Hcoppe-Seyler's Z. Physiol. Chem. Bed. 356, S. 1685 - 1692, November 1975
Purification and Characterization of a Hemagglutinin from Arachis hypogaea Tcadao Terao, Tatsuro Irimura and Toshiaki Osawa
(Received 14 July 1975)
Summary: A. hypogaea hemagglutinin was purified by ammonium sulfate fractionation and Sepharose 6 B column chromatography. The homogeneity of the purified hemagglutinin was ascertained by ultracentrifugal analysis and polyacrylamide gel electrophoresis. It has a molecular weight of 106.500 and is a tetramer of a subunit with a molecular weight of 27.000. The purified hemagglutinin agglutinated neuraminidase-treated human erythrocytes regardless
of their ABO group type, but did not agglutinate intact erythrocytes. In hapten inhibition assays with simple sugars, the so-called Mäkelä's group 2 sugars, which bear the same configuration of hydroxy groups at C-3 and C-4 as D-galactopyranose, were inhibitors for this hemagglutinin. It does not contain any carbohydrate, in contrast to most phytohemagglutinins except concanavalin A and wheat germ agglutinin.
Reinigung und Charakterisierung eines Hämagglutinins aus Arachis hypogaea Zusammenfassung: A. -hypogaea-Yiamag$.utmm wurde mittels Ammoniumsulfat-Fraktionierung und Sepharose-6 B-Chromatographie gereinigt. Das gereinigte Hämagglutinin erwies sich als einheitlich in der analytischen Ultrazentrifugation und der Polyacrylamid-Gelelektrophorese. Es hat ein Mol.-Gew. von 106500 und liegt als Tetramer vor (Mol.-Gew. der Untereinheiten 27 000). Das gereinigte Hämagglutinin agglutiniert Neuraminidase-behandelte menschliche Erythrozyten
unabhängig von ihrer Blutgruppe im ABO-System, intakte Erythrozyten werden jedoch nicht agglutiniert. Im Hapten-Hemmtest mit einfachen Zukkern erwiesen sich die Zucker der sogenannten Mäkelä-Gruppe 2, die an C-3 und C-4 dieselbe Hydroxygruppenkonfiguration aufweisen wie o-Galaktopyranose, als Inhibitoren für das Hämagglutinin. Im Gegensatz zu den meisten Phytohämagglutininen (mit Ausnahme von Concanavalin A und Weizenkeim-Agglutinin) enthält dieses Hämagglutinin keine Kohlenhydrate.
Address: Prof. T. Osawa, Division of Chemical Toxicology and Immunochemistry, Faculty of Pharmaceutical Sciences, University of Tokyo, Bunkyo-ku, Tokyo, Japan Enzymes: Neuraminidase, acylneuraminyl hydrolase (EC 3.2.1.18); Trypsin, (EC 3.4.2.4).
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1686
T. Terao, T. Irimura and T. Osawa
Many plants contain agglutinins which are capable of agglutinating erythrocytes and other cells through their surface oligosaccharide determinantsl 1 · 2 !. These hemagglutinins are most frequently found in the seeds of leguminous plants and differ from one another in their specificities!31. The presence of a hemagglutinin inArachis hypogaea seeds (peanuts) was first described by Boyd et aU 4 l, and some properties of the crude hemagglutinin were also reported by Bird^l and Uhlenbruck et alJ 6 '. Inhibition studies on A hypogaea hemagglutinin with various simple sugars and glycopeptides have already been carried out byDahretal.l 7 Undbyusl 8 l. In this paper, we report the purification and properties of A. hypogaea hemagglutinin. Materials and Methods Enzymes: Trypsin (twice recrystallized) was purchased from Worthington. Vibrio cholerae and Clostridium perfringens neuraminidases were obtained from Sigma and Boehringer Mannheim GmbH. Glycoproteins and glycopeptides: The major glycoprotein of human erythrocyte membrane (MN glycoprotein or PAS-I) was obtained from human erythrocyte stroma of blood group AB according to the method of Fukuda and Osawa^9'. Desialization of the glycoprotein was carried out by an acid hydrolysis in 0.05N H 2 SO 4 at 80 °C for l h at a glycoprotein concentration of 6 mg per ml. About 95% of the sialic acid was removed by this procedure. The hydrolysate was neutralized with solid NaHCOs, dialized against distilled water and lyophilized. The alkali treatment of the desialized MN-glycoprotein was performed with O.lN NaOH/0.lMNaBH 4 for 36 h at room temperature in a sealed tube, in the dark, under nitrogen at a concentration of 10 mg per ml. The excess borohydride was destroyed by the addition of acetic acid to pH 4.0, and a portion of the mixture was put on a Sephadex G-50 column equilibrated with lOmM Tris/ HC1, pH 7.2. The column was eluted with the same buffer, and the void volume fractions of the column eluate, containing carbohydrates linked to the protein moiety by alkali-resistant linkage, were pooled, dialyzed against distilled water and lyophilized. One of the chymotrypsin fragments of the MN-glycoprotein, Ch-3, was a gift from Dr. M. Tomita, Showa University, Tokyo. This glycopeptide has been shown'101 to contain only O-glycosidically linked sugar chains, the structure of which was suggested by Thomas and Winz-
Bd. 356(1975)
Other reagents: All other reagents were commercial preparations of the highest purity available. Acetone powder of A. hypogaea seeds: All the following procedures were carried out at room temperature. A. hypogaea seeds (100g) obtained from a local market were suspended in 300 ml of water and homogenized in a Waring Blender. Ice-cold acetone (1500 ml) was added to the homogenate with stirring. After 30 min stirring, the precipitates were collected on a B chner funnel by filtration. The filtrate was discarded. The filter cake was resuspended in acetone (1000 ml), stirred for 30 min and filtered as above. This step was then repeated four times to completely remove oily substances. The precipitates were finally air-dried and kept at - 20 °C. The yield of air-dried powder was about 30 g. Purification of A. hypogaea hemagglutinin: In a typical run, 100 g of the acetone powder was suspended in 1 / of 0.15M NaCl/15mM sodium phosphate buffer, pH 7.2, and allowed to stand overnight at 4 °C with stirring. The clear supernatant obtained by centrifugation at 20000 χ g for 30 min was subjected to (NH 4 ) 2 S0 4 fractionation. As shown in Table 1, the precipitate between 40 and 75% saturation of (NH 4 ) 2 SO 4 had most of the hemagglutinating activity. This fraction was dissolved in O.lM Tris/HCl buffer, pH 7.5, and the solution was dialyzed against distilled water. A precipitate which formed during dialysis was removed by centrifugation (12000 χ g, 30 min) and the supernatant was lyophilized. Since the hemagglutinin was known to be inhibited by D-galactosel6!, further purification of the active fraction was achieved by Sepharose 6B column chromatography as described in the legend to Fig. 1. The second protein peak eluted after the bulk of inactive proteins contained the activity against the neuraminidase-treated human erythrocytes. Active protein fractions were combined, dialyzed against distilled water and lyophilized. Table 1 summarizes data pertaining to the purification of hemagglutinin. The minimum hemagglutinating dose of the purified product obtained was 1 Mg/m/ with the desialized human erythrocytes. Molecular weight determination: The molecular weight of the purified hemagglutinin was measured by sedimentation equilibrium with a Hitachi model UCA-1A ultracentrifuge according to the method of Yphantisl 12L The concentrations of the samples tested were 0.25, 0.50, 0.75 and 1.00% in lOmM sodium phosphate buffered saline (pH 7.5). The sedimentation velocity was determined by the band sedimentation method at a speed of 56000 rpm.( 13 L Polyacrylamide gel electrophoresis: Disc electrophoresis was carried out in 7.5% polyacrylamide gel at pH 4.3 according to the method of Reisfeld et alJ 141. The sodium dodecylsulfate gel electrophoresis was performed
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Amino acid analysis: The hemagglutinin was hydrolyzed with 6N HC1 at 105 °C for 24, 48 and 72 h. The amino acid contents of the hydrolysates were determined on a JEOL-5A amino acid analyzer according to the method of S packman etalJ16l Tryptophan was measured in an unhydrolyzed protein sample by the spectrophotometric method of Goodwin and Morton!171. Half cystine and methionine were analyzed after performic acid oxidation by the method of H W 18l
~50/77*
W
1687
Peanut Hemagglutinin
Bd. 356(1975)
20
30 iO Fract. no.
