[48] Evonymus europaea lectin

552

CARBOHYDRATE-BINDING PROTEINS

[48]

[48] Evonyrnus europaea L e c t i n

By JERZY PETRYNIAK and IRWIN J. GOLDSTEIN

Introduction The arils from seeds of the spindle tree Evonymus europaea contain a lectin which agglutinates A2, B, and O erythrocytes,l-4 stimulated murine and guinea pig peritoneal cells: and Ehrlich ascites tumor cells. 6 The B and H specificity is an intrinsic property of a single lectin binding site, since absorption and elution from a blood-group type H immunoadsorbent gives an agglutinin with B as well as H specificity2 The E. europaea lectin is not inhibited by any monosaccharide. The lowest molecular weight inhibitors are disaccharides; the most convenient, lactose. Assay Methods

Ouchterlony Gel Diffusion The Ouchterlony double diffusion method is simple and convenient for qualitative testing of the reaction of the lectin with macromolecular substances. The most convenient glycoprotein used for assaying the lectin is a 0.1% solution of blood-group A + H substance obtained from porcine stomach mucin. A plate containing 1.5% agar in 0.05 M sodium barbital buffer, pH 8.2, or in 1% agar in 0.1 M phosphate buffer, pH 7.2, containing 0.02% NaN3 is prepared.

Precipitin Reactions A modification of the quantitative microprecipitin technique described by Kabat 7 can be used. Reactions are carried out in 1.5-ml polypropylene microcentrifuge tubes with caps (obtained from Curtin Matheson ScienG. Schmidt, Z. lmmunitiitsforsch. 111, 432 (1954). 2 M. Kriipe, " B l u t g r u p p e n s p e z i f i s c h e Pflanzliche EiweisskOrper (Phytagglutinine),'" p. 45. Enke, Stuttgart, 1956. 3 F. Pacfik and J. K o c o u r e k , Biochim. Biophys. Acta 400, 374 (1975). 4 j. Petryniak, M. E. A. Pereira, and E. A. Kabat, Arch. Biochem. Biophys. 178, 118 (1977). 5 j. Petryniak, D. Dug, a n d J. Podwifiska, Eur. J. Irnmunol. 13, 459 (1983). 6 U n p u b l i s h e d data. 7 E. A. Kabat, in " K a b a t and M a y e r ' s E x p e r i m e n t a l I m m u n o c h e m i s t r y , " 2nd ed., p. 542. T h o m a s , Springfield, Illinois, 1961.

METHODS IN ENZYMOLOGY, VOL. 138

Copyright © 1987by AcademicPress, Inc. All rights of reproduction in any form reserved.

[48]

Evonymus

europaea LECTIN

553

tific, Inc., Houston, Texas). Approximately 40 /xg of lectin protein is mixed with varying amounts of glycoconjugates in a final volume 250 ~1 of 0.01 M sodium phosphate buffer, pH 7.1, in 0.15 M NaCI with 0.02% azide (PBS). The tubes are incubated at 37° for 1 hr and kept at 4* for 5-7 days. Precipitates are centrifuged at 15,600 g for 10 min at 4 °, and supernatant solutions are decanted. The protein in the washed precipitates is determined by the procedure of Lowry et al. 8 Hemagglutination Procedure Hemagglutination assays are conducted using a hemagglutination plate and a 25-/xl microdiluter (Cooke Engineering Co.). After serial dilution of the lectin solution with phosphate-buffered saline (0.15 M NaC1, 0.01 M sodium phosphate, pH 7.0), 25/xl of a 3% suspension of type B or O erythrocytes is added to each well. The degree of agglutination is determined after 1 hr at room temperature. The titer is defined as the reciprocal of the highest dilution showing detectable agglutination. Purification Procedures The purification of Evonymus lectin by a conventional technique was described by Pac~tk and Kocourek. 3 There are also two procedures based upon the principle of affinity chromatography using (1) poly (L-leucine) hog A + H blood group substance as affinity adsorbent 4 and (2) asialoglycophorin coupled to Sepharose 2B as affinity adsorbent. 9 The first affinity method in which lectin is bound to blood group H determinants allows purification of relatively large quantities of lectin. Thus 2 g of polyleucyl hog A + H substance was employed to purify 230 mg of lectin. However, this technique is laborious because of the batchwise procedure and the necessity of repeated high speed centrifugations. Although the structure of the lectin-reactive oligosaccharide on glycophorin which binds Evonymous lectin is unknown, 1° this method is more convenient and faster than the first one, due to the use of column chromatography and the low lactose concentration required for elution of the lectin. The disadvantage of this method is the lower yield of lectin when compared with the first technique. Thus, 22-26 mg of lectin can be obtained from 1 run on the 150-ml column. 8 0 . H. Lowry, N. J. Rosebrough, A. L. Farr, and R. J. Randall, J. Biol. Chem. 193, 265 (1951). 9 j. Petryniak, M. Janusz, E. Markowska, and E. Lisowska, Acta Biochim. Pol. 28, 267 (1981). to j. Petryniak, B. Petryniak, K. Wa~niowska, and H. Krotkiewski, Eur. J. Biochem. 105, 335 (1980).

