IL-4 analogues with site-specific chemical modification at position 121 inhibit IL-4 and IL-13 biological activities.

IL-4 signaling into a cell occurs via assembly of a receptor complex that consists of a high-affinity IL-4Rα chain and a low affinity chain, where the low-affinity chain is either γc or IL-13Rα1. It has been previously shown that mutational disruption of the low affinity interface in the IL-4DM (double mutein) yields an antagonist that inhibits IL-4 as well as IL-13-dependent responses. The present study reveals that new types of IL-4 antagonists can be generated by site-specific chemical modification. The chemically modified IL-4 analogues consist of (1) mixed disulfides created by refolding IL-4 cysteine muteins in the presence of different thiol compounds or (2) maleimide conjugates created by modifying cysteine muteins with maleimide derivatives. IL-4 analogues chemically modified at position 121 retain marginal binding affinity to γc or IL-13Rα1 receptor ectodomains during SPR interaction analysis. The biological activity of the analogues is strongly reduced in HEK-Blue IL-4/IL-13 cells as well as in Jurkat cells. Since the IL-4 analogues modified at position 121 have the ability to inhibit γc (IL-4)- and IL13Rα1 (IL-4/IL-13)-dependent responses in Jurkat and HEK-Blue cell lines, they effectively act as IL-4 antagonists. The results of our IL-4 study provide the first example of a cytokine that is transformed into a competitive inhibitor by site-specific chemical modification.

Enzymatic deglutathionylation to generate interleukin-4 cysteine muteins with free thiol.

Interleukin-4 (IL-4) is a prototypical regulator protein of the immune system that is crucial for the pathogenesis and maintenance of asthma and other atopic diseases. It, together with IL-13, uses the IL-4 receptor α chain (IL-4Rα) to signal into immune and other cells. An IL-4 mutein acting as a dual IL-4/IL-13 receptor antagonist is in clinical development. Here, it is described how IL-4 muteins containing a single engineered cysteine with a free thiol can be prepared and used for site-specific chemical modification. The muteins were initially expressed in E. coli, refolded, and purified, but not in a fully reduced nonconjugated form. Attempts to reduce the cysteine chemically failed because the native disulfide bonds of IL-4 were also reduced under similar conditions. Therefore, an enzymatic procedure was developed to reduce glutathionylated IL-4 cysteine muteins employing glutaredoxin and reduced glutathione. Cysteine muteins engineered at four different positions around the IL-4Rα binding site were enzymatically reduced at different rates. All muteins were prepared with free thiol in reasonable yield and were modified by N-ethylmaleimide (NEM) or maleimido-PEG. The effect on IL-4Rα binding of cysteine substitution and of the site-specific modification by glutathione, N-ethylmaleimide (NEM), or a branched 2.36 kDa poly(ethylene glycol) (PEG) will be discussed.

Cloning, expression, purification, crystallization and preliminary X-ray diffraction analysis of YvoA from Bacillus subtilis.

The putative transcriptional regulator protein YvoA (BSU35030) from Bacillus subtilis was cloned and heterologously expressed in Escherichia coli. The protein was purified by immobilized metal-affinity chromatography and size-exclusion chromatography and subsequently crystallized. A complete native data set was collected to 2.50 A resolution. The crystals belonged to the monoclinic space group C2 and preliminary analysis of the diffraction data indicated the presence of approximately 12 molecules per asymmetric unit.