Preparation of erythrocyte receptors for viruses by affinity chromatography

285

Journal of Virological Methods, 1 (1980) 285 @ Elsevier/North-Holland Biomedical Press

298

PREPARATION

RECEPTORS

OF ERYTHROCYTE

FOR VIRUSES

BY AWINITY

CHROMATOGRAPHY

INGRID U. PARDOE and ALFRED T.H. BURNESS Faculty of Medicine, Memorial University of Newfoundland, AIB 3V6

St. John’s, Newfoundland,

Canada

(Accepted 4 June 1980)

A comparatively simple method for the purification of human erythrocyte receptors for encephalomyocarditis and influenza viruses is described. The procedure utilises the fact that these viruses share in common the erythrocyte receptor for wheat germ agglutinin @VGA), which enables commercially available WGA-Sepharose to be used in the purification of receptors for these viruses by affinity chromatography. Conditions are also described for introducing either rz51 into the receptor in situ, or ‘H-acetyl residues into the solubibsed receptor.

INTRODUCTION

At least two components are involved in the interaction between a virus and a cell surface: the cellular receptor and the attachment component in the virion. One way to isolate the cellular receptor is by affinity chromatography using a column containing the virion attachment component, as has been used for preparation of receptors for adenoviruses (Meager et al., 1976). Problems

with this approach

include ignorance as to which

virion component is involved in attachment, even for well-studied viruses such as the Picornaviridae, and when known, it may be difficult to produce enough component to make an adequate affinity column. A modification of this approach is to identify a more readily

available molecule

use this more abundant

which binds to the same receptor

molecule

isolation of the receptor. Preparations of glycophorin,

to prepare

an affinity

the major sialoglycoprotein

as the virus and to

chromatography in the human

column

for

erythrocyte

surface membrane (Winzler, 1969; Marchesi et al., 1976) contain receptors for influenza (Kathan et al., 1961) and encephalomyocarditis (EMC) viruses (Enegren and Burness, 1977) and, in addition, for the plant lectin, wheat germ agglutinin (,WGA) (Marchesi and Andrews, 1971). Therefore, a study was undertaken to determine whether WGA bound to an inert matrix could be used for affinity chromatographic purification, not simply of the WGA receptor, but for that of influenza and EMC viruses also.

286 MATERIALS

AND METHODS

32P-orthophosphoric acid (carrier Materials. 3 H-acetic anhydride (50 mCi/mmol), free), rzsI (carrier free), 3H-wheat germ agglutinin (1 Ci/mmol) and Aquasol- were purchased from New England Nuclear, Lachine, Quebec, Canada. Wheat germ agglutinin (WGA)-Sepharose and concanavalin A (Con A)-Sepharose (Canada) Ltd., Dorval, Quebec, Canada. Methyl mannopyranoside

