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[29] Tomato (Lycopersicon esculentum) lectin
[29]
TOMATO(Lycopersicon esculentum) LECTIN
[29] T o m a t o ( L y c o p e r s i e o n e s c u l e n t u m )
363
Lectin
By MARTIN S. NACHaAR and J. D. OPPENHEIM The purification and characterization of a lectin from the fluid or fruit juice o f the common tomato has been reported. 1~ This lectin agglutinated human erythrocytes of types A, B, O, and AB equally and also agglutinated mouse and sheep erythrocytes. Its sugar specificity is similar to that of potato lectin in that it is inhibited only by oligosaccharides containing /3-N-acetylglucosaminide sequences, a Similar to the potato lectin, the tomato lectin also contains an unusual abundance of hydroxyproline and half-cystine residues, with arabinose as the primary sugar component. 1'3 Two isolectin forms have been identified. 1 The lectin has the peculiar property of being antimitogenic for mouse and chicken lymphocytes. ~ Hemagglutination Assay The presence of the lectin in the juice of the tomato can be detected by observing agglutination of human erythrocytes of A, B, O, or AB blood types. Although protease-treated erythrocytes are somewhat more sensitive to agglutination by the lectin z'4 we have found it unnecessary to utilize such cells, as only those tomato extracts capable of agglutinating untreated cells contain enough lectin to make purification worthwhile. Hemagglutination assays were performed on microagglutination slides using serial dilutions of 0.1 ml of a lectin solution in PBS (0.01 N sodium phosphate buffer co~ataining 0.15 M NaC1) pH 7.6. To each dilution, 0.1 ml of a three times washed 4% human erythrocyte suspension was added. The slides were subsequently incubated for 30 min at room temperature on a rotary shaker. Agglutination was read macroscopically and recorded semiquantitatively using a 0 to 4+ scale (e.g., no agglutination to total agglutination). Extracts useful for purification should display at least a 1+ agglutination reaction at a 1 : 8 to 1 : 16 dilution. Hemagglutination inhibition assays were performed similarly, using serial dilutions of various sugars, glycoproteins, glycolipid or bacterial components in 0.1 ml of a lectin dilution in PBS that in the complete assay mixture would give a final hemagglutination titer of 4-8. After incubating the assay mixtures for 30 t M. S. Nachbar, J. D. Oppenheim, and J. O. Thomas,J. Biol. Chem. 255, 2056 (1980). 2 D. C. Kilpatrick,Biochem. J. 185, 269 (1980). 3 A. K. Allen and A. Neuberger,Biochem. J. 135, 307 (1973). M. S. Nachbar, unpublished observations.
METHODS IN ENZYMOLOGY, VOL. 83
Copyright © 1982 by Academic Press, Inc. All rights of reproduction in any form reserved. ISBN 0-12-181983-3
364
CARBOHYDRATE-BINDING PROTEINS
[29]
min at 25° on a rotary shaker, 0.1 ml of a 4% human erythrocyte suspension was added and the incubation was continued for an additional 30 min before reading. The minimum concentration of inhibitor causing complete inhibition was recorded. Purification Procedure The purification of tomato lectin is based upon the principle of affinity chromatography using known glycoprotein inhibitors of tomato lectin agglutination as affinity adsorbents. Elution from the affinity columns can be accomplished either by passage through the column(s) of the specific sugar inhibitor (chitobiose) or an acidic high-salt buffer.
