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Vol. 130, No. 2, 1985 BIOCHEMICAL AND BIOPHYSICAL RESEARCH COMMUNICATIONS July 31, 1985 Pages 835-840 COMPARISON OF WATER EXPOSED AREA OF CHOLERA TOXIN WHEN FREE IN SOLUTION AND BOUND TO LIPOSOMES CONTAINING THE GANGLIOSIDE GM1 Maurizio TOMASI*, Khalid M. ABU-SALAH**, and John B.C. FINDLAY*** *Laboratorio di Biologia Cellulare, Istituto Superiore di Sanitl, Viale Regina Elena 299, 00161 Roma, Italy **King Saud University, P.O. Box 2455, Biochemistry Department, Riyadh, Saudi Arabia ***Biochemistry Department, The University of Leeds, Leeds LS2 9JT, U.K. Received May 6, 1985 Membrane impermeable diazocoupling reagents were used for studying the water exposure of subunits ( a, B ,y ) of cholera toxin (CT) when bound to liposomes containing the ganglioside G interaction between CT with Lip-GM1 shielded the Mt!ind?r$%l,',bn ':E particular, since a maximum of one amino acid residue on each/l subunit was modifiable. When CT was labeled free in solution five residues of each /? subunit can be coupled, but it produced loss of binding ability. New area ofp subunit was exposed to reagents after having removed a subunit. This labeling may serve as a tool to assess the topology of CT upon binding with Lip-GMl. 0 1985 Academic Press, Inc. Cholera toxin (CT) is a multisubunit protein composed of three different types of subunit: a(MW 21000), p (MW 11400) and y (MW 9000) assembled as a aYp5 oligomer in which the a subunit is linked to the y subunit via a disulfide bond which can be splitted upon treatment with thiol reagents (1). CT initiates cell intossication by a rapid binding between p, region and the cell surface monosialoganglioside GM1 (2, 3). After a lag time of about 20' a subunit modifies irreversibly the adenylate cyclase system which is located in the cytoplasmic side of the plasmamembrane (l-3). The way whereby a subunit crosses the membrane barrier is still unclear. To understand how the interaction between CT and G Ml may induce translocation of a subunit membrane model systems have been used (4-6). In this report we examine the availability of the CT subunits to react with water soluble probes before and after binding with liposomes containing GM1 (Lip-GM1). The reagents employed 0006-291X/85 $1.50 835 Copyright 0 1985 by Academic Press, Inc. All rights of reproduction in any form reserved.

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Vol. 130, No. 2, 1985 BIOCHEMICAL AND BIOPHYSICAL RESEARCH COMMUNICATIONS

July 31, 1985 Pages 835-840

COMPARISON OF WATER EXPOSED AREA OF CHOLERA TOXIN WHEN FREE IN SOLUTION AND BOUND TO LIPOSOMES CONTAINING THE GANGLIOSIDE GM1

Maurizio TOMASI*, Khalid M. ABU-SALAH**, and John B.C. FINDLAY***

*Laboratorio di Biologia Cellulare, Istituto Superiore di Sanitl, Viale Regina Elena 299, 00161 Roma, Italy

**King Saud University, P.O. Box 2455, Biochemistry Department, Riyadh, Saudi Arabia

***Biochemistry Department, The University of Leeds, Leeds LS2 9JT, U.K.

Received May 6, 1985

Membrane impermeable diazocoupling reagents were used for studying the water exposure of subunits ( a, B ,y ) of cholera toxin (CT) when bound to liposomes containing the ganglioside G interaction between CT with Lip-GM1 shielded the Mt!ind?r$%l,',bn ':E particular, since a maximum of one amino acid residue on each/l subunit was modifiable. When CT was labeled free in solution five residues of each /? subunit can be coupled, but it produced loss of binding ability. New area ofp subunit was exposed to reagents after having removed a subunit. This labeling may serve as a tool to assess the topology of CT upon binding with Lip-GMl. 0 1985 Academic Press, Inc.

Cholera toxin (CT) is a multisubunit protein composed of three

different types of subunit: a(MW 21000), p (MW 11400) and y (MW 9000)

assembled as a aYp5 oligomer in which the a subunit is linked to the

y subunit via a disulfide bond which can be splitted upon treatment with

thiol reagents (1). CT initiates cell intossication by a rapid binding

between p, region and the cell surface monosialoganglioside GM1 (2, 3).

After a lag time of about 20' a subunit modifies irreversibly the

adenylate cyclase system which is located in the cytoplasmic side of the

plasmamembrane (l-3). The way whereby a subunit crosses the membrane

barrier is still unclear. To understand how the interaction between CT

and G Ml may induce translocation of a subunit membrane model systems

have been used (4-6). In this report we examine the availability of the

CT subunits to react with water soluble probes before and after binding

with liposomes containing GM1 (Lip-GM1). The reagents employed

0006-291X/85 $1.50

835 Copyright 0 1985 by Academic Press, Inc.

All rights of reproduction in any form reserved.