50
70
80
Fig. 1. Sepharose 6B column chromatography of crude hemagglutinin. 150 mg of the (NH 4 ) 2 SO4 fraction (40 - 75 % saturation) was dissolved in 3 ml of lOmM Tris/HCl, pH 7.2, and then applied to a column (2 χ 45 cm) which was equilibrated with the same buffer. The column was run with the same buffer. After the second protein peak (peak B) had eluted, the column was further washed with a buffer containing 0.5 Μ D-galactose. Fractions of 7 m/ were collected and the fractions corresponding to peak Β were pooled.
according to Weber and Osbornl15'. 25 Mg of protein was placed onto a 7.5% gel in 0.1M sodium phosphate buffer, pH 7.1, containing 0.1% sodium dodecylsulfate and 1 % 2-mercaptoethanol. Gels were stained with Coomassie Brilliant Blue. Isoelectric focusing: Isoelectric focusing was performed using an LKB 8 100-1 column. After the run, fractions (each 2.5 ml) were analyzed foM 2 80 and PH· The PH gradient was linear over the range pH 4 to 8.
Table 1. Purification of Arachis hypogaea hemagglutinin.
Carbohydrate determination: Total neutral sugar was determined by the phenol/H2S04 method of Dubois et alJ 191 with D-mannose as the standard. Amino sugar was determined according to Blix' ' and by means of a JEOL-5A amino acid analyzer. Hydrolysis for this assay was carried out with 4N HC1 for 8 h at 100 °C in a sealed tube. N-Terminal amino acid: The W-terminal amino acid was dansylated according to Kinoshita et alJ211, and the dansyl derivative was identified by thin-layer chromatography on a polyamide sheet. Solvent systems used were a) benzene/acetic acid, 9:1; b) 2% aqueouspyridine/methanol, 2:1; c) hexane/ethanol/acetic acid, 4:2:1. Treatment of the A. hypogaea hemagglutinin with iodo[ 2- !4C]acetic acid: 3 mg of hemagglutinin in 0.3 ml of lOmM sodium phosphate buffered saline was treated with a 100-fold excess of iodo[ 2-14C]acetic acid (5.1 mCi/mmol) at 25 °C for 3 h. The reaction mixture was put on a Sephadex G-25 column (superfine, 0.5 χ 20 cm) which was preequilibrated with lOmM sodium phosphate buffered saline (pH 7.0). The fractions containing the protein were collected and a portion of the combined fraction was used to measure the radioactivity with a Beckman model LS-230 liquid scintillation spectrophotometer. Hemagglutination assays: The titration and inhibition assays using desialized human erythrocytes were carried out according to the method previously described^ 22 ].
Fraction
Crude extract (NH4)2S04 fract. 0 - 40% 40-75% 75 - 100% Sepharose 6B column Fract. A Fract. B
Yield from 100 g seeds
Total hemagglutinin titer
[mg]
(titer χ volume [m/])
1280
92400
120 550 320
6400 76800 12800
430 85
542 87120
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1688
T. Terao, T. Irimura and T. Osawa
Lymphocyte culture for mitogenic assay: Preparation and cultivation of human peripheral lymphocytes were performed according to the method of Toyoshima et al.l23'. Incorporation of [ 3 H]thymidine into lymphocyte DNA was measured as described previously 1241. Treatment of human erythrocytes with various enzymes: Treatment of human erythrocytes with neuraminidase and trypsin were performed under the conditions previously described!251
Results Poly aery lamide gel electrophoresis The electrophoretic homogeneity of the purified hemagglutinin was confirmed by disc electrophoresis on a polyacrylamide gel. A single band was obtained at pH 4.3 (Fig. 2 a). The sodium dodecylsulfate gel electrophoresis also indicated the homogeneity of the hemagglutinin (Fig. 2 b). Molecular weight determination: The molecular weight of 106500 was calculated for the purified hemagglutinin from the sedimentation equilibrium centrifugationt 12 l Furthermore, when the purified protein was subjected to polyacrylamide gel electrophoresis in the presence of sodium dodecylsulfate, it gave a single band (Fig. 2 b) corresponding to a molecular weight of 27000 (Fig. 3). This indicates that the intact hemagglutinin consists of four subunits of molecular weight 27000.
Bd. 356(19775)
yr-flefc
\
v
\Alb.
A.hypogatu hemigg utinin
;
\
\£Ohym.A
Λ\
4
d[cmj -+
6
Fig. 3. Determination of the molecular weight of A hypogaea hemagglutinin by polyacrylamide gel electrophoresis in the presence of sodium dodecylsulfate. Running conditions were the same as in Fig. 2 b). Standard proteins used were human 7-globulin (7-Glob., MT = 160000), bovine serum albumin (Alb., MT = 67000), ovalbumin (Ον., Μτ = 45 000) and bovine chymotrypsinogen A (Chym. A, Mt = 25 000).
Chemical composition: The amino acid composition of the purified hemagglutinin is presented in Table 2. Its characteristic feature is the high proportion of acidic amino acids and the rather low proportion of basic amino acids. This is consistent with the pi value (pi 5.95) of the protein. TV-Terminal amino acid analysis was carried out by the dansyl method, and only alanine was found to be dansylated. Free sulfhydryl group determination with iodo[2-14C]acetic acid revealed that essentially no free SH group was present.
Fig. 2. Polyacrylamide gel electrophoresis of purified hemagglutinin. a) 7.5% gel in 0.35M /3-alanine/acetate, pH 4.3. b) 7.5% gel in 0.1 M sodium phosphate, pH 7.1, in the presence of sodium dodecylsulfate.
