Hapten inhibition of adsorption: Specificity of the Sophora japonica lectin

Journal of Immunological Methods, 8 (1975) 169--174 © North-Holland Publishing Company, Amsterdam -- Printed in The Netherlands

HAPTEN INHIBITION OF ADSORPTION: SPECIFICITY OF THE SOPHORA JAPONICA LECTIN*

SUE MIN CHIEN, SNEH SINGLA and R.D. PORETZ** Department of Biochemistry and Molecular Biology, University of Kansas Medical Center, Kansas City, Kansas 66103, U.S.A. (Received 4 December 1974, accepted 11 March 1975)

Since hapten inhibition of precipitation is relatively time consuming, we have developed a hapten inhibition of adsorption assay to explore the sugar binding specificity of the Sophora japonica lectin. Adsorbents were prepared with Sephadex and p-aminophenyl ~-D-galactopyranoside using the CNBr procedure. The ability of simple saccharides to inhibit the binding of lectin to the adsorbent was performed employing a fixed amount of adsorbent and lectin and varying quantities of inhibitor. Plots of the percent inhibition of adsorption with respect to the logarithm of micromoles of inhibitor yielded sigmoid shaped curves. The quantities of various saccharides required to cause 50% inhibition of the lectin--adsorbent interaction relative to D-galactose were within ± 5% of the relative inhibiting potencies of the sugars in the lectin--blood group substance precipitation reactions.

INTRODUCTION Previously, the detailed analysis o f the c a r b o h y d r a t e binding specificities o f lectins had been c o n d u c t e d either b y h a p t e n i n h i b i t i o n o f a g g l u t i n a t i o n (Watkins and Morgan, 1 9 5 2 ) or h a p t e n i n h i b i t i o n o f p r e c i p i t a t i o n (So and Goldstein, 1 9 6 7 ; P o r e t z , 1972). T h e f o r m e r p r o c e d u r e , w h i c h is relatively rapid, gives at best, o n l y s e m i - q u a n t i t a t i v e results. H a p t e n i n h i b i t i o n o f prec i p i t a t i o n yields highly r e p r o d u c i b l e and q u a n t i t a t i v e d a t a b u t requires 2--3 d a y s f o r c o m p l e t e analysis. F u r t h e r m o r e , the p r e c i p i t a t i o n p r o c e d u r e is dependent u p o n the acquisition of a carbohydrate containing substance capable o f i n t e r a c t i n g with the c a r b o h y d r a t e binding site o f and p r e c i p i t a t i n g with the lectin. Such material is o f t e n difficult t o o b t a i n or u n o b t a i n a b l e and is used in an e x p e n d a b l e m a n n e r . In an e f f o r t to s h o r t e n the analysis t i m e e m p l o y i n g a p r e c i p i t a t i n g s y s t e m , h a p t e n inhibition o f t u r b i d i t y has been r e p o r t e d for c o n c a n a v a l i n A ( G o l d s t e i n et al., 1 9 6 5 ; P o r e t z and Goldstein, * This investigation was supported in part by PHS grants AI 10471 and AI 11903. ** To whom correspondence should be directed. Present address: Biochemistry Department, Bureau of Biological Research, Rutgers University, New Brunswick, New Jersey 08903, U.S.A.

170 1970). However, turbidimetric analysis requires greater quantities of reagents than the precipitation m e t h o d (Poretz and Goldstein, 1968). In order to analyze the relative carbohydrate binding specificities of lectins in a rapid and economical manner, we have developed the method of hapten inhibition of adsorption. The procedure is based upon the ability of a lectin to reversibly bind to a carbohydrate linked to a solid phase support. Methods have been published describing the adsorption of lectins to insoluble adsorbents for the specific purification of these proteins (Lis and Sharon, 1973) as well as to determine the relative binding activity of lectin preparations after various treatments (Uchida and Matsumoto, 1972). However, we believe this is the first report of the measurement of the ability of simple soluble substrates to inhibit the binding of a lectin to a solid phase adsorbent for the quantitative determination of the binding specificity of these proteins. MATERIALS AND METHODS

Preparation of adsorbent Sephadex G-200 (Pharmacia Fine Chemicals, Piscataway, N.J.) conjugate containing ~-D-galactosyl residues was prepared by the CNBr procedure of Porath et al. (1967) as described by Cuatrecasas (1970) using p-aminophenyl ~-D-galactopyranoside (Cyclo Chemical Company, Los Angeles, Calif.) as the reactive amine. The adsorbent has been stored at 4°C as an aqueous suspension at pH 7.8 containing 0.1% sodium azide for periods of up to t w o years.

Preparation of lectin The S. japonica lectin used in all experiments was purified as previously reported (Poretz et al., 1974). The lectin was at least 95--98% precipitable by an o p t i m u m quantity of human ovarian type B blood group substance (a generous gift from Professor W.M. Watkins).

