Vol. 57, No. 11INFECTION AND IMMUNITY, Nov. 1989, P. 3324-33300019-9567/89/113324-07$02.00/0Copyright C 1989, American Society for Microbiology

Binding of Pertussis Toxin to Eucaryotic Cells and GlycoproteinsMAARTEN H. WITVLIET,12 DRUSILLA L. BURNS,'* MICHAEL J. BRENNAN,' JAN T. POOLMAN 2 AND

CHARLES R. MANCLARK'Laboratory of Pertussis, Center for Biologics Evaluation and Research, Food and Drug Administration, Bethesda,

Maryland 20892,1 and Laboratory ofBacterial Vaccines, National Institute for Public Health andEnvironmental Protection, Bilthoven, The Netherlands2

Received 26 April 1989/Accepted 24 July 1989

The binding of pertussis toxin and its subunits to cell surface receptors and purified glycoproteins wasexamined. The interaction of pertussis toxin with components of two variant Chinese hamster ovary (CHO) celllines was studied. These cell lines are deficient in either sialic acid residues (LEC 2) or sialic acid and galactoseresidues (LEC 8) on cell surface macromolecules. The binding of pertussis toxin to components of these cellsdiffered from the binding of the toxin to wild-type components. Although the toxin bound to a 165,000-daltonglycoprotein found in N-octylglucoside extracts of wild-type cells, it did not bind to components found inextracts of LEC 2 cells. In contrast, the toxin bound to components found in extracts of LEC 8 cells, which arevariant cells that contain increased amounts of terminal N-acetylglucosamine residues on cell surfacemacromolecules. These results suggest that the receptor for pertussis toxin on CHO cells contains terminalacetamido-containing sugars. The cytopathic effect of the toxin on both types of variant cells was much reducedcompared with its effects on wild-type cells. Thus, optimal functional binding of pertussis toxin appears torequire a complete sialyllactosamine (NeuAc-+GalPi4GlcNAc) sequence on surface macromolecules. Inaddition to studying the nature of the eucaryotic receptor for pertussis toxin, we examined correspondingbinding sites for glycoproteins on the toxin molecule. Binding of both S2-S4 and S3-S4 dimers of the toxin tocellular components and purified glycoproteins was observed. The two dimers bound to a number ofglycoproteins containing N-linked oligosaccharides but not 0-linked oligosaccharides, and differences in thebinding of the two dimers to some glycoproteins was noted. These data indicate that the holotoxin moleculecontains at least two glycoprotein-binding sites which may have slightly different specificities for glycoproteins.

Pertussis toxin, an exotoxin produced by the organismBordetella pertussis, displays a wide variety of biologicaleffects, including the ability to induce leukocytosis, alter thehost immune system and affect glucose homeostasis (25-27,35). These actions of the toxin result from its ability to bindto cells and subsequently ADP-ribosylate a family of GTP-binding regulatory proteins (G proteins) involved in hor-monal signal transduction (6, 16, 19, 20). When G proteinsare ADP-ribosylated by pertussis toxin, the host cell nolonger responds to a variety of hormones and neurotransmit-ters (9, 17).

Pertussis toxin has the A-B structure typical of manybacterial toxins (15, 33). The toxin comprises an enzymati-cally active A or S1 subunit and a B oligomer which is madeup of five subunits termed S2, S3, S4, and S5, found in a1:1:2:1 ratio, and which is responsible for binding of thetoxin to the eucaryotic cell surface (33, 34). The B oligomercomplex is formed by association of two dimers (S2-S4 andS3-S4) held together by an S5 subunit (33). A high degree ofhomology (67 to 70%) between S2 and S3 is predicted fromthe nucleotide sequences of the respective genes (23, 29).Despite this homology, S2 and S3 cannot substitute for eachother in the native toxin molecule (33).