were from Pharmacia was from Calbiochem,

San Diego, CA, U.S.A. Lactoperoxidase, glucose oxidase, N-acetylglucosamine, sodium deoxycholate (DQC) and sodium dodecyl sulphate (SDS) were from Sigma Chemical Co., St. Louis, MO, U.S.A. Basic fuchsin (to make Schiff’s reagent), Coomassie brilliant blue R-250, lithium diiodosalicylate and Triton X-100 were from Eastman Kodak Co., Rochester, NY, U.S.A. Other chemicals were from Fisher Scientific Co., Halifax, Nova Scotia, Canada. Outdated human type 0 blood was obtained from the Canadian Red Cross Blood Transfusion Service. Viruses. Growth of the K2 strain of EMC virus in Krebs ascites tumour cells (Sanders et al., 1958) and its purification have been described previously (Burness et al., 1974). The WSN strain of influenza virus was a gift, from Dr. R.M. Krug. Haemagglutination inhibition tests were performed on microtitre plates as described previously (Enegren and Bumess, 1977). Glycophorin preparations were made as described by Marchesi and Andrews (1971) and labelled with 3H-acetic anhydride in vitro by the procedure of Montelaro and Rueckert (1975) as follows. Approximately 1 ml of the glycophorin preparation in water at a concentration of 1 mg protein/ml was added directly to 5 mCi 3H-acetic anhydride distilled to the bottom of the breakseal tube used by the manufacturer to ship the material. After 30 min at ambient temperature, the glycophorin solution was dialysed against several changes of distilled water for at least 24 h until the radioactivity in the diffusate was at background level. Phosphorylation of erythrocytes. About 5 ml packed, freshly-drawn, washed human erythrocytes were mixed with 5 ml 20 mM HEPES buffer, pH 7.2, containing 0.14 M NaCl and 0.5 M KC1 and phosphorylated by addition of 4 mCi 32P-orthophosphoric acid (Shapiro and Marchesi, 1977). After incubationlor 90 min at 37’C, the cells were washed thoroughly with PBS until the supernatant fluid washes contained background levels of radioactivity and then erythrocyte membranes were prepared as described by Fairbanks et al. (1971). Iodination of elythrocytes.

Approximately

200 1.11packed,

freshly-drawn,

human

erythrocytes, washed with PBS, were mixed with 5 ~1 lactoperoxidase (1 mg/ml), 5 ~1 glucose oxidase (3 /d/ml), 20 111glucose (4.5% w/v) and 100 /..&i rz5 I and made up to 1 ml with PBS (Hubbard and Cohn, 1972). After incubating for 30 mm at 37°C with constant agitation, the cells were washed three times with 10 ml PBS before preparing erythrocyte membranes as described by Fairbanks et al. (197 1). Affinity chromatography. In most experiments, a bed volume of 0.9 X 8 cm WGASepharose was used. For some experiments in which membranes were dissolved in Triton

X-100, the bed volume was 1.5 X 12 cm. In either case the column was equilibrated about 50 ml of one of the following:

PBS, 05% DOC! in water (Clementson

0.05% SDS in 0.15 M sodium phosphate

buffer, pH 7.2 containing

with

et al., 1977),

0.25 M NaCl (Shapiro

and Marchesi, 1977) or 0.5% Triton X-100 in 56 mM sodium borate buffer, pH 8 (Adair and Komfeld, 1974). Preparations of glycophorin or membranes were applied to the column in the solution used for equilibration. A 25 ml wash of the same buffer was sufficient to remove unbound material and reduce radioactivity in isotopically labelled preparations in the eluate to background levels. Bound material was eluted with 25 ml equilibration solution containing 0.1 M acetylglucosamine. The whole 2.5 ml of eluate was combined, dialysed, to remove the acetylglucosamine, and then lyophilised. For radioactive preparations, samples of the 1 ml fractions collected were monitored in 10 ml Aquasolin a Beckman LS-350 scintillation spectrometer, and only the fractions comprising the radioactive peak were combined, dialysed and lyophihsed. Chromatography on Con A-Sepharose was performed in a similar way to chromatography on WGA-Sepharose in the presence of 0.5% DOC. To elute material specifically bound to the Con A-Seph~ose, the column was washed with2% methy~annopyr~oside in OS’% DOC. SDS-polyacrylamide gel electrophoresis Including the Coomassie blue and periodic acid-Schif~s reagent staining procedures were performed on 5 -6% polyac~la~de gels as described by Fairbanks et al. (1971). Gels stained by either method were scanned at 550 nm in a Gilford spectrophotometer fitted with a gel transport accessory. When radioactive material was examined by gel electrophoresis, the components were located by pulverising the gels in a Gilson gel fractionator and to each fraction was added 1 ml 0.1% SDS before measuring radioactivity in 10 ml Aquasol-2.

