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A study of the sites of glycosylation of zein proteins using the lectin concanavalin A
Plant Science, 38 (1985) 93--98
93
Elsevier Scientific Publishers Ireland Ltd.
A STUDY OF THE SITES OF GLYCOSYLATION OF ZEIN PROTEINS USING THE LECTIN CONCANAVALIN A
J.A. SMITHa, W.L. ROTTMANN b'* and I. RUBENSTEIN b aDepartment of Horticultural Sciences and Landscape Architecture and bDepartment of Genetics and Cell Biology, University of Minnesota, St. Paul, MN 55108 (U.S.A.)
(Received November 6th, 1984) (Revision received January 2nd, 1985) (Accepted January 4th, 1985) The sites of glycosylation of zeins, the maize (Zea mays L.) storage proteins, were studied using the affinity of the lectin Concanavalin A (Con A) for certain glycosides. Zeins which were extracted from kernels of Illinois High Protein (IHP), W22, W64A and Oh43 were separated by isoelectric focusing and analyzed with a radiolabeled Con A binding technique. Certain sub-groups of the zein proteins contained carbohydrate moieties which bound Con A while others did not. Zeins extracted from Oh43 kernels had a higher relative affinity for Con A than those of other maize lines. Further analyses of the zeins of Oh43 by gas chromatography demonstrated the presence of fucose, mannose and glucose. Key words: Zea mays; zeins; glycosylation; Concanavalin A
Introduction T h e zein p r o t e i n f a m i l y r e p r e s e n t s a large g r o u p o f p r o t e i n s w i t h similar p h y s i c a l a n d c h e m i c a l p r o p e r t i e s [ 1 ] . Zeins are soluble in 70% e t h a n o l and 60% o f t h e i r a m i n o acid residues consist o f leucine, g l u t a m i n e , alanine a n d p r o l i n e [ 2 ] . T h e y are low in t w o o f t h e essential a m i n o acids: lysine a n d t r y p t o p h a n . W h e n s e p a r a t e d b y s o d i u m d o d e c y l sulfate (SDS) gel e l e c t r o p h o r e s i s t h e s e p r o t e i n s exhibit two major bands with apparent m o l e c u l a r weights o f a b o u t 19 0 0 0 a n d 23 000 a n d t w o m i n o r b a n d s w h i c h have a p p a r e n t m o l e c u l a r weights o f 10 0 0 0 a n d 15 0 0 0 [ 3 ] . T h e t r u e h e t e r o g e n e i t y o f zein can be m o r e clearly d e m o n s t r a t e d w i t h isoelectric focusing ( I E F ) gels [4] a n d 2 - d i m e n sional gel analysis w h e r e m o r e t h a n 20 diff e r e n t p r o t e i n s can be identified [ 5 ] . With *Present address: 3M Center, St. Paul, MN 55144, U.S.A. Abbreviations: Con A, Concanavalin A; IEF, isoelectric focusing; IHP, Illinois High Protein; SDS, sodium dodecyl sulfate.
these techniques, a complex protein pattern can be seen w h i c h is d i f f e r e n t f o r e a c h inbred line [ 4 ] . Zeins, like m a n y o t h e r m a j o r seed-storage p r o t e i n s , are n o w k n o w n to be g l y c o p r o t e i n s . Early a t t e m p t s to s h o w t h e p r e s e n c e o f glycosides w i t h t h e periodic acid~Schiff r e a c t i o n p r o d u c e d negative results [ 4 ] . In a later r e p o r t , t h e h y b r i d W F 9 X B 3 7 has b e e n s h o w n to c o n t a i n 1 m o l o f glucose p e r m o l e o f zein [ 6 ] . H o w e v e r , it is n o t k n o w n w h a t o t h e r k i n d s o f sugars m a y be p r e s e n t in zein or w h e t h e r d i f f e r e n t i a l degrees o f g l y c o s y l a t i o n m a y a c c o u n t f o r s o m e o f t h e h e t e r o g e n e i t y in this f a m i l y . L e c t i n s are p r o t e i n s w h i c h b i n d specifically to saccharides. T h e r e f o r e , we have used C o n A, which has a specific b i n d i n g a f f i n i t y for a-D-mannopyranosides; a-D-glycopyranosides, D - f r u c t o f u r a n o s i d e s , a n d rnyo-inositol [ 7 - - 1 0 ] , as a p r o b e to i d e n t i f y sites o f glyc o s y l a t i o n o n t h e zeins. T h e p u r p o s e o f t h e s e e x p e r i m e n t s was t o initiate a s t u d y o f t h e p a t t e r n o f g l y c o s y l a t i o n o f zein p r o t e i n s . We c o n c l u d e t h a t s o m e zein
0168-9452/85/$03.30 © Elsevier Scientific Publishers Ireland Ltd. Printed and Published in Ireland