Pertussis toxin acts by first binding to receptors on thesurface of the eucaryotic cell. Binding sites for the eucary-otic receptor may be located on both the S2-S4 and S3-S4dimers of the B oligomer, since both dimers have beenreported to inhibit the action of pertussis toxin on ratadipocytes (34). Moreover, both dimers can agglutinateerythrocytes (30). However, direct binding of both of the

* Corresponding author.

dimers to eucaryotic receptors or receptorlike proteins hasnot been demonstrated.Although the exact chemical nature of the eucaryotic

receptor for pertussis toxin remains to be identified, severallines of evidence support the idea that it is a glycoprotein.First, pertussis toxin binds to glycoproteins such as hapto-globin and fetuin (31, 33). Isolated S2-S4 has also beenshown to bind to haptoglobin (8). Second, the receptor forpertussis toxin on Chinese hamster ovary (CHO) cells hasbeen shown to be a 165-kilodalton (kDa) glycoprotein (3). Inthe same study, evidence was obtained which suggested thatcertain carbohydrate residues associated with glycoproteinsbut not glycolipids may be important in the binding ofpertussis toxin to eucaryotic cells. A variant CHO cell line(clone 15B) which specifically lacks the terminal sialyllac-tosamine (NeuAc- GalP4GlcNAc) residues on N-linked oli-gosaccharides of glycoproteins was found to be resistant totoxin action (3).

In this study, we have examined both the structure of theeucaryotic receptor for pertussis toxin and the correspond-ing binding sites on the toxin molecule. Binding of pertussistoxin to several types of variant CHO cells which lackdifferent carbohydrate moieties on their surface was studiedto determine which residues on the eucaryotic cell receptorare critical for toxin action. Two variant cell lines werecompared with wild-type and 15B cells, one (LEC 2) defi-cient in sialic acid residues on cell surface macromolecules(11) and the other (LEC 8) deficient in both sialic acid andgalactose residues (10). In addition, we have examined theability of isolated S2-S4 and S3-S4 dimers to bind to cellproteins as well as purified glycoproteins in order to betterunderstand the mechanism by which pertussis toxin maybind to cellular receptors.

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MATERIALS AND METHODS

Materials. Pertussis toxin was purchased from the Michi-gan Department of Public Health. Fetuin, asialofetuin, hap-toglobin, thyroglobulin, glycophorin, and 3,3',5,5'-tetrame-thylbenzidine were purchased from Sigma Chemical Co., St.Louis, Mo., and laminin was purchased from CollaborativeResearch, Inc., Lexington, Mass. N-Octylglucoside wasfrom Boehringer Mannheim Biochemicals, Indianapolis,Ind. Mannose and lactose were from BDH, Poole, England;lactosamine was from Janssen, Beerse, Belgium; other sac-charides were from Sigma. Anti-mouse immunoglobulin G(IgG) horseradish peroxidase conjugate, 4-chloro-1-naph-thol, and Tween 20 were from Bio-Rad Laboratories, Rich-mond, Calif. Sheep anti-mouse IgG conjugated to horserad-ish peroxidase was prepared as previously described (28).Monoclonal antibody 6DX3 (specific for the S4 subunit) wasthe generous gift of James G. Kenimer, Center for BiologicsEvaluation and Research, Bethesda, Md.

Purification of S2-S4 and S3-S4 dimers. S2-S4 and S3-S4dimers were purified by chromatography on CM-Sepharoseas previously described (33). Each dimer preparation wasfree of the other dimer species as judged by sodium dodecylsulfate (SDS) polyacrylamide gel electrophoresis.CHO cell assay. Wild-type CHO cells (ATCC strain CCL

61), LEC 2 CHO cells (ATCC strain CRL 1736), LEC 8 CHOcells (ATCC strain CRL 1737), and 15B CHO cells (gener-ously provided by Nathan Sharon, Weizmann Institute,Rehovot, Israel) were cultured as previously described (3).Toxin was serially diluted (twofold) with medium (Ham F-12medium; Flow Laboratories, Inc., McLean, Va.) containing10% fetal calf serum in a 96-well microdilution plate to givevarious concentrations in 100 ,ul. CHO cells (1.0 x 104 in 100,ul of medium) were then added to each well. After incuba-tion for 24 to 48 h in a CO2 incubator at 37°C, the cells wereexamined under a microscope to determine the extent ofclustering. The minimal concentration of toxin which causedclustering of all cells is reported.

