BIOCHIMICA ET BIOPHYSICA ACTA 387

BBA Report

BBA 21332

Binding of soybean agglutinin by normal and trypsin-treated red blood cells

JULIUS A. GORDON*, NATHAN SHARON** and HALINA LIS

Department of Biophysics, Weizmann Institute o f Science, Rehovoth (Israel)

(Received February 21st, 1972)

SUMMARY

Trypsinized human erythrocytes were found to bind 3 times as much

soybean agglutinin as the untreated cells. With rabbit erythrocytes, no effect of trypsinization on the binding of the agglutinin was observed. However, the susceptibility to agglutination increased 100- to 200-fold for all erythrocytes. The

increased erythrocyte agglutinability can therefore not be explained simply by an

increase in the number of agglutinin binding sites.

Prior treatment of erythrocytes with proteolytic enzymes can dramatically

reduce the amount of agglutinin (lectin) required to produce hemagglutination ~- 4. The

increased ease of agglutination following enzymatic treatment has been attributed to the

exposure of additional agglutinin-specific receptor sites which are thought to be in a "cryptic" form on the untreated cell 3' 4 and more recently to rearrangements of pre-

existing sites s . We report here the results of our quantitative binding studies of

12 s I-labelled soybean agglutinin with human and with rabbit erythrocytes; these studies

indicate the absence of a simple relationship between the extent of binding of the agglutinin and the increased agglutinability of the erythrocyte following trypsinization.

Soybean agglutinin was isolated and purified as previously described 6. The

labelling was performed according to the chloramine-T method of Greenwood et al. 7,8

using carrier-free Na 12s I (Radiochemical Centre, Amersham, England, 20 mCi/ml). The

labelled protein was separated from the low molecular weight radioactive material by

Abbreviation: GalNAc, N-acetyl-D-galactosamine.

*Permanent address: Department of Pathology, University of Colorado Medical School, Denver, Colo., U.S.A. **To whom correspondence should be addressed.

Biochim. Biophys. Acta, 264 (1972) 387-391

388 BBA REPORT

gel filtration on a Sephadex G-150 column (1,4 cm × 45 cm) in saline; the fractions containing the labelled protein were combined and dialysed 48 tl at 4 °C against saline to remove the last traces of low molecular weight radioactive material. The preparation obtained behaved upon chromatography on columns of calcium phosphate in a fashion identical to that of unlabelled soybean agglutinin 6, and there was no change m agglutinating activity or specificity of the agglutinin after iodination. The counting

rate of the ~2 s l-labelled soybean agglutinin preparation used in the reported experiments was 4500 cpm//ag protein. Erythrocytes were prepared for use by washing 3-4 times with

saline containing 0.01 M potassium-sodium phosphate, pH 7.4 (phosphate-buffered saline) and suspending m phosphate-buffered saline (4% cell suspension, about 3.108 cells/ ml) to give an absorbance of 2 at 620 nm. The absorbance was measured in a Colernan Junior Spectrophotometer equipped with a special adaptor 2' 9 using 10 mm X 75 mm round cuvettes. Determination of agglutinating activity on untreated and trypsinized erythrocytes was perforined by the quantitative spectrophotometric method of Liener as previously described 2' 9. Trypsinization was carried out with Bacto-trypsin, Difco, I mg/ml, at 37 °C. At specified times samples were withdrawn, immediately cooled, the trypsinized erythrocytes washed 5 times with phosphate-buffered saline and re-suspended in the original volume of phosphate-buffered saline.

For binding experiments, 5 ml of cell suspension was treated with the specified amount of ~2Sl.labelled soybean agglutinin for 30 min at room temperature. The supernatant was removed from the cells which were then washed 2 times with 5 ml

phosphate-buffered saline; the cell-bound radioactivity was determined in a Packard series 5000 Auto-Gamma spectrometer. Another washing did not remove any significant amount of radioactivity. The extent of binding was found to be dependent

only on the ratio agglutinin:cells and to be invariant after 5 min of incubation. It can be seen from Fig. 1 that at a given soybean agglutinin concentration,

rabbit erythrocytes bound 5-6 times more soybean agglutinin per cell than did human erythrocytes. With human erythrocytes, the amount of soybean agglutinin bound was highest with type A, somewhat lower with type O and lowest with type B erythrocytes. When increasing amounts of ~2 s l-labelled soybean agglutinin were added to a constant

number of erythrocytes (30-300 IJg/5 ml erythrocyte suspension and in one

experiment with type A erythrocytes as much as 1500/ag/5 ml), the amount of radioactivity bound to the cells increased linearly with tire amount of soybean agglutinin added (Fig. 1 ). Our inability to saturate the erythrocyte with soybean agglutinin is in accord with the observations by Boyd e l al. J o on tire binding of lima bean agglutinin to human erythrocytes. At concentrations of soybean agglutinin greater than those reported here, hemolysis of the erythrocytes occurred before the experiment could be completed. The binding experiments to be described were therefore carried out with two amounts of soybean agglutinin (60/ag and 120 ,ug per 5 ml erythrocyte suspension) high enough to give marked agglutination under our conditions but without leading to significant hemolysis.