When 2.5 mg of the hemagglutinin was subjected to the phenol/H2S04 test for neutral sugars, and the Elson-Morgan reaction for amino sugars, no color developed in either reaction, suggesting the absence of carbohydrate. In both reactions, concanavalin A, which is known not to contain any sugar!261, was used for a blank. The absence of carbohydrate was further confirmed by the application of an acid hydrolysate of the hemagglutinin to an automatic sugar analyzer (JEOL-5A)
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Bd. 356 (1975)
for neutral sugars and an amino acid analyzer (JEOL-5A) for amino sugars. Effects of enzyme treatments of erythrocytes on the hemagglutinin titer Table 3 shows the effects of various enzyme treatments of human erythrocytes on the titer of the purified hemagglutinin. Although it does not affect intact human erythrocytes, it strongly agglutinates neuraminidase-treated cells, regardless of their blood type. The minimum hemagglutinating dose of the purified preparation was about 1 g per ml. Trypsin treatment of the erythrocytes did not make them susceptible to the hemagglutinin. Neuraminidase treatment of the trypsinized erythrocytes, however, resulted in a greater increase in their agglutinability than treatment with neuraminidase alone. On the other hand, trypsin treatment of the neuraminidase-treated erythrocytes rather decreased their agglutinability. This might be caused by the removal of some glycoprotein receptors from the cell surface during the tryptic treatment.
Table 3. Effect of enzymes on agglutinability of human erythrocytes with purified hemagglutinin. Experiments were performed as previously described'25!.
Amino Acid
Cys/2 Val Met He Leu Tyr Phe Lys His Arg Trp Carbohydrate Free SH Pi • y 20,w
1
Not detected.
Titer
Enzyme
n* n* 1/128 1/32
Untreated Trypsin V. cholerae neuraminidase Trypsin after V. cholerae neuraminidase V. cholerae neuraminidase after trypsin
1/256
No agglutination.
Table 4. Inhibition assay of purified hemagglutinin with various sugars, glycopeptides and glycoproteins Sugars, glycopeptides and MN-glycoprotein D-Galactose Lactose Melibiose Raffinose D-Galactosamine L-Arabinose jV-Acetyl-D-galactosamine D-Glucose D-Glucosamine W-Acetyl-D-glucosamine L-Fucose D-Mannose Maltose p-Nitrophenyl-a-Dgalactopyranoside p-Nitrophenyl-/3-Dgalactopyranoside Ch-3 Asialo-Ch-3 MN-glycoprotein Asialo MN-glycoprotein Alkaline borohydridetreated Asialo MN-glycoprotein
Table 2. Chemical composition and some physicochemical properties of A. hypogaea hemagglutinin.
Asp Thr Ser Glu Pro Gly Ala
1689
Peanut Hemagglutinin
Mol/mol hemagglutinin
144.3 72.4 74.0 34.8 16.6 65.8 43.7 16.6 97.8 _a
51.8 38.0 17.8 52.1 33.0 8.4 16.5 2.7 _a _a
5.95 6.05 a
Minimum concentration [mMJ completely inhibiting 4 times the hemagglutination dose
25 12.5 50 50 6 50 > 100 > 100 > 100 > 100
>100 > 100 > 100
12 5 > 1.3a 0.01a >10 a 0.0053
2.5a 2.5a
Expressed as mg/m/.
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1690
T. Terao, T. Irimura and T. Osawa
Assays of hemagglutination inhibition by simple sugars, glycopeptides and glycoproteins The results of the tests of the purified hemagglutinin with various simple sugars as haptene inhibitors are listed in Table 4. In general, the so-called Mäkelä's group 2 sugars'3' are potent inhibitors. D-Galactosamine is a particularly strong inhibitor of this hemagglutinin. As for the anomers of p-nitrophenyl-D-galactopyranoside, both compounds are inhibitory. Although Ch-3, a glycopeptide from the major sialoglycoprotein (MN-glycoprotein or PAS-1), was not inhibitory to A. hypogaea hemagglutinin, asialo Ch-3 was a strong inhibitor. The inhibitory activity of MN-glycoprotein was also examined. As is shown in Table 4, the intact MN-glycoprotein could not inhibit the hemagglutinin at a concentration of 10 mg/m/. On the other hand, removal of sialic acid from the glycoprotein made it strongly inhibitory. These observations are entirely consistent with the results shown in Table 3, and are also in agreement with the results of Uhlenbruck et aU6l Alkaline borohydride treatment of the desialized MN-glycoprotein appreciably decreased the inhibitory activity of the glycoprotein. Mitogenicity of A. hypogaea hemagglutinin The mitogenic activity of the A. hypogaea hemagglutinin on human peripheral lymphocytes was measured by the stimulation of incorporation of [ 3H]thymidine into lymphocyte DNA. As is shown in Table 5, neither intact lymphocytes nor neuraminidase-treated cells were stimulated.
Bd. 356 (197 5)
Discussion Several hemagglutinins specific for the Mäkelä's group 2 sugars have been purified by specific absorption on a Sepharose column and subsequent displacement with D-galactose or lactosel 27 ' 28 '. In the present work, the hemagglutinin from A hypogaea was also purified by taking advantage of the specific interaction of the hemagglutinin with Sepharose 6B. The active protein was effectively separated from inactive proteins by Sepharose column chromatography, and could be eluted without using the haptenic sugars. The purified hemagglutinin was found to be homogeneous by ultracentrifugal analysis, electrophoresis on polyacrylamide gel and TV-terminal amino acid determination. The sedimentation constant was 6.0 S and a molecular weight of 106500 was obtained by sedimentation equilibrium centrifugation. Some of so-called Mäkelä's group 2 sugars were found to be inhibitors of this hemagglutinin. It is interesting to note that the purified hemagglutinin was not inhibited byA^-acetyl-D-galactosamine, in contrast to the most of the other D-galactosespecific hemagglutinins. Since D-galactosamine was found to be a rather strong inhibitor, it is clear that the jV-acetyl group ofA^-acetyl-o-galactosamine hinders the binding of this sugar to the peanut hemagglutinin. The contribution of the hydroxyl or amino group at C2 of D-galactose or D-galactosamine to the binding of these sugars to the hemagglutinin is not clear from our present work. Further experiments will be necessary to clarify this point.
Table 5. Lymphocyte stimulation by hemagglutinins.
[ 3 H]Thymidine incorporation3 Hemagglutinin
a
Human peripheral lymphocytes 10s cells/ml) were used. The cells were treated with neuraminidase according to Novogradsky and Katchalskü32]
A. hypogaea hemag.
(3 b
Concanavalin A
Cone.