94 proteins are able to specifically bind Con A. Further, the patterns for the binding seen in IEF gels are distinctive for each type of zein suggesting a genetic control of post-translational modification. Materials and methods
Meal preparation Kernels of Z. mays L. (IHP of the 1971 selection) were obtained from R.L. Phillips (University of Minnesota); W22 and W64A were obtained from C.Y. Tsai (Purdue University, W. Lafayette, IN); and Oh43 was obtained from D.V. Glover (Purdue University, W. Lafayette, IN). These lines were grown at the St. Paul Campus of the University of Minnesota during the summer of 1978 and were self-pollinated. De-embryonated mature kernels were frozen in liquid nitrogen and stored at - 8 0 ° C . To prepare meal, the frozen kernels were lyophilized and placed in a Torit capsule with an abrasive bar and vibrated for 1 min on the Torit shaker [11]. One hundred milligrams of the resulting meal were used for protein extraction. Zein isolation Several studies have confirmed that the unique solubility of zein in 70% ethanol allows the direct extraction of these proteins from kernels (2--4). Dry meal samples were first defatted by mixing with chilled acetone for 30 min at 4°C, centrifuging at 2500 × g for 10 min at 0--4°C, followed by removal of the acetone. The meal was then dried and extracted twice with 5 ml of chilled 0.5 M NaC1 solution to remove albumins and globulins. The meal was then washed two times with distilled water to reduce the salt concentration. The zein fraction was extracted from the resulting pellet by mixing with 5 ml of 70% (v/v) ethanol containing 1% (v/v)~-mercaptoethanol. This extraction was repeated twice and the ethanol extracts were lyophilized in convenient aliquots and stored a t - 8 0 ° C . Protein determinations were done on replicate samples using the Lowry m e t h o d [12].
Isolation o f lectins The iodination was done according to methods described by Burridge [13]. Two milligrams of Con A (Calbiochem) were dissolved in a reaction mixture consisting of 100 ~l of lectin buffer (containing 0.15 M NaC1, 50 mM Tris--HC1, 0.5 mM CaC12, 0.5 mM MnC12, and 0.05% (w/v) Na azide adjusted to pH 7.4) and 0.2 mCi of carrier free Na1125 (New England Nuclear). The Con A reactive site was protected with a-methyl-D-mannopyranoside. The reaction was initiated with 5 ~l of a chloramine T solution (5 mg/ml) and allowed to proceed for 45 s at 0--4°C. The reaction was stopped by the addition of 5 pl of sodium metabisulfite (20 mg/ml). The resulting mixture was passed through a column of Biogel-P10 to remove free I ~2s from the iodinated lectin. This procedure resulted in a labeled Con A protein with a specific activity of 5.6 × 107 cpm/mg or greater. Electrophoresis IEF was carried out according to the technique of Righetti and co-workers with LKB 2117 Multipore equipment [14]. The polymerized gel was cooled for 1 h on the gel platform at 8--10°C. Dried protein samples were reconstituted in a solution of 6 M urea, 10 mM Tris/glycine (pH 8.5}, and 2% .6-mercaptoethanol to a concentration of 100 pg/25 pl and were applied to the surface of the gel. The pH gradients were measured by recording the pH of 1 ml vols. of distilled water containing 5-mm segments of gel and also by pI marker proteins (Calbiochem; pI marker protein kit). Sodium dodecyl sulfate electrophoresis was carried out according to the technique of Laemmli [15] using 10% (w/v) polyacrylamide gels. Lectin detection The procedure of Burridge was used [13]. Gels were equilibrated with lectin buffer. Binding specificity was demonstrated by incubating identical gels in the presence of either the Con A hapten, a-methyl-D-mannopyranoside, or D-galactose, which is not a
95 hapten, prior to the application of labeled lectin. Labeled Con A lectin was diluted to an activity of 2 × 107 cpm/ml with lectin buffer containing 0.5 mg/ml of hemoglobin which reduces non-specific protein adsorption by Con A. Two milliliters of the preparation was spread over the gel surface with a bent glass rod; the gels were then kept in a humidified chamber for 8 h at room temperature and were then washed with lectin buffer until wash solutions contained only background counts. The gel was dried onto chromatography paper and overlayed with X-ray film and the film was exposed for 18 h a t - 8 0 ° C .
