Báo cáo khoa học: Gliadain, a gibberellin-inducible cysteine proteinase occurring in germinating seeds of wheat, Triticum pdf

Gliadain, a gibberellin-inducible cysteine proteinase occurring in germinating seeds of wheat, Triticum aestivum L., specifically digests gliadin and is regulated by intrinsic cystatins Toshihiro Kiyosaki 1 , Ichiro Matsumoto 1 , Tomiko Asakura 1,2 , Junko Funaki 3 , Masaharu Kuroda 4 , Takumi Misaka 1 , Soichi Arai 5 and Keiko Abe 1 1 Department of Applied Biological Chemistry, Graduate School of Agricultural and Life Sciences, The University of Tokyo, Japan 2 Faculty of Management, Atomi University, Saitama, Japan 3 Faculty of Human Environmental Science, Fukuoka Women’s University, Japan 4 National Agricultural Research Center, Niigata, Japan 5 Department of Nutritional Science, Tokyo University of Agriculture, Japan Cysteine proteinases (CPs) exist in a wide variety of plants. These enzymes are involved in a number of physiological events, such as the post-translational pro- cessing of storage proteins into mature forms [1–8] and the liberation of amino acids to be used during germi- nation [1,9,10]. The participation of CPs in intracellu- lar protein catabolism for senescence [11,12] and programmed cell death [13,14] is of phytophysiological importance. Some CPs are induced in seeds for stress tolerance as in the case of drought [15–17] and damage Keywords cystatin; cysteine proteinase; gibberellin; gliadin; wheat Correspondence K. Abe, Department of Applied Biological Chemistry, Graduate School of Agricultural and Life Sciences, The University of Tokyo, 1-1-1,Yayoi Bunkyo-ku, Tokyo 113-8657, Japan Fax: +81 3 5841 8006 Tel: +81 3 5841 5129 E-mail: aka7308@mail.ecc.u-tokyo.ac.jp T. Asakura, Faculty of Management, Atomi University, 1-9-6, Nakano Niiza-shi, Saitama 352-8501, Japan Fax: +81 4 8478 4142 Tel: +81 4 8478 4110 E-mail: asakura@mail.ecc.u-tokyo.ac.jp (Received 9 January 2007, revised 10 February 2007, accepted 19 February 2007) doi:10.1111/j.1742-4658.2007.05749.x We cloned a new cysteine proteinase of wheat seed origin, which hydro- lyzed the storage protein gliadin almost specifically, and was named glia- dain. Gliadain mRNA was expressed 1 day after the start of seed imbibition, and showed a gradual increase thereafter. Gliadain expression was suppressed when uniconazol, a gibberellin synthesis inhibitor, was added to germinating seeds. Histochemical detection with anti-gliadain serum indicated that gliadain was present in the aleurone layer and also that its expression intensity increased in sites nearer the embryo. The enzy- mological characteristics of gliadain were investigated using recombinant glutathione S-transferase (GST)–progliadain fusion protein produced in Escherichia coli. The GST–progliadain almost specifically digested gliadin into low molecular mass peptides. These results indicate that gliadain is produced via gibberellin-mediated gene activation in aleurone cells and secreted into the endosperm to digest its storage proteins. Enzymologically, the GST–progliadain hydrolyzed benzyloxycarbonyl-Phe-Arg-7-amino- 4-methylcoumarin (Z-Phe-Arg-NH 2 -Mec) at K m ¼ 9.5 lm, which is equi- valent to the K m value for hydrolysis of this substrate by cathepsin L. Hydrolysis was inhibited by two wheat cystatins, WC1 and WC4, with IC 50 values of 1.7 · 10 )8 and 5.0 · 10 )8 m, respectively. These values are comparable with those found for GST–progliadain inhibition by E-64 and egg-white cystatin, and are consistent with the possibility that, in germina- ting wheat seeds, gliadain is under the control of intrinsic cystatins. Abbreviations CP, cysteine proteinase; GST, glutathione S -transferase; HMM, high molecular mass; LMM, low molecular mass; NH 2 -Mec, 7-amino- 4-methylcoumarin; WC, wheat cystatin; Z, benzyloxycarbonyl. 