Preparation of Affinity Absorbents Poly(L-Leucine) Hog A + H Blood Group Substance Affinity Column. Hog A + H blood group substance is purified from hog gastric mucin by an ethanol precipitation procedure) Insoluble poly(L-leucine) hog A + H blood group substance is prepared by the copolymerization of the blood group substance with the N-carboxyanhydride of L-leucine (Vega) according to Tsuyuki et al. 6 The insoluble polyleucyl hog A + H blood group substance affinity column is prepared by pouring it as a slurry of adsorbent, activated charcoal, and Celite according to Etzler. 7 Prior to use, the column is washed sequentially with PBS, pH 7.6, containing 0.02% sodium azide (PBS-azide), 0.1 M acetic acid, 1.0 M NaCI at pH 3, and again with PBS-azide. Ovomucoid Sepharose 4B. Ovomucoid-Sepharose 4B affinity beads are prepared by coupling ovomucoid (Sigma type III-O), at a concentration of 30 mg/ml, with an equal volume of hydrated, washed CNBr-activated Sepharose 4B (Pharmacia Fine Chemicals) according to the manufacturer's directions. After coupling, the remaining CNBr-active sites on the gel are blocked by treatment with 1.0 M ethanolamine. The beads are washed with alternating solutions of 0.1 M borate, 1.0 M NaC1, pH 8.0, and 0.1M acetic acid, 1.0M NaCI, pH 4, for three cycles and then washed with PBS-azide. From 60 to 80% of the ovomucoid can be expected to bind to the Sepharose 4B under these conditions. Prior to acid-salt elutions (see below), the column is washed with 0.1 M acetic acid, 1.0 NaC1, pH 3, and then with the PBS-azide. 5 E. A. Kabat, "Blood Group Substances: Their Chemistry and Immunochemistry." Academic Press, New York, 1956. 6 H. Tsuyuki, H. Van Kley, and M. A. Stahman, J. A m . Chem. Soc. 78, 764 (1956). 7 M. Etzler, this series, Vol. 28 [39].
[29]
TOMATO(Lycopersicon esculentum) LECTIN
365
Preparation of Extract Store-bought tomatoes are washed and" weighed and then homogenized in a Waring blender following the addition of 1.5 M sodium chloride (100 ml per kilogram of tomato). The slurry is then centrifuged at 11,000 g for 15 min, and the supernatant and debris are filtered through large funnels plugged with cheesecloth. The filtrate is neutralized by the dropwise addition of 10 N sodium hydroxide with vigorous stirring. To the solution sodium azide is added to a final concentration of 0.02%. The solution is filtered sequentially through glass fiber filters (Gelman Type A-E, 47 mm) and microporous filters (Amicon 0.45 ~m).
Affinity Chromatography Immediately after filtration the extract is applied to and passed through either a polyleucine hog A + H blood group substance (HGM) column I or an ovomucoid-Sepharose 4B column that has been equilibrated with PBS-azide. Although either column can be used in the affinity purification, the results presented here were obtained using the ovomucoid column. Flow rates are maintained at the highest level possible. For a typical 1.5-2.0 cm wide, 10-15 cm long column the average flow rates were 300 ml/hr and 800-900 ml/hr for the HGM and ovomucoid columns, respectively. Failure to attain these flow rates will lead to column clogging by gel particles forming in the extract with time. Such high flows inevitably lead to a minor loss of agglutinating activity in the column effluent (about 10% of the total). Samples of the effluent should be checked for hemagglutinating activity to assure that binding of the lectin to the column is occurring and to monitor spillage and saturation of the column. The column is then washed with the PBS-azide, and the effluent is monitored for hemagglutinating activity and for absorbance at 280 nm until the A2s0 falls below 0.02. Elution of the bound lectin is accomplished in one of two ways. Chitobiose Elution. Elution of agglutinating activity can be achieved by the passage of the PBS-azide buffer containing 10 mg/ml of chitobiose (i.e., the sugar is prepared according to the method of Rupley 8), at a flow rate of 50-80 ml/hr. The column is then washed with the PBS-azide buffer. A typical elution profile is illustrated in Fig. 1A. Fractions containing hemagglutinating activity at 1:10 dilution in the PBS-azide buffer are pooled, then concentrated in an Amicon cell using a PM-10 filter. The concentrated material is diluted with 9 volumes of distilled water and reconcentrated. The sequential dilution-concentration steps are repeated 5 times. The final desalted material is then lyophilized. 8 j. A. Rupley, Biochim. Biophys. Acta 83, 245 (1964).
366
CARBOHYDRATE-BINDING PROTEINS
~
A
[29]
~B
\
\
E2.0 Ciitobiose HAc'. ..... 1.0 u
[ -i
4.
z~
0
!