Intact cells
[Mg/m/]
[cpm]
10 25 50 100 250 10
1
Neuraminidase treated cellsb [cpm]
132 126 110 105 120
158 118 136 270 115
7268
13633
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Bd. 356(1975)
Peanut Hemagglutinin
Although the neuraminidase-treated erythrocytes are strongly agglutinated by the purified hemagghitinin, neither intact erythrocytes nor trypsintreated cells are. Binding experiments using 12 5 I-hemagglutinin revealed that it could not bind to the intact human erythrocytes at all (results are not shown). There are two possible explanations of these results. a) Most of the receptor sites for the A. hypogaea hemagglutinin on the intact erythrocytes are covered with sialic acid, and are thus inaccessible for steric reasons. b) As the hemagglutinin is an acidic protein (pi 5.95), the sialic acid on the cell surface may repel it from the erythrocyte surface. In our previous report' 7 ), we suggested that A hypogaea hemagglutinin might bind primarily to the 0-glycosidically linked carbohydrate chains of the membrane glycoproteins of human erythrocytes. The results shown in Table 4 support this suggestion. Thus, glycopeptide Ch-3, whose carbohydrate moieties are solely composed of 0-glycosidically linked carbohydrate chains, is not inhibitory. However, asialo-Ch-3 is a strong inhibitor of the ,4. hypogaea hemagglutinin. This is consistent with the observation that asialo MNglycoprotein, if treated with alkaline NaBH4, loses most of its inhibitory activity toward the hemagglutinin (Table 4). Recently, Dahr et all 7 ) also reported that the ]3-galactosyl group of alkalilabile oligosapcharide chains was a part of the receptor site of this hemagglutinin, and this receptor site was masked by sialic acid linked to C3 of the galactose. It is suggested that the redistribution of agglutinin receptors is important for the agglutination of fibroblasts and other cells'29). Recent investigation in this laboratory using ferritin-conjugated A. hypogaea hemagglutinin indicated that the receptors for it on human erythrocytes did not show any clustering'30). The relatively narrow specificity of the peanut hemagglutinin renders it useful for the elucidation of carbohydrate structures on the surface and organelles of various cells. Henning and Uhlenbiuck have recently applied it to the detection of carbohydrate structure on isolated subcellular oiganelles of rat liver'31!.
1691
Some of the hemagglutinins of plant origin have mitogenic activity against lymphocytes'2). Some of them, especially soy bean hemagglutinin, are mitogenic only against neuraminidase-treated lymphocytes'32). The purified A. hypogaea hemagglutinin did.not show mitogenic activity at concentrations between l(^g and 25C^g per ml with intact or neuraminidase-treated human peripheral lymphocytes. We are most grateful to professor T. Matsumura, Showa University, Tokyo for making available to us his ultracentrifuge. We would like to thank Dr. M. Tomita for the gift of glycopeptide Ch-3 and Dr. T. Kinoshita of Showa University for his advice on the determination of the W-terminal amino acid. This work was supported by research grants from the Ministry of Education of Japan, the Mitsubishi Foundation and the MishimaKaiun Memorial Foundation.
Literature 1 Toms, G.C. & Western, A. (1972) in Chemotaxonomy of the Leguminosae (Harborne, H., Boulten, D. & Turner, B.L., eds.) pp. 367 - 462, Acad. Press, New York. 2 Lis, H. & Sharon, N. (1973) Annu. Rev. Biochem 43, 541-574.
3 Mäkelä, O. (1957) Ann. Med. Exp. Biol. Fenn. 35, Suppl. 1-133.
4 Boyd, W.C., Green, D.M., Fujinaga, D.M., Drabik, J.S. & Waszczenki-Zachaxezenko, E. (1959) Vox Sang. 6, 456-467. 5 Bird, G.W.G. (1964) Vox Sang. 9, 748 - 749. 6 Uhlenbruck, G., Pardoe, G.I. & Bird, G.W.G. (1969) Z. Immun. Forsch. 138, 423 - 433. 7 Dahr, W., Uhlenbruck, G. & Bird, G.W.G. (1975) Vox Sang. 28, 133- 148. 8 Irimura, T., Kawaguchi, T., Terao, T. & Osawa, T. (1975) Carbohyd. Res. 39, 317 - 327. 9 Fukuda, M. & Osawa, T. (1973) /. Biol. Chem 248, 5100-5105. 10 Tomita, M. & Marchesi, V.T. (1975) Proc. Nat. Acad. Sei. U.S.A., in press. 11 Thomas, D.B. & Winzler, R. J. (1969) /. Biol Chem. 244, 5943 - 5946. 12 Yphantis, D.A. (1960) Ann. N. Y. Acad. Sei. 88, 586-601. 13 Vinograd, J., Brunner, R., Kent, R. & Weigle, J. (1963) Proc. Nat. Acad. Sei. U.S.A. 49, 902 - 910. 14 Reisfeld, R. ., Lewis, U. J. & Williams, D. E. (1962) Nature (London) 195, 281 - 283. 15 Weber, K. & Osborn, M. (1969)/. Biol. Chem 244, 4406-4412. 16 Spackman, D. H., Stein, W. H. & Moore, S. (1958) Anal Chem 30, 1190-1206.
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T. Terao, T. Irimura and T. Osawa
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25 Irimura, T. & Osawa, T., (1972) Arch. Biochem. Biophys. 151,475-482. 26 Agrawal, B. B. L. & Goldstein, I. J. (196T) Biochirm Acta 133, 376-319. 19 Dubois, M., Gilles, K. A., Hamilton, J. K., Rebers, R. A. Biophys 27 Tomita, M., Kurokawa, T., Onozaki, K., Ichiki, N.., & Smith, F. (1956) Anal Chem. 28, 350 - 356. Osawa, T. & Ukita, T. (1972) Expenentia 24, 84 - 85.. 20 Blix, G. (1948Mcfe Chem Scand. 2, 467 - 473. 28 Terao, T. & Osawa, T. (1973) /. Biochem 74, 21 Kinoshita, T., linuma, F. & Tsuji, A. (1974) Chem 199-201. 29 Pharm.Bull 22,2421-2426. Nicolson, G. (1914) Int. Rev. Cytol. 39, 90-174.. 22 30 Matsumoto, I. & Osawa, T. (1970) Arch. Biochem. Irimura, T., Nakajima, M., Hirano, H. & Osawa, T . Biophys. 140,484-491. (1975) Biochim. Biophys. Acta, in press. 23 31 Toyoshima, S., Osawa, T. & Tonomura, A. (1970) Henning, H. & Uhlenbruck, G. (1973) Nature Biochim. Biophys. Acta 221, 514 - 521. (London) 242, 120 - 122. 24 32 Toyoshima, S., Akiyama, Y., Nakano, K., Tonomura, Novogradsky, A. & Katchalski, E. (1973) Proc. Nat. A. & Osawa, T. (1971) Biochemistry 10, 4457 - 4463. Acad. Sei U.S.A. 70, 2515 - 2518. 17 Goodwin, T. W. & Morton, R. A. (1946) Biochem. J. 40,628-632. 18 Hirs, C.H.W. (1956) J. Biol. Chem. 219,611-621.