1908 FEBS Journal 274 (2007) 1908–1917 ª 2007 The Authors Journal compilation ª 2007 FEBS by pathogens [18,19]. Despite such information about plant CPs, little is known about how they are regula- ted in vivo. Several plant CPs have been purified from seeds, such as aleurain, EP-A and EP-B from barley [20–22], oryzain a, b and c from rice [23], a CP from maize [24–26], CysP1 and CysP2 from soybean [27], and CPR1 and CPR2 from vetch [28], some of which are known to digest storage proteins in vitro. There is also a series of processing enzymes, including CPs, which function in the maturation of synthesized storage pro- teins by limited hydrolysis at specific sites. Good exam- ples are the proteinases present in Vigna mungo seeds [29], pumpkin [30], soybean [31] and castor bean [32]. In general, the expression of CPs occurring in seeds is controlled by gibberellin, a phytohormone involved in various phytophysiological events, such as differenti- ation and development [33]. This hormone is usually synthesized in the embryo during germination and is secreted into the endosperm via the scutellum. In rice seeds, gibberellin induces expression of the three CPs, oryzain a, b and c, which respond to gibberellin in dif- ferent fashions [23]. The target enzymes, oryzains, for which the synthesis is mediated by gibberellin [34–37], are synthesized in the aleurone layer of rice seeds and are transported to the endosperm to digest the storage protein glutelin. The activity of CPs is regulated by cysteine protein- ase inhibitors, cystatins. Indeed, we purified oryzacys- tatin I and II as rice CP inhibitors [38,39]. There are also wheat cystatins, WC1, WC2, and WC4, which are expressed in various tissues at the early stages of matu- ration and germination, when many proteinases, inclu- ding CPs, begin to act [40]. These results suggested that the activities of CPs are generally regulated by cystatins. In this study, we investigated CPs in the seeds of wheat, Triticum aestivum L., which contains 10–14% protein. The protein is comprised of four components in various proportions: glutenin, 30–40%; gliadin, 40– 50%; albumin, 3–5%; and globulin, 6–10%. The major components, gliadin and glutenin, bind with each other to constitute gluten that determines the properties of dough. Some CPs actually exist in wheat seeds [41] and their purified preparations can digest Em protein [42,43]. A 23-kDa CP has been purified from dormant wheat seeds and is efficiently inhibited by intrinsic cystatins [40], although no information is available about its nucleotide sequence. This study was per- formed to define the physiological functions of glia- dain, which is a CP expressed in germinating wheat seeds, as well as to determine its effects at the molecu- lar level under the control of wheat cystatins. Results Characterization of a cDNA clone encoding wheat CP A cDNA library was constructed from 1-day germina- ting wheat seed mRNA. Use of oryzasin b and c cDNAs as probes gave 38 positive clones from 500 000 plaques. These clones comprised four groups, all of which encoded CPs belonging to the papain family. All the CPs retained the consensus sequence for the catalytic triad composed of cysteine, histidine, and asparagine residues. One of these CPs, named gliadain, was subjected to further analyses. Gliadain was so named as it almost specifically digested gliadin, as dis- cussed below. Gliadain shows 87.8% identity to barley EP-B [44] and 70.6% to rice CP [45]. The putative cat- alytic triad were found as 162Cys)301His)322Asn, and two potential N-glycosylation sites were detected in gliadain (Fig. 1). Effects of exogenously added gibberellin on the expression of gliadain mRNA and protein Northern blot analysis was performed to define the expression stage of gliadain. Although gliadain mRNA was somewhat detectable at the maturation stage, much clearer expression was observed during germina- tion (Fig. 2A). Expression during germination was dis- tinct in seeds but was not detected in shoots or roots. The enzyme concentration does, in fact, increase over time. We then investigated the effects of the phytohor- mone, gibberellin, on the expression of gliadain at both the mRNA and protein levels, as its addition is known to induce the expression of hydrolytic enzymes, such as amylase [46] and CPs in germinating seeds [33,47]. Synthesis of gibberellin begins when a seed absorbs water. We examined the time course of chan- ges in gliadain mRNA expression due to the action of gibberellin and its suppression by uniconazole, a speci- fic gibberellin-synthesis inhibitor. Although no signifi- cant effect of the inhibitor was observed in the first 12 