Isolectins from wax bean with differential agglutination of normal and transformed mammalian cells

Biochimica et Biophys~ca Acta, 31o /1973) 273-277

:.0 Elsevier Scientific Publishing Company, Amsterdam - Printed in The Netherlands BBA 36403 ISOI.ECTINS FROM WAX BEAN W I T H D I F F E R E N T I A L AGGLUTINATION OF NORMAL AND TRANSFORMED MAMMALIAN CELLS

t3EN-.\MI SEI.A, [t.\I.INA I.IS, NATItAN SH:klC.ON AXD I.EO SACItS Departments of Genetics a~ld Biophysics, IVeizmann l,stllute of Science. Rehovoth (Israel)

(lleceived November 27th, 1972)

SUMMARY Wax bean agglutinin prepared according to Liener was separated by chromatography on hydroxylapatite into three fractions, two of which were biologically active. Both fractions are glycoproteins with essentially identical amino acid composition and molecular weight, each consisting of four subunits, molecular weight 3o ooo. The isolectins agglutinated transformed cells at a concentration about IOO times lower than that required to agglutinate normal cells. Agglutination was inhibited by fetuin but not by any of the simple sugars tested.

Cell agglutinating proteins, also known as lectins, are widely distributed in plants I. Many of them combine specifically with saccharides and serve as molecular probes for the study of polysaccharides, glycoproteins and sugars on cell surfaces 2--1°. Some lectins have been found to differentially agglutinate normal and transformed cells2-~. The isolation and properties of an agglutinin from the yellow wax bean (Phaseolus vulgaris) have been reported by Liener and his coworkers n. In this communication we describe the separation of this agglutinin into three fractions, two of which are biologically active. The two isolectins are hemagglutinating and activate lymphocytes. They agglutinate transformed cells at very low concentrations (0.2-0. 5/~g/ml). The amino acid composition, sugar content and molecular weight of the two isolectins arc essentially identical and the basis of their separation on hydroxylapatite is unknown. The presence of closely related multiple fl~rms of lectins has also been observed in soybean and other plantsL The variety of Phaseolus vulgaris used in this work was stringless Brittle-Wax, obtained from Hazera Co., Haifa, Israel. The initial purification of the agglutinin was performed according to Takahashi et al. n, and involved fractionation with (NH4) 2 SO, and chromatography on columns of DEAE-cellulose and CM-cellulose. The active material eluted from the CM-cellulose column was further purified by chromatography on a column of hydroxylapatite. I00 mg in lyophilized form were dissolved in 20 ml of o.ooi M phosphate buffer, pH 6.8, and applied to a column (2.5 cm × 3o cm) of

274

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h y d r o x y l a p a t i t e (Calbiochem) e q u i l i b r a t e d with the same huffer. The column was washed with buffer until the absorbance at 28o nm of the effluent was below o.oi. E l u t i o n was carried out by stepwise increases in the concentration of p h o s p h a t e buffer, p H 6.8. F r a c t i o n s of zo ml were collected, assayed for their a b s o r b a m ' e at 280 nm a n d for their a g g l u t i n a t i n g a c t i v i t y t o w a r d s t r y p s i n i z e d r a b b i t e r y t h r o c y t e s using the s p e c t r o p h o t o m e t r i c inethod of Liener r'. The elution p a t t e r n is shown in Fig. z. "the tubes corresponding to each peak were pooled, d i a l y z e d exhaustiveh" against w a t e r and lyophilized. r