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Purification and Characterization of a Hemagglutinin from Arachis hypogaea Tcadao Terao, Tatsuro Irimura and Toshiaki Osawa
(Received 14 July 1975)
Summary: A. hypogaea hemagglutinin was purified by ammonium sulfate fractionation and Sepharose 6 B column chromatography. The homogeneity of the purified hemagglutinin was ascertained by ultracentrifugal analysis and polyacrylamide gel electrophoresis. It has a molecular weight of 106.500 and is a tetramer of a subunit with a molecular weight of 27.000. The purified hemagglutinin agglutinated neuraminidase-treated human erythrocytes regardless
of their ABO group type, but did not agglutinate intact erythrocytes. In hapten inhibition assays with simple sugars, the so-called Mäkelä's group 2 sugars, which bear the same configuration of hydroxy groups at C-3 and C-4 as D-galactopyranose, were inhibitors for this hemagglutinin. It does not contain any carbohydrate, in contrast to most phytohemagglutinins except concanavalin A and wheat germ agglutinin.
Reinigung und Charakterisierung eines Hämagglutinins aus Arachis hypogaea Zusammenfassung: A. -hypogaea-Yiamag$.utmm wurde mittels Ammoniumsulfat-Fraktionierung und Sepharose-6 B-Chromatographie gereinigt. Das gereinigte Hämagglutinin erwies sich als einheitlich in der analytischen Ultrazentrifugation und der Polyacrylamid-Gelelektrophorese. Es hat ein Mol.-Gew. von 106500 und liegt als Tetramer vor (Mol.-Gew. der Untereinheiten 27 000). Das gereinigte Hämagglutinin agglutiniert Neuraminidase-behandelte menschliche Erythrozyten
unabhängig von ihrer Blutgruppe im ABO-System, intakte Erythrozyten werden jedoch nicht agglutiniert. Im Hapten-Hemmtest mit einfachen Zukkern erwiesen sich die Zucker der sogenannten Mäkelä-Gruppe 2, die an C-3 und C-4 dieselbe Hydroxygruppenkonfiguration aufweisen wie o-Galaktopyranose, als Inhibitoren für das Hämagglutinin. Im Gegensatz zu den meisten Phytohämagglutininen (mit Ausnahme von Concanavalin A und Weizenkeim-Agglutinin) enthält dieses Hämagglutinin keine Kohlenhydrate.
Address: Prof. T. Osawa, Division of Chemical Toxicology and Immunochemistry, Faculty of Pharmaceutical Sciences, University of Tokyo, Bunkyo-ku, Tokyo, Japan Enzymes: Neuraminidase, acylneuraminyl hydrolase (EC 3.2.1.18); Trypsin, (EC 3.4.2.4).
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1686
T. Terao, T. Irimura and T. Osawa
Many plants contain agglutinins which are capable of agglutinating erythrocytes and other cells through their surface oligosaccharide determinantsl 1 · 2 !. These hemagglutinins are most frequently found in the seeds of leguminous plants and differ from one another in their specificities!31. The presence of a hemagglutinin inArachis hypogaea seeds (peanuts) was first described by Boyd et aU 4 l, and some properties of the crude hemagglutinin were also reported by Bird^l and Uhlenbruck et alJ 6 '. Inhibition studies on A hypogaea hemagglutinin with various simple sugars and glycopeptides have already been carried out byDahretal.l 7 Undbyusl 8 l. In this paper, we report the purification and properties of A. hypogaea hemagglutinin. Materials and Methods Enzymes: Trypsin (twice recrystallized) was purchased from Worthington. Vibrio cholerae and Clostridium perfringens neuraminidases were obtained from Sigma and Boehringer Mannheim GmbH. Glycoproteins and glycopeptides: The major glycoprotein of human erythrocyte membrane (MN glycoprotein or PAS-I) was obtained from human erythrocyte stroma of blood group AB according to the method of Fukuda and Osawa^9'. Desialization of the glycoprotein was carried out by an acid hydrolysis in 0.05N H 2 SO 4 at 80 °C for l h at a glycoprotein concentration of 6 mg per ml. About 95% of the sialic acid was removed by this procedure. The hydrolysate was neutralized with solid NaHCOs, dialized against distilled water and lyophilized. The alkali treatment of the desialized MN-glycoprotein was performed with O.lN NaOH/0.lMNaBH 4 for 36 h at room temperature in a sealed tube, in the dark, under nitrogen at a concentration of 10 mg per ml. The excess borohydride was destroyed by the addition of acetic acid to pH 4.0, and a portion of the mixture was put on a Sephadex G-50 column equilibrated with lOmM Tris/ HC1, pH 7.2. The column was eluted with the same buffer, and the void volume fractions of the column eluate, containing carbohydrates linked to the protein moiety by alkali-resistant linkage, were pooled, dialyzed against distilled water and lyophilized. One of the chymotrypsin fragments of the MN-glycoprotein, Ch-3, was a gift from Dr. M. Tomita, Showa University, Tokyo. This glycopeptide has been shown'101 to contain only O-glycosidically linked sugar chains, the structure of which was suggested by Thomas and Winz-
Bd. 356(1975)
Other reagents: All other reagents were commercial preparations of the highest purity available. Acetone powder of A. hypogaea seeds: All the following procedures were carried out at room temperature. A. hypogaea seeds (100g) obtained from a local market were suspended in 300 ml of water and homogenized in a Waring Blender. Ice-cold acetone (1500 ml) was added to the homogenate with stirring. After 30 min stirring, the precipitates were collected on a B chner funnel by filtration. The filtrate was discarded. The filter cake was resuspended in acetone (1000 ml), stirred for 30 min and filtered as above. This step was then repeated four times to completely remove oily substances. The precipitates were finally air-dried and kept at - 20 °C. The yield of air-dried powder was about 30 g. Purification of A. hypogaea hemagglutinin: In a typical run, 100 g of the acetone powder was suspended in 1 / of 0.15M NaCl/15mM sodium phosphate buffer, pH 7.2, and allowed to stand overnight at 4 °C with stirring. The clear supernatant obtained by centrifugation at 20000 χ g for 30 min was subjected to (NH 4 ) 2 S0 4 fractionation. As shown in Table 1, the precipitate between 40 and 75% saturation of (NH 4 ) 2 SO 4 had most of the hemagglutinating activity. This fraction was dissolved in O.lM Tris/HCl buffer, pH 7.5, and the solution was dialyzed against distilled water. A precipitate which formed during dialysis was removed by centrifugation (12000 χ g, 30 min) and the supernatant was lyophilized. Since the hemagglutinin was known to be inhibited by D-galactosel6!, further purification of the active fraction was achieved by Sepharose 6B column chromatography as described in the legend to Fig. 1. The second protein peak eluted after the bulk of inactive proteins contained the activity against the neuraminidase-treated human erythrocytes. Active protein fractions were combined, dialyzed against distilled water and lyophilized. Table 1 summarizes data pertaining to the purification of hemagglutinin. The minimum hemagglutinating dose of the purified product obtained was 1 Mg/m/ with the desialized human erythrocytes. Molecular weight determination: The molecular weight of the purified hemagglutinin was measured by sedimentation equilibrium with a Hitachi model UCA-1A ultracentrifuge according to the method of Yphantisl 12L The concentrations of the samples tested were 0.25, 0.50, 0.75 and 1.00% in lOmM sodium phosphate buffered saline (pH 7.5). The sedimentation velocity was determined by the band sedimentation method at a speed of 56000 rpm.( 13 L Polyacrylamide gel electrophoresis: Disc electrophoresis was carried out in 7.5% polyacrylamide gel at pH 4.3 according to the method of Reisfeld et alJ 141. The sodium dodecylsulfate gel electrophoresis was performed
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Amino acid analysis: The hemagglutinin was hydrolyzed with 6N HC1 at 105 °C for 24, 48 and 72 h. The amino acid contents of the hydrolysates were determined on a JEOL-5A amino acid analyzer according to the method of S packman etalJ16l Tryptophan was measured in an unhydrolyzed protein sample by the spectrophotometric method of Goodwin and Morton!171. Half cystine and methionine were analyzed after performic acid oxidation by the method of H W 18l
~50/77*
W
1687
Peanut Hemagglutinin
Bd. 356(1975)
20
30 iO Fract. no.