The results of experiments on the binding of 12s l-labelled soybean agglutinin to

Biochim. Biophys. Acta, 264 (1972) 387-391

BBA REPORT 389

~ 3

x E cL

o

4 12

'*'I

I J I L 48 56 64

ADDED(c p mxl6 5)

' 0 x E

u

I I I

0 o R

S x ~ - A

x

~V

- I I I

D o o o-

I0 30

I

6O

TRYPSIN TREATMENT (minules)

Fig. 1. The extent of binding of ] 2Sl-labelled soybean agglutinin to rabbit erythrocytes (o) and human erythrocytes of type A (x), B (-) and O (P~) plotted as a function of the amount of soybean agglutinin added. The erythrocytes were washed with phosphate-buffered saline twice after a 30-rain incubation with ~ 2SI-labelled soybean agglutinin as outlined in the text. The counting rate of the ;2 s I-labelled soybean agglu tinin was 4500 cpm/~g protein.

Fig. 2. The extent of ~251-1abelled soybean agglutinin binding to rabbit and human erythrocytes is shown plotted against time of trypsin treatment in minutes (for experimental details see text). 'Fhe symbols are as in Fig. 1. The upper figure shows the result obtained with a concentration of 120 ~g 12 s l-labelled soybean agglutinin (4500 cpm/~g) per 5 ml of erythrocyte suspension, while the lower figure is one-half this concentration.

erythrocytes and the agglutinating activity of soybean agglutinin as a func t ion of trypsin

t rea tment of the erythrocytes are shown in Fig. 2 and Table I. Two no tewor thy

features emerge from the above results: (a) with rabbit erythrocytes there is no change

whatsoever in the ex ten t of b ind ing of 12 s I-labelled soybean agglutinin as a result of

t rypsinizat ion, while with h u man erythrocytes there is a rapid 2-3-fold increase within

10 rain after exposure to t rypsin, bu t practically no change upon further t rypsinizat ion

(Fig. 2); and (b) the susceptibil i ty to agglut inat ion of all types of erythrocytes tested

increased con t inuous ly with exposure to t rypsin and after 1 h was 100- to 200-fold higher

than that of unt ryps in ized cells (Table I).

As can be seen in Table II, the radioactivi ty b o u n d to the erythrocytes could

be removed almost quant i ta t ive ly by a 1 mM solut ion of N-acetyl-D-galactosamine

(GalNAc). D-Galactose and D-galactosamine at the same concen t ra t ion were much less

efficient, while N-acetyl-D-glucosamine (not shown in Table II) did no t release any

radioacivity at all. When the b ind ing exper iments were carried out in the presence of the

monosaccharides at a concen t ra t ion of 1 raM, GalNAc prevented almost complete ly the

b inding of soybean agglutinin to the erythrocytes , while D-galactose and D-galactosamine

Biochim. Biophys. Acta, 264 (1972) 387-391

390 BBA REPORT

TABLE 1

ACTIVITY OF PURIFIED SOYBEAN AGGLUTININ AS A FUNCTION OF TRYPSINIZATION OF ERYTHROCYTES

Agglutinating units/rag soybean agglutinin *

Rabbit ttuman, O,pe

A B

Untreated 40 2 1 2 Trypsinized 10 rain 1200 65 30 60

20 rain 4000 30 rain 6000 120 65 200 60 min 8500 340 100 400

*Agglutinating activity determined by the spectrophotometric method of Liener 2' 9. Results are estimated to be plus or minus 0.5 unit for the untreated human erythrocytes and plus or minus 10% for the remaining values.