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Fig. i. Chromatography on hydroxylapatite of wax bean agglutinin prepared according to Takahashi et al. s. too mg dissolved in 2o ml t mM phosphate buffer, pH 6.8, were applied to a column (z.5 cm × 3° cm) of hydroxylapatite in equilibrium with the same buffer. Elution was carried out by stepwise increases in the concentration of phosphate buffer, pH 0.8. The arrows indicate the points of change in the buffer concentration. Three fractions were obtained. ]'he first, eluted with o.z M buffer, contained a b o u t 8o% (w/w) of the a p p l i e d m a t e r i a l and was devoid of a n y a g g l u t i n a t i n g a c t i v i t y , either t o w a r d s e r y t h r o c y t e s or fibroblasts. A c t i v i t y was, however, fi)und in F r a c t i o n s II and I11, eluted with o.2 and o. 4 M buffer and representing a b o u t I5 and 5 % of the s t a r t i n g material, respectively. Upon r e - c h r o m a t o g r a p h y of the isolated F r a c t i o n s l 1 and I I I , no change was observed in their p a t t e r n of elution or in their bi(flogical activity. Amino acid analysis of the two fractions did not show a n y significant differences. The two fractions had the same c o n t e n t of neutral sugar (about 8%) as d e t e r m i n e d b y the phenol m e t h o d of Dubois 'a with D-mannose as s t a n d a r d . P a p e r c h r o m a t o g r a p h y in ~ - b u t a n o l - p y r i d i n e - - w a t e r (6:4: 3, v/v/v) of the acid h y d r o l y s a t e s (I M I1('1, 6 h, Ioo °C) of the two fractions revealed the presence of mannose, arabinose, glucose, galactose a n d fucose. These results are in a g r e e m e n t with those r e p o r t e d by T a k a h a s h i et al. n . E s t i m a t i o n of molecular weight b y gel filtration on columns of S e p h a d e x G-z5o superfine gave values in the range of z25 ooo =:_ 5ooo for both fractions as c o m p a r e d with the value of I32 ooo r e p o r t e d b y T a k a h a s h i el al. n. E x a m i n a t i o n of the two fractions on p o l y a c r y l a m i d e gel electrophoresis in the presence of sodium dodecvl sulfate i n d i c a t e d t h a t both fractions are comprised of subunits of tool. wt 3 ° ooo (Fig. z); each of the i n t a c t fractions is thus m a d e up of fi)ur subunits.

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Fig. 2. Mobility of Fractions iI and 1[[ on polyacrylamide gel electrophoresis in the presence of sodium dodecyl sulfate. The runs were made on a 8-i5°j, linear gradient gel in o.ol M phosphate buffer, containing 2°,/o sodium dodecyl sulfate. Bovine serum albumin, ovalbumin, glyceraldehyde phosphate dehydrogenase and chymotrypsinogen were used as markers. The arrows indicate the mobilities of Fractions i1 and lII.

Tile two fractions were tested for agglutination with cultured normal golden hamster embryo fibroblasts, non-transformed cells from tile mouse cell line 3T3, hamster embryo and 3T3 cells transformed by polyoma virus or Simian virus 4 o, and hamster cells transformed after treatment with the chemical carcinogen dimethylnitrosamine. The cells were grown as described, and tested for agglutinatione, 5 three days after seeding lO6 cells per IOO m m Petri dish. At a concentration of I - 2 /~g/ml, both fractions completely agglutinated ( + + _ + , refs 2 and 5) transformed cells and trypsinized normal and 3T3 cells. Trypsinization was carried out with crystalline trypsin (I Fg/ml) for 5 min at 37 °C, and terminated by the addition of a 5-fold excess of soybean trypsin inhibitor. The concentration of Fractions 1I and I I I required to agglutinate non-trypsinized normal cells was about IOO times higher than t h a t required for the agglutination of the treated cells. The agglutinating activity of Fractions II and I i [ with trypsinized rabbit erythrocytes was 9000 and I 5 o(io hemagglutinating units/mg N (ref. I2), respectively. Both fractions activated mouse spleen lymphocytes to incorporate :aH:thymidine into DNA. (;ell agglutination was inhibited by very low concentrations of fetuin (Gibco, prepared according to Spiro method; 5/~g/ml gave 5or},o inhibition) and to a lesser extent by ovomucoid and bovine submaxillary mucin (Worthington; 50 #g/ml gave 5o°,o inhibition). None of a number of monosaccharides tested (I~-galactose, It-mannose, o-glucose, l.-fucose, N-acetyl-D-glucosamine and N-acetyl-D-galactosanline at a concentration of I mg/ml, or sialic acid and sialyl lactose at 25o/~g/ml) showed any inhibitory activity. Removal of the terminal sialic acid from fetuin by treatment with purified neuraminidase (Behringwerke; 5o #g per 5 mg fetuin per ml in acetate buffer, pH 5-5, 16 h at 37 °C) did not change the inhibitory activity of fetuin. This result differs from previous findings~4; the discrepancy may, however, be due to the use of a crude preparation of neuraminidase in the earlier work ~4. Marked enhancement of

276

B.-,\. SELA {'t al.