h after the start of imbibition, distinct suppression of mRNA occurred at 24 h and thereafter (Fig. 2B). The lack of a significant suppressive effect at this ini- tial stage may be due to the presence of a certain amount of gibberellin initially. Inhibition of gliadain mRNA expression also occurred, probably with the same time course, during the stage from 1 to 5 days after the start of imbibition (Fig. 2B). Our observa- tions indicate that the expression of gliadain mRNA is markedly affected by gibberellin. T. Kiyosaki et al. Wheat seed cysteine proteinase FEBS Journal 274 (2007) 1908–1917 ª 2007 The Authors Journal compilation ª 2007 FEBS 1909 To confirm these results, we examined the expression of gliadain protein by immunoblotting analysis using anti-gliadain serum. Our results showed that, in the absence of added uniconazole, a cross-reactive band of 30 kDa appeared approximately 1 day after the start of imbibition (Fig. 2C). This band may have been due to mature gliadain molecules with sugar chains, as the sizes of pro- and mature gliadain molecules estimated from their putative primary structures are 38.6 and 25.3 kDa, respectively. Two potential N-glycosylation sites exist in the probable maturation enzyme region. The level of this band of 30 kDa increased much more at 3 days than 1 day after the start of imbibition. No particular increase in band intensity was observed when uniconazole was added. Although gliadain was detected at low levels in the presence of the inhibitor, this may have been due to gliadain synthesis prior to its inhibition by uniconazole. These results are consis- tent with northern blotting data showing that a large quantity of gliadain mRNA was expressed 2 days after the start of imbibition (Fig. 2B). The use of unicona- zole thus indicated that the levels of an antigen- positive protein decreased 1 day after the start of imbibition, with little change in quantity for the next 4 days. In summary, gibberellin-inducible gliadain is expressed distinctly during germination. Histochemical detection of gliadain occurring in 1-day germinating seeds Compartmentalization of gliadain expression was dis- sected by immunostaining with an anti-gliadain serum. Immunopositive staining was detected in the aleurone layer and also in the endosperm region in the vicinity of the embryo (Fig. 3A). Expression of gliadain in the endosperm was limited to the region near the embryo in which protein bodies had already been degraded (Fig. 3B). No significant staining was detected in the regions in which the protein bodies remained, which are somewhat distant from the embryo (Fig. 3A). Expression of gliadain was also detected in the aleu- rone, with an increase in expression intensity at sites near the embryo. These signals were not detected with- out anti-gliadain serum (Fig. 3C,D). Significant stain- ing was observed in the embryo and epidermis, but it is not clear whether this signal is due to gliadain itself or some artificial high background induced by the sec- ondary antibody used, because the same was also Fig. 1. Comparison of the amino acid sequences of gliadain with those of EP-B and rice CP. Asterisks show the catalytic triad of CPs. Amino acids identical to gliadain are shown in reverse type. The potential N-glycosylation sites in gliadain are lined. The accession numbers of these proteins are as follows: gliadain, AB262584; EP-B, U9495; rice CP, D76415. Wheat seed cysteine proteinase T. Kiyosaki et al. 1910 FEBS Journal 274 (2007) 1908–1917 ª 2007 The Authors Journal compilation ª 2007 FEBS observed without anti-gliadain serum. The observed results strongly suggest that gliadain is involved in the proteolysis of storage proteins. Biochemical properties of recombinantly produced gliadain Immunostaining with anti-gliadain serum suggested that gliadain was involved in the degradation of stor- age proteins. To obtain a more detailed insight into the biochemical properties of gliadain in vitro,we prepared recombinant gliadain. For this, a GST–pro- gliadain expression plasmid was constructed and intro- duced into Escherichia coli. The expected fusion protein was produced as an insoluble inclusion body. The product was then purified according to the method of Matsumoto et al. [48]. The molecular mass of GST–progliadain is 60 kDa (Fig. 4A). To identify the endogenous substrate of gliadain, we reacted GST–progliadain with gliadin and glutenin, both of which are the prolamins according to the Osborne classification. Gliadin is comprised of five elements, high molecular mass (HMM)-gliadin species of 104 000–125 000 Da, a-, b-, and c-gliadin that migrate together on SDS ⁄ PAGE, and x-gliadin as a minor component. In this experiment, x-gliadin and HMM-gliadin were not extracted, and a-, b-, and c-gliadin of 25 000–30 000 Da, were examined. 