- Email: [email protected]
Analysis of the inhibition of Clq binding to aggregated immunoglobulin
Moleculur fmmunolog~,Vol. 22, No. 4, pp. 503-504. Pnnted in Great Britain
1985
LETTER
0161-5890/85 $3.00 + 0.00 1985 Pergamon Press Ltd
c
TO THE EDITOR
ANALYSIS OF THE INHIBITION OF Clq BINDING TO AGGREGATED IMMUNOGLOBULIN (First received 29 February 1984; accepted in revised form 12 September 1984)
Many ionic compounds have been shown to inhibit the binding of complement component Clq to aggregated immunoglobulin (Sledge and Bing, 1973; Lin and Fletcher, 1978; Hughes-Jones and Gardner, 1978; Burton et al., 1980; Williams et al., 1983). In some cases [e.g. Sledge and Bing (1973) and Hughes-Jones and Gardner (1978)) direct binding studies, using radioactive inhibitors, were used in attempts to establish whether the inhibitors acted by binding to Clq or to aggregated immunoglobulin. However, in other cases [e.g. Lin and Fletcher (1978) and Williams CI al. (1983)] it has been claimed that it is possible to deduce the mode of action of an inhibitor from the results of experiments in which the dependence of the concn of aggregated immunoglobulin-bound Clq on the concn of Clq free in solution was determined as a function of inhibitor concn. From such experiments it was concluded that: “Polyanions.. .inhibited competitively Clq binding, probably through charge interactions with Clq” (Lin and Fletcher. 1978), and “_.. the Dixon plots in Fig. 3 demonstrated that both cationic polypeptides compete with IIA [insoluble IgG aggregates] for Clq binding” (Williams et al., 1983). The analysis outlined below shows that such conclusions are unwarranted, in that identical inhibition patterns will be obtained in such experimental systems, regardless of whether an inhibitor acts by binding to Clq or aggregated immunoglobulin. Thus, it is not possible to establish the mode of action of an inhibitor unless the results of direct binding studies, of the type reported by Sledge and Bing ( 1973) and Hughes-Jones and Gardner (1978) are available. The case of an inhibitor acting by binding to the aggregated immunoglobulin may be specified by two equilibrium constants:
The case of an inhibitor acting by binding represented by the equilibrium constants.
Ko=-
’ (6)
IC,,Clq, Clq,=-----. KD + Clq,
(7)
However, in experiments of the type reported by Lin and Fletcher (1978) and Williams et al. (1983) it is only possible to measure the concns of Clq in solution (Clq,) and Clq bound to the aggregated immunoglobulin (Clq,). In this case, the concn of Clq free in solution is not equal to the concn of Clq not bound to the aggregated immunoglobulin (Clqs, but rather: Clq, = Clq,+ From
equation
(6) it follows Clq,=
Hence,
(3)
give: IC,.Clq,.K,
K, f K,.f f K,Clq;.
Clq,
where Clql represents the concn of immunoglobulinbinding sites of Clq blocked by inhibitor, and the other terms are as specified above. From equation (5), with the conservation equation IC, = IC,+ Clq, (in this case ICI = 0):
where Clq,, IC,, Clq,, I and ICI represent the concns of Clq not bound to the aggregated immunoglobulin, unoccupied Clq-binding sites on the aggregated immunoglobulin, occupied Clq-binding sites on the aggregated immunoglobulin, inhibitor and Clq-binding sites on the aggregated immunoglobulin which have been blocked with inhibitor. In this case the concn of Clq not bound to the aggregated immunoglobulin (C lq,) is equal to the concn of Clq in solution (Clq,). These equations, together with the conservation equation for the total Clq-binding sites on the aggregated immunoglobulin (IC,):
Clq,=-----Ku.
Clq,,IC,
K _ C’%.f ’ Clql’
(1)
Ic,=Ic,+cly,+IcI
to Clq may be
(4) 503
from equations
Clq,
=
Clql.
(8)
+ I/K,)
(9)
that:
Clq,/(l
(7) and (9): Clqs.IC,.