Vol. 130, No. 2, 1985 BIOCHEMICAL AND BIOPHYSICAL RESEARCH COMMUNICATIONS

diazocouple specifically to tyrosine, histidine and lysine side chains

exposed to the acqueous medium (7). The labeling was utilised to compare

the number of amino acid residues of CT modified by probes when free in

solution and when bound to Lip-GMl. Three different sets of polypeptide

side chains were recognized on ~85 region: the first set is the most

reactive one and it is available to reagents in any condition; the

second set regards four residues for each fi subunit which can be labeled

only when CT is free in solution; the third one is disclosed after

removing of a subunit from 785 region.

MATERIALS AND METHODS

Highly purified (Istituto di Chimica

of Prof. G. Tettamanti Milano). Cholera toxin

wT15a kind gift of Dr. .J3.5L. c

Tayot (Institut.Merieux, Lyon France). 11 iodoaniline and[ Slsulphanilic acid were obtained from Amersham

Inter-national, U.K. All other reagents were and were

ealytical grade or better, obtained from BDH CheT&als. [: SI-DABS was prepared as

described by Tinberg et al. (8). I II-DDISA was prepared according to the method of Helmkamp and Sears (7). The specifif2Fctivity of prepared probe was 6.0 Ci/mol. Labeling of CT with[ II-DDISA arc'% DABS was performed after having interacted 6Opg of CT dissolved in 30 ~1 of 100 mM sodium phosphate, pH 8.0, at 37'C for 1 h with 200 ~1 of liposomes containing 1 mg of mixed lipids and 19 pg of described in (4, 5). Further labeling experiments were free and bound, where a subunit was splitted from yf15 region upon

1 h at 37°C y+th 30 mM glutathione dissolved in the same II-DDISA or.[ Sl-DABS was added to give the desired molar

ratios of probe. The mixtures were incubated at 4°C for 2 h followed by dialysis against 100 mM Tris-HCl buffer pH 8.2 for 36 h at 4°C to remove the unreacted probe. Aliquots of dialysed material were divided into equal portions and the protein precipitated with acetone (5). One sample was used for determining the protein content by amino acid analysis; the other one was examined with SDS-polyacrylamide gel electrophoresis (SDS-PAGE) performed according to Laemli (9). The total number of

modified amino acid residues was estimated from the ratio of moles of probe incorporated per mole of protein as determined by amino acid analysis. The number of modified residues in the a subunit and rb, region was obtained from the relative distribution of radioactivity in the two species as revealed by SDS-PAGE. was estimated as reported in (4).

The binding of CT to Lip-GM1

RESULTS

The pattern of labeling of CT with c12511 -DDISA as obtained from

SDS-PAGE indicated that both a subunit and rb, region were covalently

modified by the reagent (data not shown). To further investigate this

phenomenon labeling of CT at molar ratios of probe to protein that range

836

Vol. 130, No. 2, 1985 BIOCHEMICAL AND BIOPHYSICAL RESEARCH COMMUNICATIONS

TABLE I

EFFECT OF BINDING BETWEEN CHOLERA TOXIN AND GM1 CONTAINING LIPOSOMES ON DISTRIBUTION

OF LABELING WITH [ '=I]-DDISA IN (Y-SUBUNIT AND +S REGION

Cholera Toxin in Buffer Cholera Toxin bound to lisosomes-GM1

Modified Ability to bind Modified Residual Bound Residues in (a) to liposomes-GM Residues in (a) Toxin after

Molar ratio of after labeling [b)

[l*'Il -DDISA: Toxin a

labeling

Y4 (%) Q Yfls (%)

10 0.4 2.2 98 0.1 0.4 97

20 1.4 4.7 95 0.8 1.1 95

30 4.6 11.0 42 2.1 3.9 98

40 6.1 14.9 5 2.8 5.0 96

80 11.0 26.2 3 3.9 5.4 94

120 14.2 25.3 3 6.2 5.5 98

(a) The number of modified amino acid residues was estimated as described in Materials and Methods.

(b) The binding was assayed by electrophoresis as reported in (4).

