Rice seed globulin: A protein similar to wheat seed glutenin

Phyrochemistry, Vol. 31, No. 10, pp. 3455-3459,1992

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Printed in Great Britain .

0031-9422/92 $5 .00 + 0.00 1992 Pergamon Press Ltd

RICE SEED GLOBULIN : A PROTEIN SIMILAR TO WHEAT SEED GLUTENIN and H .

S . KOMATSU

Department of Molecular Biology,

HIRANO

Institute of Agrobiological Resources, Kannondai 2-1-2, Tsukuba, Ibaraki 305, Japan (Received in revised form I1 March

Key Word Index-Oryza

sativa;

1992)

Gramineae; rice; seed endosperm protein ; globulin ; amino acid sequences .

Abstract-The globulin of the seed endosperms of rice has an isoelectric point of 6 .4 and a M, of 26000. There is one intra-disulphide bond, but no N-linked oligosaccharide chain . The amino acid sequence of the N-terminal and internal regions of the globulin was determined and found to be homologous with that of glutenin, the storage protein in the seed endosperms of wheat . Rice globulin and wheat glutenin were rich in glycine, and glutamic acid or glutamine, and in addition, wheat glutenin cross-reacted with antibody raised against rice globulin, These results suggest that rice seed globulin represents a protein similar to wheat seed glutenin.

INTRODUCTION

The major storage protein in endosperms of rice (Oryza sativa) is an acid and/or alkaline-soluble protein, glutelin, which accounts for 80% of the total protein [1] . Saltsoluble protein, globulins and alcohol-soluble protein, prolamins, are also present in relatively low amounts in rice endosperms and account for 2-8% and 1-5% of the total protein in the endosperms, respectively [2]. Rice storage proteins are accumulated in two different kinds of protein bodies during seed development with both globulins and glutelins being stored in protein body type II and prolamins in protein body type I [3]. The amino acid sequences of the glutelin and prolamin have been determined by protein sequencing and have also been deduced from nucleotide sequence data of the DNA encoding these proteins [4, 5] . Although rice prolamin has been found to be structurally homologous with the prolamin from seed endosperms of both wheat and barley, rice glutelin is highly homologous in amino acid sequence with IIS globulin, named legumin, which is a major seed storage protein in legumes [4, 6] . Thus, a question arises as to which category the rice globulin is structurally classified. In the present study, we have analysed the amino acid sequence, amino acid composition and immunological properties of rice globulin, and found that the rice globulin is structurally similar to the glutenin wheat seed storage protein .

RESULTS AND DISCUSSION

Proteins extracted from the endosperms of mature rice seeds were separated by 2D-PAGE (Fig . 1). The globulin which had a M, of 26000 [7] was found to have an isoelectric point of 6 .4 (Fig. 1, Spot 8) . Based on the intensity of Coomassie blue staining, this protein consisted of 6 .3% of the total seed endosperm protein in var . Norin 29 . The proteins of spots 1-7 shown in Fig. 1 were glutelin a subunits [8] .

After separation by SDS-PAGE, the globulin was electroblotted and directly sequenced by Edman degradation in a gas-phase sequencer, but was found to have a blocking group at the N-terminus . Consequently, the electroblotted globulin was digested on the polyvinylidene difluoride (PVDF) membrane with pyroglutamyl peptidase and subjected to N-terminal amino acid sequence analysis, to enable the sequence determination of the globulin by Edman degradation . The N-terminal pyroglutamic acid of globulin was thought to be formed by cyclization of either glutamine or glutamic acid . The sequences of 10 residues from the N-terminus of globulin were determined (Fig . 2). The endosperm proteins were separated by 2D-PAGE, and the globulin was electroeluted from the gel . After the electroelution, the globulin was digested with Staphylococcus aureus V8 protease on the SDS-PAGE gel and electroblotted from the gel on to the PVDF membrane to determine the internal amino acid sequences (Fig . 2). Four major fragments of globulin were obtained ; one was from the N-terminal region (Fig . 2, No. 1), the others (Fig. 2, Nos 1-3) from the internal regions of the globulin, In total, the sequences of 39 amino acid residues were determined and a structural homology search indicated that the amino acid sequence of globulin was homologous with that of the high-molecular-weight (HMW) subunit of the glutenin storage protein in wheat seeds, which was deduced by the cDNA sequences [9]. The wheat seed glutenin HMW subunit is one of the important determinants of the bread-making quality of wheat flour [11]. The amino acid composition of rice globulin is shown in Table 1 . The globulin was rich in glycine (ca 76 residues), and glutamic acid or glutamine (ca 32 residues), accounting for 20.6 and 1.6 .9%, respectively . The glutenin HMW subunit is also rich in glycine (20%) and glutamine (36%) [9] and both proteins can thus be considered to be glycine-rich, and glutamic acid- or glutamine-rich . In addition, the wheat endosperm proteins were separated by SDS-PAGE and electroblotted on to a PVDF membrane, the wheat glutenin HMW subunit was shown to

3455



3456

S. KOMATSU

and

H . HIRANO

6 .4

8 .5

V

V

a

14 }-

AGE attern of total proteins of rice (var . Norin 29) seed endosperms . Right to left, electric fo the first dimension; top to bottom, SDS-PAGE for the second dimension . Detected by Coomassie blue staining . (1-7) glutelin a subunits; (8) globulin.

