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

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  • 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 of 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 el 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

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    ADDED(c p mxl6 5)

    ' 0 x E

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    S x ~-A

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    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 function of trypsin

    treatment of the erythrocytes are shown in Fig. 2 and Table I. Two noteworthy

    features emerge from the above results: (a) with rabbit erythrocytes there is no change whatsoever in the extent of binding of 12 s I-labelled soybean agglutinin as a result of

    trypsinization, while with human erythrocytes there is a rapid 2-3-fold increase within 10 rain after exposure to trypsin, but practically no change upon further trypsinization

    (Fig. 2); and (b) the susceptibility to agglutination of all types of erythrocytes tested

    increased continuously with exposure to trypsin and after 1 h was 100- to 200-fold higher

    than that of untrypsinized cells (Table I).

    As can be seen in Table II, the radioactivity bound to the erythrocytes could

    be removed almost quantitatively by a 1 mM solution of N-acetyl-D-galactosamine (GalNAc). D-Galactose and D-galactosamine at the same concentrat ion were much less

    efficient, while N-acetyl-D-glucosamine (not shown in Table II) did not release any

    radioacivity at all. When the binding experiments were carried out in the presence of the

    monosaccharides at a concentrat ion of 1 raM, GalNAc prevented almost completely the

    binding 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 of ~ 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: U

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