50
70
80
Fig. 1. Sepharose 6B column chromatography of crude hemagglutinin. 150 mg of the (NH 4 ) 2 SO4 fraction (40 - 75 % saturation) was dissolved in 3 ml of lOmM Tris/HCl, pH 7.2, and then applied to a column (2 χ 45 cm) which was equilibrated with the same buffer. The column was run with the same buffer. After the second protein peak (peak B) had eluted, the column was further washed with a buffer containing 0.5 Μ D-galactose. Fractions of 7 m/ were collected and the fractions corresponding to peak Β were pooled.
according to Weber and Osbornl15'. 25 Mg of protein was placed onto a 7.5% gel in 0.1M sodium phosphate buffer, pH 7.1, containing 0.1% sodium dodecylsulfate and 1 % 2-mercaptoethanol. Gels were stained with Coomassie Brilliant Blue. Isoelectric focusing: Isoelectric focusing was performed using an LKB 8 100-1 column. After the run, fractions (each 2.5 ml) were analyzed foM 2 80 and PH· The PH gradient was linear over the range pH 4 to 8.
Table 1. Purification of Arachis hypogaea hemagglutinin.
Carbohydrate determination: Total neutral sugar was determined by the phenol/H2S04 method of Dubois et alJ 191 with D-mannose as the standard. Amino sugar was determined according to Blix' ' and by means of a JEOL-5A amino acid analyzer. Hydrolysis for this assay was carried out with 4N HC1 for 8 h at 100 °C in a sealed tube. N-Terminal amino acid: The W-terminal amino acid was dansylated according to Kinoshita et alJ211, and the dansyl derivative was identified by thin-layer chromatography on a polyamide sheet. Solvent systems used were a) benzene/acetic acid, 9:1; b) 2% aqueouspyridine/methanol, 2:1; c) hexane/ethanol/acetic acid, 4:2:1. Treatment of the A. hypogaea hemagglutinin with iodo[ 2- !4C]acetic acid: 3 mg of hemagglutinin in 0.3 ml of lOmM sodium phosphate buffered saline was treated with a 100-fold excess of iodo[ 2-14C]acetic acid (5.1 mCi/mmol) at 25 °C for 3 h. The reaction mixture was put on a Sephadex G-25 column (superfine, 0.5 χ 20 cm) which was preequilibrated with lOmM sodium phosphate buffered saline (pH 7.0). The fractions containing the protein were collected and a portion of the combined fraction was used to measure the radioactivity with a Beckman model LS-230 liquid scintillation spectrophotometer. Hemagglutination assays: The titration and inhibition assays using desialized human erythrocytes were carried out according to the method previously described^ 22 ].
Fraction
Crude extract (NH4)2S04 fract. 0 - 40% 40-75% 75 - 100% Sepharose 6B column Fract. A Fract. B
Yield from 100 g seeds
Total hemagglutinin titer
[mg]
(titer χ volume [m/])
1280
92400
120 550 320
6400 76800 12800
430 85
542 87120
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1688
T. Terao, T. Irimura and T. Osawa
Lymphocyte culture for mitogenic assay: Preparation and cultivation of human peripheral lymphocytes were performed according to the method of Toyoshima et al.l23'. Incorporation of [ 3 H]thymidine into lymphocyte DNA was measured as described previously 1241. Treatment of human erythrocytes with various enzymes: Treatment of human erythrocytes with neuraminidase and trypsin were performed under the conditions previously described!251
Results Poly aery lamide gel electrophoresis The electrophoretic homogeneity of the purified hemagglutinin was confirmed by disc electrophoresis on a polyacrylamide gel. A single band was obtained at pH 4.3 (Fig. 2 a). The sodium dodecylsulfate gel electrophoresis also indicated the homogeneity of the hemagglutinin (Fig. 2 b). Molecular weight determination: The molecular weight of 106500 was calculated for the purified hemagglutinin from the sedimentation equilibrium centrifugationt 12 l Furthermore, when the purified protein was subjected to polyacrylamide gel electrophoresis in the presence of sodium dodecylsulfate, it gave a single band (Fig. 2 b) corresponding to a molecular weight of 27000 (Fig. 3). This indicates that the intact hemagglutinin consists of four subunits of molecular weight 27000.
Bd. 356(19775)
yr-flefc
\
v
\Alb.
A.hypogatu hemigg utinin
;
\
\£Ohym.A
Λ\
4
d[cmj -+
6
Fig. 3. Determination of the molecular weight of A hypogaea hemagglutinin by polyacrylamide gel electrophoresis in the presence of sodium dodecylsulfate. Running conditions were the same as in Fig. 2 b). Standard proteins used were human 7-globulin (7-Glob., MT = 160000), bovine serum albumin (Alb., MT = 67000), ovalbumin (Ον., Μτ = 45 000) and bovine chymotrypsinogen A (Chym. A, Mt = 25 000).
Chemical composition: The amino acid composition of the purified hemagglutinin is presented in Table 2. Its characteristic feature is the high proportion of acidic amino acids and the rather low proportion of basic amino acids. This is consistent with the pi value (pi 5.95) of the protein. TV-Terminal amino acid analysis was carried out by the dansyl method, and only alanine was found to be dansylated. Free sulfhydryl group determination with iodo[2-14C]acetic acid revealed that essentially no free SH group was present.
Fig. 2. Polyacrylamide gel electrophoresis of purified hemagglutinin. a) 7.5% gel in 0.35M /3-alanine/acetate, pH 4.3. b) 7.5% gel in 0.1 M sodium phosphate, pH 7.1, in the presence of sodium dodecylsulfate.