RESULTS

Before receptors

for viruses could be isolated

necessary to learn something

about their properties

by afftity

c~omatography,

so that they could be identified

it was once

isolated . SDS-gel electrophoresis of human erythrocyte membranes More than 10 components were detected by Coomassie blue staining when human erythrocyte membranes were subjected to SDS-gel electrophoresis (Fig. la), in agreement with previous reports (Fa~b~s et al., 1971; Steck, 1974). When similar gels were stained with periodic acid-Schiffs reagent (Fig. lb), four components, PAS-1 to 4 (nomenclature of Fairbanks et al., 1971; Steck, 1974) were seen together with variable amounts of other components, including one reported to contain lipids (Fairbanks et al., 1971), glycolipids possibly being responsible for its PAS-positive staining (Lenard, 1970). PAS-l and PAS-2, which are probably interconvertible and represent the dimer and

288

15 10 5 0 15 cu 10 0 x 52

v

0 0 2 4 6 0 10 DISTANCE FROM ORIGIN

15 10 5 0

0 20 40 60 80 FRACTION NUMBER

Fig. 1. SDS-gel electrophoreslB o f human a) Coomassie paration

blue,

of membranes

were fractionated

erythrocyte

membranes.

The gels were stained

with either

or b) periodic with

acid-Schiffs reagent; or the erythrocytes were labelled prior to preanhydride, in which case the gels c) 12’1, d) “*PO, and e) 3H-acetic

for radioactivity

measurements

(see Methods).

monomer forms of glycophorin A (Marton and Gavin, 1973; Furthmayr and Marchesi, 1976), together constitute about 80% of the total PAS-positive material, excluding the variable ‘lipid’ component (Fig. la and b). Radioactivity associated with r2’ I-labelled

membranes

subjected

to SDS-gel electro-

phoresis gave a pattern (Fig. lc) which resembled the PAS-stained gels, the peaks corresponding to PAS- 1 and PAS-2 comprising about 90% of the total radioactivity, excluding that in the ‘lipid’ region of the gel. In contrast, membranes from whole erythrocytes labelled with 32P04 (Fig. Id), or ery throcyte membranes labelled with 3H-acetic anhydride

(Fig. le) contained

little radioactivity

the SDS-gels, most of the radioactivity

corresponding

to the PAS-l to 4 region of

running with the ‘lipid’ component.

SDS-gel electrophoresis of glycophonk preparations Glycophorin, prepared by the lithium diiodosalicylate procedure (Marchesi and Andrews, 197 1) and examined by SDS-gel electrophoresis gave identical patterns whether stained with Coomassie blue (Fig. 2a) or PAS (Fig. 2b) suggesting that all of the components present in the glycophorin preparation were glycoproteins. Profiles of glycophorin (Fig. 2b) or of erythrocyte membranes (Fig. lb) were very similar when stained

289

1

I

I

I

1

a

C

1 PAS

-2

1 \ LIPID

b

. 0

6

DISTANCE4 2 FROM

8 10 ORIGIN km)

0

40 60 20 FRACTION NUMBER

80

Fig. 2. SDS-gel electrophoresis of glycophorin preparations. The gels were stained with either a) Coomassie blue or b) periodic acid-Schiffs reagent, or were fractionated for radioactivity measurements, in which case the 3H-acetylated glycophorin preparation was c) unextracted, or d) extracted with chloroform/methanol before electrophoresis.

with PAS, except that the ‘lipid’ component of membranes was greatly reduced in the glycophorin preparation suggesting that the lithium diiodosalicylate procedure extracts most of the glycoproteins present in human erythrocyte membranes. Although erythrocyte membrane glycoproteins were not readily acetylated in situ (Fig. le), glycophorin preparations were acetylated when treated with 3H-acetic anhydride. Acetylated and non-acetylated preparations were indistinguishable on gels stained with Coomassie blue or PAS (result not shown). The radioactivity profile of 3Hacetylated phoresed

glycophorin

was similar to the distribution

in parallel, except that a sizeable proportion

of material on stained gels electroof the radioactivity

the ‘lipid’ region of the gel (Fig. 2~). The latter radioactivity electropherograms

of 3 H-acetylated

(2 : 1, v/v) (Saito and Hakomori, nature of the material.