21 31 41 51 glutenin AEGEASEQLQ CERELQELQE RELKACQQVM DQQLRDISPE globulin


RELFAFQQQL QVQL 2)

81 91 151 161 glutenin KGGSFYPGET TPPQQLQQRI GYYPTSPQQP GQWQQ globulin

S MPP 3)

GYYGEQQQQP G 4)

Fig. 2. Amino acid sequence homology between rice seed globulin and wheat seed glutenin . < Q represents pyroglutamic acid .

cross-react with anti-globulin antibody (Fig . 3). Finally, neither rice globulin nor wheat glutenin are glycoproteins that can be identified by the concanavalin A-peroxidase method. Taken together, these results, the amino acid sequence, the amino acid composition and the immunological properties of the rice globulin indicate that rice globulin is structurally similar to glutenin, the seed storage protein in wheat. There are two cysteine residues in the globulin molecule (Table 1) which suggests that disulphide bonding may be present . The methods utilized in this study separated proteins under both reducing and non-reducing conditions (Fig . 4), which as shown by Allore and Barber [11]

can be used to detect the presence of intramolecular disulphide bonds. The rice globulin had a lower M, in the nonreduced track than in the reduced one, thus indicating the presence of one putative intrachain disulphide bond . However, no interdisulphide bonds in globulin were detected, in contrast to the case of wheat glutenin, which has many interdisulphide bonds [12] . Rice glutelin has been found to be ho amino acid sequence with the legume 11S globu this study, the rice globulin was found to have a sequence homologous with glutenin . Recently, the N-terminal amino acid sequence of the wheat globulin [13], which has a similar immunological property to the oat globulin

3457

Globulin of rice seed [14], was determined . However, the sequence is completely different from that of the rice globulin. Thus, there is a gap between the classifications of seed storage proteins based on structural homology and solubility [15] . On the other hand, the solubility of seed storage proteins is known to often vary depending on the presence of reducing agents in extraction buffers, temperature for the extraction [16] and ionic strength of the buffers [17] . Since confusion is inevitable, classification of seed storage proteins based on solubility [15] might be reconsidered . EXPERIMENTAL

Plant materials. Mature seeds of rice (0. sativa L. var. Norin 29) and wheat (Triticum aestivum L. var. Norin 60) harvested in the previous year were used in this study . Gel electrophoresis. A portion (10 mg) of endosperms from dry mature seeds was removed, homogenized with 100 p1 of lysis buffer [18] and centrifuged at 15 000 g for 5 min. The supernatant was subjected to 2D-PAGE [19] . SDS-PAGE of the total endosperm protein was performed with 17% seen and 5% stacking gels [20]. The proportion of protein was determined by densitometry . Removal of blocked N-terminal amino acid. After sepn by 2DPAGE, proteins were electroblotted on to a PVDF membrane (Problott, Applied Biosystems, Foster City) [20] and detected by Ponceau 3R staining . Portions of the PVDF membrane containing the proteins were cut out and soaked in 100 mM HOAc containing 0.5% w/v PVP-40 at 37° for 30 min. The membrane

