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[42] Bauhinia purpurea agglutinin
[42]
Bauhinia purpurea AGGLUTININ
367
nonreducing D-galactose residue as well as the penultimate Nacetyl-o-galactosamine residue (which itself is not inhibitory). Glycoproteins that contain unsubstituted o-Gal /3(1 ~ 3) o-GalNAc sequences, such as asialoglycophorin,or the antifreeze glycoprotein, are precipitated readily by the lectin. As mentioned above, rat spleen lymphocytes are stimulated only after removal of sialic acid from the cells by neuraminidase. The stimulation is inhibited by o-galactose but not by N-acetyl-D-galactosamine. Sequential treatment of rat lymphocytes with neuraminidase followed by fl-galactosidase abolishes the response of the cells to PNA.18 This indicates that binding of the lectin to D-galactosyl residues on lymphocyte surface is the initial step in stimulation of these cells. Applications of Peanut Agglutinin The lectin can be used for clinical determination of T-polyagglutinability of erythrocytes, which has been detected by crude peanut extracts.l'"'-' Mouse thymocytes have been separated into two subpopulations by differential agglutination with PNA, since only the immature thymocytes, which contain less sialic acid on their surface, are agglutinated by the lectin. 19More recently separation by PNA of cloned embryonal cells into two subpopulation has been achieved. 23 This method may be useful in separating subpopulations of other cells if they differ in their content of surface sialic acid. Immobilized PNA may be used for fractionation and purification of water-soluble 24 or detergent-solubilized glycoproteins, z4"25 By this method, asialoglycophorin has been isolated from the human erythrocyte membrane. 25 22 G. W. G. Bird and J. Wingham, Scand. J. Haematol. 8, 307 (1971). 23 y. Reisner, G. Gachelin, P. Dubois, J.-F. Nicolas, N. Sharon, and F. Jacob, Dev. Biol. 61, 20 (1977). z4 R. Lotan, G. Beattie, W. Hubbell, and G. L. Nicolson, Biochemistry 16, 1787 (1977). 25 W. G. Carter and N. Sharon, Arch. Biochem. Biophys. 180, 570 (1977).
[42] B a u h i n i a p u r p u r e a
Agglutinin
B y TOSHIAKI O S A W A , TATSURO IRIMURA, a n d TSUTOMU K A W A G U C H I
Crude extract of Bauhinia purpurea seeds was first described as a satisfactory anti-N reagent by Boyd et al. ~ Purification of B. purpurea W. C. Boyd, D. L. Everhart, and M. H. McMaster, J. lrnrnunoL 81,414 (1958).
368
CARBOHYDRATE-BINDING PROTEINS
[42]
hemagglutinin (BPH) can be readily achieved by a two-step process involving (a) ammonium sulfate fractionation and (b) affinity chromatography on a column made of bovine submaxillary mucin (BSM) covalently attached to Sepharose 4B. The purified BPH is an N-acetyl-D-galactosamine-binding protein and nonspecific regarding M,N blood groups.'-' It combines preferentially with O-fl-D-galactopyranosyl-(l~3)-N-acetylD-galactosamine sugar sequence in the sugar chains like those of mucins (mucin-type sugar chains) on human erythrocyte membranes. 3-5
Assay Method Titration. The diluent and the erythrocyte-suspending solution in all tests is 0.15 M NaCl--0.025 M sodium phosphate buffer (pH 7.0). H u m a n erythrocytes freshly obtained from a donor are washed three times and used for routine assays as a 3 % suspension. To each 0.05 ml of 2-fold serial dilutions of thc B P H solution, in a tube, an equal volume of human erythrocyte suspension is added. The mixture is kept for I hr at room temperature and then examined for agglutination. Sugar Inhibition. The inhibition assays are carried out in tubes as follows. To 0.05 ml of sugar solution is added an equal volume of the BPH solution carefully diluted to have four minimum hemagglutinating doses. After incubation for 2 hr at room temperature, 0.05 ml of human erythrocyte suspension is added. The mixture is kept for 1 hr at room temperature and then examined for agglutination.