from IO to 120, was carried out (Table 1). The results show that the

extent of labeling was constantly higher for unbound toxin than for the

bound one. The number of modified amino acid residues in the a subunit

and the yf15 region was estimated in each case (Table 1): in the yfl5

region they reached a maximum value of approx. 26 at a molar ratio of

[12511-DDISA/protein of 40/l when labeling was carried out subsequent to

binding. On the other hand, the number of modified amino acid residues

in the a subunit increased over the whole range of 1125

11 -DDISA

concentrations. When the labeling of CT was carried out in buffer only,

however, the extent of labeling of both a and p subunits increased with

increasing molar ratios of probe to protein. It is worth noting that the

binding capacity of CT to the Lip-GM1 was abolished when more than five

amino acid residues were modified in the yj5 region prior to binding

(Table 1). In the case the labeling was performed on bound toxin,

however, the percentage of binding was not altered irrespective of the

molar ratio of probe/toxin used and no more than 5 residues resulted to

837

Vol. 130, No. 2, 1985 BIOCHEMICAL AND BIOPHYSICAL RESEARCH COMMUNICATIONS

TABLE 2

EFFECT OF DISULFIDE BRIDGE SPLITTING ON DISTRIBUTION OF LABELING WITH

['25I1 -DDISA IN Q!-SUBUNIT AND & REGION

Cholera Toxin bound to liposomes-GM1

Cholera Toxin in Buffer

Modified residues Modified residues My&r Ratio of 1 II-DDISA: Toxin a r4 01 r&

20 2.1 4.2 0.8 3.8

40 8.0 16.0 1.2 9.8

80 12.2 30.3 1.4 14.6

The conditions were the same as in Table 1 and the splitting of disulfide bridge was performed with dithiothreitol as reported in (5).

be modified on y fl, region. The above results were confirmed using

another diazocoupling reagent, [35SlDABS (data not shown). Both free and

vesicle bound toxins were exposed, after reduction, to the action of

1125~l-~~~~~. Since the above results did not clarify the topology of

a subunit further experiments were carried out on reduction conditions

which previous work has been reported to dissociate c subunit from rj5

region (4). In Table 2 it is shown that when the labeling was performed

on the bound and reduced CT, it was obtained a decrease in the a subunit

and an increase in the yf15 region. In contrast, labeling of the free

form of the toxin led to a slight increase in the radioactivity content

of the rp5 region with little or no change in the a subunit.

DISCUSSION

Our results indicate that when CT interacts with its receptor,

inserted into liposomes, about 80% of water exposed surface of binding

region is shielded by lipids. This percentage is inferred by comparing

the labeling (expressed as number of modified residues) which resulted

on a subunit and 785

region before and after binding to Lip-GM1

(Table 1). It is assumed that reactive residues are uniformely

838

Vol. 130, No. 2, 1985 BIOCHEMICAL AND BIOPHYSICAL RESEARCH COMMUNICATIONS

distributed on the whole binding region surface. Although this

evaluation may be quite roughly it emerges that the binding of CT to

Lip-GM1 produces an increase of hydrophobicity because the water was

removed out from the most hydrophilic components of interacting

molecules, namely the b5 region and oligosaccharide moiety of GML.

However this phenomenon does not account for lipid insertion of

a subunit (5), which was instead induced by reduction of the disulfide

bridge joining a subunit to y f15 region. Here we report that identical

reduction conditions lead to a significant shielding of a subunit to

probes, while Yfi5 region unmasks new reactive residues. The results

further support the idea that splitting of that disulfide bond causes

lipid insertion of a subunit while yB5 region remains located at level

of lipid polar groups.

Finally the methodology of CT labeling provides a mean to identify

three different peptides of ,B subunit. One peptide may be recognized

by the unique residue which is modified when CT is bound to Lip-GML.

This fragment of ,6 subunit should not be involved in any interaction

except water solvent. Another one appears to belong to that proteic area

in contact withc subunit, since one modifiable residue of ;d subunit is

disclosed after removing of n subunit from ~8, region. The third one,

is labeled at molar ratio probe protein 30/l, only when CT is free in

solution, and this peptide seems to be settled into binding site.

ACKNOWLEDGEMENTS

The authors express their deep gratitude to Mr. Tony Thackrah for carrying out the[l33imo acid analyses, and to Mr. Paul Barclay for the preparation of II-DDISA. M.T. was the recipient of a short term fellowship of Accademia Nazionale dei Lincei.

REFERENCES

1. fill, D.M. (1977) Adv. Cyclic. Nucl. Res. g, 87-118. 2. Bennet, V. and Cuatrecasas, P. (1977). In: "Specificity and Action

of Animal, Bacterial and Plant Toxin". Chapman and Hall, London. pp. 3-65.

3. Fishman, P.H. (1982) .I. Membr. Biol. 2, 85-97.

839

Vol. 130, No. 2, 1985 BIOCHEMICAL AND BIOPHYSICAL RESEARCH COMMUNICATIONS

4. Tomasi, M., Ausiello, C., Battistini, A. and D'Agnolo, G. (1979) FEBS Lett. 106, 309-312.

5. Tomasi, M. and Montecucco, C. (1981) J. Biol. Chem. 256, 11177-11181.

6. Wisniesky, B.J. and Bramhall, J.S. (1981) Nature 2, 319-321. 7. Helmkamp, R.W. and Sears, D.A. (1970) Int. J. Appl. Radiat. Isot.

2-l, 683-685. 8. Tindberg, H.M., Melnick, R.L., Maquire, .I. and Packer, L. (1974)

Biochim. Biophys. Acta 345, 118-128. 9. Laemli U.K. (1970) Nature (London) 227, 680-685.

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