Carbohydrate analysis The total neutral sugar content of zein extracted from Oh43 was quantitatively determined with the anthrone m e t h o d [17]. As supporting evidence for the presence of neutral sugars, samples of Oh43 were hydrolyzed for 6 h in 2 N HC1 at 100°C. Hydrolyzed samples were passed through a Dowex 50-X8 (H ÷ form) column coupled to a Dowex 1-X8 (formate form) column. Neutral sugars were eluted with distilled water. Under these conditions inorganic ions, sugars with charged groups, amino acids and peptides remain bound. Eluted samples were alternately dried and dissolved in distilled water 6 times in a rotary evaporator at 45°C to volatilize formic acid [17]. Samples were subjected to reduction with sodium borohydride followed by acetylation with acetic anhydride in pyridine. The alditol acetates were analyzed by gasliquid chromatography on an OV-225 column at 225°C [18]. Since certain sugars are differentially degraded under the hydrolytic conditions employed and since only neutral sugars were analyzed, the results are only considered qualitative evidence for the presence of neutral sugars. Results Zein proteins isolated from IHP, W22, W64 and Oh43 kernels were analyzed by SDS and IEF polyacrylamide gel electrophoresis.
The protein bands showed differential patterns of labeling with Con A. In all samples, the 19 000-dalton proteins bound relatively more Con A than the 23 000-dalton proteins. Shown in Fig. 1 are the results of the procedure applied to IHP, W22 and W64A. Con A attached to only a few of the bands seen in the IEF gels; the intensity of the lectin binding does not always reflect the intensity of the protein bands seen in the gel (l~ig. 2). In IHP zein proteins, bands at pH 6.8, 7.0 and 7.1 bound Con A, while those at pH 7.2 and 7.4 did not. In W22, all of the visible protein bands reacted with Con A. In the W64A sample, bands at pH 7.0 and 7.2 bound Con A while the others did not. In the case of Oh43, there was a higher affinity for the Con A labeled reagent than seen in the other types of zeins (Figs. 2 and 3). In the SDS gels, when 100 ttg of Oh43 protein was applied, the binding was so intense that the two large bands were not distinguishable. In those IEF which contained 100 ttg of protein, there were Con A reactive bands which were not visible with Coomassie blue stain. The IEF and SDS gels were repeated with 10 pg of Oh43 protein. When this reduced a m o u n t of protein was applied to the SDS gels, the 19 000
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Fig. 1. Comparison of Con A binding properties of zein proteins from different lines of maize. IEF gels of (A) IHP zeins, (B) W22 zeins, and (C) W64A zeins are shown. Lane 1, stained with Coomassie blue; Lane 2, fluorographed after adding radioactive Con A; Lane 3, fluorographed after adding radioactive Con A to a gel pre-treated with D-galactose; Lane 4, fluorographed after adding radioactive Con A to a gel pre-treated with the hapten, amethyl-D-mannopyranoside.
pg of neutral sugar per pg of protein. Assuming that average molecular weight of zein to be 20 000, this represents an average of 2 mol of neutral sugar per mole of zein. Further analysis of the Oh43 zein extract by gas
chromatography revealed the presence of fucose, mannose and glucose. The latter two are known hapten binding sites on glycoproteins for Con A.
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Fig. 2. Comparison of zein protein IEF gel patterns and amounts of Con A binding to zeins from Oh43, W22, W64A and IHP. Right panel, gel stained with Coomassie blue. Middle panel, fluorographed for 24 h after adding radioactive Con A. Left panel, same as middle panel-fluorographed for 12 h.
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Fig. 3. SDS and IEF gels showing Con A binding to zeins isolated from Oh43. (A) 100 #g protein loaded per lane. (B) 10 ug protein loaded per lane. Lane 1, SDS gel stained with Coomassie blue; Lane 2, SDS gel fluorographed after adding radioactive Con A; Lane 3, IEF gel stained with Coomassie blue; Lane 4, IEF gel fluorographed after adding radioactive Con A; Lane 5, IEF gel fluorographed after adding radioactive Con A to a gel pre-treated with D-galactose; Lane 6, IEF gel fluorographed after adding radioactive Con A to a gel pre-treated with the hapten, ~-methyl-D-mannopyranoside.
Discussion The glycosylation of plant proteins represents post-translational modification of gene products. Many of the major seed storage proteins of legumes are glycoproteins [ 7 ] . Zeins, as the major storage protein found in maize kernels, are important as the source of nitrogen for the germinating seed as well as providing nutritional food to humans and livestock. An understanding of their production and construction on the molecular level has been the object of study in this laboratory for many years. The zein proteins are highly hydrophobic and the amino acid composition is conserved
from one genotype to the next. The biological significance of the glycosylation of some of the zein proteins is not understood at present. Since zeins constitute the nitrogen source for the germinating seed, their glycosylation may play a role in the catabolism of the zeins and/or the structure of the protein b o d y . There are relatively few types of glycopeptide linkages [ 1 9 ] . One type, referred to as the N-glycosidic linkage, usually involves the amide group of an asparagine residue and the carbonyl group of N-acetyl glucosamine. Specific amino acid sequences or sequons [20] are correlated with the glycosylation of asparagine residues; the sequon Asn-X-Ser/Thr is most c o m m o n l y involved in glycosylation.