123 45601235 3535 seeds shoots roots Maturation (weeks) Germination (days) 048121235 Hours Days (+) (–) uniconazol KIM-112 175 83 62 47.5 32 25 16.5 6.5 kDa 135 135 (days) A B C Fig. 2. Expression of gliadain in maturing and germinating wheat seeds. (A) Northern blotting analysis of gliadain in maturing and germinating seeds. Aliquots of 10 lg of total RNA were loaded into each lane. Maturation stage is shown in weeks after flowering and germination stage in days after imbibition. Shoots and roots were harvested from seeds 3 and 5 days after imbibition. (B) Nor- thern blotting analysis of gliadain in germina- ting seeds with the gibberellin synthetic inhibitor, uniconazole, in the medium. Absence (–) and presence (+) of uniconazole is indicated. Numbers indicate hours and days after imbibition. (C) Western blotting analysis of gliadain in germinating seeds. Anti-gliadain serum was raised against GST–progliadain. Numbers indicate days after imbibition. SDS ⁄ PAGE was performed with 80% ammonium sulfate-precipitated fractions from 1-, 3-, and 5-day imbibed seeds. ‘–‘ and ‘+’ are as in (B). T. Kiyosaki et al. Wheat seed cysteine proteinase FEBS Journal 274 (2007) 1908–1917 ª 2007 The Authors Journal compilation ª 2007 FEBS 1911 Reacting GST–progliadain, we found that a-, b-, and c-gliadin were degraded into smaller fractions, while the enzyme had no appreciable effect on HMM-glute- nin or low molecular mass (LMM)-glutenin (Fig. 4B). These results suggested that gliadain digests gliadin specifically, and so we named this proteinase gliadain. To confirm the substrate specificity of gliadain, we attempted to react GST–progliadain with rice protein composed of glutelin, globulin, and prolamin. We extracted rice protein bodies from seeds, and then incubated them with GST–progliadain; none of these proteins was digested by GST-progliadain (data not shown). Taken together, these results indicated that gliadain digests only wheat seed gliadin. Next, we investigated the substrate specificity using synthetic peptides and found that GST–progliadain hydrolyzed benzyloxycarbonyl benzyloxycarbonyl-Phe- Arg-7-amino-4-methylcoumarin (Z-Phe-Arg-NH 2 -Mec) much better than Z-Arg-Arg-NH 2 -Mec and Arg-NH 2 - Mec. The K m value for the hydrolysis of Z-Phe-Arg- NH 2 -Mec was 9.5 lm, which was comparable with the case when the same substrate was treated with cath- epsin L. In addition, GST–progliadain was most active at pH 4–6, as in cathepsin L. These observations, together with the amino acid sequence similarity (data not shown), suggest that gliadain belongs to the cath- epsin L family. Inhibitory activity of cystatins against gliadain Information is available on the inhibition of CPs by cystatins. As expected, GST–progliadain was inhibited by E-64 and egg white cystatin at IC 50 concentrations in the order of 10 )8 m, as well as by the two wheat cystatins, WC1 and WC2, at IC 50 concentrations in the order of 10 )8 m (Table 1). AB CD Fig. 3. Immunohistochemical detection of gliadain in germinating seeds. Seeds were imbibed on moist cotton cloth at 25 °C for 1 day and pieces were frozen with O.C.T. compound in liquid nitrogen. Sections were sliced longitudinally at a thickness of 4 lm. (A) Section stained with anti-gliadain serum. (B) Higher magnification of endosperm stained with anti-gliadain serum. (C) Section stained without anti-gliadain serum. (D) Higher magnification of endosperm without anti-gliadain serum. Arrowheads indicate the signal of gliadain. Ale, aleurone layer; Emb, embryo; End, endosperm of wheat seed. GST-progliadain 60 kDa + – 97 66 42 30 20 kDa HMM- glutenin LMM- glutenin + – 97 66 42 30 20 kDa α α -, β β -, γ γ - gliadin