Purification Procedures
Preparation ofDesialyzed BSM--Sepharose 4B. To Sepharose 4B swollen in water (100 ml) is added 50 g of cyanogen bromide in 100 ml of water under stirring. The pH of the mixture is kept at 11.0 for 10 rain. The product is then washed with ice water and ice cold 0.5 M NaHCO3 (pH 8.5). The activated Sepharose is immediately suspended in 0.1 M NaHCO3, to which is added 600 mg of BSM prepared by the method of Tettamanti and Pigman, n and left to react overnight at room temperature under stirring. The product is then washed with 0.1 M NaHCOz and water and suspended in 100 ml of 50 mM sodium phosphate buffer (pH 6.0). To this suspension is added 0.2 unit of Clostridium perfringens neuraminidase z T. Irimura and T. Osawa, Arch. Biochern. Biophys. 151, 475 (1972). 3T. Irimura, T. Kawaguchi,T. Terao, and T. Osawa, Carbohydr. Res. 39, 317 (1975). 4T. Kawaguchi and T. Osawa, Biochemistry 13, 3169(1974). T. Kawaguchi and T. Osawa, Biochemistry 15, 4581 (1976). G. Tettamanti and W. Pigman, Arch. Biochem. Biophys. 124, 41 (1968).
[42]
Bauhinia purpurea AGGLUTININ
369
(Boehringer-Mannheim GmbH). After incubation at 37° for 3 hr, the product is washed thoroughly with 0.15 M NaC1-0.15 mM sodium phosphate buffer (pH 7.0). Step 1. Finely powdered B. purpurea alba seeds (100 g, F. W. Schumacher, Sandwich, Massachusetts) is suspended in I liter of 0.15 M NaC1--0.15 mM sodium phosphate buffer (pH 7.0) and allowed to stand overnight at 4° with continuous stirring. The clear supernatant fraction (crude extract) obtained by centrifugation at 12,000 g for 20 min is directly subjected to (NH4)2SO4 fractionation. As shown in Table I, the precipitate that results between 0.4 and 0.7 saturation with (NH4)2SO4 has hemagglutinating activity. It is dialyzed against distilled water until free of NH4 ÷ and then lyophilized (crude BPH). Step 2. One hundred milligrams of crude BPH are dissolved in 5 ml of 0.15 M NaCI-0.15 mM sodium phosphate buffer (pH 7.0), dialyzed overnight against the same buffer, and then applied to a column (2 x 32 cm) of desialyzed BSM-Sepharose 4B equilibrated with the same buffer. Elution is carried out with the same buffer and, after a large protein peak is eluted out, subsequent elution is performed with the buffer containing 0.1 M lactose. Fractions of l0 ml are collected at l0 ml per hour at 4 °. The lactose-eluted protein is dialyzed against distilled water and lyophilized (purified BPH). A typical elution profile is illustrated in Fig. 1. The details of purification of BPH are shown in Table I. 1.2 0.1 M Lactose 1.0 ~--t 0.8
o 0.6 0.4 0.2 0
~
_.J 0
~0
20
30
40
50
froction number
FIG. 1. Affinity chromatography of crude Bauhinia purpurea agglutinin on desialyzed
bovine submaxillary mucin-Sepharose 4B.
370
CARBOHYDRATE-BINDING
[42]
PROTEINS
TABLE I DETAILS OF PURIFICATION OF B a u h i n i a p u r p u r e a AGGLUTININ (BPH)
Fraction
Minimum hemagglutinating dose (/zg/ml)
Yield from 100 g of seeds (rag)
Dialyzed crude extract (NH4)2SO4 fractions 0 - 0 . 4 satn. 0 . 4 - 0 . 7 satn. (crude BPH) 0 . 7 - 1 . 0 satn. Purified B P H
3550
5,000
1000 750 50 80
10,000 625 > 10,000 40
Properties of Purified BPH
Physical and Chemical Properties. z The purified BPH forms a single peak in the ultracentrifuge when tested at concentratior~s as high as 10 mg/ml. The sedimentation coefficient (sg0,w) calculated from the sedimentation velocity data is 7.5 S. The molecular weight of BPH as determined by sedimentation equlibrium assuming a partial specific volume of 0.73 is 195,000. The purified BPH is also homogeneous in polyacrylamide gel disc electrophoresis at pH 4.3 as well as at pH 8.3. It gives a single discrete band in polyacrylamide gel disc electrophoresis in the presence of
T A B L E II BINDING CONSTANTS OF B a u h i n i a p u r p u r e a AGGLUTININ TO HUMAN ERYTHROCYTES AND LYMPHOCYTES Ko a
nb
Cells
( × 107)
( x 106)
Erythrocyte Normal Sialidase-treated c
1.5 1.5
0.4 2.0
Lymphocyte Normal Sialidase-treated c
1.1 1.2
1.6 6.0
a Apparent association constant ( M - l ) for major receptor sites. b N u m b e r o f major receptor sites. c A p p r o x i m a t e l y 40% of sialic acids on the cell surface is removed.
