Quantitative Binding of 125I-Concanavalin A to Normal and Transformed Cells

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Quantitative

Binding of

125I-Concanavalin

A to

Normal and

Transformed Cells

DONNA J. ARNDT-JOVIN' AND PAUL BERG

Department of Biochemistry,Stanford University School of Medicine, Stanford, California 94305

Received for publication 2 August 1971

We have measured the quantitative binding of the radioactively labeled ag-glutinin1251-concanavalinA to normal mammalian cells and simian virus 40- and polyoma virus-transformed cells from tissue culture. Parallel measurements ofthe amountof 125I-concanavalin Anecessary to causeagglutinationof thecellsin sus-pensionwerecarriedout. Thetransformedand nontransformedcellsused for these

experiments showlarge differences in their ability to be agglutinated by

12qI-con-canavalin A. However, these cell lines have the same number ofspecific binding sites and similar affinities for the agglutininwhether transformed, trypsinized, or nontransformed. We conclude that the differential capacityofconcanavalin A to agglutinate transformed cells relativetonormalcells does not result fromdifferences in the number ofbinding sites betweenthetwo types of cells.

A number of proteins, termed

phytohemag-glutinins becausetheyagglutinateredbloodcells, have beenisolatedfromplants (2).Low concen-trations of several of these proteins agglutinate

cells transformed chemically or by

deoxyribo-nucleic acid (DNA) tumor viruses, whereas ag-glutination of nontransformed cellsrequires rela-tively high concentrations of the proteins (1, 4, 10,20). Theconcentration ofagglutinin required

to agglutinate cells is

inversely

related to the

saturationdensities reached in culture (18).

The phytohemagglutinins precipitate different

macromolecular carbohydrates andprobably in-teractwiththecellsurfacedat "exposed"

carbo-hydrate moieties; as

expected,

the agglutinin

activity can be inhibited or even reversed by carbohydrate haptens specific for each phyto-hemagglutinin (4, 10). Nontransformed cells be-come agglutinable by the activity ofproteases; this observation suggested that nontransformed cellscontain"cryptic"

binding

sites for the

phyto-hemagglutinins and that these sitescanbeexposed byproteasesor byarearrangementin the archi-tecture of the cell surface

by

transformation (3,

11).

This hypothesis was based upon quantitative differences in the agglutination of different cell lines (3) and from areport that the amount of 63Ni-labeled concanavalin A (con A) bound to

IFellow of the Jane Coffin ChildsMemorialFund forMedical Research. Present address: Abteilung fur Molekulare Biologie, Max-Planck-Institut fUrBiophysikalischeChemie,D-3400 Goet-tingen, West Germany.

transformed cells was considerably greater than that bound to nontransformed cells (11). How-ever, the quantitative relationship between the amount of phytohemagglutin from red kidney beans neededto cause cellagglutinationand the amount ofthe agglutinin bound tothe cell sur-face has been obscure (20). Moreover, in the study with the 63Ni-labeledconA (11), the radio-active marker was not covalently linked to the protein and thus it is difficultto be certainthat thedeterminations of 88Ni bindingtothe cells ac-tually measured complex formation betweenthe con A and the cell surface; the fact that the amount of 65Ni binding was inhibited less than

50%

by

an excess ofthe

hapten

is

particularly

unsettling.

Wefelt that moredirect measurements of ag-glutinin bindingtocells and the correlation of that

bindingtothe phenomenon ofagglutinationwere needed to substantiate the "cryptic-site" hy-pothesis(11). SinceconA canbeeasily purifiedto physical homogeneity and many ofits physical and carbohydrate bindingproperties arealready

known (12, 14, 15, 19, 21), it was selected for iodination with 125I (17) to produce a stable covalent linkage between the labeled group and the agglutinin. Although the amount of 1251-labeled con A needed to agglutinate nontrans-formed cellswas muchgreater than that needed for nontransformedcells,wecouldnotdetectany significant difference in the binding of labeled proteintothetwotypesofcells.

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MATERIALS AND METHODS Tissue culture. Mouse fibroblast cell lines 3T3M-T20 (clonedfrom aline fromR. Dulbecco) and SV-3T3-26-d2 (alsofrom R.Dulbecco)andbaby hamster kidney cell lines BHK-21 (original clone from labora-toryof M. Stoker) and MT-1 (apolyoma-transformed clonederivedfrom thisline) were cultured in plastic petri dishes (Nunc, 100 by 20 mm, orFalcon, 35by 10 mm) in Dulbecco's modified Eagle's medium (GrandIslandBiologicalCo.)supplementedwith10% calfserum and 25,g ofchlortetracycloneperml.