the i n h i b i t o r y a c t i v i t y was obtaine.d when fetuin (5 mg/ml in a<'etate lmffer, pH 5-5) was heated for 3 min at i o o ~C. On the other hand, digestion by pronase reduced the i n h i b i t o r y a c t i v i t y of fetuin t)3, a factor of 5 - I o . The residual a c t i v i t y was h m n d to be associated with the crude g l y c o p e p t i d e fraction isolated by gel filtration on Sephadex (;-5o of the pronase digest. These results suggest t h a t the i n h i b i t o r y a c t i v i t y of fetuin is not due to the interaction of the agglutinin with the t e r m i n a l sialic acid but r a t h e r with some other p a r t of the c a r b o h y d r a t e chain. The e x t e n t of inhibition appears to depend on some s t r u c t u r a l features of the fetuin molecule. A similar phenomenon was observed in the s t u d y of glycoprotein inhibitors of the influenza virus h e m a g g l u t i n a t i o n reaction is. Thus, proteolytic digestion of ovine s u b m a x i l l a r y glyc o p r o t e i n almost c o m p l e t e l y abolished its i n h i b i t o r y a c t i v i t y . On the other hand, p o l y m e r i z a t i o n of cq-acid glycoprotein (orosomucoid) by cr~sslinking or by heating, resulted in a 6oo-fi)ld increase in its i n h i b i t o r y a c t i v i t y . It is possible t h a t the enh a n c e m e n t of the i n h i b i t o r y a c t i v i t y o b t a i n e d upon heating of fetuin is due to polym e r i z a t i o n ; a l t e r n a t i v e l y , the p a r t of the c a r b o h y d r a t e chain of t~tnin for which the wax bean agglutinins are specific, becomes more accessible to the lectins upon heating. T h a t lectins do not necessarily i n t e r a c t with t e r m i n a l sugars, b u t can bind to inner portions of saccharide chains, has been d e m o n s t r a t e d recently in a n u m b e r of cases ~<~r Concanavalin A can inhibit t u m o r developnaent in z,iz.0~s,l:~ and Fraction I I I was tested for this p r o p e r t y . T u m o r s were produced in 5 b-week-old h a m s t e r s bv subc u t a n e o u s inoculation of IO6 cells of p o l y o m a - or dimethvlnitrosanaine-transfl~rnwd h a m s t e r cells TM.4 IO d a y s after inoculation, p a l p a b l e t u m o r s developed, ranging from 3 6 m m in diameter. Injection of r m g of F r a c t i o n I I I into the t u m o r s c o m p l e t e l y inhibited t u m o r developnaent in 34 out of 4 ° animals (85~'{,) up to IbO d a y s after the last control animal with t u m o r s had died. S u b c u t a n e o u s injection of hamsters without t u m o r s with I m g of F r a c t i o n I I I led to local necrosis and ulceration of the normal tissues, which was r e p a i r e d within 3 weeks, leaving a scar. The inhibition of t u n m r d e v e l o p m e n t b y the wax bean lectin thus required a lower c o n c e n t r a t i o n than t h a t required with concanavalin A Is for the same tumors. The work was s u p p o r t e d by ( ' o n t r a c t No. 6q-o214 with the Special Virus Cancer Program of the National Cancer I n s t i t u t e , United States Public Health Service. We are i n d e b t e d to Mr R. l . . t a n for the poly'acrylamide gel electrophoresis and t,) l.)r A. Novogrodski for the d e t e r m i n a t i o n of mitogenic a c t i v i t y . I¢.EFEhl F_NCES Sharon, N. and l.is, H. (rc)7-) Science 177, q49-95q - lnbar, M. aml Sachs, L. (1969) Proc. Natl. Acad..b'ci. U.N. %~, l.tlS-t4- 5 3 lnbar, M. and Sact~s, 1.. (r969) Nature 223, 71o 7t2 .it [¢urger, 3t..M. (1909) Proc. Natl. Acad. Nci. l.'.S. 62, 994-loot 5 Sela, B., IAs, t4, Sharon, N. and Sachs, L. (197o) J. Membrane Bug. 3, 2f'7 27q 6 Sela, B., Lis, H., Sharon, N. anti Sachs, L. (i97 t) Biochim. Biophys..-tcta 24q, 504 5~>8 7 Inbar, M., Ben-13mssat, H. and Sachs, L. (t972) Nat. New Biol. 23¢), 3-t~ 8 S h o h a m , J. and Sachs, l.. (x972) Proc. Natl. Acad. Sci. U.S. 69, 2479-2482 9 Nicolson, G. I.. and [31austein, J. (1972) Biochim. Bi,~phys. Acta 260, 543-.5.17 xo Vlodavsky, 1., Inbar, M. anti Sachs, L. (1972) Biochim. Biophys. Acta 274, 3t~4 309 I 1 Takahashi, T., Ramachandramurthy, P. and Liener, 1. E. (I9(~7) Biochint. Biophys. Acta t33, 123--I33 12 IAener, I. E. (I955) Arch. Biochem. Biophys. 54, 223-23t I3 l)ubois, M., Gilles, 1(. A., Hamilton, J. K., Rebers, I'. A. and Smith, F. (t956) .4,lal. Chem. 28, 3.50--356

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