When 2.5 mg of the hemagglutinin was subjected to the phenol/H2S04 test for neutral sugars, and the Elson-Morgan reaction for amino sugars, no color developed in either reaction, suggesting the absence of carbohydrate. In both reactions, concanavalin A, which is known not to contain any sugar!261, was used for a blank. The absence of carbohydrate was further confirmed by the application of an acid hydrolysate of the hemagglutinin to an automatic sugar analyzer (JEOL-5A)
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Bd. 356 (1975)
for neutral sugars and an amino acid analyzer (JEOL-5A) for amino sugars. Effects of enzyme treatments of erythrocytes on the hemagglutinin titer Table 3 shows the effects of various enzyme treatments of human erythrocytes on the titer of the purified hemagglutinin. Although it does not affect intact human erythrocytes, it strongly agglutinates neuraminidase-treated cells, regardless of their blood type. The minimum hemagglutinating dose of the purified preparation was about 1 g per ml. Trypsin treatment of the erythrocytes did not make them susceptible to the hemagglutinin. Neuraminidase treatment of the trypsinized erythrocytes, however, resulted in a greater increase in their agglutinability than treatment with neuraminidase alone. On the other hand, trypsin treatment of the neuraminidase-treated erythrocytes rather decreased their agglutinability. This might be caused by the removal of some glycoprotein receptors from the cell surface during the tryptic treatment.
Table 3. Effect of enzymes on agglutinability of human erythrocytes with purified hemagglutinin. Experiments were performed as previously described'25!.
Amino Acid
Cys/2 Val Met He Leu Tyr Phe Lys His Arg Trp Carbohydrate Free SH Pi • y 20,w
1
Not detected.
Titer
Enzyme
n* n* 1/128 1/32
Untreated Trypsin V. cholerae neuraminidase Trypsin after V. cholerae neuraminidase V. cholerae neuraminidase after trypsin
1/256
No agglutination.
Table 4. Inhibition assay of purified hemagglutinin with various sugars, glycopeptides and glycoproteins Sugars, glycopeptides and MN-glycoprotein D-Galactose Lactose Melibiose Raffinose D-Galactosamine L-Arabinose jV-Acetyl-D-galactosamine D-Glucose D-Glucosamine W-Acetyl-D-glucosamine L-Fucose D-Mannose Maltose p-Nitrophenyl-a-Dgalactopyranoside p-Nitrophenyl-/3-Dgalactopyranoside Ch-3 Asialo-Ch-3 MN-glycoprotein Asialo MN-glycoprotein Alkaline borohydridetreated Asialo MN-glycoprotein
Table 2. Chemical composition and some physicochemical properties of A. hypogaea hemagglutinin.
Asp Thr Ser Glu Pro Gly Ala
1689
Peanut Hemagglutinin
Mol/mol hemagglutinin
144.3 72.4 74.0 34.8 16.6 65.8 43.7 16.6 97.8 _a
51.8 38.0 17.8 52.1 33.0 8.4 16.5 2.7 _a _a
5.95 6.05 a
Minimum concentration [mMJ completely inhibiting 4 times the hemagglutination dose
25 12.5 50 50 6 50 > 100 > 100 > 100 > 100
>100 > 100 > 100
12 5 > 1.3a 0.01a >10 a 0.0053
2.5a 2.5a
Expressed as mg/m/.
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1690
T. Terao, T. Irimura and T. Osawa
Assays of hemagglutination inhibition by simple sugars, glycopeptides and glycoproteins The results of the tests of the purified hemagglutinin with various simple sugars as haptene inhibitors are listed in Table 4. In general, the so-called Mäkelä's group 2 sugars'3' are potent inhibitors. D-Galactosamine is a particularly strong inhibitor of this hemagglutinin. As for the anomers of p-nitrophenyl-D-galactopyranoside, both compounds are inhibitory. Although Ch-3, a glycopeptide from the major sialoglycoprotein (MN-glycoprotein or PAS-1), was not inhibitory to A. hypogaea hemagglutinin, asialo Ch-3 was a strong inhibitor. The inhibitory activity of MN-glycoprotein was also examined. As is shown in Table 4, the intact MN-glycoprotein could not inhibit the hemagglutinin at a concentration of 10 mg/m/. On the other hand, removal of sialic acid from the glycoprotein made it strongly inhibitory. These observations are entirely consistent with the results shown in Table 3, and are also in agreement with the results of Uhlenbruck et aU6l Alkaline borohydride treatment of the desialized MN-glycoprotein appreciably decreased the inhibitory activity of the glycoprotein. Mitogenicity of A. hypogaea hemagglutinin The mitogenic activity of the A. hypogaea hemagglutinin on human peripheral lymphocytes was measured by the stimulation of incorporation of [ 3H]thymidine into lymphocyte DNA. As is shown in Table 5, neither intact lymphocytes nor neuraminidase-treated cells were stimulated.
Bd. 356 (197 5)
Discussion Several hemagglutinins specific for the Mäkelä's group 2 sugars have been purified by specific absorption on a Sepharose column and subsequent displacement with D-galactose or lactosel 27 ' 28 '. In the present work, the hemagglutinin from A hypogaea was also purified by taking advantage of the specific interaction of the hemagglutinin with Sepharose 6B. The active protein was effectively separated from inactive proteins by Sepharose column chromatography, and could be eluted without using the haptenic sugars. The purified hemagglutinin was found to be homogeneous by ultracentrifugal analysis, electrophoresis on polyacrylamide gel and TV-terminal amino acid determination. The sedimentation constant was 6.0 S and a molecular weight of 106500 was obtained by sedimentation equilibrium centrifugation. Some of so-called Mäkelä's group 2 sugars were found to be inhibitors of this hemagglutinin. It is interesting to note that the purified hemagglutinin was not inhibited byA^-acetyl-D-galactosamine, in contrast to the most of the other D-galactosespecific hemagglutinins. Since D-galactosamine was found to be a rather strong inhibitor, it is clear that the jV-acetyl group ofA^-acetyl-o-galactosamine hinders the binding of this sugar to the peanut hemagglutinin. The contribution of the hydroxyl or amino group at C2 of D-galactose or D-galactosamine to the binding of these sugars to the hemagglutinin is not clear from our present work. Further experiments will be necessary to clarify this point.
Table 5. Lymphocyte stimulation by hemagglutinins.
[ 3 H]Thymidine incorporation3 Hemagglutinin
a
Human peripheral lymphocytes 10s cells/ml) were used. The cells were treated with neuraminidase according to Novogradsky and Katchalskü32]
A. hypogaea hemag.
(3 b
Concanavalin A
Cone.
