CELL AGGLUTINATION MEDIATED BY CONCANAVALIN A AND THE DYNAMIC STATE OF THE CELL SURFACE

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1 J. Cell Set. 14, (1974) 197 Printed in Great Britain CELL AGGLUTINATION MEDIATED BY CONCANAVALIN A AND THE DYNAMIC STATE OF THE CELL SURFACE M. INOUE Department f Pathlgy, Okayama University Medical Schl, Okayama 700, Japan SUMMARY The binding f 131 I-labelled cncanavalin A ( 131 I-Cn A) t the cell surface has been studied in Ehrlich ascites tumur cells (EATC) and beef erythrcytes under varius cnditins. The binding f cncanavalin A (Cn A) t the cell surface was very specific and the available binding sites were saturated within a few minutes. The amunt f 131 I-Cn A bund t EATC was 4-14 x i 7 mlecules/cell at 37 C and 212 x i 7 mlecules/cell at C. Under these cnditins, cell agglutinatin was bserved nly at 37 C and nt at C. Hwever, the binding sites measured at C were als effective fr agglutinatin at 37 C. Beef erythrcytes were agglutinated by Cn A nly after treatment f cells with papain. The number f binding sites fr Cn A n the cell surface was decreased by this treatment t abut half the number present n untreated cells. Varius reagents such as clchicine, mnidacetic acid, dinitrphenl, rtenne, sdium azide and carbxyl cyanide-m-flurphenylhydrazne (FCCP) had n effect n Cn A-mediated cell agglutinatin. In cntrast, peridate treatment prduced a remarkable decrease in the agglutinability f cells. Frm these data, it is cncluded that the cell agglutinatin induced by Cn A was due t the tpgraphical distributin f the surface receptrs fr the lectin, and nt the result f energy-dependent r micrtubule-dependent reactin prcesses. The number and the state f Cn A receptrs n the cell surface were in a dynamic cnditin, their cnfrmatin, rientatin, and/r tpgraphical distributin changing under different cnditins. INTRODUCTION Certain lectins selectively agglutinate transfrmed cells r prtease-treated nrmal cells (Inbar & Sachs, 1969). Varius factrs are imprtant in cell agglutinatin, including the binding frces f the agglutinating mlecules, the number f mlecules invlved in agglutinatin, the surface net charge, cnfiguratin and flexibility f the cell surface. It has been pstulated that the difference in agglutinability by lectins between prtease-treated and untreated nrmal cells r nrmal and transfrmed cells is due t unmasking f cryptic agglutinin-binding sites n the membrane (Burger, 1969). This explanatin is nt in accrd with the recent finding that nrmal cells bind iadiistpe-labelled agglutinins t apprximately the same extent as transfrmed r prtease-treated nrmal cells (Cline & Livingstn, 1971; Ozanne & Sambrk, 1971; Arndt-Jvin & Berg, 1971; Sela, Lis, Sharn & Sachs, 1971). Recently, Inbar, Ben-Bassat & Sachs (1971^,6) reprted that cell agglutinatin mediated by Cn A was temperature-dependent, and that it was prbably due t structural r metablic change n the surface membrane. It is cnceivable that the mechanism f cell agglutinatin induced by Cn A des nt fllw a cmmn pattern

