Binding of R121D/Y124D

Concentration Rate constants Equilibrium constant ( Kd)

from BIAcore from previous

K a s s K diss experiments binding data

200nM 2.64x10® 3.05x10® 1.16x10'"® 3x10'"®

1G0nM 3.91x10® 2.98x10® 7.62x10'"®

194

the range 230-500pM*^ and R121D/Y124D in the range 750-1 OOOpM'V These

numbers are all within a 10-fold range and are in good agreement with receptor

binding studies showing that the mutations around 121-125 on the IL-4

molecule do not affect binding to IL-4Ra.

The experiments were also carried out using IL-2 as a negative control y

since this is known not to bind to the IL-4Ra chain. The sensorgrams are

shown in figure 6.7. The small change in resonance units seen is due to the

change in buffer in the cell when the IL-2 was added. Since the cytokine was

diluted in MBS running buffer, as the concentrations of cytokine decreased, the

change in concentration of the buffer became smaller and the change in

resonance units due to the buffer change also decreased. The sensorgram

obtained was typical of the result expected when no binding was occurring.

IL-13 was also added to the sensor chip under exactly the same

conditions as IL-2 and IL-4 (figure 6.8). The same change in buffer

concentration as observed with IL-2 meant that the resonance units increased

by a small amount, especially at high concentrations of IL-13. At concentrations

sufficient to show good binding of IL-4 to the IL-4Ra chain, no binding of IL-13

could be seen. This again agreed with results obtained by receptor binding

studies which show that IL-13 is unable to bind to the IL-4Ra chain (Zurawski

et al., 1993b). Since no binding to the IL-4Ra chain could be seen for either

200nM lOOnM 50nM 25nM 12.5nM 6.25nM 340-- 280-■ Z)

£

&

Figure 6.7: Measurement of IL-2 binding to the IL-4Ra chain. IL-4Ra protein was immobilized to the chip and IL-2 (200-6.25nM) was passed through the flow chamber at a flow rate of Spi/min. After 3 minutes, the

flow was switched to MBS and dissociation was allowed to proceed for 8 minutes before regeneration of the chip with 10mM NaOH (not shown).

CD

IL-13 concentrations 370 200nM lOOnM SOnM 25nM 12.5nM 6.25nM 330 290 g - 250 Û:

%

Figure 6.8 : Measurement of IL-13 binding to the IL-4Ra chain. IL-4Ra protein was immobilized to the chip via

biotin-streptavidin coupling and IL-13(200-6.25nM) was passed through the flow chamber at a flow rate of 5pl/min. After 3 minutes, the flow was switched to MBS and dissociation was allowed to proceed for 8 minutes before regeneration of the chip with lOmM NaOH (not shown).

6.3. DISCUSSION.

The results presented in chapters 4 and 5 have indicated that the IL-4Ra

chain may be the common component of the IL-4 and IL-13 receptors.

However, Scatchard analysis has shown no binding of IL-13 to the IL-4Ra

chain in murine cells transfected with this chain (Zurawski et al., 1993b). In this

chapter, interactions between the IL-4Ra chain and IL-13 were investigated

using BIA technology‘s^. W hen the IL-4Ra chain is immobilized to a BIAcore's^

chip, IL-4 is able to bind with similar affinity to that calculated from Scatchard

analysis. No binding of IL-13 to the IL-4Ra chain could be seen in these

experiments.

This does not rule out the IL-4Ra chain as a component of the IL-13

receptor. A model can be proposed where IL-13 binds to one receptor chain

and a change in conformation then allows binding of the IL-4Ra chain and

formation of a functional IL-13 receptor complex. This has been previously

observed for the IL- 6 receptor complex, where IL- 6 binding to the IL- 6 binding

chain allows association with the gp130 chain and signalling through the

receptor. In principle, the model could be tested using the BIAcore™. If IL-13

was allowed to associate with the proposed IL-13 binding chain (for example by

preincubation with B cell membrane extracts) before passing over the BIAcore™

chip, this might allow association with the IL-4Ra chain. Similar experiments

have been carried out using purified IL-6 R and gp130, and have proved an

198

The BIAcore^^ can also be used to calculate rates of reaction for ligand

binding. Such rates were calculated for IL-4 and for the site II signalling

mutants Y124D and R121D/Y124D. The constants obtained show good

agreement with those obtained from Scatchard plots, and indicate that

mutations in site II of the IL-4 molecule do not affect binding to the IL-4Ra

chain.

At lower concentrations, all of the kinetic constants obtained are affected

by the mass transport limitations of the system, described in the results section.

This is due to an excess of ligand binding sites compared to the amount of

ligand available and meant that the association and dissociation rates could not

be calculated accurately. This limitation could be avoided by increasing the

concentrations of the cytokine, or by decreasing the amount of IL-4Ra protein

bound to the sensor chip. However, increasing the cytokine concentrations

becomes expensive and decreasing the number of ligand binding sites lowers

the total RU change obtained in the experiment and introduces further

inaccuracies. The kinetic constants obtained here were in good agreement with

those from Scatchard analysis. Also, the most important question to be asked

by the experiments - whether IL-13 would bind to the IL-4Ra chain - was clearly

answered by the results obtained. Due to these considerations, further

experiments to fully characterize the kinetics of the interactions were not