Con A.Con A was extracted fromjack beanmeal (Sigma ChemicalCo.) andcrystallized bythe proce-dureof Sumner andHowell (21). Somepreparations were treatedwith 1Macetic acidfor 20 min and dia-lyzed to render the protein water-soluble and free from any carbohydrates (14). Allpreparations were adsorbedto aSephadexG150columnandelutedwith buffers atlowpH asdescribed by Olsonand Liener (14).

1251-labeled Con A. Specific iodination oftyrosine residues with lactoperoxidase (the kind gift of M. Morrison)wascarriedout asdescribedbyPhillipsand Morrison (17) with modifications ofconcentrations asfollows.The con A at a tetramerconcentrationof 10-4 Mto 1.6 X 10-4M wasdissolved in0.1 Msodium phosphatebuffer (pH7.4) containing Na125I at4 X 10-4to4 X 10- M(specific activity,200 to 500IuCi/

,umole).The reactionwasinitiatedbytheaddition of lactoperoxidase to aconcentration of 7.7 X 10-7 M and hydrogenperoxideat 8 X 10-5 to 1.6 X 10-4M. The incorporation of radioactivity was followed by precipitation of the protein with 5% trichloroacetic acidforcounting.

1251 counting. A Nuclear-Chicago model 1085 gamma counter was used for determining the 1251 radioactivity.

Agglutination assays.Cellagglutinationwasassayed byamodification of theproceduredescribedby Burger and Noonan (5). Subconfluent cells were removed from theplateswith 4 X 1o-4M ethylenediaminetetra-acetic acid (EDTA) in buffered saline at 37 C and washed oncewith buffered salinecontainingMg2+and Ca2+beforebeingwashed and suspendedin buffered salinelacking Mg2+and Ca2+. A100-jslitersolution of 2 X 105 to 106 cells/ml was mixed with an equal sample ofvarious concentrations of agglutinin dis-solvedinbuffered salinein the wells ofaporcelainspot plate.Theplatewaskeptingentlemotionat22C,and samplesof 20to50,literswereremovedat5,10,and and 30 min and observed in thewellsofplastic spot plates (Linbro Chemicals FB48) with an inverted microscope.

Bindingassays. Method 1was asfollows. Subcon-fluentcellswereremoved fromplateswith4 X 14M

EDTA in buffered saline lacking Mg2+ and Ca2+, washedoncewith buffered salinecontainingMg2+and Ca2+ and then washed and suspended in buffered salinewithoutMg2+and Ca2+at adensityof 5 X 105 to 106cells/ml. Different concentrationsof125I-labeled con Awithorwithout 0.3 M a-methyl-D-glucopyrano-side,ahaptenforconA,wereadded,and the cellswere

incubatedwith slowagitationfor 15 minat0C.After

centrifugation at 700 X g for 1min,the cell pellet was washed twotimes with coldbufferedsalineand then solubilizedwith 0.5 N NaOH; a sample was counted directlyinagamma counterand assayed for total pro-tein by the method ofLowry et al. (13). W

Method 2 was as follows. The cells remained at-tached to the culture plates. Subconfluent cells on 35-mmplastic petri plates were washed two times with cold buffered saline lacking Mg2+ andCa2+and then wereincubated with 0.3 ml of a solution containing different concentrations of'25I-con A with and without hapten. After 15minat 0C, the liquid was aspirated and theplates were washed twice with cold buffered saline.After the cells were solubilized in 0.5 N NaOH, they were analyzed as mentioned above. Control plates incubatedwithoutagglutininweretrypsinized, and the cellswerecountedin a hemocytometer.

RESULTS

Radioactivelabelingofcon A.Afterreactionof conAwith1251 in thepresence oflactoperoxidase and peroxide, the proteinwas dialyzed and then adsorbedto aSephadex G150 column. Figure 1 shows the elution profile of the radioactivity. Acrylamide gels of the material specifically bound toandeluted fromSephadexgaveonlyonepeak in the normalposition for purified conA. From the specific activity of the iodine, it was

deter-mined that one residue of tyrosine per 27,000 molecular weighthadbeeniodinated. This molec-ularweight isassumed to be theintactmonomer (22); however, atthepH of thebinding studies,

ml

FIG. 1. Dextranadsorptionandelutionof5 mg of 125I-con A (acetic acid-treated). The proteini was

adsorbedtoacolumn(I by27cm) ofG-150Sephadex

in 0.01 m tris(hydroxymethyl)aminomethane (Tris)-hydrochloride buffer (pH 7.2) containing 103 M

CaCl2and10-3 M MgCl2. Theeffluent wasmonitored for 125I radioactivity in a gamma counter. Adsorbed materialwaseluted with 0.02 Mglycine-HCI (pH 2.0) and dropped into tubes containing 0.075 M Tris-hydrochloride (pH9.0) to bring the solution to

neu-trality.

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ARNDT-JOVIN AND BERG

the con A probably exists as a tetramer (15, 21, 22).