98 However, n o t all s e q u o n s o f this t y p e are g l y c o s y l a t e d . S o m e o f the k n o w n a m i n o acid sequences o f zein p r o t e i n s d e d u c e d f r o m their n u c l e o t i d e sequences c o n t a i n s e q u o n s w h e r e g l y c o s y l a t i o n o f the asparagine residues m i g h t occur [21]. For example the A20 cDNA w h i c h c o d e s f o r a 19 0 0 0 < l a l t o n zein c o n t a i n s such a s e q u o n . A s e c o n d t y p e o f linkage, Oglycosidic, usually involves t h e h y d r o x y l f u n c t i o n o f h y d r o x y p r o l i n e , serine or t h r e o i n e a n d a c a r b o n y l g r o u p o f a sugar. T o date, t h e r e is n o k n o w n s e q u e n c e o f a m i n o acids w h i c h directs O - g l y c o s y l a t i o n [ 1 9 ] . Serine and t h r e o n i n e residues, h o w e v e r , are f r e q u e n t l y f o u n d in zeins [ 3 , 1 9 ] . T h e results o f these e x p e r i m e n t s (Figs. 2 and 3) s h o w e d t h a t s o m e o f the s u b - g r o u p s o f zein were g l y c o p r o t e i n s and t h a t t h e p a t t e r n o f g l y c o s y l a t i o n was specific f o r each inbred line. U n d e r s t a n d i n g o f t h e zein p r o t e i n s m u s t include t h e c o m p l e x i t y i n t r o d u c e d b y sites o f g l y c o s y l a t i o n . A p r o t e i n w h i c h o n c e appeared simple in SDS e l e c t r o p h o r e s i s has b e e n d e m o n s t r a t e d t o have m a n y levels o f m o d i f i cation exerting control on the structures.
References 1 J.S. Wall, Proteins and their reactions, in: H.W. Schultz and A.F. Anglemier (Eds.), Symposium on Food, AVI, West Port, 1964, p. 315. 2 E. Gianazza, V. Viglienghi, P.G. Righetti, F. Salam ini and C. Soave, Phytochemistry, 16 ( 1977 ) 315. 3 E. Gianazza, P.G. Righetti, F. Pioli, E. Galante and C. Soave, Maydica, 21 (1976) 1. 4 P.G. Righetti, E. Gianazza, A. Viotti and C. Soave, Planta, 136 (1977) 115. 5 G. Hagen and I. Rubenstein, Plant Sci. Lett., 19 (1980) 217.
6 F.A. Burr and B. Burr, Zein synthesis in maize endosperms, in: I. Rubenstein, R.L. Phillips, C.E. Green and B.G. Gengenbach (Eds.), The Plant Seed: Development, Preservation, and Germination, Academic Press, New York, 1979, p. 27. 7 S.M.M. Basha and R.M. Roberts, Plant Physiol., 67 (1981) 936. 8 N. Sharon and H. Lis, Science, 177 (1972) 848. 9 H. Lis and N. Sharon, Annu. Rev. Biochem., 42 (1973) 541. 10 I.J. Goldstein and C.E. Hayes, Adv. Carbohydr, Chem. Biochem., 35 (1978) 128. 11 D.E. Culley, B.G. Gengenbach, J.A. Smith, I. Rubenstein, J.A. Connelly and W.D. Park, Plant Physiol., 174 (1984) 389. 12 O.H. Lowry, N.J. Rosebrough, A.L. Farr and R.J. Randall, J. Biol. Chem., 19 (1951) 193. 13 K. Burridge, Direct identification of specific glycoproteins and antigens in sodium dodecyl sulfate gels, in: V. Ginsburg (Ed.), Methods in Enzymology, Voh 50, Academic Press, New York, 1978, p. 54. 14 P.G. Righetti and A. Righetti, High voltage analytical and preparative isoelectric focusing, J.P. Arbuthnott and J.A. Breley (Eds.), Butterworths, London, 1975, p. 114. 15 U.K. Laemmli, Nature, 227 (1970) 680. 16 R.A. Laskey and A.D. Mills, Eur. J. Biochem., 56 (1975) 335. 17 R.G. Spiro, Analysis of sugars found in glycoproteins, in: E.F. Neufeld and V. Ginsburg (Eds.), Methods in Enzymology, Vol. 8, Academic Press, New York, 1966, p. 3. 18 B. Lindberg, Methylation analysis of polysaccharides, in: V. Ginsburg (Ed.), Methods in Enzymology, Vol. 28, Academic Press, New York, 1972, p. 178. 19 D.T.A. Lamport, Structure and function of plant glycoproteins, in : J. Preiss (Ed.), The Biochemistry of Plants, a Comprehensive Treatise, Vol. 3, Carbohydrates: Structure and Function, Academic Press, 1981, p. 501. 20 R.D. Marshall, Biochem. Soc. Symp., 40 (1974) 17. 21 D.E. Geraghty, J. Messing and I. Rubenstein, EMBO J, 11 (1982) 1329.