Quantitative adsorption of the lectin A 20% suspension of galactose--Sephadex (gal--Seph) conjugate was prepared by suspending a given volume of packed adsorbent (prepared from a suspension which was centrifuged at 800 g for 15 min) in 4 times its volume of 0.01 M phosphate buffer pH 7.8 containing 0.12 M NaC1 (PBS). Increasing amounts of adsorbent were introduced into a series of 3 ml conical centrifuge tubes by adding the appropriate volume of a 20% suspension of adsorbent followed by centrifugation at 800 g for 15 rain and removal of the supernatant. Lectin (56.7 pg) was added to the packed gal--Seph, the final volume adjusted to 0.7 ml with PBS and the reactants were thoroughly mixed. After incubation for 1 hr at 4 ° C, the suspension was centrifuged at

171 8 0 0 g for 15 min and the supernatant removed. The remaining lectin--gal-Seph complex was resuspended in 0.5 ml of cold PBS, centrifuged as above and the supernatant removed. After two additional wash cycles, the adsorbed protein was eluted with 0.6 ml of 0.02 M NaOH and the protein was quantified by the modified Lowry method (Mage and Dray, 1965). The adsorbed protein was calculated as the protein found in the NaOH eluate times the ratio of the total volume (0.6 ml + volume of packed adsorbent) divided by the aliquot (0.6 ml). A control of 0.24 ml of packed Sephadex, lacking ~-galactosyl residues, was run in a parallel fashion. Less than 3% of the protein added was found in the eluate of the control.

Effect o f pH on the adsorption o f lectin S. japonica lectin (0.24 ml containing 175.9 pg of protein) was added to 0.24 ml of packed gal--Seph suspended in 0.22 ml of 0.01 M phosphate buffer containing 0.12 M sodium chloride or 0.01 M Tris buffer containing 0.13 M sodium chloride at pH values ranging from 6.5 to 8.9. The reaction mixtures were treated as described above. Hapten inhibition of adsorption Increasing amounts of inhibitor were added to a series of 3-ml conical centrifuge tubes, each containing the o p t i m u m proportion of lectin (88 pg) and gal--Seph (0.24 ml, packed). The volume of each tube was adjusted to 0.7 ml with PBS. After incubation at 4°C for 1 hr the suspensions were manipulated as described above.

Quantitative hapten inhibition o f precipitation Quantitative measurement of the inhibition of the precipitate formed upon the interaction of h u m a n ovarian type B blood group substance and the S. japonica lectin was determined by a procedure described previously (Poretz, 1972). RESULTS

Adsorption o f lectin The binding capacity of the adsorbent was evaluated at pH 7.8 by determining the a m o u n t of lectin adsorbed by increasing quantities of adsorbent. Figure 1 shows the saturation curve of such an analysis. The m a x i m u m adsorption (93% of the available lectin) was accomplished with 160 pl of packed adsorbent; resulting in a ratio of 0.35 pg of lectin/pl of adsorbent.

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Fig. 1. Adsorption of purified S. japonica lectin (56.7 /~g) by increasing quantities of gaI--Seph adsorbent.

Adsorption of lectin as a function o f p H Previous work in this laboratory (Poretz et al., 1974) established that the optimum pH for the agglutination of human erythrocytes by the S. japonica lectin ranged from pH 7.8 to pH 9. Similarly, the pH curve (fig. 2) for the binding of lectin to gal--Seph produced a pH optimum in the same range. Furthermore, the diminution of activity at pH 7.0 and general shape of the pH profile for the adsorption procedure parallels the results obtained for the agglutination of red blood cells by the lectin.

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Fig. 2. pH profile of the binding of purified S. japonica leetin (175.9 pg) by 0.24 ml of packed gal--Seph adsorbent. (e) phosphate-buffered saline; (A) Tris-buffered saline.

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Fig. 3. Hapten inhibition curves of: (e) N-acetyl-D-galaetosamine; (a) lactose; (~)) melibiose; (A) D-galactose. S. japonica leetin, 88 pg. Gal--Seph adsorbent, 0.24 ml packed.

Hapten inhibition of adsorption The sigmoidal inhibition of adsorption curves obtained by plotting the percent inhibition with respect to the logarithm of micromoles of inhibitor is shown in fig. 3. Each point represents the average of duplicate analyses. Similarly shaped curves are obtained by hapten inhibition of precipitation of the S. japonica lectin with human blood group substance (Poretz et al., TABLE 1 Inhibition of the S. japonica lectin by various saccharides.

Compound

Adsorption 50% inhibition

D-Galactose 2-Acetamido-2-deoxy-D-galactose

30.0

RIP*

50% inhibition

RIP*

25.0

1.00

5.17

4.6

5.44

1.54

20.0

1.25

8.6

3.50

6.6

3.80

6.70

3.9

6.40

5.8 19.5

Lactose

Melibiose

Precipitation

1.00

Phenyl ~-D-galactopyranoside

4.58

p-Nitrophenyl-2-acetamido-2-deoxy/3-D-galact opyranoside

0.19

158.0

0.21

119.0

p-Nitrophenyl/3-D-galactopyranoside

1.44

20.8

1.20

20.8

* See text.

