shown (left columns) (B) Detection of TGase activity in wild-type mouse skin sections using increasing concentrations (0.1–100 lM) of FITC-labeled pepK5 or FITC-cadaverine Scale bar = 50 lm B concentration However, in the skin sections, there was no significant TGase activity because FITC-labeled T26 showed no signal (data not shown) To date, detection of cross-linked TGase products using tissue sections has utilized an FITC-labeled primary amine (FITC-cadaverine) or FITC-labeled substrate peptides [42,43] The pattern of TGase activity that we observed was consistent with that seen in the skin using FITC-cadaverine [42], although cadaverine is not an isozyme-specific probe, and thus detects total TGase activity in keratinocytes However, in our study, the sensitivity of K5 was approximately 100–1000 times higher than that of FITC-cadaverine (Fig 6B) Thus, the method that we have established for the detection of TGase exhibits superior specificity and reactivity TGase expression is not limited to the skin epidermis It is also expressed in the kidney, hair follicle and liver [40,42] However, the precise roles of TGase in these tissues are unknown In addition, in the epithelial cells, activated TGase is concentrated at adherent junctions, but the substrates and biological significance of the enzyme are not fully resolved [40] A specific and sensitive detection probe, such as FITC-labeled pepK5, would be useful for investigating the role of TGase 1-mediated cross-linking reactions in keratinocytes and in other cells and tissues In ongoing experiments, we are attempting to characterize the reactivity and to identify the preferred substrate sequences of other skin TGases, as our developed screening system is applicable to other TGases [33,44] Recombinant TGase is poorly expressed as a soluble active form [17,45], thus we could not examine its reactivity in this study However, it is unlikely that the selected peptides cross-react with TGase 5, as there was no obvious reactivity of FITC-pepK5 in skin sections from a TGase knockout mouse Furthermore, the cross-linking products containing pepK5 localized primarily at the periphery of the cell, which is distinct from the location of TGase [17,18] In conclusion, we have identified several preferred substrate sequences for TGase Our findings provide valuable insight into substrate specificity in the crosslinking processes of the epidermis, and possibly other epithelial tissues as well Specific and sensitive detection of TGase activity using FITC-labeled K5 peptide could be used for clinical diagnosis of congenital ichthyoses Additional studies, particularly FEBS Journal 275 (2008) 5667–5677 ª 2008 The Authors Journal compilation ª 2008 FEBS 5673 Preferred substrate sequences for TGase Y Sugimura et al co-crystallization of TGase with the K5 peptide, are required to refine the TGase reaction mechanism, and to develop specific regulatory molecules Experimental procedures Transglutaminases Human recombinant TGases and were obtained by expression and purification from baculovirus-infected insect cells, as described previously [46,47] For activation of TGase 3, the zymogen was proteolyzed by treatment with dispase Guinea pig liver TGase was kindly provided by K Ikura (Graduate School of Science and Technology, Kyoto Institute of Technology, Japan) [48] Human Factor XIII (FibrogamminÒ P; ZLB Behring, Marburg, Germany) was activated to Factor XIIIa by treatment with bovine thrombin (Sigma, St Louis, MO, USA) Screening of a phage-displayed peptide library Screening was carried out as previously described, using an M13 Ph.D.