K,.K,+
K,
Ko,I + K;Clq,
(10)
This equation, which describes the dependence of Clq, on the experimentally measurable solution Clq concn (Clq,) is of the same form as equation (4). Hence, inhibition data of the type reported by Lin and Fletcher (1978) and Williams ef a/. (1983) will give the same patterns in either doublereciprocal form (l/Clq, vs l/Clq,) or Dixon plots (l/Clq, vs I), regardless of whether the inhibitor acts by binding to Clq or the aggregated immunoglobulin. This arises because, in the case of an inhibitor binding to Clq, it is not generally possible to measure the concns of free Clq and the soluble Clq-inhibitor complex separately; only the total concn of Clq in solution can be measured. Thus, inhibition data of the type reported by Lin and Fletcher (1978) and Williams et a/. (1983) are insufficient to allow definition of the site of action of an inhibitor. Although the above models have been developed in terms of Clq binding to insoluble aggregated immunoglobulins, the same analysis will probably be appropriate to other systems in which a soluble inhibitor acts on the equilibrium between a soluble macromolecule and a particulate system, such as a cell suspension.
504
Letter to the Editor
Tropical Medicine Unit,
M.-R.
VAN SCHRAVENDUK
Nufi‘eld Department qf
Clinical Medicine, John Radclife Hospital. O@rd OX3 9DU, U.K. and Department of Biochemistrv, University of Sydney, Sydney, N.S. W. 2006, Australia
S. B. EASTERBROOK-SMITH*
Acknol~,ledgements-This work was supported in part by the Australian Research Grants Scheme. We are grateful to Dr R. A. Dwek for his encouragement and helpful discussions during this investigation.
__~~_ *To whom
____ correspondence
should
be addressed.
REFERENCES Burton D. R., Boyd J., Brampton A. D., Easterbrook-Smith S. B., Emmanuel E. .I., Novotny J., Rademacher T. W., van Schravendijk M.-R., Sternberg M. .I. and Dwek R. A. (1980) The Clq receptor site on immunoglobulin G. Nature, Lond. 288, 338-344. Hughes-Jones N. C. and Gardner B. (1978) The reaction between complement subcomponent Clq, IgG complexes, and polyionic molecules. Immunology 34, 459-463. Lin T.-Y. and Fletcher D. S. (1978) Interaction of human Clq with insoluble immunoglobulin aggregates. Immunochemistry 15, 107-l 17. Sledge C. R. and Bing D. H. (1973) Binding properties of human complement component Clq. J. hiol. Chem. 248, 2818-2823. Williams H. R., Fletcher D. S., Harris E. E. and Lin T.-Y. (1983) Inhibition of immune aggregate-induced activation of the first complement component by cationic polypeptides. Biochim. biophys. Acta 151, 69-16.
1985
LETTER
0161-5890/85 $3.00 + 0.00 1985 Pergamon Press Ltd
c
TO THE EDITOR
ANALYSIS OF THE INHIBITION OF Clq BINDING TO AGGREGATED IMMUNOGLOBULIN (First received 29 February 1984; accepted in revised form 12 September 1984)
Many ionic compounds have been shown to inhibit the binding of complement component Clq to aggregated immunoglobulin (Sledge and Bing, 1973; Lin and Fletcher, 1978; Hughes-Jones and Gardner, 1978; Burton et al., 1980; Williams et al., 1983). In some cases [e.g. Sledge and Bing (1973) and Hughes-Jones and Gardner (1978)) direct binding studies, using radioactive inhibitors, were used in attempts to establish whether the inhibitors acted by binding to Clq or to aggregated immunoglobulin. However, in other cases [e.g. Lin and Fletcher (1978) and Williams CI al. (1983)] it has been claimed that it is possible to deduce the mode of action of an inhibitor from the results of experiments in which the dependence of the concn of aggregated immunoglobulin-bound Clq on the concn of Clq free in solution was determined as a function of inhibitor concn. From such experiments it was concluded that: “Polyanions.. .inhibited competitively Clq binding, probably through charge interactions with Clq” (Lin and Fletcher. 1978), and “_.. the Dixon plots in Fig. 3 demonstrated that both cationic polypeptides compete with IIA [insoluble IgG aggregates] for Clq binding” (Williams et al., 1983). The analysis outlined below shows that such conclusions are unwarranted, in that identical inhibition patterns will be obtained in such experimental systems, regardless of whether an inhibitor acts by binding to Clq or aggregated immunoglobulin. Thus, it is not possible to establish the mode of action of an inhibitor unless the results of direct binding studies, of the type reported by Sledge and Bing ( 1973) and Hughes-Jones and Gardner (1978) are available. The case of an inhibitor acting by binding to the aggregated immunoglobulin may be specified by two equilibrium constants:
The case of an inhibitor acting by binding represented by the equilibrium constants.
