comparison of water exposed area of cholera toxin when free in solution and bound to liposomes...
TRANSCRIPT
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.
840