554

CARBOHYDRATE-BINDING PROTEINS

[48]

Preparation of Extract Extract can be prepared from whole seeds or from arils of the seeds. Whole Seeds Extract. The seeds of Evonymus europaea are ground in a mortar to a fine slurry and extracted with 9 volumes of phosphatebuffered saline, pH 7.1, containing 0.2% sodium azide. The suspension is kept overnight at 4 ° with occasional stirring. The precipitate is centrifuged at 2000 rpm for 1 hr at 4 °, and the supernatant solution is decanted and cleared of fat by filtration on a Bfichner funnel, using Whatman No. 1 paper, and then through a 0.45-/zm Millipore filter. The precipitate is reextracted twice with PBS; a fourth extract is free of hemagglutinating activity. The three extracts show a decrease in agglutination of human B and O erythrocytes from an initial titer of 16. Arils Extract. Arils are peeled off from seeds by pressing seeds in hands, followed by shaking on a Petri dish. Fifty grams of arils is defatted by treatment with 150 ml of cold methanol at 4 ° for 1 hr with occasional stirring. The defatted arils are extracted with 9 ml of PBS per gram of arils. The initial four extracts, containing over 90% of the hemagglutinating activity, were combined and used for lectin purification. The hemagglutinating titer of the combined extracts varied from 1 : 64 to 1 : 512.

Preparation o f Affinity Absorbents Poly(L-leucine) Hog A + H Blood-Group Substance. Hog A + H blood-group substance is purified from hog gastric mucin (Sigma) by an ethanol precipitation procedure.ll Insoluble poly (L-leucine) hog A + H blood-group substance is prepared by the copolymerization of the bloodgroup substance with L-leucine N-carboxyanhydride according to Tsuyuki et al., ~z as described by Kaplan and Kabat. ~3The polyleucyl hog A + H is mixed with Celite in the ratio 1 : 1 w/w, and washed extensively with PBS until Azso nm -< 0.03. Washings are separated from the immunoadsorbent by centrifugation at 12,000 rpm for 1 hr at 15° in a Beckman L-2 centrifuge using an LW 19 rotor; this avoids loss of fine particles. Preparation o f Asialoglycophorin A-Sepharose 2B Conjugate. Glycophorin was obtained from human blood-group O,MN erythrocyte membranes by phenol-water extraction. ~4Sialic acid is released by hydrolysis 11 E. A. Kabat, "Blood Group Substances: Their Chemistry and Immunochemistry." Academic Press, New York, 1956. 12 H. Tsuyuki, H. Van Kley, and M. A. Stahmann, J. Am. Chem. Soc. 78, 764 (1956). 13 M. E. Kaplan and E. A. Kabat, J. Exp. Med. 123, 1061 (1966). ~4T. Baranowski, E. Lisowska, A. Morawiecki, E. Romanowska, and K. Str6iecka, Arch. lmmunol. Ther. Exp. 7, 15 (1959).