-12 phage-display system (New England Biolabs Inc., Ipswich, MA, USA) [33] Briefly, approximately 1.5 · 1011 (1st round panning) or · 1012)13 (2nd to 5th round panning) phage clones were incubated at 37 °C with TGase (10 ngỈlL)1) in 10 mm Tris ⁄ HCl pH 8.0, 150 mm NaCl ⁄ Tris buffer containing mm dithiothreitol, mm CaCl2 and mm Bio-Cd [EZ-linkÔ 5-(biotinamido)pentylamine] (Pierce Biotechnology, Rockford, IL, USA) After termination of the catalytic reaction by addition of EDTA, the phage particles were precipitated in the presence of polyethyleneglycol and NaCl with salmon sperm DNA as a carrier Next, the phage clones that covalently incorporated Bio-Cd were selected by affinity chromatography using mono-avidin gel (SoftLinkÔ soft release avidin resin; Promega Corp., Madison, WI, USA) After washing for with NaCl ⁄ Tris containing 0.1% Tween-20 and mm EDTA and then with NaCl ⁄ Tris twice, the bound phage particles were eluted by NaCl ⁄ Tris containing mm biotin The entire eluate was used to infect Escherichia coli ER2738 host bacteria to amplify the phage The phage particles were concentrated by precipitation with polyethyleneglycol–NaCl and then used for subsequent rounds of amplification After panning five times in all, the DNA sequences of the peptides in the selected phage clones were determined Construction of the expression vector for GST fusion proteins The vector plasmid pET24d-GST(QN) was used to express modified GST in which all the glutamine residues had been substituted by asparagine residues, fused to a peptide at the N-terminus [33] The DNA of each phage was isolated, and 5674 the sequences of the 12-mer peptides were amplified by PCR Amplified PCR products were digested and inserted into pET24d-GST(QN) For generation of peptide mutants in which each amino acid was substituted by alanine or asparagine, PCR-based mutagenesis was carried out Either E coli BL21(DE3)LysS or BL21(DE3)LysE were transformed with expression plasmids, and expression in bacteria was induced by the addition of isopropyl b-d-thiogalactoside Recombinant proteins were purified using TALON metal affinity resin according to the manufacturer’s instructions (BD Biosciences, San Jose, CA, USA) The concentration of the purified protein was determined by the Bradford method (Bio-Rad Laboratories Inc., Hercules, CA, USA) Evaluation of preferred sequences using recombinant proteins The reactivities of recombinant GST(QN) fusion proteins were evaluated by incorporation of Dansyl-Cd (Sigma), a fluorescence-labeled pentylamine Recombinant protein (200 ngỈlL)1) and 0.5 mm Dansyl-Cd were incubated in NaCl ⁄ Tris buffer containing mm CaCl2 and mm dithiothreitol in the presence of TGase (0.75 ngỈlL)1) Dimethylcasein (200 ngỈlL)1) was used as a positive control The reaction mixture was incubated at 37 °C and then separated by 12.5% SDS–PAGE A fluorograph of the gel was obtained by UV irradiation (254 nm) to visualize the amount of incorporated Dansyl-Cd To quantify the results, the fluorescence intensity of each product was analyzed using scion imaging software (Scion Corp., Frederick, MD, USA) For incorporation of radiolabeled putrescine, [3H]-putrescine (1.0 mm) was incubated in NaCl ⁄ Tris buffer containing mm CaCl2 and mm dithiothreitol with the recombinant proteins (1 lgỈlL)1) in the presence of TGase (37.5 ngỈlL)1), TGase (2.5 ngỈlL)1), TGase (8 ngỈlL)1) or Factor XIIIa (12.5 ngỈlL)1), so that similar amounts of incorporation into dimethylcasein (1 lgỈlL)1) were achieved The reaction mixture was precipitated by addition of the 10% trichloroacetic acid and then subjected to centrifugation at 10 000 g for 15 at °C The precipitate was dissolved and radioactivity was determined using a scintillation counter Evaluation of synthetic peptides as a substrate The 12-amino-acid peptide pepK5 (YEQHKLPSSWPF) was synthesized and biotinylated at the N-terminus A mutant peptide in which glutamine was substituted by asparagine was also synthesized as pepK5QN (YENHKLPSSWPF) and biotinylated Biotinylated pepT26 (HQSYVDPWMLDH) was used as the TGase preferred substrate