2 198 M. Inue in every case. With this cnsideratin in mind the authr has attempted t clarify the mechanism f cell agglutinatin induced by Cn A and fund several interesting phenmena. MATERIALS AND METHODS Ehrlich ascites tumur cells (EATC) were transplanted in the peritneal cavity f ddn mice and harvested frm ascites fluid 7 days after transplantatin. Tumur cells r beef erythrcytes were washed 3 times with glucse-free Hanks's balanced salt slutin (HBS) by centrifuging at 2000 rev/min fr 30 s. Cncanavalin A (Cn A) was purified frm Jack bean meal (Sigma Chemical C.) by the methd f Agrawal & Gldstein (1967), and stred at 20 C in HBS cntaining 1 mm MnCli as a stabilizer. Sialidase (Type V), hyalurnidase (Type I) and papain were btained frm Sigma Chemical C. Other reagents used were f analytical grade. All experiments were carried ut in HBS. The number f tumur cells used was i 7 /ml, and f beef erythrcytes, i 8 /ml. Agglutinatin tests were carried ut after incubating the cells with agglutinin fr 10 min at rm temperature, fllwing the methd f Burger & Gldberg (1967). The binding f 131 I-Cn A was carried ut at and 37 C. Cells suspended in 3 ml f HBS were incubated with different cncentratins f 131 I-Cn A, with r withut 100 mm a-methyl- D-glucside (a-mg), a haptenic inhibitr f Cn A. After varius perids f incubatin they were washed 3 times with 10 ml f cld HBS, and cell-bund radiactivity was then determined by use f a well-type scintillatin cunter (Tshiba Medical C. Ltd, Type UDS-24210). The binding number f Cn A was calculated by subtracting cell-bund radiactivity in the presence f u-mg (100 nun) frm the ttal radiactivity f bund Cn A in the absence f the haptenic inhibitr. The mlecular weight f Cn A used fr the calculatin f the binding number was m I-Cn A used in this experiment was prepared by idinatin f native Cn A with Na lsl l after the methd described by Marchalnis (1969). The labelled lectin was further purified by Sephadex G-200 clumn chrmatgraphy in the presence f 100 mm a-mg. 131 I-Cn A (i* cpm/mml Cn A) was fund t be identical with the native agglutinin as regards agglutinatin activity. Prtein cncentratin was determined by the methd f Lwry, Rsebrugh, Farr & Randall (1951) using bvine serum albumin as a standard. RESULTS Binding f 131 I-Cn A t the surface f EATC Fig. 1 shws the time curse f binding f 131 I-Cn A t the surface f EATC at 37 C; binding was cmplete within abut 5 min. Fig. 2 shws the binding f 131 I-Cn A t EATC at and 37 C. The amunt f Cn A bund t the cell increased until saturatin as a functin f Cn A cncentratin. The amunt f 131 I-Cn A bund was 4-14 x i 7 mlecules/cell at 37 C and 2-12 x i 7 mlecules/cell at C. Hwever, cell agglutinatin was bserved nly at 37 C and nt at C. Abut twice as much Cn A was bund t the cell surface at 37 C as at C. If cells pretreated with 131 I-Cn A at C were washed with cld HBS t remve free Cn A frm the reactin mixture and the temperature was raised frm t 37 C, then cell agglutinatin was bserved withut additin f any mre lectin t the system. The results suggest that the binding sites fr Cn A n the cell surface changed their state depending n temperature. Fig. 3 shws the changes in agglutinability and the number f Cn A binding sites f beef erythrcytes after treatment f the cells with papain, which rendered them agglutinable with Cn A. Hwever, the number f binding sites fr Cn A n the erythrcyte surface was decreased t half the riginal number by the enzyme treatment. The data suggest that there was n direct relatinship

3 Cell agglutinatin and surface receptr 199 x CD Time, min Fig. 1. Binding f 131 I-cncanavalin A t Ehrlich ascites tumur cells. The cells (i 7 cells/ml) were incubated with m I-Cn A (123 fig/m\) at 37 C fr varius times, and cell-bund radiactivity f 131 I-Cn A determined as described in text. 10 Bund Cn A/cell, x10" 7 Fig. 2. Binding f m I-cncanavalin A t Ehrlich ascites tumur cells at and 37 C. EATC (i 7 cells/ml) were incubated with varius cncentratins f 131 I-Cn A at and 37 C C fr 30 min, and cell-bund radiactivity f 131 I-Cn A determined as described in text. O O, C;, 37 C.