[48]

Evonymus europaea LECTIN

555

in 0.025 M H2SO4 at 80° for 40 min and removed by dialysis. ~5 Sepharose 2B (Pharmacia Fine Chemicals, Uppsala, Sweden) was activated by the cyanogen bromide technique. ~6 To the activated Sepharose (150 ml) is added 150 mg of the asialoglycophorin. The coupling procedure is carried out according to the instructions of Pharmacia Fine Chemicals; 92% of the desialized glycophorin is coupled to the gel. The conjugate is stored at 4°. Affinitiy Chromatography Affinity Chromatography on Poly(L-leucine) Hog A + H Blood-Group Substance. 4 A batchwise technique is used. The washed affinity adsorbent (2 g of polyleucyl hog A + H mixed with 2 g of Celite) is mixed with 1.2 liters of crude extract and left for 1 hr in the cold with occasional stirring. The suspension is then subjected to ultracentrifugation at 12,000 rpm for 1 hr, and the supernatant is discarded, if flee of hemagglutinating activity. The lectin is completely adsorbed. Additional Evonymus extract is added to the 2 g of immunoadsorbent, but saturation is not reached even with 10 liters of crude extract. The polyleucyl hog A + H substance containing the lectin is then washed with PBS, pH 7.1, and centrifuged at 12,000 rpm until the A280,m washings decreased to 0.03. Specific elution is carried out by suspending in 90 ml of 0.5 M lactose in PBS containing 0.02% sodium azide, pH 7.1, and incubation for 15 min at 37°, followed by centrifuging at 20,000 rpm for 1 hr at 15° in a Beckman L-2 centrifuge using an SW 25.1 rotor. The eluates are decanted and combined. Elution is repeated until the A280nmof the eluate is decreased to 0.05 above that of the lactose. The yield of lectin varied for different preparations from 120 to 230 mg per 370 g of Evonymus seeds. Eluates are concentrated and dialyzed against 4 changes each of 50 volumes of PBS until periodate-positive material was not found in a 24-hr dialyzate; three additional 24-hr dialyzates were carried out before the material was used. During dialysis 2456% of the protein precipitated. Affinity Chromatography on Asialoglycophorin A-Sepharose 2B Conjugate. 9 All procedures were performed at 4°. Before each run, the column containing the conjugate is washed consecutively with 0.15 M NaCI/0.01 M NaHCO3, 0.15 M NaC1/0.01 M acetate buffer, pH 4, and finally with PBS. The E. europaea extract is centrifuged for 1 hr at 2000 rpm directly before application to the affinity column to remove traces of precipitate which might impair or stop the flow of extract on the column. J5 E. Lisowska and M. Duk, Arch. lmmunol. Ther. Exp. 24, 39 (1976). 16 p. Cuatrecasas, J. Biol. Chem. 245, 3059 (1970).

556

CARBOHYDRATE-BINDING PROTEINS

[48]

jj

-

02

20 ~_

15

c ._o

B a a a a m a D a

I'lnt~oa

2:4 2B Elution volume I FIG. 1. Affinity chromatography o f the E. europaea lectin on an asialoglycophorin A 0

0.'4

0'8 " 2.1

Sepharose 2B column. 9 The crude lectin extract (400 ml, hemagglutinating titer 1 : 64) was applied to the 1.6 × 55 cm column, followed by washing with PBS until the absorbance at 280 nm was less than 0.005; the flow rate was 56 ml/hr, and 14-ml fractions were collected. The lectin was elpted with 0. I M lactose in PBS at a flow rate of 30 ml/hr; 7.5-ml fractions were collected. The arrow shows the point of application o f the lactose solution. For determination o f hemagglutinating activity the aliquots of fractions eluted with lactose were dialysed against PBS. Agglutination score was counted as follows: 4+ = 10; 3+ = 8; 2+ = 5; 1+=3.