glycophorin

extracted

was found in

was missing (Fig. 2d) from with chloroform/methanol

1971) prior to electrophoresis,

confirming

the lipid

Affinity chromatography of glycophorin on WGA-Sepharose When 3H-acetylated glycophorin was chromatographed on WGA-Sepharose in PBS (Fig. 3a), about 10% of the recovered radioactivity was not retained whereas 90% was bound and subsequently eluted by PBS containing 0.1 M acetylghrcosamine, the sugar required for WGA binding to erythrocytes (Burger and Goldberg, 1967). A similar dis-

290

-PBS

-- +

3

3

P 92

2; ;

;

b

5 1

1

0

L

0 25

25

50 FRACTION

Fig. 3. Affinity

chromatography

DOC, was transferred respectively,

and

glycophorin washed

then

with

preparation

with

0.5%

0.5% DOC containing

NUMBER

of 3 H-acetylated

to a column these

of WGA-Sepharose same

solutions

in 0.5% DOC was transferred

DOC,

then

with

v

5

glycophorin.

Glycophorin

which was washed containing

0.1 M acetylglucosamine.

to a column

0.5% DOC containing

in a) PBS, or b) 0.5%

first with PBS or 0.5% DOC, c) A similar

of Con A-Sepharose

0.1 M acetylglucosamine

which was first and finally

with

2% methylmannoside.

tribution of radioactivity was obtained when 0.5% DOC (Fig. 3b), 0.5% Triton X-100 or 0.05% SDS were present in the buffer (results not shown), demonstrating that the presence of detergents apparently had little effect on the binding of WGA to this receptor. To prove that binding was through the WGA moiety rather than through non-specific adsorption to the Sepharose, 3H-acetylated gly co p horin in 0.5% DOC was chromatographed on a Con A-Sepharose column. Close to 100% of the recovered radioactivity was not retained by the column (Fig. 3~). No radioactivity

was eluted by subsequent

washings

of the column with either 0.5% DOC containing 0.1 M acetylglucosamine which eluted material bound to WGA-Sepharose (Fig. 3b) or with 0.5% DOC containing 2% methylmamroside

(Fig. 3c), the sugar required

for binding

Con A to erythrocytes

(Goldstein

et al., 1965). These observations proved that glycophorm was binding specifically to the WGA moiety on WGA-Sepharose and at the same time suggested that our preparation of glycophorin Affnio

was free of contamination

with receptor for Con A.

chromatography of neuraminidase- treated glycophorin

Although N-acetylglucosamine is accepted as the major sugar haptene involved in binding WGA to cell surfaces, sialic acid residues are also apparently involved in the interaction (Burger and Goldberg, 1967). Treatment of glycophorin preparations with neuraminidase to remove terminal sialic acid had a marked effect on the binding of the lectin

291

DOC_

-

a

e&l glucosamine b

i

I

i

0

20

40 0 20 FRACTION NUMBER

40

Fig. 4. Affinity c~omato~aphy of neur~~idase-treated glycophorin. 3H-acetylated glycophorin (7 pg) was treated at a) with 5 units neuraminidase or b) with PBS, and both samples were incubated for 30 min at 37°C followed by heating for 10 min at lOO”C, made 0.5% with DOC and chromatographed separately on WGA-Sepharose.

to WGA-Sepharose. About 60% of the radioactivity in 3H-acetylated ~ycopho~, which had been treated with neuraminidase previously and then boiled to destroy the enzyme, failed to bind to WGA-Sepharose (Fig. 4a). The behaviour of desialylated glycophorin was not due to the heating step since 80% of a boiled, but non-enzyme treated preparation became bound to WGA-Sepharose and was released with acetylglucosamine (Fig. 4b). Affinity chromatography of solubilised erythrocyte membrane