[42]
Bauhinia purpurea AGGLUTININ
371
TABLE III COMPARISON OF HEMAGGLUTINATION INHIBITORY ACTIVITIES OF SIMPLE SUGARS AND GLYCOPEPTIDES
Sugars
Minimum concentration (mM) completely inhibiting 4 hemagglutinating doses
D-Glucose D-Galactose D-Mannose L-Fucose N-Acetyl-D-glucosamine N-Aeetyl-D-galactosamine Lactose Melibiose Maltose N-Acetyllactosamine Di-N-acetylchitobiose Phenyl a-D-galactopyranoside Phenyl fl-D-galactopyranoside Methyl N-acetyl-a-D-galactosaminide Methyl N-acetyl-/3-o-galactosaminide PSMa: Glycopeptide SA c SA, GalNAc a BSM: Glycopeptide SA PTb: Glycopeptide SA SA, Gale
> 100 1.6 > 100 >100 > 100 0.8 1.6 1.6 > 100 3.2 > 100 3.2 1.6 0.4 0.4 0.9 0.6 0.3 ND ~ 0.M >2.7 1.7 >3.4
Porcine submaxillary mucin. b Porcine thyroglobulin. c Sialidase-treated. a a-N-Acetylgalactosaminidase-treated. e/3-Galactosidase-treated. f Not determined. o Expressed as millimoles of GalNAc residue of BSM-glycopeptide. a
sodium dodecyl sulfate 7 with or without/3-mercaptoethanol treatment. The molecular weight of the subunit is estimated to be 44,000. BPH is a glycoprotein. It contains 7.7% of neutral sugars and 3.4% of N-acetyl-o-glucosamine. The major neutral sugar constituent is mannose (4.9%), and the remainder of the neutral sugar is made up of smaller amounts of xylose, glucose, fucose, and galactose. The amino acid com7 G. Fairbanks, T. L. Steck, and D. F. H. Wallach,
Biochemistry 10, 2606 (1971).
372
CARBOHYDRATE-BINDING PROTEINS
[42]
position of BPH is characterized by its relatively high content of aspartic acid, serine, and threonine, and by the absence of methionine. Binding Affinity. 4.5 BPH binds to both human erythrocytes and lymphocytes with almost the same association constants. Table II shows the association constants for the major receptor sites (K0) and the average number of major receptor sites per cell determined for human erythrocytes and lymphocytes using 12~I-labeled BPH prepared by the chloramine-T method of Hunter. 8 Specificity. 2,3-~ The results of the inhibition assays on the purified BPH with simple sugars and glycopeptides are shown in Table III. Among the simple sugars tested, the most active inhibitor is N-acetyl-o-galactosamine, and, in general, the so-called M~ikel~i's group 2 sugars are potent inhibitors. Furthermore, it is seen from Table III that BPH is strongly inhibited by a glycopeptide obtained from porcine submaxillary mucin and its sequential degradation products, all of which contain Gal---> GalNAc sugar sequence, but it is not significantly inhibited by a glycopeptide obtained from porcine thyroglobulin which contains Gal---> GIcNAc sugar sequence. BPH can thereby serve as a tool for detecting mucin-type sugar chains on the cell surface.