93
Elsevier Scientific Publishers Ireland Ltd.
A STUDY OF THE SITES OF GLYCOSYLATION OF ZEIN PROTEINS USING THE LECTIN CONCANAVALIN A
J.A. SMITHa, W.L. ROTTMANN b'* and I. RUBENSTEIN b aDepartment of Horticultural Sciences and Landscape Architecture and bDepartment of Genetics and Cell Biology, University of Minnesota, St. Paul, MN 55108 (U.S.A.)
(Received November 6th, 1984) (Revision received January 2nd, 1985) (Accepted January 4th, 1985) The sites of glycosylation of zeins, the maize (Zea mays L.) storage proteins, were studied using the affinity of the lectin Concanavalin A (Con A) for certain glycosides. Zeins which were extracted from kernels of Illinois High Protein (IHP), W22, W64A and Oh43 were separated by isoelectric focusing and analyzed with a radiolabeled Con A binding technique. Certain sub-groups of the zein proteins contained carbohydrate moieties which bound Con A while others did not. Zeins extracted from Oh43 kernels had a higher relative affinity for Con A than those of other maize lines. Further analyses of the zeins of Oh43 by gas chromatography demonstrated the presence of fucose, mannose and glucose. Key words: Zea mays; zeins; glycosylation; Concanavalin A
Introduction T h e zein p r o t e i n f a m i l y r e p r e s e n t s a large g r o u p o f p r o t e i n s w i t h similar p h y s i c a l a n d c h e m i c a l p r o p e r t i e s [ 1 ] . Zeins are soluble in 70% e t h a n o l and 60% o f t h e i r a m i n o acid residues consist o f leucine, g l u t a m i n e , alanine a n d p r o l i n e [ 2 ] . T h e y are low in t w o o f t h e essential a m i n o acids: lysine a n d t r y p t o p h a n . W h e n s e p a r a t e d b y s o d i u m d o d e c y l sulfate (SDS) gel e l e c t r o p h o r e s i s t h e s e p r o t e i n s exhibit two major bands with apparent m o l e c u l a r weights o f a b o u t 19 0 0 0 a n d 23 000 a n d t w o m i n o r b a n d s w h i c h have a p p a r e n t m o l e c u l a r weights o f 10 0 0 0 a n d 15 0 0 0 [ 3 ] . T h e t r u e h e t e r o g e n e i t y o f zein can be m o r e clearly d e m o n s t r a t e d w i t h isoelectric focusing ( I E F ) gels [4] a n d 2 - d i m e n sional gel analysis w h e r e m o r e t h a n 20 diff e r e n t p r o t e i n s can be identified [ 5 ] . With *Present address: 3M Center, St. Paul, MN 55144, U.S.A. Abbreviations: Con A, Concanavalin A; IEF, isoelectric focusing; IHP, Illinois High Protein; SDS, sodium dodecyl sulfate.
these techniques, a complex protein pattern can be seen w h i c h is d i f f e r e n t f o r e a c h inbred line [ 4 ] . Zeins, like m a n y o t h e r m a j o r seed-storage p r o t e i n s , are n o w k n o w n to be g l y c o p r o t e i n s . Early a t t e m p t s to s h o w t h e p r e s e n c e o f glycosides w i t h t h e periodic acid~Schiff r e a c t i o n p r o d u c e d negative results [ 4 ] . In a later r e p o r t , t h e h y b r i d W F 9 X B 3 7 has b e e n s h o w n to c o n t a i n 1 m o l o f glucose p e r m o l e o f zein [ 6 ] . H o w e v e r , it is n o t k n o w n w h a t o t h e r k i n d s o f sugars m a y be p r e s e n t in zein or w h e t h e r d i f f e r e n t i a l degrees o f g l y c o s y l a t i o n m a y a c c o u n t f o r s o m e o f t h e h e t e r o g e n e i t y in this f a m i l y . L e c t i n s are p r o t e i n s w h i c h b i n d specifically to saccharides. T h e r e f o r e , we have used C o n A, which has a specific b i n d i n g a f f i n i t y for a-D-mannopyranosides; a-D-glycopyranosides, D - f r u c t o f u r a n o s i d e s , a n d rnyo-inositol [ 7 - - 1 0 ] , as a p r o b e to i d e n t i f y sites o f glyc o s y l a t i o n o n t h e zeins. T h e p u r p o s e o f t h e s e e x p e r i m e n t s was t o initiate a s t u d y o f t h e p a t t e r n o f g l y c o s y l a t i o n o f zein p r o t e i n s . We c o n c l u d e t h a t s o m e zein
0168-9452/85/$03.30 © Elsevier Scientific Publishers Ireland Ltd. Printed and Published in Ireland
94 proteins are able to specifically bind Con A. Further, the patterns for the binding seen in IEF gels are distinctive for each type of zein suggesting a genetic control of post-translational modification. Materials and methods