The'251-conAretained itshaptenpropertiesas demonstrated by its ability to be adsorbed onto Sephadex and eluted by either 0.1 M glucose or low pH and by the fact that the bindingtocells can be competed for or reversed by a-methyl-D-glucopyranoside butnotby N-acetylglucosamine. Competition experiments were done by binding '251-con A to cells in the presence of cold, un-modified con A. Allof the '25I-con A was com-peted by unmodifiedconA, and thecompetition experiments gave the same binding constants for 1251-con A asunmodified conA.

Agglutination of transformed and nontrans-formed cells. Table 1 givesthe levels of'251-con A and unlabeled conArequired to obtain aggluti-nationofmousefibroblasts [3T3 M and SV-3T3 (simian virus40-transformed) or 3T6 (non-viral-transformed)] and baby hamster kidney cells [BHK-21 (nontransformed) or MA-8 (abortively transformed) and MT-1 (polyoma virus-trans-formed)] and the levels required after mild trypsinization.

TABLE 1. Levels of '251-coni A or tlnlabeled conl A

requiredforagglutinationi of mouisefibroblasts andbaby hamsterkidney cells

Cellline

Concnof

'251-conA

orunlabeled

conA (,ug/ml) for half-maxi-mal

aggluti-nation

Mouse Normal

3T3M...

3T3M after0.01%trypsin for

10min...

Transformed

SV3T3...

SV3T3 after 0.01% trypsin for

10 min...

3T6...

3T6 after0.01% trypsin for

10 min...

Hamster Normal

BHK-21...

BHK-21 after0.01% trypsin for

10min...

MA-8...

MA-8 after0.01% trypsin for

10min... Polyomavirus-transformed

MT-1...

MT-1 after0.01% trypsinfor

10min...

70-120

7 7-15

7 7-10

7-10

25-30 7

100-120

10 7 7-10

16

12

cpm xio-3 8

4

16

12

cpm t10-3 ₈

4 A

0 40 80, 2,~ 120. 160 200

ggIml"''I-con A

B

//

II

0 40 80 120 160 200

[image:3.485.255.444.65.367.2]

ggl1m251-conA

FIG. 2. Binidinigof"5I-coniA to cells in

solutiont

by method 1. (A) 3T3M cells (105) at 0 C; (B) SV3T3 cells (105) at 0 C. Symbols: X, untreated cellsplus 'I5I-con A, counts perminuiite bouind; Oi, cells treated with 0.01%'o trypsillfor 10millplus 125I-conA, counts perminute bounid; A, cellsplus 0.3 -4f a-methylgluco-pyraniosideplus '251-con A, colltits per

miniute

bounlid.

The transformedand nontransformed cells used for these experiments exhibited significant dif-ferencesintheir ability to be agglutinated by con A. Thesamedifferenceswereobserved for wheat germ agglutinin activity with these cells (unpub-lisheddata), andtheyconfirmthedifferencesseen inagglutinabilityby others (1, 3, 4, 10, 20). How-ever, these cell lines have the same number of con A binding sites available whether trans-formed, trypsinized, or nontransformed as mea-suredbythespecificbindingof1251-labeledconA, calculatedonthebasis ofequal concentrations of cellprotein, orhave a slightlygreater number of binding sites for nontransformed cells compared totransformed or trypsinized cells calculated on the basis ofequalcellnumbers.

Binding of 1251-concanavalin A to transformed and nontransformed cells. Figure 2 shows typical

bindingcurvesfor1251-conA to 3T3and SV-3T3 cells in solution. The half-maximal binding of

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TABLE 2. Half-maximal binding ofi5I-con A to 3T3 versus SV-3T3 anidBHK versuspolyoma-BHK cells

Concnof 125I-con A Cell line Cell number Method ofbindine_bnig (ug/ml)for

half-maximalbinding

Mouse Normal

3T3M...X...SX 105 1 22 40-50

3T3M... 5 X1011 0 60-80

3T3M... 105 1 0 60-&0

3T3M, 0.01%trypsin for 10 min 105 1 0 60

3T3M... 3 X 105 2 0 75

3T3M... 105 2 0 50-60

Simian virus 40-transformed

SV3T3.... 5 X 105 1 22 40

SV3T3... 5X105 1 0 60-80

SV3T3... ... 105 1 0 60-80

SV3T3, 0.01% trypsin for 10 min.... 10, 1 0 60-80

SV3T3... 3X105 2 0 60-80

SV3T3 ..105 2 0 50

Hamster Normal

BHK,MA-8... 2 X 105 1 22 30

BHK,MA-8 ... ... 105 1 0 35-40

Polyomavirus-transformed

Polyoma-BHK,MT-1.... 2 X 105 1 22 40

Polyoma-BHK, MT-1...1 105 1 0 35-40

125I-con A (at different cell concentrations, at temperaturesof22or0C, withor without mild trypsindigestion)to3T3versusSV-3T3and BHK

versuspolyoma-BHKcells isgiveninTable2. Since proteolytic digestion ofcells resulted in

greater agglutinability, it was conceivable that

small amounts of proteolytic enzyme released fromthecellswhentheywereremovedfromthe plates couldcause erroneousinterpretation of the

specific binding results. To overcome this prob-lem, the binding assay was done with cells still

attached totheplatesaswellaswiththecellsin solution.Typical bindingcurvesfor 251I-conAto