W. M. Hunter, in "Handbook of Experimental Immunology" (D. M. Weir, ed.), p. 608. Blackwell, London, 1967.
Bauhinia purpurea AGGLUTININ
367
nonreducing D-galactose residue as well as the penultimate Nacetyl-o-galactosamine residue (which itself is not inhibitory). Glycoproteins that contain unsubstituted o-Gal /3(1 ~ 3) o-GalNAc sequences, such as asialoglycophorin,or the antifreeze glycoprotein, are precipitated readily by the lectin. As mentioned above, rat spleen lymphocytes are stimulated only after removal of sialic acid from the cells by neuraminidase. The stimulation is inhibited by o-galactose but not by N-acetyl-D-galactosamine. Sequential treatment of rat lymphocytes with neuraminidase followed by fl-galactosidase abolishes the response of the cells to PNA.18 This indicates that binding of the lectin to D-galactosyl residues on lymphocyte surface is the initial step in stimulation of these cells. Applications of Peanut Agglutinin The lectin can be used for clinical determination of T-polyagglutinability of erythrocytes, which has been detected by crude peanut extracts.l'"'-' Mouse thymocytes have been separated into two subpopulations by differential agglutination with PNA, since only the immature thymocytes, which contain less sialic acid on their surface, are agglutinated by the lectin. 19More recently separation by PNA of cloned embryonal cells into two subpopulation has been achieved. 23 This method may be useful in separating subpopulations of other cells if they differ in their content of surface sialic acid. Immobilized PNA may be used for fractionation and purification of water-soluble 24 or detergent-solubilized glycoproteins, z4"25 By this method, asialoglycophorin has been isolated from the human erythrocyte membrane. 25 22 G. W. G. Bird and J. Wingham, Scand. J. Haematol. 8, 307 (1971). 23 y. Reisner, G. Gachelin, P. Dubois, J.-F. Nicolas, N. Sharon, and F. Jacob, Dev. Biol. 61, 20 (1977). z4 R. Lotan, G. Beattie, W. Hubbell, and G. L. Nicolson, Biochemistry 16, 1787 (1977). 25 W. G. Carter and N. Sharon, Arch. Biochem. Biophys. 180, 570 (1977).
[42] B a u h i n i a p u r p u r e a
Agglutinin
B y TOSHIAKI O S A W A , TATSURO IRIMURA, a n d TSUTOMU K A W A G U C H I
Crude extract of Bauhinia purpurea seeds was first described as a satisfactory anti-N reagent by Boyd et al. ~ Purification of B. purpurea W. C. Boyd, D. L. Everhart, and M. H. McMaster, J. lrnrnunoL 81,414 (1958).
368
CARBOHYDRATE-BINDING PROTEINS
[42]
hemagglutinin (BPH) can be readily achieved by a two-step process involving (a) ammonium sulfate fractionation and (b) affinity chromatography on a column made of bovine submaxillary mucin (BSM) covalently attached to Sepharose 4B. The purified BPH is an N-acetyl-D-galactosamine-binding protein and nonspecific regarding M,N blood groups.'-' It combines preferentially with O-fl-D-galactopyranosyl-(l~3)-N-acetylD-galactosamine sugar sequence in the sugar chains like those of mucins (mucin-type sugar chains) on human erythrocyte membranes. 3-5
Assay Method Titration. The diluent and the erythrocyte-suspending solution in all tests is 0.15 M NaCl--0.025 M sodium phosphate buffer (pH 7.0). H u m a n erythrocytes freshly obtained from a donor are washed three times and used for routine assays as a 3 % suspension. To each 0.05 ml of 2-fold serial dilutions of thc B P H solution, in a tube, an equal volume of human erythrocyte suspension is added. The mixture is kept for I hr at room temperature and then examined for agglutination. Sugar Inhibition. The inhibition assays are carried out in tubes as follows. To 0.05 ml of sugar solution is added an equal volume of the BPH solution carefully diluted to have four minimum hemagglutinating doses. After incubation for 2 hr at room temperature, 0.05 ml of human erythrocyte suspension is added. The mixture is kept for 1 hr at room temperature and then examined for agglutination.