I
,
I
10 30 50 70
t
" - i ' " ~t I
I
10 30
i
50
3*~
I
70
0
FRACTION NUMBER FIG. 1. Elution profiles of agglutinating activity of tomato extract. An extract (10 liters) of tomatoes (3.4 kg) in 0.15 M NaCI, pH 7.0, was divided into two aliquots. Each aliquot was applied to a 2.5 x 7.5 cm column of ovomucoid-Sepharose 4B beads at a flow rate of 900 ml/hr. The columns were subsequently washed with phosphate-buffered saline (PBS), pH 7.0, until the absorbance at 280 nm was <0.01; 25-ml fractions were collected. One column (profile A) was then eluted with a 10 mg of chitobiose per milliliter of solution in PBS, and the other column (profile B) was eluted with 0.1 N acetic acid containing 1.0 M NaC1; 5-ml fractions were collected. The eluted fractions from each column were monitored for absorbarley at 280 nm and hemagglutinating activity (the acid-eluted fractions were neutralized with 0.1 N NaOH prior to assay). All fractions from each column that contained such activities were pooled. The inset demonstrates a sodium dodecyl sulfate-polyacrylamide electrophoresis slab gel comparing tomato lectin prepared on (a) a poly(L-leucine) hog A + H blood group substance affinity column; (b) an ovomucoid-Sepharose 4B affinity column eluted with chitobiose; (c) an ovomucoid-Sepharose 4B affinity column eluted with 0.1 N acetic acid containing 1.0 M NaCI.
Acid-Salt Elution. A g g l u t i n a t i n g a c t i v i t y c a n also b e r e l e a s e d f r o m t h e o v o m u c o i d - S e p h a r o s e 4B c o l u m n b y e l u t i o n w i t h 0.1 N a c e t i c acid cont a i n i n g 1.0 M NaC1, p H 3.0. E l u t i o n b y this m e t h o d is i l l u s t r a t e d i n Fig. l B . T h o s e f r a c t i o n s o f t h e e l u a t e that e x h i b i t a b s o r b a n c e at 280 n m are n e u t r a l i z e d w i t h 0.1 N N a O H . A s a m p l e f r o m e a c h f r a c t i o n is d i l u t e d 1 to 10 w i t h distilled w a t e r a n d c h e c k e d for h e m a g g l u t i n a t i n g activity. F r a c t i o n s d e m o n s t r a t i n g a c t i v i t y are p o o l e d a n d d i a l y z e d e x t e n s i v e l y a g a i n s t distilled w a t e r , c o n c e n t r a t e d in a n A m i c o n cell u s i n g a P M - 1 0 m e m b r a n e , and lyophilized.
[29]
TOMATO(Lycopersicon esculentum) LECTIN
367
Comments. Comparison by sodium dodecyl sulfate-polyacrylamide gel electrophoresis (see Fig. 1, inset), sugar:protein ratios, and hemagglutinating activity per milligram dry weight show that the materials eluted from either of the two affinity columns (HGM or ovomucoid) by either method are identical. Between 25 and 50 mg of purified tomato lectin can be prepared from 4-5 kg of tomatoes using columns containing either 200 mg of polyleucine hog A + H blood group substance or 75 ml of ovomucoid-Sepharose-4B. Recovery of total activity from the initial extract ranges from 70 to 85% depending upon the method employed. The lectin is purified 250-300 times by the single affinity chromatographic procedure. The affinity-eluted material appears to be homogeneous and requires no further purification steps. An alternative method of purification employing Pronase-treated glutaraldehyde-fixed human type B erythrocytes has been r e p o r t e d / T h i s latter method is not adequate for large-scale preparation and runs the risk of contaminating the eluate with erythrocyte products.