After exhaustive washing of the column with PBS, the bound lectin was eluted with 0.1 M lactose (Sigma). Active fractions showing hemagglutinating titer not lower than I : 2 were pooled, concentrated and dialyzed in a Diaflo chamber, using a UM I0 membrane Amicon (Holland), until no periodate-positive material 7 was found in the ultrafiltrate. The maximal lectin binding capacity of the adsorbent was about 4 ml of the extract with hemagglutinating titer 1 : 256 per 1 ml of the conjugate. The elution pattern of E. europaea extract from the affinity column is shown in Fig. I, and results of purification are presented in Table I.

Properties

o f E v o n y r n u s europaea L e c t i n

Stability During 8 years of storage at 4 ° (the longest period observed) the purified lectin solution retained its activity. Lyophilized preparations are also stable for at least 2 years.

Evonymus europaea LECTIN

[48]

557

TABLE 1 PURIFICATION OF THE

Purification step

Volume (mi)

Crude extract Lactose eluatea

400 18

E.,europoea

LECTIN ON AN ASIALOGLYCOPHORIN A - S E P H A R O S E 2 B COLUMN 9

Total hemagglu- Specific Total tinating activity Recovery Purifiprotein activity (units/mg of activity cation (mg) (units) protein) (%) factor 820 22

25 600 18 400

31 836

100 72

27

After dialysis and concentration.

Homogeneity lmmunochemical. Evidence was obtained supporting immunochemical homogeneity of Evonymus lectin. The lectin displaced by elution with lactose from poly(L-leucine) hog A + H blood-group substance was 78% specifically precipitated by blood-group B substances, 4 the remainder probably being in the form of soluble complexes since such precipitates have appreciable solubility. Physicochemical. By physicochemical criteria the lectin shows several distinct populations of protein molecules. In the analytical ultracentrifuge using schlieren optics, the purified lectin in PBS, pH 7.1, sedimented as 2 peaks: a large symmetrical peak with S°0,w = 7.8 (calculated by extrapolation to zero concentration) and a small, diffuse, slow-moving peak. The slow-moving peak is a subunit of the fast-moving peak as it was observed that an increase of pH of the lectin solution up to pH 9 caused the disappearance of fast-moving peak and an increase in the amount of the slow-moving peak. 6 Thus the lectin shows pH-dependent dissociation into subunits. Upon immunoelectrophoresis against rabbit antisera to the crude extract, the isolated hemagglutinin gave three bands as compared with nine in the crude extract. These bands all migrated toward the anode. The intrinsic viscosity ~ was 0.057 dl/g. Disk gel electrophoresis in polyacrylamide, pH 9.4, showed three distinctive bands; two intense fast-moving bands, the faster being more diffuse, and one slow-moving band. Three additional very faint bands of intermediate mobility were seen. Analytical isoelectric focusing revealed heterogeneity of the purified lectin. Six very close bands with isoelectric points in the range 4.3-4.7 were obtained on ampholine, pH 3-6 and 3-10.

558

CARBOHYDRATE-BINDING PROTEINS

[48]

Molecular Weight. The molecular weight of the E. europaea lectin calculated from the intrinsic viscosity 0.057 dl/g, sedimentation coefficient S°0,w7.8, and fi of 0.71 was approximately 166,000. 4 However, Pacfik and Kocourek, 3 using the Yphantis method, reported 126,700 and 119,200 for two preparations of Evonymus: phytohemagglutinin I and a mixture of isophytohemagglutinins, respectively. Subunit Structure On sodium dodecyl sulfate-polyacrylamide gels the purified lectin revealed two intense bands of Mr 17,000 and 35,000 and two faint bands of 53,000 and 67,000. This pattern changed on reduction with 0.1% 2-mercaptoethanol. The 53,000 and 67,000 bands disappeared, the intensity of the 35,000 decreased, and the 17,000 band increased, indicating some subunit structure partly covalently and partly noncovalently linked. Composition. The Evonymus lectin is an acid glycoprotein containing 4.8% D-galactose, 2.9% D-glucose, and 2.8% N-acetyl-D-glucosamine. The amino acid composition is noteworthy for its high content of aspartic acid (13.5%) and glutamic acid (7.1%) as well as threonine, proline, glycine, alanine, valine, leucine, and half-cystine. Specificity