Ko=-
’ (6)
IC,,Clq, Clq,=-----. KD + Clq,
(7)
However, in experiments of the type reported by Lin and Fletcher (1978) and Williams et al. (1983) it is only possible to measure the concns of Clq in solution (Clq,) and Clq bound to the aggregated immunoglobulin (Clq,). In this case, the concn of Clq free in solution is not equal to the concn of Clq not bound to the aggregated immunoglobulin (Clqs, but rather: Clq, = Clq,+ From
equation
(6) it follows Clq,=
Hence,
(3)
give: IC,.Clq,.K,
K, f K,.f f K,Clq;.
Clq,
where Clql represents the concn of immunoglobulinbinding sites of Clq blocked by inhibitor, and the other terms are as specified above. From equation (5), with the conservation equation IC, = IC,+ Clq, (in this case ICI = 0):
where Clq,, IC,, Clq,, I and ICI represent the concns of Clq not bound to the aggregated immunoglobulin, unoccupied Clq-binding sites on the aggregated immunoglobulin, occupied Clq-binding sites on the aggregated immunoglobulin, inhibitor and Clq-binding sites on the aggregated immunoglobulin which have been blocked with inhibitor. In this case the concn of Clq not bound to the aggregated immunoglobulin (C lq,) is equal to the concn of Clq in solution (Clq,). These equations, together with the conservation equation for the total Clq-binding sites on the aggregated immunoglobulin (IC,):
Clq,=-----Ku.
Clq,,IC,
K _ C’%.f ’ Clql’
(1)
Ic,=Ic,+cly,+IcI
to Clq may be
(4) 503
from equations
Clq,
=
Clql.
(8)
+ I/K,)
(9)
that:
Clq,/(l
(7) and (9): Clqs.IC,.
K,.K,+
K,
Ko,I + K;Clq,
(10)
This equation, which describes the dependence of Clq, on the experimentally measurable solution Clq concn (Clq,) is of the same form as equation (4). Hence, inhibition data of the type reported by Lin and Fletcher (1978) and Williams ef a/. (1983) will give the same patterns in either doublereciprocal form (l/Clq, vs l/Clq,) or Dixon plots (l/Clq, vs I), regardless of whether the inhibitor acts by binding to Clq or the aggregated immunoglobulin. This arises because, in the case of an inhibitor binding to Clq, it is not generally possible to measure the concns of free Clq and the soluble Clq-inhibitor complex separately; only the total concn of Clq in solution can be measured. Thus, inhibition data of the type reported by Lin and Fletcher (1978) and Williams et a/. (1983) are insufficient to allow definition of the site of action of an inhibitor. Although the above models have been developed in terms of Clq binding to insoluble aggregated immunoglobulins, the same analysis will probably be appropriate to other systems in which a soluble inhibitor acts on the equilibrium between a soluble macromolecule and a particulate system, such as a cell suspension.
504
Letter to the Editor
Tropical Medicine Unit,
M.-R.
VAN SCHRAVENDUK
Nufi‘eld Department qf
Clinical Medicine, John Radclife Hospital. O@rd OX3 9DU, U.K. and Department of Biochemistrv, University of Sydney, Sydney, N.S. W. 2006, Australia
S. B. EASTERBROOK-SMITH*
Acknol~,ledgements-This work was supported in part by the Australian Research Grants Scheme. We are grateful to Dr R. A. Dwek for his encouragement and helpful discussions during this investigation.
__~~_ *To whom
____ correspondence
should
be addressed.
REFERENCES Burton D. R., Boyd J., Brampton A. D., Easterbrook-Smith S. B., Emmanuel E. .I., Novotny J., Rademacher T. W., van Schravendijk M.-R., Sternberg M. .I. and Dwek R. A. (1980) The Clq receptor site on immunoglobulin G. Nature, Lond. 288, 338-344. Hughes-Jones N. C. and Gardner B. (1978) The reaction between complement subcomponent Clq, IgG complexes, and polyionic molecules. Immunology 34, 459-463. Lin T.-Y. and Fletcher D. S. (1978) Interaction of human Clq with insoluble immunoglobulin aggregates. Immunochemistry 15, 107-l 17. Sledge C. R. and Bing D. H. (1973) Binding properties of human complement component Clq. J. hiol. Chem. 248, 2818-2823. Williams H. R., Fletcher D. S., Harris E. E. and Lin T.-Y. (1983) Inhibition of immune aggregate-induced activation of the first complement component by cationic polypeptides. Biochim. biophys. Acta 151, 69-16.