was washed with deionized H 2O x 10. After addition of pyroglutamyl peptidase (10 µg) (Boehringer Mannheim, Germany) in 0 .1 M Pi buffer pH 8 containing 5 mM dithiothreitol and 10 mM EDTA, the reaction soln was incubated at 30° for 24 hr to remove the N-terminal pyroglutamic acid [8] . The membrane was then washed with deionized H 2 0, dried and subjected to gas-phase sequencing. Internal amino acid sequence analysis. Proteins were sepd by 2D-PAGE and stained with Coomassie Brilliant blue R250 . Gel pieces containing the protein were removed and the protein electroeluted from the get pieces using an Electrophoretic Concentrator (M1759, ISCO, Lincoln) at 2 W constant power for 2 hr . After electroelution, the protein soln was dialysed against deionized H 2 O for 2 days and dried . Proteins were dissolved in 20 p1 of SDS sample buffer (pH 6 .8) [18] and overlaid with 20141 of a soln containing a 10 µ1 of S . aureus V8 protease (Pierce, Rockford) (0.1-0.2 pg µl - ') in deionized H 2 0 and 10,ul of SDS sample buffer (pH 6 .8) containing 0.001 % w/v Bromophenol Blue. Electrophoresis was performed until the sample was stacked in the upper gel and interrupted for 30 min to digest the protein [21] . Electrophoresis was then continued and the sepd digests electroblotted on to the PVDF membrane, dried and subjected to gas-phase sequencing [22] . Amino acid sequence analysis . The membrane was applied to the upper glass block of the reaction chamber in a gas-phase protein sequencer (477A, Applied Biosystems). Edman degradation was performed according to the standard program supplied by Applied Biosystems. The released phenylthiohydantoin amino acid derivatives were identified by the on-line system of HPLC . The amino acid sequences obtained were compared with sequences of over 7,967 proteins contained in the amino acid sequence data base (National Biomedical Research Foundation, Protein Identification Resource ; Release 28 .0, 1991). Amino acid composition analysis . After S-pyridylethylation [23], the protein samples were hydrolysed for 24 hr with 6 M Table 1 . Amino acid composition of rice seed globulin

1

2

1

2

Fig. 3. Wheat glutenin cross-reacted with anti-globulin antibody . (A) Rice endosperms ; (B) wheat endosperms; (1) detection with Coomassie blue staining; (2) immunodetection with antiglobulin antibody . (a, b) Position of glutenin in wheat ; (c) position of globulin in rice .

Amino acid

Amino acid residues

Asx Thr Ser Glx Gly Ala Cys Val Met lie Leu Tyr Phe Lys His Arg Pro Trp Total

4.2 1 .9 10.0 31 .9 76.3 28.8 2.0 14.0 4.2 10.9 10.0 5.5 7.5 10.0 0.9 10.9 6.4 n.d. 245

°la

2.02 0.80 3.78 16.86 20.59 9.21 0.88 5 .90 2.22 5.12 9.36 3.57 4.45 5.23 0.50 6.83 2.64 n.d. 100

The number of each amino acid is an average number calculated from three amino acid analyses . n.d.=Not determined.



3458

S . KOMATSU

kDa

kDa

A

94 67 r

r

and H.

43 r

HIRANO

30 r

20 Y

14 r

C

94 67> +ME

43)W-

30

201"-

14A--

-ME Fig. 4. 2D SDS-PAGE pattern of total protein of rice seed endosperms . (A) ID separations of reduced extracts ; (B) 1D separations of nonreduced extracts ; (C) 2D-PAGE under nonreducing conditions in the first dimension and reducing conditions in the second dimension . Arrow indicates the position of the spot related to globulin . HCI containing 0 .1% phenol . Amino acid composition was determined on an amino acid analyser . Detection of glycoprotein with an N-linked oligosaccharide chain . Proteins from seed endosperms were sepd by SDS-PAGE,

electroblotted on to a PVDF membrane and reacted with peroxidase-coupled concanavalin A (Honen, Tokyo) [24] . Detection of disulphide bonding . Proteins were extd in the SDS sample buffer pH 6 .8 without 2-mercaptoethanol [25] and separated in the SDS polyacrylamide disk gel in the first dimension . The gel was removed from the tube and incubated for 30 min in the SDS sample buffer, pH 6 .8, containing 1% 2mercaptoethanol to achieve protein reduction . The disk containing the proteins gel was subjected to SDS-PAGE in the second dimension [12] . Preparation of polyclonal antibody and unmunoblot. Rice globulin with M, of 26000 [21], was sepd by SDS-PAGE and elctroeluted as described above . After electroelution, the protein soln was dialysed against deionized H 2O for 2 days and dried up . Using this protein, antibody against rice globulin was raised in adult rabbits [26] . The purified globulin and an equal vol . of a complete adjuvant was injd into rabbits x 3 at one-month intervals. The anti-serum obtained was used directly in the protein-blotting expt . Gel-fractionated proteins from wheat endosperms were blotted on the PVDF membrane . The blots were blocked with 3% gelatin in Tris-buffered saline (TBS ; 20 mM

Tris-HCI, pH 7.5, 500 mM NaCl) . The primary anti-globulin serum was used at a working diln of 1 : 1000 with 16 hr incubation . After washing, anti-rabbit-IgG horseradish peroxidase (GSR-HRP, Bio-Rad, Richmond) dild to 1 :1000 for 1 hr at room temp. was used as an indirect label . The membrane was stained by the 'HRP Color Development' (Bio-Rad) . thank Dr R. N. Waterhouse for her reading of the manuscript . We thank M . Kuroishi for her assistance. This work was supported partly by the Grant from Basic Research Core System of Science, Science and Technology Agency in Japan . Acknowledgements-We

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