Meal preparation Kernels of Z. mays L. (IHP of the 1971 selection) were obtained from R.L. Phillips (University of Minnesota); W22 and W64A were obtained from C.Y. Tsai (Purdue University, W. Lafayette, IN); and Oh43 was obtained from D.V. Glover (Purdue University, W. Lafayette, IN). These lines were grown at the St. Paul Campus of the University of Minnesota during the summer of 1978 and were self-pollinated. De-embryonated mature kernels were frozen in liquid nitrogen and stored at - 8 0 ° C . To prepare meal, the frozen kernels were lyophilized and placed in a Torit capsule with an abrasive bar and vibrated for 1 min on the Torit shaker [11]. One hundred milligrams of the resulting meal were used for protein extraction. Zein isolation Several studies have confirmed that the unique solubility of zein in 70% ethanol allows the direct extraction of these proteins from kernels (2--4). Dry meal samples were first defatted by mixing with chilled acetone for 30 min at 4°C, centrifuging at 2500 × g for 10 min at 0--4°C, followed by removal of the acetone. The meal was then dried and extracted twice with 5 ml of chilled 0.5 M NaC1 solution to remove albumins and globulins. The meal was then washed two times with distilled water to reduce the salt concentration. The zein fraction was extracted from the resulting pellet by mixing with 5 ml of 70% (v/v) ethanol containing 1% (v/v)~-mercaptoethanol. This extraction was repeated twice and the ethanol extracts were lyophilized in convenient aliquots and stored a t - 8 0 ° C . Protein determinations were done on replicate samples using the Lowry m e t h o d [12].
Isolation o f lectins The iodination was done according to methods described by Burridge [13]. Two milligrams of Con A (Calbiochem) were dissolved in a reaction mixture consisting of 100 ~l of lectin buffer (containing 0.15 M NaC1, 50 mM Tris--HC1, 0.5 mM CaC12, 0.5 mM MnC12, and 0.05% (w/v) Na azide adjusted to pH 7.4) and 0.2 mCi of carrier free Na1125 (New England Nuclear). The Con A reactive site was protected with a-methyl-D-mannopyranoside. The reaction was initiated with 5 ~l of a chloramine T solution (5 mg/ml) and allowed to proceed for 45 s at 0--4°C. The reaction was stopped by the addition of 5 pl of sodium metabisulfite (20 mg/ml). The resulting mixture was passed through a column of Biogel-P10 to remove free I ~2s from the iodinated lectin. This procedure resulted in a labeled Con A protein with a specific activity of 5.6 × 107 cpm/mg or greater. Electrophoresis IEF was carried out according to the technique of Righetti and co-workers with LKB 2117 Multipore equipment [14]. The polymerized gel was cooled for 1 h on the gel platform at 8--10°C. Dried protein samples were reconstituted in a solution of 6 M urea, 10 mM Tris/glycine (pH 8.5}, and 2% .6-mercaptoethanol to a concentration of 100 pg/25 pl and were applied to the surface of the gel. The pH gradients were measured by recording the pH of 1 ml vols. of distilled water containing 5-mm segments of gel and also by pI marker proteins (Calbiochem; pI marker protein kit). Sodium dodecyl sulfate electrophoresis was carried out according to the technique of Laemmli [15] using 10% (w/v) polyacrylamide gels. Lectin detection The procedure of Burridge was used [13]. Gels were equilibrated with lectin buffer. Binding specificity was demonstrated by incubating identical gels in the presence of either the Con A hapten, a-methyl-D-mannopyranoside, or D-galactose, which is not a
95 hapten, prior to the application of labeled lectin. Labeled Con A lectin was diluted to an activity of 2 × 107 cpm/ml with lectin buffer containing 0.5 mg/ml of hemoglobin which reduces non-specific protein adsorption by Con A. Two milliliters of the preparation was spread over the gel surface with a bent glass rod; the gels were then kept in a humidified chamber for 8 h at room temperature and were then washed with lectin buffer until wash solutions contained only background counts. The gel was dried onto chromatography paper and overlayed with X-ray film and the film was exposed for 18 h a t - 8 0 ° C .