Evonymus lectin exhibits a complex type of carbohydrate-binding specificity. The purified lectin precipitates well with B, H, and Aa bloodgroup substances but not with A~ substance. Inhibition of precipitation with milk and blood-group oligosaccharides showed the lectin to be most specific for blood-group B oligosaccharides having the basic structure aoGal(1 ~ 3)[aLFuc(1 ~ 2)]/3DGaI(1 ---> 3 or 4)/3DGlcNAc. The lectin does not discriminate between type 1 and type 2 oligosaccharide chains, having the core /3DGaI(1 ~ 3)flDGIcNAc or/3oGal(l --~ 4)/3oGIcNAc, respectively, and both types are equally active, both in inhibition and precipitation tests. 4,n7Evonymus lectin is also inhibited by blood-group H oligosaccharides, but to a lesser degree. For 50% inhibition of precipitation, 3.5,850, and 290,000 nmol of B and H oligosaccharides and lactose, respectively, are required. 4 Studies with synthetic glycoconjugate shows that Evonymus europaea lectin precipitated with aDGal(1 ~ 3)13oGal(1 ~ 4)/3DGIcNAc-BSA, aLFuc(1 ~ 2) /3DGaI(1---> 3)I3DGIcNAc-BSA, aLFuc(l ~ 2)/3DGaI(I t7 j. Petryniak and I. J. Goldstein, Biochemistry., 25, 2829 (1986)

Evonymus europaea

148]

'~

LECTIN

559

~

~

~1

t

t

t~-~

•.

"~ ~ . , @

e-

E I* e~

e-

E

< 0

z

Z < Z

o

x--

E

._= I-,

Z

'C" e.,

t

.m

0

°

t

<

,,,,,

N e~

0 :o

I-

z

0

e~

E e~ e-

~m

0 0

tt

b

m

e-

r~

e-.

e~

b-

eL. e~

560

CARBOHYDRATE-BINDING PROTEINS

[48]

4)DGIcNAc, and aDGal(1 --~ 3)[ctLFUC(I --> 2)]BDGaI-BSA. However, the lectin neither precipitated with otLFuc(1 --~ 2)BDGaI-BSA, aDGal(1 --~ 3)BDGaI-BSA, or BDGaI(1 --~ 4)BDGIcNAc-BSA nor agglutinated erythrocytes of Oh phenotype having multiple terminal BDGaI(I --~ 4) BDGicNAc residues.J7 These results indicate that the minimal structural requirement for glycoprotein precipitation or for cell agglutination by the lectin includes any of the 3 trisaccharides (fucosylated or nonfucosylated) derived from the blood-group B tetrasaccharide (see Table II). The monosaccharides linked to the B-D-galactosyl residue in the blood-group B tetrasaccharide (a-D-galactose, a-L-fucose, and B-N-acetyl-D-glucosamine) participate almost equally in binding to the lectin inasmuch as removal of any one of these sugars reduces the inhibiting potency of the resulting trisaccharide to the same degree, aLFuc(1 --> 2)BDGaI(1 --~ 3)BDGIcNAc-BSA (H type 1) and aLFuc(l --~ 2)BDGaI(I --* 4)BDGIcNAc (H type 2) were precipitated to the same extent. The E. europaea lectin neither precipitated aDGal(l --~ 4)BDGaI(I ---> 4)BDGIcNAc-BSA, Lea-BSA, Leb-BSA, or BDGIcNAc(1 --~ 4)[otLFuc(1 --~ 6)]BoGIcNAc-BSA nor agglutinated Oh,Le a and Oh,Le b erythrocytes, demonstrating that terminal D-galactose linked a(l --~ 4) to subterminal B-D-galactose or a-L-fucose linked to N-acetylglucosamine prevents lectin binding. CPK molecular models, built on the basis of data from IH-NMR and HSEA calculations provided by Lemieux et al., j8 show that these a-D-galactosyl and O~-L-fUcosyl groups act to sterically hinder lectin binding to these oligosaccharides; these observations also suggest that the lectin binds to the B-side of these oligosaccharides. The B-sides, both on the blood-group H type I and type 2 oligosaccharides, provide a similar contour which can fully account for their equal reactivity with E. europaea lectin. Among the L-fucose binding lectins (Evonymus europaea, L o t u s tetragonolobus, and Ulex europaeus I) which react with blood-group H oligosaccharides, E v o n y m u s lectin is the only one which shows the unique ability to react with H type 1 oligosaccharides, E v o n y m u s lectin also precipitates a number of other glycoproteins containing some oligosaccharides of unknown structure. These include asialoglycophorin from human erythrocytes of both O and Oh ("Bombay") phenotypes, l°,19 laminin from EHS sarcoma tumor cells, 19 as well as many blood-group glycoproteins obtained from human ovarian cysts of HLeb,Lea activity, I active precursor blood-group substance. 4 18R. U. Lemieux, K. Bock, L. T. J. Delbaere, S. Koto, and V. S. Rao, Can. J. Chem. 58~ 631 (1980). ~9In preparation.