Properties of Tomato Lectin Homogeneity. 1 The purified tomato lectin \J--" ~r1% 1 c m = 4.8) migrates as a single symmetrical peak in ultracentrifugation and gel filtration. It produces a linear In C versus r 2 plot in sedimentation equilibrium analysis in either 7 M guanidine-HC1 or in 0.1 M NaCl, 0.01 M sodium phosphate, pH 6.8, further indicating no gross heterogeneity. NH2-terminal analysis yielded only a single amino acid, methionine. The lectin migrates as a single band in sodium dodecyl sulfate-polyacrylamide electrophoresis in both acidic and alkaline systems. Isoelectrofocusing indicated that there are two major isolectins with pI values of 8.8 and 10.0, respectively. Molecular Weight. 1 The molecular weight of the lectin, as determined by gel filtration and sedimentation equilibrium ultracentrifugation is 71,000. The intrinsic sedimentation coefficient (sg0,w), as determined by sedimentation velocity, is 3.9 S. Composition. 1 Tomato lectin is a basic glycoprotein composed of equal amounts of carbohydrate and protein. Arabinose (85%) and galactose (15%) are the only detectable sugar moieties. The amino acid composition is noteworthy for the high content of hydroxyproline (16%) and halfcysteine (11%) and the absence of valine and leucine residues. Specificity. ~The hemagglutinating activity of tomato lectin is inhibited by oligosaccharides of N-acetyl-D-glucosamine, but not by the monosaccharide itself. The most effective inhibitor is the four-sugar oligosaccharide ofN-acetyl-D-glucosamine. A sequence of this sugar, however, is not a prerequisite for inhibition, in that an alternating sequence of N-acetyl-D-glucosamine and muramic acid has also been shown to be an
368
CARBOHYDRATE-BINDING PROTEINS
[30]
effective inhibitor. Tomato lectin exhibits a similar sugar inhibitory specificity to potato lectin. 1"9 Distinct differences between the two, however, are observed for inhibition by glycoproteins. The most notable were the effectiveness o f glycophorin and T a m m - H o r s f a l l glycoprotein to inhibit tomato lectin while having no effect on potato lectin. Stability. When lyophilized, the lectin is stable indefinitely (at least 3 years). It is stable in PBS at - 2 0 ° for 5 - 6 months, at 4° for several weeks, and at room temperature for up to 96 hr. It is relatively resistant to heat denaturation at 100° in its crude state, but not in the purified state. Exposure to citrate or E D T A does not change its activity. 2 It is relatively resistant to proteolytic digestion, l'z One hour o f exposure to p H 1.5 decreases its activity by only 25%. N o change in its activity is observed over the p H range 4 - 9 for up to 1 hr. 1 Periodate oxidation inactivates the lectin. 2 Immunochemistry. The lectin displays immunological cross-reactivity with Datura stramonium (thorn apple) 2 and with Solanum tuberosum (potato) 3 lectins, both also from the plant family Solanaceae. Mitogenicity. ~ Tomato lectin antagonizes the action of T (concanavalin A and phytohemagglutinin) and B (pokeweek mitogen and Escherichia coli lipopolysaccharide) lymphocyte mitogens and depresses background mitogenicity in a dose-dependent manner. In this regard it resembles wheat germ agglutinin, which has a similar but not identical sugar specificity. 1 The mechanism of its antimitogenic effect remains to be determined. It is not toxic to the cells. Acknowledgments Part of this work was supported by United States Public Health Service Institutional Grant RR05399 and by United States Public Health Service Grant HL21328. a A. K. Allen and A. Neuberger, this series, Vol. 50 [37].
[30] D a t u r a
stramonium Lectin
By JANE F. CROWLEY and IRWIN J. GOLDSTEIN
The seeds ofDatura stramonium contain a lectin that is specific for oligomers o f N-acetyl-D-glucosamine. 1~ The Datura lectin is one o f a family o f chitin binding lectins that have been discovered in seeds o f the 1 M. Eisler and L. Portheim, Z. lmrnunitaetsforsch. Exp. Ther. 1 1, 151 (1908). 2 G. I. Pardoe, G. W, G. Bird, G. Uhlenbruck, I. Sprenger, and M. Heggen, Z. Immunitaetsforsch. Allerg. Klin. Immunol. 140, 374 (1970).
