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.
Lehrstuhl für Physiologische Chemie II, Theodor-Boveri-Institut für Biowissenschaften (Biozentrum) der Universität Würzburg, Würzburg, Germany. viswa@live.de
Viswanadham
V
Duppatla
Maja
M
Gjorgjevikj
Werner
W
Schmitz
Mathias
M
Kottmair
Thomas D
TD
Mueller
Walter
W
Sebald
D003545:Cysteine / Q000737:chemistry / Q000378:metabolism*; D005978:Glutathione / Q000378:metabolism*; D005980:Glutathione Reductase / Q000378:metabolism*; D006801:Humans; D015847:Interleukin-4 / Q000737:chemistry* / Q000378:metabolism*; D008958:Models, Molecular; D015394:Molecular Structure; D015202:Protein Engineering; D013438:Sulfhydryl Compounds / Q000032:analysis* / Q000737:chemistry
D013438:Sulfhydryl Compounds; D015847:Interleukin-4; D005980:Glutathione Reductase; D005978:Glutathione; D003545:Cysteine