Having established that glycophorin preparations behaved as expected on WGASepharose, attempts were made to recover WGA receptor directly from solubilised ery throcyte membranes. ~embmnes, prepared from about 0.1 ml packed cells labelled with ‘2~I, were stirred for 2 h at 0°C in 7 ml 0.5% Triton X-100 in 56 mh4 borate buffer, pH 8.0 (Adair and Kornfeld, 1974). After centrifugation at 10,000 g for 60 min to remove undissolved material, the preparation was transferred directly to a WGA-Sepharose column which was washed with 0.5% Triton X-100 in 56 mh4 borate buffer, pH 8.0, until radioactivity in the 1 ml fractions eluting from the column had reached background levels. The column was then washed with 0.1% Triton X-100 in 56 mM borate buffer, pH 8.0, containing 0.1 M N-acetylglucosamine and the distribution of radioactivity in the fractions measured. Of the recovered radioactivity, about 30% was not retained by the column and the remaining 70% became adsorbed but was released by the N-acetylglucosamine (Fig. 5).

292

15

.Triton x -100

TritonX~lOO -~--t acetyl glucosamlne

SJ 0

x

10

Ix a 0

C

25 FRACTION

50 NUMBER

Fig. 5. Affinity chromatography of detergent-solubilised human erythrocyte membranes. A lz51labelled erythrocyte membrane preparation, solubilised in Triton X-100, was transferred to a column of WGA-Sephaxose which was washed first with Triton X-100 in borate buffer and then with the same solution containing 0.1 M acetylglucosamine.

Fractions comprising the unbound and bound peaks of radioactivity from tne WGASepharose column were combined separately and the two samples dialysed extensively against water and concentrated before analysis by SDS-gel electrophoresis. Almost all of the material in the bound sample was glycophorin, predominantly in the monomeric (PAS-2) form (Fig. 6). PAS-3 was also evident in the bound sample but in amounts varying in duplicate experiments. The remainder of the PAS-3 together with all of the ‘lipid’ component was present in the unbound sample (Fig. 6b). From these experiments, it appears possible to prepare glycophorin direjtly by affinity chromatography of Triton X-100~solubilised membranes on WGA-Sepharose. Furthermore, it is clear that most of the rzs I incorporated into erythrocytes becomes associated with glycophorin thus providing a convenient method for labelling this molecule. In contrast to iodination, attempts to label glycophorin with 32P04 were not encouraging, as described above (Fig. Id). The possibility was considered that glycophorin could not be phosphorylated, despite a previous report (Shapiro and Marchesi, 1977) and this was investigated as follows. 32P-labelled erythrocyte membranes, to which 3Hacetylated glycophorin was added as a marker, were dissolved in 0.5% DOC and chromatographed on WGA-Sepharose. The vast excess of 32P-radioactivity was not bound and the column required extensive washing with 0.5% DOC to reduce 32P-radioactivity in the eluate to background levels. When the eluting buffer was changed to 0.1 M acetylgluco-

293

a

FRACTION

Fig. 6.

SDS-gel electrophoresis of human

matography. described ponents

NUMBER

Triton

X-100

solubilised

in Fig. 5 by affinity present

erythrocyte

membrane

‘ZSI-labelled

membranes

chromatography

in each preparation

analysed

into b) unbound

fractions were

separated

by affinity

a) untreated,

and c) bound

chro-

or separated

fractions

as

and the com-

by SDS-gel electrophoresis.