Preparation of cell extracts. Extracts of CHO cells wereprepared by using 0.2 M N-octylglucoside as previouslydescribed (3). Goose erythrocyte ghosts were prepared (12)and extracted with N-octylglucoside as described above.

Polyacrylamide gel electrophoresis. Samples were preparedfor SDS-polyacrylamide gel electrophoresis by addition ofdithiothreitol and SDS to give final concentrations of 50 mMand 1% (wt/vol), respectively. Each sample was then heatedat 100°C for 1 min. SDS-gel electrophoresis was performedessentially as described by Laemmli (21). Gels were stainedwith Coomassie brilliant blue R250.

ADP-ribosylation assay. CHO cells were harvested, andCHO wild-type or variant cell membrane proteins wereprepared for ADP-ribosylation as previously described (7).Membranes were incubated with [32P]NAD (10 to 50 Ci/mmol; Du Pont, NEN Research Products, Boston, Mass.),and incorporation of radiolabel into the substrate for pertus-sis toxin was detected by SDS-polyacrylamide gel electro-phoresis followed by autoradiography (6).Immunoblot analysis. Proteins separated on SDS-poly-

acrylamide gels were electrophoretically transferred to ni-trocellulose membranes by the procedure of Burnette (5).After transfer, the membranes were washed in phosphate-buffered saline (PBS; pH 7.4) containing 0.05% Tween 20(PBS-Tween) and were then incubated with a 1:1,000 dilu-tion of the appropriate monoclonal antibody in PBS-Tweenfor 1 h. The membranes were washed three times withPBS-Tween and were then incubated for 1 h with goat

anti-mouse IgG conjugated to horseradish peroxidase. Themembranes were washed three times with PBS, and bandswere visualized by addition of the color reagent (30 mg of4-chloro-1-naphthol in 10 ml of methanol combined with 50ml of PBS containing 0.015% hydrogen peroxide).

Detection of binding of pertussis toxin or dimers toproteins which had been electrophoretically transferred tonitrocellulose was conducted as follows. Nitrocellulosesheets were incubated overnight with PBS-Tween containing0.5% bovine serum albumin. The strips were then incubatedfor 2 h with pertussis toxin or dimers in PBS-Tween con-taining 0.5% bovine serum albumin. After being washedthree times with PBS-Tween, the strips were incubated for 1h with a monoclonal antibody specific for the S4 subunit(6DX3) in PBS-Tween (1:1,000 dilution of ascites). Themembranes were then incubated for 1 h with goat anti-mouseIgG conjugated to horseradish peroxidase. After the mem-branes had been washed three times with PBS, the bandswere visualized by the addition of the color reagent asdescribed above.Dot blot analysis. Proteins in PBS were applied directly to

nitrocellulose. The membranes were then incubated over-night in PBS containing 0.5 mg of P-casein per ml, incubatedwith dimers for 2 h, and washed three times with PBS-Tween. The membranes were then incubated for 1 h withmonoclonal antibody 6DX3 (1:1,000 dilution of ascites inPBS-Tween). Binding was visualized with anti-mouse IgGhorseradish peroxidase conjugate as described above.

Inhibition of binding of pertussis toxin and dimers to fetuinby saccharides. Microdilution plates were incubated over-night with fetuin (2.5 ,ug/ml) in 5 mM sodium phosphate (pH7.2) containing 37.5 mM NaCl. The plates were then washedwith PBS, and PBS containing 0.5% bovine serum albuminwas added as a blocking agent. After 1 h, the plates werewashed with PBS-Tween. Pertussis toxin (2.5 ,ug/ml), S2-S4(1 ,ug/ml), or S3-S4 (2 ,ug/ml) in PBS-Tween containing 0.2%bovine serum albumin either with or without the indicatedsaccharide was added. After 90 min, the plates were washedwith PBS-Tween and the monoclonal antibody 6DX3 wasadded (1:1,000 dilution of ascites in PBS-Tween). After theplates had been washed, color was developed by the additionof sheep anti-mouse IgG conjugated to horseradish peroxi-dase for 90 min followed by three washes with PBS andaddition of the substrate 3,3',5,5'-tetramethylbenzidine. TheA450 was read.