3T3M and SV-3T3 cells at 105 cells per 35-mm

plate are given in Fig. 3 and reveal the same

numberofsitespercell for normalortransformed cells, 10' to3 x 101. Thehalfmaximalbinding

of125I-conA tothesecelllinesand toBHK

nor-mal and polyoma-transformed cell lines is

con-stantfor 3 X 104to105cellsper35-mmplate and

occursat a conA concentration of -70

Ag/ml;

the binding is inhibited by 0.03 M

a-methyl-D-glucopyranoside and is completely competed for by unmodifiedconA.Nodifferenceintheamount

ofbinding to normal and transformed cells was

observed for con A labeled with

'4C-iodoaceta-mide.

DISCUSSION

Ourdata indicate that there isanequal number

ofconAbinding sitesonthe cell surfaceof

trans-formed andnontransformed cells.Therefore, the differential capacity of con A to agglutinate transformed cells relativetonontransformed cells doesnotresult fromdifferences in the number of binding sites between thetwo typesofcells.

If there isindeed no difference in the number ofconA binding sites or indeed of other agglu-tinin-binding sites (20) between transformed and nontransformed cells, then how can one explain the differential agglutinability of the cell lines? Three models can be generated to explain the phenomenon: (i) steric interference, (ii) allosteric conversion, and (iii) ionic interaction. Steric inter-ference wouldrequire that theagglutinin-binding

site be buriedonthe surface ofthe normal, non-transformed cell, such that the second hapten binding site of the agglutinin is unavailable for bindingto asiteonanadjacent cell. Conversely, the transformed cell binding site would be more external, allowing the second hapten binding of theagglutininsitetoattachtoanother cell.

Allosteric conversion would require that the binding ofagglutinintothetransformedcellcause a rearrangement in the structure of the surface, such that the lectin is ableto interact with

adja-centcells,orthatthebinding ofagglutininto the nontransformedcellcause arearrangementinthe

cellsurface such that the second hapten siteonthe agglutinin cannot participate in second binding interactions with anothercell, orboth.

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ARNDT-JOVIN AND BERG

2C

16

cpm 12 x10-3

8

4

A

I

/

0 40 80 120

g/ml"'1-conA

20F

16

12

4

0

B

grees oftrypsinization causethe cells to become monodisperse.

Although we do not know enough about the x actual differences in cell surfaces between trans-formed and normal cells to distinguish clearly a mechanism foragglutination,wehave shown that

transformation does not create or uncover new binding sites for the agglutinin con A, nor has such a difference been observed for the

phyto-hemagglutininfrom redkidney beans;perhapsno difference exists for any ofthe agglutinins. The models which can explain suchobservations re-quire more sophisticated understanding of the 1: 2{i0 interaction of cell surfaces and their structural

makeup.

Similar results for thespecific binding of lectins totransformed and normal cells havebeenfound concurrentlyinother laboratories (6,16).

0

40 80 120

.igtmi 1251-conA

FIG. 3. Binding of'251-conA tocellsi (A) 3T3Mcells (101) at 0C; (B) SV31

at 0C. Symbols: X, cellsplus '25I-con,

minute bound; A, cells plus 0.03 M

az-pyranoside plus '25I-con A, counts per

The argument of ionic interactic that theability of cells to growon t

cellsortobe contact-inhibitedbythe adjacentcells andtheabilityofcellst

oneanother inthepresenceofcopoly

specific proteins may both depend (

figuration and localized charge whici

on adjacent cells. It is possible that membrane potential common to cel ionically masked on the surface of

formedcell. This modelrequires noc

number ofbinding sites foragglutinil formedand normalcellsbutsimplya(

thecharge repulsionofthecells.The

cellsurfacesby proteolyticenzymesd( the amount ofspecific binding of cc

described above but could drastical ionicmakeupofthe cells. Indeed, so] trypsinization alone can cause cells

sticky and adhere to one another i

presence of external proteins, whereE

ACKNOWLEDGMENTS

This investigation was supported by Public Health Service grantGM 13235 fromtheNationalInstitute of General Medical Sciencesand byAmerican Cancer SocietygrantVC-23A. It was alsoaided bya grantfrom theJane CoffinMemorial Fund for Medical Research.

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