4 2OO M. Inue between the number f binding sites available n the cell surface and the agglutinability f cells, and it therefre appears that ther factrs are imprtant fr inducing cell agglutinatin. X u Papaln treatment, h Fig. 3. Effect f papain treatment f beef erythrcytes n agglutinability (O O) and number f cncanavalin A-binding sites (# ). Beef erythrcytes (i 8 cells/ ml) were pretreated with -1 % papain at 37 C fr varius perids. The cncentratin f 131 I-Cn A used fr determining the amunt f cell-bund radiactivity and the agglutinability f cells was 83 /ig/ml. Fr further details, see text. Effect f varius reagents n cell agglutinatin induced by Cn A It has been reprted that certain reagents such as clchicine r vinblastine inhibit the agglutinatin f plymrphnuclear leukcytes induced by a lw cncentratin f Cn A (Berlin & Ukena, 1972). Table 1 shws the effect f varius reagents n Cn A-mediated cell agglutinatin f EATC. A metablic inhibitr f glyclysis such as mnidacetic acid, and inhibitrs f the energy transfer reactin f mitchndria such as rtenne, FCCP, dinitrphenl and sdium azide had n effect n the agglutinatin. Furthermre, clchicine, a specific inhibitr f micrtubule actin, had n effect n agglutinatin under the cnditins f this experiment. In cntrast, treatment with ptassium peridate prduced a remarkable decrease in the agglutinability f cells by Cn A. All these reagents had the same effect n the agglutinatin f papain-treated beef erythrcytes. Table 2 shws the effect f chemical fixatin f EATC n the agglutinability f cells by Cn A. Native EATC were agglutinated by Cn A at 37 C and nt at C. Hwever, the cells were agglutinated by Cn A at bth temperatures if they had been fixed by glutaraldehyde. The data suggest that energy-transducing reactins r the actin f micrtubules are excluded frm a leading rle in cell agglutinatin mediated by Cn A.

5 Cell agglutinatin and surface receptr 201 Table 1. Effect f varius reagents n cell agglutinatin induced by cncanavalin A Pretreatment Agglutinatin Cntrl + + Mnidacetic acid, 1 mm + + Rtenne, 0-2 mm + + FCCP, -i HIM + + Dinitrphenl, 1 mm + + Sdium azide, 10 mm + + Clchicine, -i mg/ml + + Ptassium peridate, 0-5 % Cells were pretreated with each reagent fr 30 min at rm temperature. Agglutinatin was carried ut with 200 /Jg/ml f Cn A fr 10 min in the presence f the reagent. Table 2. Effect f chemical fixatin f EAT cells n agglutinatin induced by cncanavalin A Temperature C 37 C Cntrl N Yes Glutaraldehyde-fixed cells Yes Yes EATC (i 7 cells/ml) were fixed with 0-25 % glutaraldehyde fr 30 min at rm temperature, then washed 3 times with 10 ml f cld HBS. Other cnditins as in Table 1. DISCUSSION In this reprt, evidence has been btained which indicates that there is n crrelatin between the number f lectin-receptr sites available and agglutinatin behaviur. The agglutinability f beef erythrcytes increased remarkably even when the number f binding sites fr Cn A n the cell surface was reduced by papain treatment, which liberated Cn A-binding substances frm the surface (Inue, Mri & Sen, 1972; Inue, Mri, Utsumi & Sen, 1972). There was n further increase in the amunt f Cn A bund t the cell surface at 37 C after the receptrs n the cell surface were saturated by Cn A. Therefre, pincytsis f Cn A mlecules by the cells is unlikely t accunt fr the difference between the amunt f Cn A bund t the cell surface at and at 37 C. Rather, it seems t shw a difference between the number f expsed binding sites fr Cn A available at the 2 temperatures. It is als suggested that receptrs fr Cn A may change their state f mlecubr cnfrmatin and/r f rientatin n the cell surface. In the case f the actin f inhibitrs r uncuplers f the energy-transducing reactin, they shwed n effect n agglutinatin. Therefre, energy-dependent prcesses were excluded as essential factrs in this agglutinatin. Cell agglutinatin was als induced even when EATC was pretreated by glutaraldehyde. Furthermre, EATC r papain-treated beef erythrcytes adhered