Protein determination. Protein was measured by themethod of Bradford (2), with ovalbumin as the standard.

RESULTS

Nature of the CHO cell receptor for pertussis toxin. WhenCHO cells are exposed to small amounts of pertussis toxin,they grow in a clustered pattern owing to toxin-catalyzedADP-ribosylation of the G proteins in the cells (7, 18). Aseries of variant CHO cell lines which were selected for theirresistance to lectin-mediated toxicity (4, 32) and which havealtered carbohydrate structures on surface macromolecules(Fig. 1) were examined for their ability to cluster whenexposed to petussis toxin. Significant differences in suscep-tibility to pertussis toxin action were seen with the variants.Wild-type CHO cells were clustered by pertussis toxin at aminimum concentration of 0.06 ng/ml. In contrast, LEC 2,LEC 8, and 15B cells were clustered by the toxin atconcentrations of 250, 31, and 250 ng/ml, respectively.When membrane preparations of wild-type, LEC 2, and

LEC 8 cells were incubated with pertussis toxin, the G

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3326 WITVLIET ET AL.

NeuAc NeuAc

IGal Gal

IGICNAC GIcNAc

IMan Man

\ /Man

IGIcNAc

Fuc - GIcNAc

ASN

WILD-TYPE

Man Man\M /Man

\ /

Man

IGIcNAc

IGIcNAc

ASN

15B

FIG. 1. Alterations in carbohydrate structures on wild-type andvariant CHO cells. Variations in carbohydrate structures are illus-trated by the structures of biantennary Asn-linked complex-typeoligosaccharide side chains of the indicated cell types (22).

proteins of all cell types were ADP-ribosylated by the toxin(Fig. 2). Thus, the lack of response of the variant cells topertussis toxin was not due to an inability of the toxin toADP-ribosylate the G proteins of these cells. The ability ofpertussis toxin to ADP-ribosylate G proteins in intact cellswas measured by exposing intact cells to pertussis toxin for24 h, harvesting the cells, preparing a membrane fraction,and incubating this fraction with [32P]NAD and pertussistoxin. Cellular substrates not ADP-ribosylated by the toxinwithin the cells are free to be modified in vitro and thusbecome radiolabeled. In contrast, any protein ADP-ribosy-lated within the CHO cell can no longer serve as a toxin

4 1,00 0-_r>. .Mbst,:.ofwl

1 2 3 4 5 6 7 8 9

FIG. 2. ADP-ribosylation of 41,000-Da substrates of wild-typeand variant CHO cells. Confluent cultures of wild-type and variantCHO cells were incubated without (lanes 1, 4, and 7) or withpertussis toxin at concentrations of either 10 ng/ml (lanes 2, 5, and8) or 100 ng/ml (lanes 3, 6, and 9) for 24 h. Cells were harvested, andmembrane proteins (100 ,ug per lane) from wild-type CHO cells(lanes 1 to 3), LEC 2 cells (lanes 4 to 6), and LEC 8 cells (lanes 7 to9) were prepared and subsequently incubated with pertussis toxin(1.5 ,ug) and [32P]NAD as described in Materials and Methods.Radiolabeled toxin substrate was visualized after SDS-polyacryl-amide gel electrophoresis and autoradiography. The position of the41,000-Da toxin substrate is indicated.