Purification Procedures
Preparation ofDesialyzed BSM--Sepharose 4B. To Sepharose 4B swollen in water (100 ml) is added 50 g of cyanogen bromide in 100 ml of water under stirring. The pH of the mixture is kept at 11.0 for 10 rain. The product is then washed with ice water and ice cold 0.5 M NaHCO3 (pH 8.5). The activated Sepharose is immediately suspended in 0.1 M NaHCO3, to which is added 600 mg of BSM prepared by the method of Tettamanti and Pigman, n and left to react overnight at room temperature under stirring. The product is then washed with 0.1 M NaHCOz and water and suspended in 100 ml of 50 mM sodium phosphate buffer (pH 6.0). To this suspension is added 0.2 unit of Clostridium perfringens neuraminidase z T. Irimura and T. Osawa, Arch. Biochern. Biophys. 151, 475 (1972). 3T. Irimura, T. Kawaguchi,T. Terao, and T. Osawa, Carbohydr. Res. 39, 317 (1975). 4T. Kawaguchi and T. Osawa, Biochemistry 13, 3169(1974). T. Kawaguchi and T. Osawa, Biochemistry 15, 4581 (1976). G. Tettamanti and W. Pigman, Arch. Biochem. Biophys. 124, 41 (1968).
[42]
Bauhinia purpurea AGGLUTININ
369
(Boehringer-Mannheim GmbH). After incubation at 37° for 3 hr, the product is washed thoroughly with 0.15 M NaC1-0.15 mM sodium phosphate buffer (pH 7.0). Step 1. Finely powdered B. purpurea alba seeds (100 g, F. W. Schumacher, Sandwich, Massachusetts) is suspended in I liter of 0.15 M NaC1--0.15 mM sodium phosphate buffer (pH 7.0) and allowed to stand overnight at 4° with continuous stirring. The clear supernatant fraction (crude extract) obtained by centrifugation at 12,000 g for 20 min is directly subjected to (NH4)2SO4 fractionation. As shown in Table I, the precipitate that results between 0.4 and 0.7 saturation with (NH4)2SO4 has hemagglutinating activity. It is dialyzed against distilled water until free of NH4 ÷ and then lyophilized (crude BPH). Step 2. One hundred milligrams of crude BPH are dissolved in 5 ml of 0.15 M NaCI-0.15 mM sodium phosphate buffer (pH 7.0), dialyzed overnight against the same buffer, and then applied to a column (2 x 32 cm) of desialyzed BSM-Sepharose 4B equilibrated with the same buffer. Elution is carried out with the same buffer and, after a large protein peak is eluted out, subsequent elution is performed with the buffer containing 0.1 M lactose. Fractions of l0 ml are collected at l0 ml per hour at 4 °. The lactose-eluted protein is dialyzed against distilled water and lyophilized (purified BPH). A typical elution profile is illustrated in Fig. 1. The details of purification of BPH are shown in Table I. 1.2 0.1 M Lactose 1.0 ~--t 0.8
o 0.6 0.4 0.2 0
~
_.J 0
~0
20
30
40
50
froction number
FIG. 1. Affinity chromatography of crude Bauhinia purpurea agglutinin on desialyzed
bovine submaxillary mucin-Sepharose 4B.
370
CARBOHYDRATE-BINDING
[42]
PROTEINS
TABLE I DETAILS OF PURIFICATION OF B a u h i n i a p u r p u r e a AGGLUTININ (BPH)
Fraction
Minimum hemagglutinating dose (/zg/ml)
Yield from 100 g of seeds (rag)
Dialyzed crude extract (NH4)2SO4 fractions 0 - 0 . 4 satn. 0 . 4 - 0 . 7 satn. (crude BPH) 0 . 7 - 1 . 0 satn. Purified B P H
3550
5,000
1000 750 50 80
10,000 625 > 10,000 40
Properties of Purified BPH
Physical and Chemical Properties. z The purified BPH forms a single peak in the ultracentrifuge when tested at concentratior~s as high as 10 mg/ml. The sedimentation coefficient (sg0,w) calculated from the sedimentation velocity data is 7.5 S. The molecular weight of BPH as determined by sedimentation equlibrium assuming a partial specific volume of 0.73 is 195,000. The purified BPH is also homogeneous in polyacrylamide gel disc electrophoresis at pH 4.3 as well as at pH 8.3. It gives a single discrete band in polyacrylamide gel disc electrophoresis in the presence of
T A B L E II BINDING CONSTANTS OF B a u h i n i a p u r p u r e a AGGLUTININ TO HUMAN ERYTHROCYTES AND LYMPHOCYTES Ko a
nb
Cells
( × 107)
( x 106)
Erythrocyte Normal Sialidase-treated c
1.5 1.5
0.4 2.0
Lymphocyte Normal Sialidase-treated c
1.1 1.2
1.6 6.0
a Apparent association constant ( M - l ) for major receptor sites. b N u m b e r o f major receptor sites. c A p p r o x i m a t e l y 40% of sialic acids on the cell surface is removed.