Carbohydrate analysis The total neutral sugar content of zein extracted from Oh43 was quantitatively determined with the anthrone m e t h o d [17]. As supporting evidence for the presence of neutral sugars, samples of Oh43 were hydrolyzed for 6 h in 2 N HC1 at 100°C. Hydrolyzed samples were passed through a Dowex 50-X8 (H ÷ form) column coupled to a Dowex 1-X8 (formate form) column. Neutral sugars were eluted with distilled water. Under these conditions inorganic ions, sugars with charged groups, amino acids and peptides remain bound. Eluted samples were alternately dried and dissolved in distilled water 6 times in a rotary evaporator at 45°C to volatilize formic acid [17]. Samples were subjected to reduction with sodium borohydride followed by acetylation with acetic anhydride in pyridine. The alditol acetates were analyzed by gasliquid chromatography on an OV-225 column at 225°C [18]. Since certain sugars are differentially degraded under the hydrolytic conditions employed and since only neutral sugars were analyzed, the results are only considered qualitative evidence for the presence of neutral sugars. Results Zein proteins isolated from IHP, W22, W64 and Oh43 kernels were analyzed by SDS and IEF polyacrylamide gel electrophoresis.
The protein bands showed differential patterns of labeling with Con A. In all samples, the 19 000-dalton proteins bound relatively more Con A than the 23 000-dalton proteins. Shown in Fig. 1 are the results of the procedure applied to IHP, W22 and W64A. Con A attached to only a few of the bands seen in the IEF gels; the intensity of the lectin binding does not always reflect the intensity of the protein bands seen in the gel (l~ig. 2). In IHP zein proteins, bands at pH 6.8, 7.0 and 7.1 bound Con A, while those at pH 7.2 and 7.4 did not. In W22, all of the visible protein bands reacted with Con A. In the W64A sample, bands at pH 7.0 and 7.2 bound Con A while the others did not. In the case of Oh43, there was a higher affinity for the Con A labeled reagent than seen in the other types of zeins (Figs. 2 and 3). In the SDS gels, when 100 ttg of Oh43 protein was applied, the binding was so intense that the two large bands were not distinguishable. In those IEF which contained 100 ttg of protein, there were Con A reactive bands which were not visible with Coomassie blue stain. The IEF and SDS gels were repeated with 10 pg of Oh43 protein. When this reduced a m o u n t of protein was applied to the SDS gels, the 19 000
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Fig. 1. Comparison of Con A binding properties of zein proteins from different lines of maize. IEF gels of (A) IHP zeins, (B) W22 zeins, and (C) W64A zeins are shown. Lane 1, stained with Coomassie blue; Lane 2, fluorographed after adding radioactive Con A; Lane 3, fluorographed after adding radioactive Con A to a gel pre-treated with D-galactose; Lane 4, fluorographed after adding radioactive Con A to a gel pre-treated with the hapten, amethyl-D-mannopyranoside.
pg of neutral sugar per pg of protein. Assuming that average molecular weight of zein to be 20 000, this represents an average of 2 mol of neutral sugar per mole of zein. Further analysis of the Oh43 zein extract by gas
chromatography revealed the presence of fucose, mannose and glucose. The latter two are known hapten binding sites on glycoproteins for Con A.
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Fig. 2. Comparison of zein protein IEF gel patterns and amounts of Con A binding to zeins from Oh43, W22, W64A and IHP. Right panel, gel stained with Coomassie blue. Middle panel, fluorographed for 24 h after adding radioactive Con A. Left panel, same as middle panel-fluorographed for 12 h.
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Discussion The glycosylation of plant proteins represents post-translational modification of gene products. Many of the major seed storage proteins of legumes are glycoproteins [ 7 ] . Zeins, as the major storage protein found in maize kernels, are important as the source of nitrogen for the germinating seed as well as providing nutritional food to humans and livestock. An understanding of their production and construction on the molecular level has been the object of study in this laboratory for many years. The zein proteins are highly hydrophobic and the amino acid composition is conserved
from one genotype to the next. The biological significance of the glycosylation of some of the zein proteins is not understood at present. Since zeins constitute the nitrogen source for the germinating seed, their glycosylation may play a role in the catabolism of the zeins and/or the structure of the protein b o d y . There are relatively few types of glycopeptide linkages [ 1 9 ] . One type, referred to as the N-glycosidic linkage, usually involves the amide group of an asparagine residue and the carbonyl group of N-acetyl glucosamine. Specific amino acid sequences or sequons [20] are correlated with the glycosylation of asparagine residues; the sequon Asn-X-Ser/Thr is most c o m m o n l y involved in glycosylation.