1 2 3 4FIG. 3. Binding of pertussis toxin to CHO cell extracts. N-

Octylglucoside extracts (equivalent to 105 cells per lane) of wild-type CHO cells (lane 1), LEC 2 cells (lane 2), LEC 8 cells (lane 3),and 15B cells (lane 4) were separated on a 4 to 20% gradient gelcontaining SDS and electrophoretically transferred to nitrocellulose.Nitrocellulose strips were incubated with pertussis toxin (2.5 ,ug/ml). Binding of pertussis toxin to immobilized proteins was visual-ized with an anti-S4 monoclonal antibody (6DX3) by using theimmunoblot techniques described in Materials and Methods. Mo-lecular weight markers are indicated (in thousands).

substrate and will not incorporate radiolabel. Treatment ofLEC 2 cells with concentrations of toxin as high as 100 ng/mldid not result in ADP-ribosylation of the G proteins in intactcells. The G proteins of intact LEC 8 cells were ADP-ribosylated only at toxin concentrations of greater than 10ng/ml. These results are consistent with the lack of ability ofpertussis toxin to cluster variant cells and suggest thatpertussis toxin does not gain access to G proteins in intactvariant cells.The ability of pertussis toxin to bind to proteins of the

variant cells was next examined by using immunoblot tech-niques. Pertussis toxin detected a 165-kDa component ofwild-type cells (Fig. 3), as has been previously reported (3).Binding of pertussis toxin to components in extracts fromLEC 8 cells was also seen. Holotoxin bound to a number ofbands which had molecular masses lower than those ob-served when the extract from wild-type cells was examined.Binding of pertussis toxin to components in extracts fromeither LEC 2 or 15B cell extracts was not detected.

Location of the glycoprotein-binding site on pertussis toxin.Receptor-binding sites on the pertussis toxin molecule werestudied by examining the ability of the S2-S4 and S3-S4dimers to bind to extracts of CHO cells. Very weak bindingof S3-S4 to the 165-kDa protein of wild-type CHO cells wasobserved (Fig. 4). Binding of S2-S4 to this component wasnot detected. Both S2-S4 and S3-S4 did bind, however, to alower-molecular-mass band which was not detected by per-tussis toxin and which therefore may not be a true toxinreceptor. The binding of pertussis toxin and dimers tocomponents found in goose erythrocyte membrane extracts

Gal

GIcNAc

Man

Gal

GIcNAc

Man

Man

G1cNAc

Fuc - GIcNAc

ASN

LEC 2

-200K

-92K

-69K

Man

GIcNAc GIcNAc

Man Man\Man

G1cNAc

Fuc - G1cNAcIASN

LEC 8

-46K

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BINDING OF PERTUSSIS TOXIN TO GLYCOPROTEINS 3327

II.,-.

- 69K

- 46K

1 2 3FIG. 4. Binding of pertussis toxin and dimers to CHO cell

extracts. N-Octylglucoside extracts (15 ,ug of protein per lane) ofwild-type CHO cells were separated on 4 to 20% gradient gelscontaining SDS and electrophoretically transferred to nitrocellulose.Nitrocellulose strips were incubated with pertussis toxin (2.5 jig/ml;lane 1), S2-S4 (2 jig/ml; lane 2), or S3-S4 (2 jig/ml; lane 3). Bindingwas visualized with an anti-S4 monoclonal antibody (6DX3) by usingthe immunoblot techniques described in Materials and Methods.Molecular weight markers are indicated (in thousands).

was also studied, since goose erythrocytes are readily agglu-tinated by pertussis toxin and both dimers. In contrast to theresults obtained with CHO cell extracts, both S2-S4 andS3-S4 bound components found in goose erythrocyte mem-brane extracts (Fig. 5). S2-S4 bound better than S3-S4 to amajor band which has a Mr of approximately 115,000. Wenext used dot blot techniques to examine the ability of thedimers to bind to various purified glycoproteins. Binding ofholotoxin and both dimers to a number of glycoproteinscontaining N-linked oligosaccharides was detected (Fig. 6).Little or no binding to glycophorin, which contains mostly0-linked oligosaccharides (14), was observed. Slight differ-ences in the binding of the two dimers were apparent. Mostnotably, in this assay, S2-S4 bound to fetuin better than toasialofetuin, whereas S3-S4 bound to asialofetuin better thanto fetuin.