peptide for comparison The peptides were FEBS Journal 275 (2008) 5667–5677 ª 2008 The Authors Journal compilation ª 2008 FEBS Y Sugimura et al dissolved in dimethylsulfoxide (Me2SO) To evaluate the activity and specificity of the peptides, a microtiter plate assay was performed as described previously, with some modifications [33] Briefly, 200 ngỈlL)1 bovine casein (Nacalai Tesque Inc., Kyoto, Japan) was incubated with each peptide (0.01 mm peptide) in the presence of TGase (1.9 ngỈlL)1) or TGase (2.0 ngỈlL)1) in NaCl ⁄ Tris buffer containing mm CaCl2 and mm dithiothreitol at 37 °C When comparing the reactivities of TGase and TGase 3, the molar concentration of recombinant enzyme in each reaction mixture was the same (20 nm: TGase 1, 1.8 ngỈlL)1; TGase 3, 1.5 ngỈlL)1) At the indicated times, 50 lL of the reaction mixture was transferred to a 96-well microtiter plate (Maxisorp; Nunc, New York, NY, USA) The microtiter plate was incubated for h at 37 °C to allow casein in the reaction mixture to attach to the plate After blocking with skim milk and washing with 0.1 m Tris buffer (pH 8.0), streptavidin– peroxidase (Rockland Immunochemicals Inc., Gilbertsville, PA, USA) was added, and the plates were incubated for h at 37 °C The plates were washed in 0.1 m Tris buffer with and without 0.01% Triton-X 100, and conjugation of streptavidin–peroxidase to the biotinylated peptide was detected by the addition of H2O2 and o-phenylenediamine An equal volume of 2.5 n H2SO4 was added, and the absorbance at 492 nm was measured Detection of in situ TGase activity in mouse skin sections Fluorescence-labeled peptides (FITC-pepK5 and FITCpepK5QN) were synthesized, and skin sections were prepared from wild-type and TGase 1-null mice using standard methods [20] The frozen sections were dissected into 3-lm slices and frozen until use Sections were dried and then blocked with 1% BSA in NaCl ⁄ Pi at room temperature The sections were incubated for 90 with a solution containing 100 mm Tris ⁄ HCl pH 8.0, mm CaCl2 or mm EDTA, and mm dithiothreitol, in the presence of FITC-labeled peptide or FITCcadaverine (Sigma) After washing with NaCl ⁄ Pi three times for min, anti-fading solution was added to the sections, which were then covered with a cover glass For hematoxylin and eosin staining, tissue sections were fixed and stained using standard methods For immunostaining, skin tissue sections were analyzed using a monoclonal anti-mouse TGase IgG [40] The skin sections were fixed in a solution containing methanol and acetone (1 : 1) and then blocked in NaCl ⁄ Pi containing 0.1% BSA The sections were incubated with the antibody at 37 °C for 90 min, and the immunoreactive proteins were detected using biotinylated secondary antibody (anti-rat IgG) and FITC-avidin (Zymed, San Francisco, CA, USA) Fluorescence was analyzed by 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(TGase 3): regulation of its activity by proteolysis and guanine nucleotides J Biochem (Tokyo) 125, 1048–1054 Ikura K, Yokota H, Sasaki R & Chiba H (1989) Amino acid sequence of guinea pig liver transglutaminase from its cDNA sequence Biochemistry 28, 2344–2348 FEBS Journal 275 (2008) 5667–5677 ª 2008 The Authors Journal compilation ª 2008 FEBS 5677 ... sequence for transglutaminase J Biotechnol 13 1, 12 1? ?12 7 36 Tarcsa E, Candi E, Kartasova T, Idler WW, Marekov LN & Steinert PM (19 98) Structural and transglutaminase substrate properties of the small... whereas there was a significant increase in incorporation in the presence of TGase pepK5QN also failed to react with casein in the presence of TGase (data not shown) These results indicate that the. .. or in the presence of EDTA, which indicated that the cross-linking reaction was catalyzed specifically by TGase To further evaluate the specificity of the assay for TGase 1, skin sections from a