METHODS IN ENZYMOLOGY,VOL. 83
Copyright © 1982by Academic Press, Inc. All rights of reproductionin any formreserved. ISBN 0-12-181983-3
TOMATO(Lycopersicon esculentum) LECTIN
[29] T o m a t o ( L y c o p e r s i e o n e s c u l e n t u m )
363
Lectin
By MARTIN S. NACHaAR and J. D. OPPENHEIM The purification and characterization of a lectin from the fluid or fruit juice o f the common tomato has been reported. 1~ This lectin agglutinated human erythrocytes of types A, B, O, and AB equally and also agglutinated mouse and sheep erythrocytes. Its sugar specificity is similar to that of potato lectin in that it is inhibited only by oligosaccharides containing /3-N-acetylglucosaminide sequences, a Similar to the potato lectin, the tomato lectin also contains an unusual abundance of hydroxyproline and half-cystine residues, with arabinose as the primary sugar component. 1'3 Two isolectin forms have been identified. 1 The lectin has the peculiar property of being antimitogenic for mouse and chicken lymphocytes. ~ Hemagglutination Assay The presence of the lectin in the juice of the tomato can be detected by observing agglutination of human erythrocytes of A, B, O, or AB blood types. Although protease-treated erythrocytes are somewhat more sensitive to agglutination by the lectin z'4 we have found it unnecessary to utilize such cells, as only those tomato extracts capable of agglutinating untreated cells contain enough lectin to make purification worthwhile. Hemagglutination assays were performed on microagglutination slides using serial dilutions of 0.1 ml of a lectin solution in PBS (0.01 N sodium phosphate buffer co~ataining 0.15 M NaC1) pH 7.6. To each dilution, 0.1 ml of a three times washed 4% human erythrocyte suspension was added. The slides were subsequently incubated for 30 min at room temperature on a rotary shaker. Agglutination was read macroscopically and recorded semiquantitatively using a 0 to 4+ scale (e.g., no agglutination to total agglutination). Extracts useful for purification should display at least a 1+ agglutination reaction at a 1 : 8 to 1 : 16 dilution. Hemagglutination inhibition assays were performed similarly, using serial dilutions of various sugars, glycoproteins, glycolipid or bacterial components in 0.1 ml of a lectin dilution in PBS that in the complete assay mixture would give a final hemagglutination titer of 4-8. After incubating the assay mixtures for 30 t M. S. Nachbar, J. D. Oppenheim, and J. O. Thomas,J. Biol. Chem. 255, 2056 (1980). 2 D. C. Kilpatrick,Biochem. J. 185, 269 (1980). 3 A. K. Allen and A. Neuberger,Biochem. J. 135, 307 (1973). M. S. Nachbar, unpublished observations.
METHODS IN ENZYMOLOGY, VOL. 83
Copyright © 1982 by Academic Press, Inc. All rights of reproduction in any form reserved. ISBN 0-12-181983-3
364
CARBOHYDRATE-BINDING PROTEINS
[29]
min at 25° on a rotary shaker, 0.1 ml of a 4% human erythrocyte suspension was added and the incubation was continued for an additional 30 min before reading. The minimum concentration of inhibitor causing complete inhibition was recorded. Purification Procedure The purification of tomato lectin is based upon the principle of affinity chromatography using known glycoprotein inhibitors of tomato lectin agglutination as affinity adsorbents. Elution from the affinity columns can be accomplished either by passage through the column(s) of the specific sugar inhibitor (chitobiose) or an acidic high-salt buffer.