Binding of both dimers as well as holotoxin to fetuin wasaffected by certain mono-, di-, and trisaccharides (Table 1).Sialic acid and the trisaccharide siallylactose (NeuAc->Gal,4Glc) markedly inhibited the ability of pertussis toxin andS3-S4 to bind to fetuin. Sialyllactose had less effect on S2-S4binding to fetuin.

DISCUSSIONA CHO cell variant (clone 15B) has previously been

shown to be resistant to pertussis toxin action (3). Since thiscell line lacks the terminal NeuAc->Gal14GlcNAc oligosac-charide side chains on N-linked glycoproteins, this sequenceof carbohydrates may be a part of the structure of thereceptor for pertussis toxin. Furthermore, since these cells

-200K

_MO lo *_ --116K- 97K

- 66K

- 43K

1 2 3 4FIG. 5. Binding of pertussis toxin and dimers to goose erythro-

cyte extracts. N-Octylglucoside extracts of goose erythrocyteghosts (15 ,ug of protein per lane) were separated by SDS-polyacryl-amide gel electrophoreis, and the proteins on the gel were eitherstained with Coomassie blue (lane 1) or electrophoretically trans-ferred to nitrocellulose (lanes 2 to 4). Nitrocellulose strips wereincubated with pertussis toxin (2.5 jig/ml; lane 2), S2-S4 (2 ,jg/ml;lane 3), or S3-S4 (2 ,ug/ml; lane 4). Binding was visualized with ananti-S4 monoclonal antibody (6DX3) by using the immunoblottechniques described in Materials and Methods. Molecular weightmarkers are indicated (in thousands).

vary from wild-type cells only in the structure of theirN-linked oligosaccharide chains, the receptor for pertussistoxin is most probably a glycoprotein. In this study, we haveexamined the ability of pertussis toxin to act on two othervariant CHO cell lines. The first of these cell lines, LEC 2, isdefective in its ability to translocate CMP-sialic acid acrossGolgi vesicle membranes and therefore is deficient in sialicacid residues on cell surface glycoproteins and glycolipids(11). The second cell line, LEC 8, is unable to translocateUDP-galactose into the lumen of the Golgi apparatus (10).Thus, both glycoproteins and glycolipids on the surface ofthese cells are deficient in galactose residues and also exhibita sialic acid deficiency which is secondary to the galactosedeficiency.

All variant cell types were much less sensitive to toxinaction than wild-type cells were. The lack of ability ofpertussis toxin to ADP-ribosylate G proteins in intact 15Bcells (3) or LEC 2 and LEC 8 cells was not due simply to analteration in the G proteins in membrane fractions isolatedfrom these cells, since these G proteins can be ADP-ribosylated in vitro when exposed to pertussis toxin. Morelikely, the inability of the toxin to ADP-ribosylate G proteinsin intact cells and thus cluster the variant CHO cells is due toaltered binding of the toxin to the surface components ofthese cells. This hypothesis is supported by the finding thatbinding of pertussis toxin to components extracted fromLEC 2 or 15B cells was not detected. Although binding toproteins of LEC 8 cells was observed, the apparent molec-ular masses of these proteins were lower than that of thereceptor on wild-type CHO cells (165 kDa). These alter-ations may represent differences in the oligosaccharideslinked to a single polypeptide, the presence of more than one

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PT S2-S4 S3-S4

HAPTOGLOBIN

THYROGLOBULI N

FETUIN

ASIALOFETUIN

LAMININ

GLYCOPHORIN

1 2 3 1 2 3 1 2 3FIG. 6. Binding of dimers to glycoproteins. Either 1 pg (lanes 1), 0.25 jxg (lanes 2), or 0.0625 ,ug (lanes 3) of the indicated glycoprotein was

applied directly to nitrocellulose. Dot blots were then incubated with pertussis toxin (2.5 ,ug/ml), S2-S4 (1 p.g/ml), or S3-S4 (1 ,ug/ml). Bindingof toxin species to the glycoproteins was visualized as described in Materials and Methods.