samine in 05% DOC, the 3H-acetylated glycophorin was eluted together with a coinciin glycophorin redent peak of 32P-labelled glycophorin (Fig. 7). The 32P-radioactivity presented less than 0.06% of the total radioactivity added to the column and yet this material was selectively bound by the WGA-Sepharose illustrating the specificity of the procedure. From this experiment it was also learned that phosphorylation was not as effective as iodination for 1abeIling glycophorin and that solubilisation of erythrocyte membranes

for chromatography

on WGA-Sepharose

could be accomplished

as effect-

ively with 0.5% DOC as with 0.5% Triton X-100. Biological properties of WGA-Sepharose purified preparations Glycophorin

preparations

inhibit

WGA, influenza

virus (Marchesi and Andrews,

1971)

and EMC virus (Enegren and Burness, 1977) haemagglutination. It was important to establish whether these inhibitory properties survived WGA chromatography. For this purpose, glycophorin preparations were chromatographed on WGA-Sepharose and the HA inhibitory activity of the bound fraction was compared, after dialysis, with the original, non-chromatographed sample. Close to 100% of the inhibitory activity of the original material was recovered in the bound fraction when influenza virus and WGA were used for assay (Table I) (EMC virus was not tested in these experiments).

294

20

40

FRACTION

60

80

NUMBER

Fig. 7. Affinity chromatography of “P-labelled human erythrocyte membranes. Membranes prepared from freshly drawn blood labelled with “ZPO, were dissolved in 0.5% DOC, mixed with ‘Hacetylated glycophorin and chromatographed on WGA-Sepharose as described in Fig. 3; ‘*P, continuous line; ‘H, broken line.

TABLE 1 HA inhibitory properties of glycophorm following chromatography

on WGA-Sepharose

Agent

Glycophorin preparation

Glycophorin dilution to inhibit 1 HAU

Influenza virus Influenza virus Wheat germ agglutinin Wheat germ agglutinin

Original Bound Original Bound

lull1 1 in 16 1 in 36 1 in 33

Glycophorin was chromatographed on WGA-Sepharose as described in Fig. 2, but in the presence of 0.5% Triton X-100 in 56 mM borate buffer, pH 8.0. The HA inhibitory activity of the original, unchromatographed sample and the fraction bound in chromatography were compared after adjusting to the same protein concentrations.

One purpose of the present study was to determine whether the HA inhibitory activity of glycophorin preparations was due to glycophorin per se or to some other minor component such as glycolipids (Fig. 2c) contaminating the preparation. We have already shown that during WGA-Sepharose chromatography glycophorin (PAS-l and PAS-2) is bound but other PAS-positive components, as revealed by SDS-gel electrophoresis, are not retained (Fig. 5). To test which component contained the HA inhibitory activity, a 3H-acetylated glycophorin preparation was chromatographed on WGA-Seph-

295

TABLE

2

Inhibition

of EMC virus haemagglutination

Sample

by WGA-Sepharose

HAU inhibited

c.p.m.

and unbound

fractions

HAU/c.p.m.

Ratio

80

3440

0.023

1

640

3487

0.184

8

Unbound Bound

bound

arose and the bound and unbound fractions rechromatographed separately. The twice bound and twice unbound fractions were dialysed against distilled water, lyophilised, and made up to the same number of c.p.m./min/ml. The bound fraction was about 8 times more effective at reducing the HA titre for EMC virus compared with the unbound fraction (Table 2). This finding, which suggests that glycolipids in the glycophorin preparation play at best a minor role in the inhibitory properties of the preparation, was supported in another way. Extraction of glycophorin preparations with chloroform/ methanol, which removed the lipid component (Fig. 2d), had little effect or even enhanced the HA inhibitory properties against WGA, and influenza and EMC viruses (Table 3). TABLE Effect

3 of lipid extraction

of glycophorin

preparation

on haemagglutination

inhibitory

% HA inhibition

Agent

Unextracted EMC virus Influenza

virus

Wheat germ agglutinin a

activity

Preparation

extracted

with chloroform/methanol

Extracted8

100

127

100

202

100

99 (2

: 1, v/v) (Saito and Hakomori,

1971).

