Application note

Oligomerization of Interleukin-2

Interleukin-2 (IL-2) is a lymphokine with a range of crucial roles in immune activation and homeostasis (1). It is produced by a subset of activated T-lymphocytes and acts as a growth factor for T cells and NK cells (2), and promotes proliferation, activation-induced cell death, antibody secretion, and cytokine production in various cell types (3). Intravenous treatment with high dose IL-2 has been used in patients with certain cancers (4) (5).

IL-2 has a molecular weight of 15.5 kDa and is globular, which corresponds to a size of 2.04 nm for a monomeric form (6).

Here we use Microfluidic Diffusional Sizing (MDS) on a Fluidity One instrument to assess the hydrodynamic radius (Rh) over a dilution series. The tests use a commercially available lyophilized form with no additives. We observe that the Rh increases with increasing concentration, in a manner which indicates a monomer-trimer equilibrium with positive cooperativity.

This result challenges much of the modern literature which assumes a monomeric form (7) (8) (9), or in some instances a dimer (10). One study by Siddarth et al uses analytical ultracentrifugation and produced data which cannot rule out a monomer-trimer equilibrium, though a monomer-dimer model is eventually concluded (11). It should be noted that a different IL-2 source and buffer is used in this study.

Methods

50 μg of lyophilized human IL-2 recombinantly expressed in E. coli (Sigma-Aldrich code SRP3085) was dissolved in 10 mM acetic acid at 1 mg/mL.

A 10 μL aliquot of this stock was diluted with 23.3 μL of 138.6 mM acetic acid to produce a solution of nominally 300 μg/mL in 100 mM acetic acid. (The subsequent measured concentrations of the dilution series being consistently higher than expected suggests that this initial stock was > 300 μg/mL, likely due to the purchased 50 μg containing more than expected.)

This 300 μg/mL solution was diluted in 2-fold dilution series using 100 mM acetic acid to give 7 varying concentrations, with a lower limit of 4.7 μg/mL.

Each concentration was analysed using a Fluidity One prototype instrument to measure the Rh. Tests were performed in triplicate, with the values reported below being an average of the repeats.

The observed Rh change over changing concentration was then assessed against different models to infer the nature of the oligomerization.

Results

The concentrations of the dilution series along with the measured Rh and concentration are shown in Table 1.

Given the molecular weight of IL-2, the theoretical Rh for a monomer is 2.04 nm, dimer is 2.62 nm and trimer is 3.03 nm. As such the sizes observed at higher concentrations are consistent with trimer formation.

Figure 1 shows fitting of the data points to a monomer-dimer equilibrium model (see appendix 1), which does not accurately represent the data.

Table 1: Results of MDS analysis on the IL-2 dilution series
Nominal Concentration (µg/mL) Average Measured Rh (nm) Average Measured Concentration (µg/mL) Notes
4.7 1.87 4.32 -
9.4 2.00 10.75 Average of 2 runs
18.8 2.29 22.53 -
37.5 2.85 40.47 -
75 2.98 88.90 -
150 2.91 190 Average of 2 runs
300 3.05 411

-

Figure 1: Results of dilution series analysis fitted to both a monomer-dimer equilibrium model, and a Hill Equation
Figure 1: Results of dilution series analysis fitted to both a monomer-dimer equilibrium model, and a Hill Equation

It is clear that the monomer-dimer fitting fails at capturing the steepness of the curve, indicating that there may be cooperative behaviour.

Fitting the data instead to a Hill equation provides a more representative fit, as shown in Figure 1. Akaike’s Information Criterion Test also shows that the Hill equation is ~3000-fold more likely than the monomer-dimer equilibrium model.

The steepness of the slope observed in this fitting indicates there is positive cooperativity in the oligomerisation, as indicated in Table 2.

Table 2: Hill equation fitting of the dilution series data.
No cooperativity; n = 1  / Positive Cooperativity; n>1
y = MRh + (DRh - MRh)*xn/(Kdn+xn) Result
MRh (nm) 1.88 ±0.05
DRh (nm) 3.00 ±0.04
Kd (µg/mL) 22 ±2
Kd (µM) 1.42 ±0.13
n 3.3 ±0.7
R2 0.961

Conclusion

A change in the Rh of IL-2 on dilution has been observed using microfluidic diffusional sizing on a Fluidity One instrument. The Rh changed from 3.1 nm at 300 μg/mL concentration, to 1.9 nm at 4.7 μg/mL.

The change in size indicates that oligomerization occurs at concentrations above 20 μg/mL. The size observed at 300 μg/mL is consistent with a trimer.

When the data is analysed, a Hill equation provides a better fit compared to a monomer-dimer equilibrium model. The steepness of the curve on the Hill equation fit indicates positive cooperativity in the assembly of the trimers.

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