protein containing sugars reactive with pertussis toxin, orproteolytic degradation of a larger species.The finding that pertussis toxin does not bind to proteins

found in extracts of LEC 2 cells suggests that sialic acidresidues contribute to binding of the toxin. This result isconsistent with the finding that sialic acid inhibits pertussistoxin binding to fetuin and with previous results demonstrat-ing that sialidase treatment of the 165-kDa CHO cell receptorabolishes pertussis toxin binding to this component (3).Interestingly, the toxin does bind to proteins found inextracts of LEC 8 cells which are deficient in both sialic acidand galactose residues on surface glycoconjugates. Thesecells probably have increased amounts of terminal N-acetyl-glucosamine residues on cell surface proteins (4), which maycontribute to binding. These results are consistent with

TABLE 1. Effect of saccharides on the binding of pertussis toxinand dimers to fetuina

Addition'% Binding' to fetuin of:

PT S2-S4 S3-S4

None lood looe iOOfNeuAc 40 50 22Gal 86 67 60GlcNAc 84 65 66Man 83 70 61Glc 91 100 80Fuc 100 100 88NeuAc- GalP4Glc 20 63 14GalP4Glc 71 100 60GalP4GlcNAc 65 85 64

a The ability of pertussis toxin and dimers to bind to fetuin in the presenceof the indicated saccharides was measured by using an enzyme-linkedimmunosorbent assay technique as described in Materials and Methods. Theconcentration of saccharides used was 100 mM for monosaccharides and 12.5mM for di- and trisaccharides.

b Abbreviations: NeuAc, sialic acid; Gal, galactose; GlcNAc, N-acetylglu-cosamine; Man, mannose; Glc, glucose; Fuc, fucose; NeuAc--Ga1,4Glc,sialyllactose; Gall4Glc, lactose; GalP4GlcNAc, N-acetyllactosamine.

c Values are expressed as the percentage binding in the presence of theindicated saccharide compared with binding in the absence of any sugar andare means of duplicate assays.d100%: A450 = 0.69.100%o: A450 = 0.65.

f 100%: A450 = 0.57.

previous studies in which the carbohydrate groups involvedin the binding of pertussis toxin to fetuin were examined (1).Those studies demonstrated that asialofetuin was less effec-tive than fetuin in competing with 1251-fetuin for binding topertussis toxin. Removal of terminal galactose residues onasialofetuin (thus unmasking N-acetylglucosamine residues)partially restored the ability of this fetuin derivative tocompete with the radiolabeled fetuin for binding to pertussistoxin. Thus, pertussis toxin appears to bind best to CHO cellreceptors with acetamido-containing sugars in the nonreduc-ing terminal position of the oligosaccharide units.Although pertussis toxin binds to proteins from LEC 8

cells, the amount of pertussis toxin needed to cluster thesecells was dramatically increased over the amount needed tocluster wild-type CHO cells. These results, together withthose obtained when the effect of pertussis toxin on LEC 2cells was examined, suggest that the presence of a completeNeuAc-*Galp4GlcNAc sequence may be important for op-timal functional binding of the toxin. We have determinedthat pertussis toxin does not bind to sialoparaglobosidewhich has been chromatographed on a thin-layer plate (datanot shown). This molecule terminates in a NeuAc--Gal,4-GlcNAc sequence, suggesting that the branched nature ofthe oligosaccharide side chains on glycoproteins or thepolypeptide backbone may contribute to optimal lectinlikebinding of pertussis toxin to glycoproteins.