TABLE II

RELEASE OF ,2 S I_LABELLED SOYBt';AN AGGLUTIN1N FOLLOWING EXPOSURE OF ERYTHROCYTI".S TO SUGARS

Washed erythrocytes with a known amount of bound radioactive soybean agglutinin were suspended in 5 ml of a 1 mM solution of the indicated sugar in phosphate-buffered saline. After 30 rain incubation at room temperature, the erythrocytes were centrifuged, washed 2 times with 5 ml of phosphate-buffered saline and the residual bound radioactivity determined. The release of radioactivity is expressed as % of radioactivity originally bound. Not shown is the finding that the time of trypsin treatment from 10 rain to 1 h is without effect on the percent release of the 12 S l.labelled soybean agglutinin.

Erythrocytes Release o f ~ 2 Si.labelled soybean agglutinin (%)

D-GalNAc D-Galactosambte D-Galaetose added added added

Rabbit Untreated 90 10 6 Trypsinized 74 13 7

Hi,matt Type A: Untreated 84 34 37

Trypsinized 93 Type B: Untreated 85 32 39

Trypsinized 94 20 30 Type O: Untreated 90 32 40

Trypsinized 97 20 25

caused only 30 -40% inhibi t ion . These results are in comple t e agreement wi th the

previous findings on the saccharide-specif ic i ty o f soybean agglutinin 2.

I1 seems clear f rom these studies that the increased agglutinabil i ty o f rabbit

e ry th rocy t e s fol lowing t ryps in t r ea tmen t is no t the direct result o f an increase in the

n u m b e r o f b inding sites for soybean agglutinin on the cell. With h u m a n e ry th rocy t e s we do

Bioehim. Biophys. Aeta, 264 (1972) 387-391

BBA REPORT 391

observe an increase in the number of sites with binding affinities apparently similar to

those of the sites present on the untreated erythrocytes, yet even here the increase in

the number of sites for soybean agglutinin does not parallel the progressive and dramatic

increase in agglutinability upon continuing exposure of the cells to trypsin. We conclude,

therefore, that factors other than an increase in the number of soybean agglutinin binding

sites are generally controlling the increased agglutinability of trypsin-treated erythrocytes.

Similar conclusions have recently been reached from studies on the binding of

radioactively labelled agglutinins to normal and transformed somatic cells s ' 11,12

Soybean agglutmin interacts with human blood cells of type A, O and B and

with rabbit erythrocytes; these interactions are specifically inhibited by GalNAc and

related sugars. This observation can be explained along the usual lines 13 by suggesting

that soybean agglutinin interacts with erythrocyte surface receptors containing GalNAc

which need not necessarily be part of the blood group substance of type A. Moreover,

the lack of b lood type specificity may be the result of the inabili ty of soybean agglutinin

to distinguish between a and/3 linked GalNAc. In type A blood group substance,

GalNAc is c~ linked, whereas it is possible that on the erythrocyte surface it occurs both

a and/3 linked. Yet one should not overlook the possibility that soybean agglutinin

contains functionally linked but topographically independent sites for binding GalNAc

and for at tachment to the erytbrocyte surface.

This study was supported by Grant FG-Is-247 from the U.S. Department of

Agriculture. One of us (J.A.G.) would like to thank Professor E. Katchalski for support

and facilities and also to thank the American Cancer Society for fellowship support

graciously given.

REFERENCES

1 0 . M~kel~,Ann. Med. Exp. Biol. Fenn., 35 (1957) suppl. 11. 2 H. Lis, B. Sela, L. Sachs and N. Sharon, Biochim. Biophyx. Aeta, 211 (1970) 582. 3 G.I. Pardoe and G. Uhlenbruck, J. Med. Lab Teehnol., 27 (1970) 249. 4 0 . Prokop, G. Uhlenbruck and W. Kohler, Vox Sang., 24 (1968) 321. 5 G.L. Nicolson,NatureNew Biol., 233 (1971) 244. 6 H. Lis, N. Sharon and E. Katchalski, J. Biol. Chem., 241 (1966) 684. 7 F.C. Greenwood, H.H. Hunter and J.S. Glover, Bioehem. J., 89 (1965) 114. 8 B. Sela, H. Lis, L. Sachs and N. Sharon, Biochim. Biophys. Acta, 249 (1971) 564. 9 I.E. Liener, Arch. Biochem. Biophys., 54 (1954) 223.

10 W.C. Boyd, H.M. Bhata, M.A. Diamond and S. Matsubara, J. lmmunol., 89 (1962) 463. 11 M.J. Cline and D.C. Livingstone, Nature, 232 (1971) 155. 12 B. Ozanne and J. Sambrook, Nature, 232 (1971) 156. 13 G.I. Pardoe and G. Uhlenbruck, Med. Lab. Technol., 28 (1971) 1.

Biochim. Biophys. Acta, 264 (1972) 387-391