Detergents such as SDS, Triton X-100 and DOC are notoriously difficult to remove from protein preparations. Moreover, they create difficulties in testing for biological activity when cells or membranes form part of the assay procedure. Removal of such detergents from glycophorin preparations can be accomplished with ease by affinity chromatography as the following experiment illustrates. Erythrocyte membranes containing 25 mg protein were solubilised in 25 ml of 0.5% Triton X-100 in 56 mM borate buffer, pH 8.0 and transferred to a WGA-Sepharose column of bed volume 1.5 X 12 cm. The column was then washed with about 25 ml 0.5% Triton X-100 in borate buffer to remove unbound material followed by about 50 ml PBS to remove the detergent which was monitored by extinction measurements at 275 run. The material bound to the

296 TABLE 4 HA inhibitory activity of glycophorin prepared by WGA-Sepharose chromatography of Triton X-100 solubilised membranes Agent

Glycophorin dilution to inhibit 1 HAU

EMC virus Influenza virus Wheat germ agglutinin _-

1 in72 1 in58 lin112

column was displaced with 0.1 M acetylglucosamine in PBS, dialysed to remove the hexosamine, lyophilised and finally dissolved in PBS for testing. The bound material was effective at inhibiting haemagglutination by WGA, EMC and influenza viruses (Table 4), but, even at high concentrations, did not cause lysis of erythrocytes, unlike some earlier preparations produced by affinity chromatography without the step involving the PBS column wash to remove the detergent. DISCUSSION Affinity chromatography is a particularly because the method is presumably mimicking

attractive method for isolating receptors the receptor interactions existing in nature

and, therefore, can be expected to show high specificity. Detergents apparently had no gross effect on the binding of receptor of WGA-Sepharose since identical profiles were obtained when glycophorin was chromatographed in their presence or absence. Most of the affinity chromatography experiments described in this report were performed in the presence of detergents for three reasons. Firstly, detergents were used to solubilise membranes and their use enabled the solubilised material to be transferred directly

to the column

without

intermediate

purification.

Secondly,

the components

present in glycophorin preparations undergo aggregation in the absence of detergent (Pardoe and Burness, unpublished results) leading to the possibility that material lacking the specific WGA binding site could become bound to the affinity column by being in a complex with the WGA receptor. Thirdly, although glycophorin. is soluble in simple aqueous buffers, many virion and membrane proteins require detergents for solubility and it was important to establish that the specific interaction of WGA with glycophorin takes place in the presence of detergents. That specific interaction does take place suggests that affinity chromatography should be a useful technique for isolating proteins requiring detergents for solubility. Three methods were investigated for making the WGA and k&s erythrocyte receptors radioactive in vitro. 125Iodination of membranes yielded highly radioactive yet biologic-

297

ally active preparations acetic anhydride

and appears to be the method

was also found to be a convenient

of choice. Acetylation

and particularly

with 3H-

simple method

but

for purified receptors rather than those in situ in the membrane. It has been membrane

suggested

of human

that

glycophorin,

erythrocytes,

the major sialoglycoprotein

is the receptor

in the surface

for WGA (Marchesi and Andrew%

1971) influenza virus (Kathan et al., 1961; Marchesi and Andrew% 1971) and EMC virus (Enegren and Burness, 1977). These conclusions were based on the ability of glycophorin preparations to inhibit haemagglutination by these three agents. However, the preparations tested contained, in addition to glycophorin, minor components which could have been responsible for the HA inhibition. In the study reported here, we have shown that WGA, attached to an inert matrix, selectively binds the major sialoglycoprotein of the erythrocyte surface. Moreover, the purified glycophorin so obtained is still very effective at causing HA inhibition of both EMC and influenza viruses. This suggests that both viruses and WGA do share a common receptor on the erythrocyte surface and that the minor components in the original preparations are not responsible for the interactions. ACKNOWLEDGEMENTS

This work was supported by grants from the Medical Research Council of Canada and by the National Cancer Institute of Canada. We are grateful to Dr. R. Krug for the influenza cells.

virus and to the Canadian

Red Cross, St. John’s, Newfoundland

for supplying

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