98 However, n o t all s e q u o n s o f this t y p e are g l y c o s y l a t e d . S o m e o f the k n o w n a m i n o acid sequences o f zein p r o t e i n s d e d u c e d f r o m their n u c l e o t i d e sequences c o n t a i n s e q u o n s w h e r e g l y c o s y l a t i o n o f the asparagine residues m i g h t occur [21]. For example the A20 cDNA w h i c h c o d e s f o r a 19 0 0 0 < l a l t o n zein c o n t a i n s such a s e q u o n . A s e c o n d t y p e o f linkage, Oglycosidic, usually involves t h e h y d r o x y l f u n c t i o n o f h y d r o x y p r o l i n e , serine or t h r e o i n e a n d a c a r b o n y l g r o u p o f a sugar. T o date, t h e r e is n o k n o w n s e q u e n c e o f a m i n o acids w h i c h directs O - g l y c o s y l a t i o n [ 1 9 ] . Serine and t h r e o n i n e residues, h o w e v e r , are f r e q u e n t l y f o u n d in zeins [ 3 , 1 9 ] . T h e results o f these e x p e r i m e n t s (Figs. 2 and 3) s h o w e d t h a t s o m e o f the s u b - g r o u p s o f zein were g l y c o p r o t e i n s and t h a t t h e p a t t e r n o f g l y c o s y l a t i o n was specific f o r each inbred line. U n d e r s t a n d i n g o f t h e zein p r o t e i n s m u s t include t h e c o m p l e x i t y i n t r o d u c e d b y sites o f g l y c o s y l a t i o n . A p r o t e i n w h i c h o n c e appeared simple in SDS e l e c t r o p h o r e s i s has b e e n d e m o n s t r a t e d t o have m a n y levels o f m o d i f i cation exerting control on the structures.
References 1 J.S. Wall, Proteins and their reactions, in: H.W. Schultz and A.F. Anglemier (Eds.), Symposium on Food, AVI, West Port, 1964, p. 315. 2 E. Gianazza, V. Viglienghi, P.G. Righetti, F. Salam ini and C. Soave, Phytochemistry, 16 ( 1977 ) 315. 3 E. Gianazza, P.G. Righetti, F. Pioli, E. Galante and C. Soave, Maydica, 21 (1976) 1. 4 P.G. Righetti, E. Gianazza, A. Viotti and C. Soave, Planta, 136 (1977) 115. 5 G. Hagen and I. Rubenstein, Plant Sci. Lett., 19 (1980) 217.
6 F.A. Burr and B. Burr, Zein synthesis in maize endosperms, in: I. Rubenstein, R.L. Phillips, C.E. Green and B.G. Gengenbach (Eds.), The Plant Seed: Development, Preservation, and Germination, Academic Press, New York, 1979, p. 27. 7 S.M.M. Basha and R.M. Roberts, Plant Physiol., 67 (1981) 936. 8 N. Sharon and H. Lis, Science, 177 (1972) 848. 9 H. Lis and N. Sharon, Annu. Rev. Biochem., 42 (1973) 541. 10 I.J. Goldstein and C.E. Hayes, Adv. Carbohydr, Chem. Biochem., 35 (1978) 128. 11 D.E. Culley, B.G. Gengenbach, J.A. Smith, I. Rubenstein, J.A. Connelly and W.D. Park, Plant Physiol., 174 (1984) 389. 12 O.H. Lowry, N.J. Rosebrough, A.L. Farr and R.J. Randall, J. Biol. Chem., 19 (1951) 193. 13 K. Burridge, Direct identification of specific glycoproteins and antigens in sodium dodecyl sulfate gels, in: V. Ginsburg (Ed.), Methods in Enzymology, Voh 50, Academic Press, New York, 1978, p. 54. 14 P.G. Righetti and A. Righetti, High voltage analytical and preparative isoelectric focusing, J.P. Arbuthnott and J.A. Breley (Eds.), Butterworths, London, 1975, p. 114. 15 U.K. Laemmli, Nature, 227 (1970) 680. 16 R.A. Laskey and A.D. Mills, Eur. J. Biochem., 56 (1975) 335. 17 R.G. Spiro, Analysis of sugars found in glycoproteins, in: E.F. Neufeld and V. Ginsburg (Eds.), Methods in Enzymology, Vol. 8, Academic Press, New York, 1966, p. 3. 18 B. Lindberg, Methylation analysis of polysaccharides, in: V. Ginsburg (Ed.), Methods in Enzymology, Vol. 28, Academic Press, New York, 1972, p. 178. 19 D.T.A. Lamport, Structure and function of plant glycoproteins, in : J. Preiss (Ed.), The Biochemistry of Plants, a Comprehensive Treatise, Vol. 3, Carbohydrates: Structure and Function, Academic Press, 1981, p. 501. 20 R.D. Marshall, Biochem. Soc. Symp., 40 (1974) 17. 21 D.E. Geraghty, J. Messing and I. Rubenstein, EMBO J, 11 (1982) 1329.