occurring in germinating seeds of wheat, Triticumaestivum L., specifically digests gliadin and is regulated by intrinsic cystatins Toshihiro Kiyosaki1, Ichiro Matsumoto1, Tomiko Asakura1
Trang 1occurring in germinating seeds of wheat, Triticum
aestivum L., specifically digests gliadin and is regulated
by intrinsic cystatins
Toshihiro Kiyosaki1, Ichiro Matsumoto1, Tomiko Asakura1,2, Junko Funaki3, Masaharu Kuroda4, Takumi Misaka1, Soichi Arai5and Keiko Abe1
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-NH2-Mec) at Km¼ 9.5 lm, which is equi-valent to the Km value for hydrolysis of this substrate by cathepsin L Hydrolysis was inhibited by two wheat cystatins, WC1 and WC4, with
IC50 values of 1.7· 10)8 and 5.0· 10)8m, 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.
Trang 2by 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 germinagermina-tion 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
Trang 3To 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.
Trang 4observed 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
(+) (–)
uniconazol KIM-112
175 83 62 47.5 32 25 16.5 6.5
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).
Trang 5Reacting 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)-gluteHMM-glute-nin (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-NH2-Mec)
much better than Z-Arg-Arg-NH2-Mec and Arg-NH2 -Mec The Km value for the hydrolysis of
Z-Phe-Arg-NH2-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 IC50concentrations
in the order of 10)8m, as well as by the two wheat cystatins, WC1 and WC2, at IC50 concentrations in the order of 10)8m(Table 1)
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
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
1 m M EDTA and 2.5 m M b-mercaptoethanol GST–progliadain was added at 4.01 · 10)3 units prior to incubation at 37 C for 24 h.
Trang 6In 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 55C for 24 h with a mixture of oryzain a and
c cDNA fragments labeled with [32P]CTP[aP] as a probe for screening Membranes were washed at 55C in 2· 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-NH2-Mec and GST-progliadain in 100 m M 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.
Trang 7germinated at 26C in the dark Germinating seeds were
treated with the following plant hormones at various
con-centrations in the medium: gibberellin A3 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 42C
with [32P]CTP[aP]-labeled gliadain cDNA as a probe
Preparation of anti-gliadain serum
Two primers, 5¢-TCCGGATCCGGACAATGACCTGG
AG-3¢ (P1) and 5¢-AAAGAATTCAGACACGCATA-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 37C, 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 reacdilu-ted 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% H2O2⁄ 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 4C, 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
Na2HPO4, and 1.8 mm KH2PO4(NaCl⁄ Pi), and then loaded onto a glutathione–Sepharose 4B column (GE Healthcare), and washed with NaCl⁄ Pi 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 lL of 1 mm Z-Phe-Arg-NH2-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-NH2-Mec and Arg-NH2-Mec were meas-ured at various pH values The reaction was performed for 10 min at 37C and stopped by adding 500 lL of ethanol⁄ HCl (473 : 27, v ⁄ v) Fluorometry was conducted at
Trang 8excitation and emission wavelengths of 370 and 461 nm,
respectively Each reaction mixture contained 1.0· 10)5m
Z-Phe-Arg-NH2-Mec and 6.0· 10)7mGST–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 IC50
Expression and purification of wheat seed
cystatins
Two wheat cystatins, WC1 and WC4, were expressed in
E coli andpurified 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 fracfrac-tion 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)3units) was added to 100 lL of each protein suspension
prior to incubation for 24 h at 37C One unit of
proteo-lytic activity of gliadain is defined as that which liberated
1 lmol of NH2-Mec from Z-Phe-Arg-NH2-Mec in 1 min
under the conditions described above
Acknowledgements
This work was supported by the Elizabeth Arnold
Foundation, the Iijima Memorial Foundation for the
Promotion of Food Science and Technology, and by a
Grant-in-Aid for Scientific Research from the Ministry
of Education, Science, Sports, and Culture of Japan
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