Preparation of Affinity Absorbents Poly(L-Leucine) Hog A + H Blood Group Substance Affinity Column. Hog A + H blood group substance is purified from hog gastric mucin by an ethanol precipitation procedure) Insoluble poly(L-leucine) hog A + H blood group substance is prepared by the copolymerization of the blood group substance with the N-carboxyanhydride of L-leucine (Vega) according to Tsuyuki et al. 6 The insoluble polyleucyl hog A + H blood group substance affinity column is prepared by pouring it as a slurry of adsorbent, activated charcoal, and Celite according to Etzler. 7 Prior to use, the column is washed sequentially with PBS, pH 7.6, containing 0.02% sodium azide (PBS-azide), 0.1 M acetic acid, 1.0 M NaCI at pH 3, and again with PBS-azide. Ovomucoid Sepharose 4B. Ovomucoid-Sepharose 4B affinity beads are prepared by coupling ovomucoid (Sigma type III-O), at a concentration of 30 mg/ml, with an equal volume of hydrated, washed CNBr-activated Sepharose 4B (Pharmacia Fine Chemicals) according to the manufacturer's directions. After coupling, the remaining CNBr-active sites on the gel are blocked by treatment with 1.0 M ethanolamine. The beads are washed with alternating solutions of 0.1 M borate, 1.0 M NaC1, pH 8.0, and 0.1M acetic acid, 1.0M NaCI, pH 4, for three cycles and then washed with PBS-azide. From 60 to 80% of the ovomucoid can be expected to bind to the Sepharose 4B under these conditions. Prior to acid-salt elutions (see below), the column is washed with 0.1 M acetic acid, 1.0 NaC1, pH 3, and then with the PBS-azide. 5 E. A. Kabat, "Blood Group Substances: Their Chemistry and Immunochemistry." Academic Press, New York, 1956. 6 H. Tsuyuki, H. Van Kley, and M. A. Stahman, J. A m . Chem. Soc. 78, 764 (1956). 7 M. Etzler, this series, Vol. 28 [39].
[29]
TOMATO(Lycopersicon esculentum) LECTIN
365
Preparation of Extract Store-bought tomatoes are washed and" weighed and then homogenized in a Waring blender following the addition of 1.5 M sodium chloride (100 ml per kilogram of tomato). The slurry is then centrifuged at 11,000 g for 15 min, and the supernatant and debris are filtered through large funnels plugged with cheesecloth. The filtrate is neutralized by the dropwise addition of 10 N sodium hydroxide with vigorous stirring. To the solution sodium azide is added to a final concentration of 0.02%. The solution is filtered sequentially through glass fiber filters (Gelman Type A-E, 47 mm) and microporous filters (Amicon 0.45 ~m).
Affinity Chromatography Immediately after filtration the extract is applied to and passed through either a polyleucine hog A + H blood group substance (HGM) column I or an ovomucoid-Sepharose 4B column that has been equilibrated with PBS-azide. Although either column can be used in the affinity purification, the results presented here were obtained using the ovomucoid column. Flow rates are maintained at the highest level possible. For a typical 1.5-2.0 cm wide, 10-15 cm long column the average flow rates were 300 ml/hr and 800-900 ml/hr for the HGM and ovomucoid columns, respectively. Failure to attain these flow rates will lead to column clogging by gel particles forming in the extract with time. Such high flows inevitably lead to a minor loss of agglutinating activity in the column effluent (about 10% of the total). Samples of the effluent should be checked for hemagglutinating activity to assure that binding of the lectin to the column is occurring and to monitor spillage and saturation of the column. The column is then washed with the PBS-azide, and the effluent is monitored for hemagglutinating activity and for absorbance at 280 nm until the A2s0 falls below 0.02. Elution of the bound lectin is accomplished in one of two ways. Chitobiose Elution. Elution of agglutinating activity can be achieved by the passage of the PBS-azide buffer containing 10 mg/ml of chitobiose (i.e., the sugar is prepared according to the method of Rupley 8), at a flow rate of 50-80 ml/hr. The column is then washed with the PBS-azide buffer. A typical elution profile is illustrated in Fig. 1A. Fractions containing hemagglutinating activity at 1:10 dilution in the PBS-azide buffer are pooled, then concentrated in an Amicon cell using a PM-10 filter. The concentrated material is diluted with 9 volumes of distilled water and reconcentrated. The sequential dilution-concentration steps are repeated 5 times. The final desalted material is then lyophilized. 8 j. A. Rupley, Biochim. Biophys. Acta 83, 245 (1964).
366
CARBOHYDRATE-BINDING PROTEINS
~
A
[29]
~B
\
\
E2.0 Ciitobiose HAc'. ..... 1.0 u
[ -i
4.
z~
0
!