[42]
Bauhinia purpurea AGGLUTININ
371
TABLE III COMPARISON OF HEMAGGLUTINATION INHIBITORY ACTIVITIES OF SIMPLE SUGARS AND GLYCOPEPTIDES
Sugars
Minimum concentration (mM) completely inhibiting 4 hemagglutinating doses
D-Glucose D-Galactose D-Mannose L-Fucose N-Acetyl-D-glucosamine N-Aeetyl-D-galactosamine Lactose Melibiose Maltose N-Acetyllactosamine Di-N-acetylchitobiose Phenyl a-D-galactopyranoside Phenyl fl-D-galactopyranoside Methyl N-acetyl-a-D-galactosaminide Methyl N-acetyl-/3-o-galactosaminide PSMa: Glycopeptide SA c SA, GalNAc a BSM: Glycopeptide SA PTb: Glycopeptide SA SA, Gale
> 100 1.6 > 100 >100 > 100 0.8 1.6 1.6 > 100 3.2 > 100 3.2 1.6 0.4 0.4 0.9 0.6 0.3 ND ~ 0.M >2.7 1.7 >3.4
Porcine submaxillary mucin. b Porcine thyroglobulin. c Sialidase-treated. a a-N-Acetylgalactosaminidase-treated. e/3-Galactosidase-treated. f Not determined. o Expressed as millimoles of GalNAc residue of BSM-glycopeptide. a
sodium dodecyl sulfate 7 with or without/3-mercaptoethanol treatment. The molecular weight of the subunit is estimated to be 44,000. BPH is a glycoprotein. It contains 7.7% of neutral sugars and 3.4% of N-acetyl-o-glucosamine. The major neutral sugar constituent is mannose (4.9%), and the remainder of the neutral sugar is made up of smaller amounts of xylose, glucose, fucose, and galactose. The amino acid com7 G. Fairbanks, T. L. Steck, and D. F. H. Wallach,
Biochemistry 10, 2606 (1971).
372
CARBOHYDRATE-BINDING PROTEINS
[42]
position of BPH is characterized by its relatively high content of aspartic acid, serine, and threonine, and by the absence of methionine. Binding Affinity. 4.5 BPH binds to both human erythrocytes and lymphocytes with almost the same association constants. Table II shows the association constants for the major receptor sites (K0) and the average number of major receptor sites per cell determined for human erythrocytes and lymphocytes using 12~I-labeled BPH prepared by the chloramine-T method of Hunter. 8 Specificity. 2,3-~ The results of the inhibition assays on the purified BPH with simple sugars and glycopeptides are shown in Table III. Among the simple sugars tested, the most active inhibitor is N-acetyl-o-galactosamine, and, in general, the so-called M~ikel~i's group 2 sugars are potent inhibitors. Furthermore, it is seen from Table III that BPH is strongly inhibited by a glycopeptide obtained from porcine submaxillary mucin and its sequential degradation products, all of which contain Gal---> GalNAc sugar sequence, but it is not significantly inhibited by a glycopeptide obtained from porcine thyroglobulin which contains Gal---> GIcNAc sugar sequence. BPH can thereby serve as a tool for detecting mucin-type sugar chains on the cell surface.
W. M. Hunter, in "Handbook of Experimental Immunology" (D. M. Weir, ed.), p. 608. Blackwell, London, 1967.