174 1974). The m o s t active i n h i b i t o r s h o w n on the graph is N-acetyl-D-galact o s a m i n e , requiring 5.8 p m o l to cause 50% i n h i b i t i o n of a d s o r p t i o n . Tabulation o f the relative inhibiting p o t e n c i e s (RIP) o f various saccharides in the h a p t e n i n h i b i t i o n o f a d s o r p t i o n and p r e c i p i t a t i o n analyses is p r e s e n t e d in table 1. The RIP is d e f i n e d as the ratio o f the Is 0 value o f D-galactose to t h a t o f saccharide. T h e Is 0 value is the m i c r o m o l e s o f c a r b o h y d r a t e r e q u i r e d for 50% inhibition of a d s o r p t i o n or p r e c i p i t a t i o n o f lectin. A parallel relat i o n s h i p is a p p a r e n t b e t w e e n the RIP o f a sugar as a n a l y z e d by the adsorpt i o n and p r e c i p i t a t i o n p r o c e d u r e s . The RIP o f a series o f c o m p o u n d s , as varied as m o n o - and disaccharides and a r o m a t i c glycosides, d e t e r m i n e d by b o t h empirical m e t h o d s agree within +4.5%. It is also evident t h a t the degree o f sensitivity for b o t h analyses is virtually identical. DISCUSSION H a p t e n i n h i b i t i o n o f a d s o r p t i o n as described in this p a p e r offers a rapid, r e p r o d u c i b l e and a c c u r a t e p r o c e d u r e for the specificity analysis o f the carboh y d r a t e binding sites o f lectins. The r e c y c l i n g o f the s y n t h e t i c a d s o r b e n t allows this t o be an attractive, e c o n o m i c a l t e c h n i q u e . T h o u g h the sensitivity o f the a d s o r p t i o n m e t h o d is equivalent to h a p t e n i n h i b i t i o n o f p r e c i p i t a t i o n , sensitivity m a y be greatly increased b y use o f m e t h o d s capable o f d e t e c t i n g smaller quantities o f protein, t h a n can be d e t e c t e d by the m i c r o - L o w r y p r o c e d u r e . Use o f r a d i o c h e m i c a l l y labeled lectin allows the a c c u r a t e m e a s u r e m e n t o f eluted p r o t e i n at levels considerably l o w e r t h a n the c o l o r i m e t r i c m e t h o d . I n d e e d , Auer et al. ( 1 9 7 2 ) have r e p o r t e d a solid phase r a d i o i m m u n e assay for adrenal specific antigen based u p o n the specific i n h i b i t i o n o f binding o f ~ 25 I labeled a n t i b o d y to antigen c o a t e d tubes. Similarly, Engvall and P e r l m a n n ( 1 9 7 2 ) d e v e l o p e d an e n z y m e linked solid phase i m m u n o a s s a y e m p l o y i n g antigen c o a t e d tubes. H o w e v e r , unlike antigen c o a t e d tubes, use o f a suspensive a d s o r b e n t , c o m p a r a b l e to the use o f gal--Seph in this p u b l i c a t i o n , allows easy and rapid m a n i p u l a t i o n o f a d s o r b e n t resulting in increased flexibility o f a d s o r p t i o n conditions. REFERENCES Auer, I.O., Y. Yagi, R. Kasukawa and F. Milgrom, 1972, Int. Arch. Allergy Appl. Immunol. 42,816. Cuatrecasas, P., 1970, J. Biol. Chem. 245, 3059. Engvall, E. and P. Perlmann, 1972, J. Immunol. 109, 129. Goldstein, I.J., C.E. Hollerman and E.E. Smith, 1965, Biochemistry 4, 876. Lis, H. and N. Sharon, 1973, Ann. Rev. Biochem. 42, 541. Mage, R. and S. Dray, 1965, J. Immunol. 95, 525. Porath, J., R. Axen and S. Ernback, 1967, Nature 215, 1491. Poretz, R.D., 1972, Meth. Enzymol. 28, 349. Poretz, R.D. and I.J. Goldstein, 1968, Immunology 14, 165. Poretz, R.D. and I.J. Goldstein, 1970, Biochemistry 9, 2890. Poretz, R.D., H. Riss, J.W. Timberlake and S.M. Chien, 1974, Biochemistry 13, 250. So, L.L. and I.J. Goldstein, 1967, J. Biol. Chem. 242, 1617. Uchida, T. and T. Matsumoto, 1972, Biochim. Biophys. Acta 257, 230. Watkins, W.M. and W.T.J. Morgan, 1952, Nature 169,825.