I
,
I
10 30 50 70
t
" - i ' " ~t I
I
10 30
i
50
3*~
I
70
0
FRACTION NUMBER FIG. 1. Elution profiles of agglutinating activity of tomato extract. An extract (10 liters) of tomatoes (3.4 kg) in 0.15 M NaCI, pH 7.0, was divided into two aliquots. Each aliquot was applied to a 2.5 x 7.5 cm column of ovomucoid-Sepharose 4B beads at a flow rate of 900 ml/hr. The columns were subsequently washed with phosphate-buffered saline (PBS), pH 7.0, until the absorbance at 280 nm was <0.01; 25-ml fractions were collected. One column (profile A) was then eluted with a 10 mg of chitobiose per milliliter of solution in PBS, and the other column (profile B) was eluted with 0.1 N acetic acid containing 1.0 M NaC1; 5-ml fractions were collected. The eluted fractions from each column were monitored for absorbarley at 280 nm and hemagglutinating activity (the acid-eluted fractions were neutralized with 0.1 N NaOH prior to assay). All fractions from each column that contained such activities were pooled. The inset demonstrates a sodium dodecyl sulfate-polyacrylamide electrophoresis slab gel comparing tomato lectin prepared on (a) a poly(L-leucine) hog A + H blood group substance affinity column; (b) an ovomucoid-Sepharose 4B affinity column eluted with chitobiose; (c) an ovomucoid-Sepharose 4B affinity column eluted with 0.1 N acetic acid containing 1.0 M NaCI.
Acid-Salt Elution. A g g l u t i n a t i n g a c t i v i t y c a n also b e r e l e a s e d f r o m t h e o v o m u c o i d - S e p h a r o s e 4B c o l u m n b y e l u t i o n w i t h 0.1 N a c e t i c acid cont a i n i n g 1.0 M NaC1, p H 3.0. E l u t i o n b y this m e t h o d is i l l u s t r a t e d i n Fig. l B . T h o s e f r a c t i o n s o f t h e e l u a t e that e x h i b i t a b s o r b a n c e at 280 n m are n e u t r a l i z e d w i t h 0.1 N N a O H . A s a m p l e f r o m e a c h f r a c t i o n is d i l u t e d 1 to 10 w i t h distilled w a t e r a n d c h e c k e d for h e m a g g l u t i n a t i n g activity. F r a c t i o n s d e m o n s t r a t i n g a c t i v i t y are p o o l e d a n d d i a l y z e d e x t e n s i v e l y a g a i n s t distilled w a t e r , c o n c e n t r a t e d in a n A m i c o n cell u s i n g a P M - 1 0 m e m b r a n e , and lyophilized.
[29]
TOMATO(Lycopersicon esculentum) LECTIN
367
Comments. Comparison by sodium dodecyl sulfate-polyacrylamide gel electrophoresis (see Fig. 1, inset), sugar:protein ratios, and hemagglutinating activity per milligram dry weight show that the materials eluted from either of the two affinity columns (HGM or ovomucoid) by either method are identical. Between 25 and 50 mg of purified tomato lectin can be prepared from 4-5 kg of tomatoes using columns containing either 200 mg of polyleucine hog A + H blood group substance or 75 ml of ovomucoid-Sepharose-4B. Recovery of total activity from the initial extract ranges from 70 to 85% depending upon the method employed. The lectin is purified 250-300 times by the single affinity chromatographic procedure. The affinity-eluted material appears to be homogeneous and requires no further purification steps. An alternative method of purification employing Pronase-treated glutaraldehyde-fixed human type B erythrocytes has been r e p o r t e d / T h i s latter method is not adequate for large-scale preparation and runs the risk of contaminating the eluate with erythrocyte products.