S3-S4 may contain a binding site for the CHO cell recep-tor, since weak binding of this species to the 165-kDareceptor was visualized by immunoblot techniques. Nobinding of S2-S4 was detected. S2-S4 may play a role inbinding of the toxin to CHO cells, but its affinity for the CHOcell receptor may be sufficiently weak that binding would notbe detected by the techniques used. Binding of pertussistoxin to goose erythrocytes may involve both dimers. S2-S4binds with an apparently higher affinity to the major toxin-binding protein of these cells. These results are consistentwith previous results demonstrating that both dimers arecapable of agglutinating erythrocytes. In those studies, S2-S4 agglutinated the cells at a concentration of 62.5 ng/ml,whereas S3-S4 agglutinated cells at a concentration of 250ng/ml (30). A more complete understanding of the apparentdifferences in the binding of the two types of dimers to CHO

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cell and goose erythrocyte receptors awaits further analysisof the major toxin-binding glycoproteins on these cells.The relative abilities of pertussis toxin dimers to bind to

proteins appear to be dependent on the specific targetprotein. The two dimers may bind optimally to differentglycoproteins. This idea is supported by the finding thatalthough weak binding of S3-S4 to components from wild-type CHO cells was detected, no binding of S2-S4 to thesecomponents was observed. S2-S4 appeared to bind better tocomponents from goose erythrocytes than did S3-S4. S2-S4bound to fetuin better than to asialofetuin, whereas theopposite result was obtained when the binding of S3-S4 tothese proteins was examined. Thus, the purified dimers haveslightly different specificities for certain glycoproteins. Inter-estingly, both dimers as well as the holotoxin bind tolaminin, a glycoprotein that is localized to the basementmembranes of mammalian tissues (24). Such interactionscould potentiate the movement of pertussis toxin throughblood vessels and across the blood-brain barrier, resulting inmore extensive effects on the host.The finding that both the S2-S4 and S3-S4 dimers can bind

to cellular components as well as to purified glycoproteinssuggests that the two dimers of pertussis toxin each containat least one receptor-binding site. Although the structures ofthe receptors remain to be determined, the studies reportedhere provide some information about which carbohydratestructures may play a role in binding of the toxin to recep-tors. Binding of S3-S4 to fetuin was inhibited best by thetrisaccharide sialyllactose (NeuAc-+Gal,4Glc). Sialic acidwas the most potent monosaccharide tested in inhibiting thebinding of S3-S4 to fetuin. These results suggest that areceptor for S3-S4 might contain a NeuAc--GalP4Glc moi-ety or a similar structure. Although the saccharide inhibitionstudies suggest that sialic acid may play a role in the bindingof S3-S4 to receptors, a contradictory result was obtainedwith the dot blot assay. In those studies, S3-S4 bound toasialofetuin better than to fetuin. Although the reason for thediscrepancy between the two assays is unknown, the resultsfrom the dot blot assay suggest that sialic acid residues arenot absolutely required for S3-S4 to bind to a glycoprotein.Specific inhibition by saccharides of the interaction of S2-S4with fetuin was less apparent. This finding suggests thatS2-S4 might bind to a more complex sugar sequence onglycoproteins or that the polypeptide chain may contributeto the interaction.Although many similarities in the binding of the two

dimers to receptors can be seen, the two dimers appear tohave slightly different modes of binding. Since S2 and S3share considerable homology, the glycoprotein-binding sitesmay be located on these subunits and differences in bindingof the two dimers might be due to altered amino acidsequence at the binding sites. In fact, recent evidencesuggests that isolated S2 can bind to haptoglobin (13).Differences in binding specificities of the dimers wouldincrease the target cell repertoire of pertussis toxin, allowpertussis toxin to exert its effects on a number of cell types,and therefore increase toxin-induced damage to the host.

ACKNOWLEDGMENTSWe thank Howard Krivan for helpful discussions. We also thank

Frederick D. Johnson and Betsy Kuipers for expert technicalassistance.

This work was supported in part by Participating Agency SupportAgreement BST-5947-P-HI-4265 between the U.S. Agency for In-ternational Development and the U.S. Public Health Service andgrant 28-1431 from the Dutch Praeventiefonds.

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