AB Fig. 4. SDS ⁄ PAGE of purified GST–proglia- dain and digestion patterns of wheat storage proteins. (A) Purified recombinant GST–pro- gliadain produced in E. coli. (B) Digestion pat- terns of gliadin and glutenin after treatment with GST–progliadain. Each of the wheat pro- tein samples fractionated according to their solubility were solubilized or suspended at a concentration of 1.0% (w ⁄ v) in 100 m M sodium acetate buffer (pH 4.5) containing 1m M EDTA and 2.5 mM b-mercaptoethanol. GST–progliadain was added at 4.01 · 10 )3 units prior to incubation at 37 °C for 24 h. Wheat seed cysteine proteinase T. Kiyosaki et al. 1912 FEBS Journal 274 (2007) 1908–1917 ª 2007 The Authors Journal compilation ª 2007 FEBS Discussion In seeds, CPs are known to play roles in storage protein digestion during germination, catabolysis of intracellular proteins, and in the processing of prepro- proteins in maturation [1–10]. In germination, many hydrolytic enzymes are expressed to produce glucose and amino acids for seedling growth [49–51]. The synthesis of some CPs is regulated by gibberel- lin, which is synthesized in the embryo, secreted into epithelial scutellum cells, and diffuses throughout the seeds where it induces the genes encoding these enzymes. In this study, gliadain mRNA was detected 1 day after imbibition, with a steady increase up to 5 days. Gliadain expression was detected only in shoots and roots of germinating seeds, and its expression was suppressed when the gibberellin synthesis inhibitor, uniconazole, was added to the medium. Gliadain was detected 12 h after the addition of uniconazole. This suggests that the effect of uniconazole appears at least 12 h after its addition, prior to which it is likely that endogenous gibberellin may be functional. Gliadain produced in the aleurone layer migrates into the endosperm to digest protein bodies, as shown by our observation of a strong expression signal of gliadain near the embryo 1 day after germination (Fig. 3A). As germination processes, the site of glia- dain expression is likely transferred to a distant part of the endosperm. Recombinant GST–progliadain diges- ted the wheat storage protein gliadin into small molecular mass fractions, suggesting that gliadain digests the protein body gliadin in the embryo from the surface toward the inner part (Fig. 4B). Endogenous cystatins must function to inhibit the proteolysis of storage proteins coming across wheat cysteine proteinases by chance. This is supported by a previous report on the purification of a cystatin–CP complex from corn kernels [26]. Three types of cysta- tin, WC1, WC2, and WC4, exist in wheat seeds; two of these molecules, WC1 and WC2, inhibited gliadain strongly in vitro (Table 1). These results indicate the possibility that gliadain is regulated by these cystatins. This hypothesis was confirmed by the observation that the sites and stages of gliadain expression are coinci- dent with those of cystatins, as shown by northern blotting analysis [37]. It is inferred that gliadain syn- thesized in the aleurone layer forms an inactive com- plex with each of the cystatins in an equimolar ratio; the complex then interacts with storage protein bodies. As a result, pH and ion strength are changed, with the result that the complex is dissociated into gliadain and cystatin. Studies of such a CP-cystatin system in plant seeds are important to understand the biological regu- lation of their protein anabolism and catabolism. In this study, we found the novel CP, gliadain, expressed only in germinating seeds to digest gliadin. This is the first report of the regulation of a CP by intrinsic cystatins in germinating wheat seeds. Further investigation of this mode of enzyme–inhibitor interac- tion will contribute to fundamental knowledge on the biochemistry and physiology of plant seeds, in general, as well as that of wheat seeds, in particular. Experimental procedures Wheat seeds Seeds of T. aestivum L., cultivar Norin 61, were harvested on the Tama Experimental Farm at the University of Tokyo, Japan. Construction of a cDNA library and isolation of wheat CP cDNA clones Total RNA was extracted from 1-day germinating wheat seeds using the phenol–SDS method, and mRNA was puri- fied using an