[49]

LECTIN FROM HOG PEANUT

561

Binding of E. europaea Lectin to Glycolipids Evonymus lectin binds to blood-group B and H active glycolipids as detected by autoradiography, with ~25I-labeled lectin, on thin-layer chromatograms. 19

[49] L e c t i n f r o m H o g P e a n u t , A m p h i c a r p a e a bracteata

By M.

MALIARIK,

D. ROBERTS, and I. J. GOLDSTEIN

The anti-A seed lectin from Amphicarpaea bracteata ~is one of several Leguminosae lectins specific for terminal nonreducing N-acetyl-D-galactosamine (D-GalNAc) 2 groups. The species Arnphicarpaea exhibits two types of seeds, one above ground and one which develops at the base of the stem, near or below the soil surface. The lectin purified from both seeds appears to be identical. The lectin, originally purified by affinity chromatography on N-acetyl-D-galactosamine coupled to epoxy-Sepharose 6B, was reported to be homogeneous by SDS-polyacrylamide electrophoresis, and an initial characterization was reported. 3 We describe here a new purification procedure and further characterization of the physical and chemical properties of the A. bracteata lectin. Assay Methods

Hemagglutination. Hemagglutination assays are conducted in a hemagglutination titer plate using 50-/xl serial dilutions of lectin solution in phosphate-buffered saline (PBS: 0.1 M sodium phosphate, 0.15 M NaCI, pH 7.1). Fifty microliters of 2-3% suspension of type A~ erythrocytes in 0.9% NaC1, 10 mM potassium phosphate, pH 7.4, are added to each well. The agglutination titer is determined after 1 hr at room temperature and defined as the reciprocal of the highest dilution causing agglutination. Quantitative Precipitation Inhibition Assay. The carbohydrate-binding specificity of the lectin was determined by a precipitation inhibition assay. Lectin (30/zg) was mixed with type A substance (20/zg) to which O. M~kelii and P. Makel/i, Ann. Med. Exp. Biol. Fenn. 34, 403 (1956). 2 Abbreviations: D-GalNAc, 2-acetamido-2-deoxy-o-galactopyranose; Mea- and Me/3GalNAc, methyl 2-acetamido-2-deoxy-a- and -/3-D-galactopyranosides, respectively; PBS, phosphate-buffered saline. 3 L. J. Blacik, M. Breen, H. G. Weinstein, B. A. Sittig, and M. Cole, Biochim. Biophys. Acta 538, 225 (1978).

METHODS IN ENZYMOLOGY, VOL. 138

Copyright © 1987 by Academic Press, Inc. All rights of reproduction in any form reserved.