Intact cells
[Mg/m/]
[cpm]
10 25 50 100 250 10
1
Neuraminidase treated cellsb [cpm]
132 126 110 105 120
158 118 136 270 115
7268
13633
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Bd. 356(1975)
Peanut Hemagglutinin
Although the neuraminidase-treated erythrocytes are strongly agglutinated by the purified hemagghitinin, neither intact erythrocytes nor trypsintreated cells are. Binding experiments using 12 5 I-hemagglutinin revealed that it could not bind to the intact human erythrocytes at all (results are not shown). There are two possible explanations of these results. a) Most of the receptor sites for the A. hypogaea hemagglutinin on the intact erythrocytes are covered with sialic acid, and are thus inaccessible for steric reasons. b) As the hemagglutinin is an acidic protein (pi 5.95), the sialic acid on the cell surface may repel it from the erythrocyte surface. In our previous report' 7 ), we suggested that A hypogaea hemagglutinin might bind primarily to the 0-glycosidically linked carbohydrate chains of the membrane glycoproteins of human erythrocytes. The results shown in Table 4 support this suggestion. Thus, glycopeptide Ch-3, whose carbohydrate moieties are solely composed of 0-glycosidically linked carbohydrate chains, is not inhibitory. However, asialo-Ch-3 is a strong inhibitor of the ,4. hypogaea hemagglutinin. This is consistent with the observation that asialo MNglycoprotein, if treated with alkaline NaBH4, loses most of its inhibitory activity toward the hemagglutinin (Table 4). Recently, Dahr et all 7 ) also reported that the ]3-galactosyl group of alkalilabile oligosapcharide chains was a part of the receptor site of this hemagglutinin, and this receptor site was masked by sialic acid linked to C3 of the galactose. It is suggested that the redistribution of agglutinin receptors is important for the agglutination of fibroblasts and other cells'29). Recent investigation in this laboratory using ferritin-conjugated A. hypogaea hemagglutinin indicated that the receptors for it on human erythrocytes did not show any clustering'30). The relatively narrow specificity of the peanut hemagglutinin renders it useful for the elucidation of carbohydrate structures on the surface and organelles of various cells. Henning and Uhlenbiuck have recently applied it to the detection of carbohydrate structure on isolated subcellular oiganelles of rat liver'31!.
1691
Some of the hemagglutinins of plant origin have mitogenic activity against lymphocytes'2). Some of them, especially soy bean hemagglutinin, are mitogenic only against neuraminidase-treated lymphocytes'32). The purified A. hypogaea hemagglutinin did.not show mitogenic activity at concentrations between l(^g and 25C^g per ml with intact or neuraminidase-treated human peripheral lymphocytes. We are most grateful to professor T. Matsumura, Showa University, Tokyo for making available to us his ultracentrifuge. We would like to thank Dr. M. Tomita for the gift of glycopeptide Ch-3 and Dr. T. Kinoshita of Showa University for his advice on the determination of the W-terminal amino acid. This work was supported by research grants from the Ministry of Education of Japan, the Mitsubishi Foundation and the MishimaKaiun Memorial Foundation.
Literature 1 Toms, G.C. & Western, A. (1972) in Chemotaxonomy of the Leguminosae (Harborne, H., Boulten, D. & Turner, B.L., eds.) pp. 367 - 462, Acad. Press, New York. 2 Lis, H. & Sharon, N. (1973) Annu. Rev. Biochem 43, 541-574.
3 Mäkelä, O. (1957) Ann. Med. Exp. Biol. Fenn. 35, Suppl. 1-133.
4 Boyd, W.C., Green, D.M., Fujinaga, D.M., Drabik, J.S. & Waszczenki-Zachaxezenko, E. (1959) Vox Sang. 6, 456-467. 5 Bird, G.W.G. (1964) Vox Sang. 9, 748 - 749. 6 Uhlenbruck, G., Pardoe, G.I. & Bird, G.W.G. (1969) Z. Immun. Forsch. 138, 423 - 433. 7 Dahr, W., Uhlenbruck, G. & Bird, G.W.G. (1975) Vox Sang. 28, 133- 148. 8 Irimura, T., Kawaguchi, T., Terao, T. & Osawa, T. (1975) Carbohyd. Res. 39, 317 - 327. 9 Fukuda, M. & Osawa, T. (1973) /. Biol. Chem 248, 5100-5105. 10 Tomita, M. & Marchesi, V.T. (1975) Proc. Nat. Acad. Sei. U.S.A., in press. 11 Thomas, D.B. & Winzler, R. J. (1969) /. Biol Chem. 244, 5943 - 5946. 12 Yphantis, D.A. (1960) Ann. N. Y. Acad. Sei. 88, 586-601. 13 Vinograd, J., Brunner, R., Kent, R. & Weigle, J. (1963) Proc. Nat. Acad. Sei. U.S.A. 49, 902 - 910. 14 Reisfeld, R. ., Lewis, U. J. & Williams, D. E. (1962) Nature (London) 195, 281 - 283. 15 Weber, K. & Osborn, M. (1969)/. Biol. Chem 244, 4406-4412. 16 Spackman, D. H., Stein, W. H. & Moore, S. (1958) Anal Chem 30, 1190-1206.
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1692
T. Terao, T. Irimura and T. Osawa
Bd. 356(19'75)
25 Irimura, T. & Osawa, T., (1972) Arch. Biochem. Biophys. 151,475-482. 26 Agrawal, B. B. L. & Goldstein, I. J. (196T) Biochirm Acta 133, 376-319. 19 Dubois, M., Gilles, K. A., Hamilton, J. K., Rebers, R. A. Biophys 27 Tomita, M., Kurokawa, T., Onozaki, K., Ichiki, N.., & Smith, F. (1956) Anal Chem. 28, 350 - 356. Osawa, T. & Ukita, T. (1972) Expenentia 24, 84 - 85.. 20 Blix, G. (1948Mcfe Chem Scand. 2, 467 - 473. 28 Terao, T. & Osawa, T. (1973) /. Biochem 74, 21 Kinoshita, T., linuma, F. & Tsuji, A. (1974) Chem 199-201. 29 Pharm.Bull 22,2421-2426. Nicolson, G. (1914) Int. Rev. Cytol. 39, 90-174.. 22 30 Matsumoto, I. & Osawa, T. (1970) Arch. Biochem. Irimura, T., Nakajima, M., Hirano, H. & Osawa, T . Biophys. 140,484-491. (1975) Biochim. Biophys. Acta, in press. 23 31 Toyoshima, S., Osawa, T. & Tonomura, A. (1970) Henning, H. & Uhlenbruck, G. (1973) Nature Biochim. Biophys. Acta 221, 514 - 521. (London) 242, 120 - 122. 24 32 Toyoshima, S., Akiyama, Y., Nakano, K., Tonomura, Novogradsky, A. & Katchalski, E. (1973) Proc. Nat. A. & Osawa, T. (1971) Biochemistry 10, 4457 - 4463. Acad. Sei U.S.A. 70, 2515 - 2518. 17 Goodwin, T. W. & Morton, R. A. (1946) Biochem. J. 40,628-632. 18 Hirs, C.H.W. (1956) J. Biol. Chem. 219,611-621.
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