6 202 M. Inue t the surface f Cn A-treated Sephadex particles (Inue et al. 1972). Amng varius reagents used fr treating the cells, nly ptassium peridate was effective in inhibiting the agglutinatin f cells mediated by Cn A. The data suggest that the agglutinatin f cells induced by Cn A depends n the tpgraphical r micr-envirnment arrangement f glycprtein receptrs fr Cn A n the cell surface which are dynamically changing their mlecular cnfrmatin r rientatin n the cell surface accrding t the physicchemical envirnment f the cells. The authr wuld like t express deep thanks t Dr G. M. W. Ck, Strangeways Research Labratry, Cambridge, England, and Dr K. Utsumi, Department f Bichemistry, Cancer Institute, Okayama University Medical Schl, Okayama, Japan, fr many stimulating discussins and suggestins. The authr als thanks Prfessr S. Sen, Department f Pathlgy, Okayama University Medical Schl, Okayama, Japan, fr his kind guidance and many helpful discussins during this wrk. REFERENCES AGRAWAL, B. B. L. & GOLDSTEIN, I. J. (1967). Prtein-carbhydrate interactin. VI. Islatin f cncanavalin A by specific adsrptin n crss-linked dextran gels. Bichim. biphys. Acta 147, ARNDT-JOVIN, D. J. & BERG, P. (1971). Quantitative binding f lt5 I-cncanavalin A t nrmal and transfrmed cells. J. Virl. 8, BERLIN, R. D. & UKKNA, T. E. (1972). Effect f clchicine and vinblastine n the agglutinatin f plymrphnuclear leukcytes by cncanavalin A. Nature, New Bil. 238, BURGER, M. M. & GOLDBERG, A. R. (1967). Identificatin f a tumr-specific determinant n neplastic cell surfaces. Prc. natn. Acad. Sci. U.S.A. 57, BURGER, M. M. (1969). A difference in the architecture f the surface membrane f nrmal and virally transfrmed cells. Prc. natn. Acad. Sci. U.S.A. 62, CLINE, M. J. & LIVINGSTON, D. C. (1971). Binding f 3 H-cncanavalin A by nrmal and transfrmed cells. Nature, New Bil. 232, INBAR, M., BEN-BASSAT, H. & SACHS, L. (1971a). Specific metablic activity n the surface membrane in malignant cell-transfrmatin. Prc. natn. Acad. Sci. U.S.A. 68, INBAR, M., BEN-BASSAT, H. & SACHS, L. (19716). Membrane changes in transfrmed cells in viv. Nature, New Bil. 236, 3-4. INBAR, M. & SACHS, L. (1969). Interactin f the carbhydrate-binding prtein cncanavalin A with nrmal and transfrmed cells. Prc. natn. Acad. Sci. U.S.A. 63, INOUE, M., MORI, M. & SENO, S. (1972). Cell recgnitin f macrphage. Sympsium fr Cell Bilgy 24 (in Press). INOUE, M., MORI, M., UTSUMI, K. & SENO, S. (1972). Rle f cncanavalin A in tumr cell agglutinatin and adhesin t macrphages. Gann 63, LOWRY, O.H., ROSEBROUGH, N. J., FARR, A. L. & RANDALL, R.J. (1951). Prtein measurement with the Flin phenl reagent. J. bil. Chem. 193, MARCHALONIS, J. J. (1969). Enzymic methd fr the trace idinatin f immunglbulins and ther prteins. Bichem. J. 113, OZANNE, B. & SAMBROOK, J. (1971). Binding f radiactively labelled cncanavalin A and wheat germ agglutinin t nrmal and virus-transfrmed cells. Nature, New Bil. 232, SELA, B., LIS, H., SHARON, N. & SACHS, L. (1971). Quantitatin f AT-acetyl-D-galactsaminelike sites n the surface membrane f nrmal and transfrmed mammalian cells. Bichim. biphys. Acta 249, {Received 25 April 1973)