Properties of Tomato Lectin Homogeneity. 1 The purified tomato lectin \J--" ~r1% 1 c m = 4.8) migrates as a single symmetrical peak in ultracentrifugation and gel filtration. It produces a linear In C versus r 2 plot in sedimentation equilibrium analysis in either 7 M guanidine-HC1 or in 0.1 M NaCl, 0.01 M sodium phosphate, pH 6.8, further indicating no gross heterogeneity. NH2-terminal analysis yielded only a single amino acid, methionine. The lectin migrates as a single band in sodium dodecyl sulfate-polyacrylamide electrophoresis in both acidic and alkaline systems. Isoelectrofocusing indicated that there are two major isolectins with pI values of 8.8 and 10.0, respectively. Molecular Weight. 1 The molecular weight of the lectin, as determined by gel filtration and sedimentation equilibrium ultracentrifugation is 71,000. The intrinsic sedimentation coefficient (sg0,w), as determined by sedimentation velocity, is 3.9 S. Composition. 1 Tomato lectin is a basic glycoprotein composed of equal amounts of carbohydrate and protein. Arabinose (85%) and galactose (15%) are the only detectable sugar moieties. The amino acid composition is noteworthy for the high content of hydroxyproline (16%) and halfcysteine (11%) and the absence of valine and leucine residues. Specificity. ~The hemagglutinating activity of tomato lectin is inhibited by oligosaccharides of N-acetyl-D-glucosamine, but not by the monosaccharide itself. The most effective inhibitor is the four-sugar oligosaccharide ofN-acetyl-D-glucosamine. A sequence of this sugar, however, is not a prerequisite for inhibition, in that an alternating sequence of N-acetyl-D-glucosamine and muramic acid has also been shown to be an
368
CARBOHYDRATE-BINDING PROTEINS
[30]
effective inhibitor. Tomato lectin exhibits a similar sugar inhibitory specificity to potato lectin. 1"9 Distinct differences between the two, however, are observed for inhibition by glycoproteins. The most notable were the effectiveness o f glycophorin and T a m m - H o r s f a l l glycoprotein to inhibit tomato lectin while having no effect on potato lectin. Stability. When lyophilized, the lectin is stable indefinitely (at least 3 years). It is stable in PBS at - 2 0 ° for 5 - 6 months, at 4° for several weeks, and at room temperature for up to 96 hr. It is relatively resistant to heat denaturation at 100° in its crude state, but not in the purified state. Exposure to citrate or E D T A does not change its activity. 2 It is relatively resistant to proteolytic digestion, l'z One hour o f exposure to p H 1.5 decreases its activity by only 25%. N o change in its activity is observed over the p H range 4 - 9 for up to 1 hr. 1 Periodate oxidation inactivates the lectin. 2 Immunochemistry. The lectin displays immunological cross-reactivity with Datura stramonium (thorn apple) 2 and with Solanum tuberosum (potato) 3 lectins, both also from the plant family Solanaceae. Mitogenicity. ~ Tomato lectin antagonizes the action of T (concanavalin A and phytohemagglutinin) and B (pokeweek mitogen and Escherichia coli lipopolysaccharide) lymphocyte mitogens and depresses background mitogenicity in a dose-dependent manner. In this regard it resembles wheat germ agglutinin, which has a similar but not identical sugar specificity. 1 The mechanism of its antimitogenic effect remains to be determined. It is not toxic to the cells. Acknowledgments Part of this work was supported by United States Public Health Service Institutional Grant RR05399 and by United States Public Health Service Grant HL21328. a A. K. Allen and A. Neuberger, this series, Vol. 50 [37].
[30] D a t u r a
stramonium Lectin
By JANE F. CROWLEY and IRWIN J. GOLDSTEIN
The seeds ofDatura stramonium contain a lectin that is specific for oligomers o f N-acetyl-D-glucosamine. 1~ The Datura lectin is one o f a family o f chitin binding lectins that have been discovered in seeds o f the 1 M. Eisler and L. Portheim, Z. lmrnunitaetsforsch. Exp. Ther. 1 1, 151 (1908). 2 G. I. Pardoe, G. W, G. Bird, G. Uhlenbruck, I. Sprenger, and M. Heggen, Z. Immunitaetsforsch. Allerg. Klin. Immunol. 140, 374 (1970).
METHODS IN ENZYMOLOGY,VOL. 83
Copyright © 1982by Academic Press, Inc. All rights of reproductionin any formreserved. ISBN 0-12-181983-3