oligo(dT) cellulose column. Double-stranded cDNA was constructed with a Time Saver cDNA synthesis kit (GE Healthcare, Chalfont St Giles, UK). The cDNA was ligated into a kgt10 (EcoRI ⁄ CIAP) phage vector (Stratagene, La Jolla, CA). After in vitro packaging, phages were grown on E. coli C600Hfl. Recombinant plaques were transferred onto nylon membranes (Hybond N, GE Healthcare) and hybridized at 55 °C for 24 h with a mixture of oryzain a and c cDNA fragments labeled with [ 32 P]CTP[aP] as a probe for screening. Membranes were washed at 55 °Cin2· NaCl ⁄ Cit containing 0.1% SDS and exposed to X-ray film. Germination of wheat seeds and treatment with plant hormones Wheat seeds were soaked in 5% hypochlorous acid contain- ing 0.02% Triton X-100 and then washed in water. Seeds were placed on a fully moistened vermiculite sheet and Table 1. Inhibition of GST–progliadain by cysteine proteinase inhibi- tors. Each reaction mixture contained 1.0 · 10–5 M inhibitor in Z-Phe-Arg-NH 2 -Mec and GST-progliadain in 100 mM Mes-NaOH buffer (pH 6.2). The reaction was carried out at 37 °C for 10 min. IC 50 was determined as described in Experimental procedures. Inhibitor IC 50 (M) Wheat cystatin 1 (WC1) 1.7 · 10 )8 Wheat cystatin 2 (WC2) 5.0 · 10 )8 Egg white cystatin 2.1 · 10 )8 E-64 3.0 · 10 )8 T. Kiyosaki et al. Wheat seed cysteine proteinase FEBS Journal 274 (2007) 1908–1917 ª 2007 The Authors Journal compilation ª 2007 FEBS 1913 germinated at 26 °C in the dark. Germinating seeds were treated with the following plant hormones at various con- centrations in the medium: gibberellin A 3 at 3.1 mm; and abscisic acid at 1 mm; and uniconazole as a gibberellin syn- thesis inhibitor at 30 mgÆL )1 . Northern blotting analysis Total RNA was extracted from mature, immature, and ger- minated seeds using the phenol–SDS method. Shoots and roots were taken from germinated seeds and then total RNA fractions were also extracted. Aliquots of 10 lg of the RNA were electrophoresed on 1% agarose gels and blotted onto nylon membranes. Hybridization was performed at 42 °C with [ 32 P]CTP[aP]-labeled gliadain cDNA as a probe. Preparation of anti-gliadain serum Two primers, 5¢-TCCGGATCCGGACAATGACCTGG AG-3¢ (P1) and 5¢-AAA GAATTCAGACACGCATA-3¢ (P2), were synthesized. P1 corresponding to the N-terminal region of gliadain has a BamHI site at the 5¢-end (under- lined). P2, corresponding to the C-terminal region has an EcoRI site at the 5¢-end (underlined). PCR was performed using these two primers and gliadain cDNA as a template. The amplified fragments were digested with both BamHI and EcoRI prior to insertion into pUC19. The resultant gliadain expression plasmid was introduced into E. coli strain YA21. The transformant was grown for 24 h at 37 °C, and cultured for a further 2 h after addition of 1 mm isopropyl thio-b-d- galactoside. Cells were collected by centrifugation, lyzed by ultrasonication, and subjected to SDS ⁄ PAGE. A band with the expected molecular mass of gliadain was cut from the gel and used to immunize a rabbit. An anti-gliadain serum was purified from the serum according to the method of Matsu- moto et al. [48]. Animals were treated in accordance with cri- teria established by the Animal Care and Use Committee at the University of Tokyo. Western blotting analysis Germinated wheat seeds were harvested at 1, 3, and 5 days after imbibition, washed in water, and stored at )80 °C. Frozen samples were crushed in solid carbon dioxide and suspended in buffer A (50 mm phosphate buffer, pH 6.6, containing 150 mm NaCl, 5 mm EDTA, and 0.1 mm E-64). The suspension was centrifuged at 3000 g and the precipi- tate was discarded. Ammonium sulfate was dissolved at a concentration of 80% in the supernatant and the solution was centrifuged at 10 000 g for 10 min. The precipitate was collected and dialyzed against buffer A. The resulting dif- fusible fraction was concentrated and electrophoresed. Sam- ples were electroblotted onto polyvinylidene fluoride membranes, immunoblotted with anti-gliadain serum dilu- ted 1000-fold, and then reacted with horseradish per- oxidase-conjugated anti-(rabbit IgG) for visualization with ECL Plus (GE Healthcare). Immunohistochemical analysis Wheat seeds germinating for 1 day were stored at )80 °C. Frozen seeds were each sliced at a thickness of 3 mm and fixed in 10% formaldehyde. Each slice was washed three times with 50 mm phosphate buffer (pH 7.3), embedded in O.C.T. compound, embedding medium for frozen tissue specimens reagent, and further sectioned at a thickness of 4 lm. The sections were soaked in 0.3% H 2 O 2 ⁄ methanol for 30 min, blocked in 5% normal goat serum, and reacted with 1 : 1000-fold diluted anti-gliadain serum at room tem- perature for 30 min. Visualization was performed with streptavidin and biotinylated horseradish peroxidase. Expression of GST–progliadain The expression plasmid for gliadain was digested with BamHI and EcoRI, and inserted into the pGEX-3X expres- sion vector (GE Healthcare). The resultant plasmid, pGEX– progliadain, was introduced into the ad 202 E. coli strain. The transformed cells were cultured in LB medium contain- ing 100 mgÆmL )1 ampicillin until the medium reached an absorbance of 0.6 at 600 nm. Then, isopropyl thio-b-d-gal- actoside was added at a final concentration of 1 mm and the resultant mixture was cultured for a further 2 h. Cells were collected by centrifugation, washed once with 20 mm Tris ⁄ HCl (pH 8.0) containing 5 mm EDTA, and resuspended in the same buffer containing 1 mm phenylmethanesulfonyl fluoride, 5 mm b-mercaptoethanol, and 1% N-lauroyl- sarcosyl. After sonication, a 20% volume of 10% Triton X-100 was added, mixed with a vortex at 4 °C, and centri- fuged at 18 000 g for 20 min. The supernatant was diluted with a mixture of 140 mm NaCl, 2.7 mm KCl, 10 mm Na 2 HPO 4 , and 1.8 mm KH 2 PO 4 (NaCl ⁄ P i ), and then loaded onto a glutathione–Sepharose 4B column (GE Healthcare), and washed with NaCl ⁄ P i . The GST fusion protein was eluted with 50 mm Tris ⁄ HCl (pH 8.0) containing 10 mm glutathione and confirmed for purity by SDS ⁄ PAGE. Assay of proteinase activity and inhibition of GST–progliadain by cystatins To detect the proteinase activity of affinity-purified GST– progliadain, 5 lLof1mm Z-Phe-Arg-NH 2 -Mec and 250 lL of 200 mm Mes-NaOH (pH 6.2) were added to 500-lL aliquots of the reaction mixture. The hydrolytic activities toward Z-Arg-Arg-NH 2 -Mec and Arg-NH 2 -Mec were meas- ured at various pH values. The reaction was performed for 10 min at 37 °C and stopped by adding 500 lLof ethanol ⁄ HCl (473 : 27, v ⁄ v). Fluorometry was conducted at Wheat seed cysteine proteinase T. Kiyosaki et al. 1914 FEBS Journal 274 (2007) 1908–1917 ª 2007 The Authors Journal compilation ª 2007 FEBS excitation and emission wavelengths of 370 and 461 nm, respectively. Each reaction mixture contained 1.0 · 10 )5 m Z-Phe-Arg-NH 2 -Mec and 6.0 · 10 )7 m GST–progliadain in 100 mm Mes-NaOH buffer (pH 6.2). Florescence intensity was measured in the presence of each inhibitor at 10 nm to 10 lm to obtain the IC 50 . Expression and purification of wheat seed cystatins Two wheat cystatins, WC1 and WC4, were expressed in E. coli and purified by DE52, MonoQ and Superose 12 col- umn chromatography according to the methods of Kuroda et al. [40]. The homogeneity of these proteins was also checked by SDS ⁄ PAGE. Extraction of wheat storage proteins and digestion of them by GST–progliadain Wheat gliadin and glutenin were extracted primarily by the method of Osborne [52] with some modifications. Wheat flour was purchased from Nisshin Flour Milling Corpora- tion (Tokyo, Japan) and proteins were extracted with 0.1 m Tris ⁄ HCl (pH 8.6) containing 4 m urea. The extract was dialyzed against water and then lyophilized. The lyophilized sample was dissolved in distilled water and the soluble frac- tion was discarded. The remaining fraction was washed with water, and dissolved in 0.15 m NaCl to extract the sol- uble fraction. The residue was washed twice with water and once with 70% ethanol. Gliadin was obtained by extracting the residue with 70% ethanol. The resulting residue was used as glutenin. Gliadin and glutenin were lyophilized and suspended in 100 mm sodium acetate buffer (pH 4.5) con- taining 1.0 mm EDTA, 2.5 mm b-mercaptoethanol at a concentration of 1.0% (w ⁄ v). GST–progliadain (4.01 · 10 )3 units) was added to 100 lL of each protein suspension prior to incubation for 24 h at 37 °C. 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