Insufficient (sub-native) helix content in soluble/solid aggregates of recombinant and engineered forms of IL-2 throws light on how aggregated IL-2 is biologically active.
Interleukin 2 (IL-2) is an extremely aggregation-prone, all-alpha helical cytokine. In its receptor-bound state, ~72 % of the polypeptide chain adopts helical structure and there is no beta sheet content whatsoever. In the past, recombinant IL-2 has been formulated and used therapeutically in humans, following production in E. coli. Therapeutic IL-2 consists entirely of functionally-active soluble aggregates with ~30 subunits per aggregate particle. Side-effects attributed to aggregation resulted in discontinuation of usage over a decade ago. Structurally, and biochemically, activity in IL-2 aggregates can potentially be explained in one of two ways : (a) individual IL-2 chains exist in sterically-accessible, receptor binding-competent (native) structures, allowing aggregates to bind directly to IL-2 receptors (IL-2R); alternatively, (b) IL-2 chains dissociate from aggregates, become free to adopt native structure, and then bind to IL-2R. We produced native IL-2 and numerous engineered forms in E. coli with the objective of obtaining insights into these possibilities. Each IL-2 variant was subjected to size exclusion chromatography, circular dichroism (CD) and Fourier transform infrared spectroscopy (FTIR). All forms produced and studied (including those with native IL-2 sequences) turned out to aggregate and also display less than ~50 % helix content as well as significant beta sheet content. No conditions were found that obviate aggregation. Aggregated IL-2 is thus insufficiently native-like to bind to IL-2R. Activity in aggregates thus probably owes to adoption of receptor binding-competent structures by chains that have already dissociated from aggregates.
Institute of Microbial Technology, Sector 39-A, Chandigarh, 160036, India.
Uzma
U
Fatima
Balvinder
B
Singh
Karthikeyan
K
Subramanian
Purnananda
P
Guptasarma
D000595:Amino Acid Sequence; D002850:Chromatography, Gel; D002942:Circular Dichroism; D003001:Cloning, Molecular; D004926:Escherichia coli / Q000235:genetics / Q000378:metabolism; D006801:Humans; D007376:Interleukin-2 / Q000096:biosynthesis / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism; D008958:Models, Molecular; D008969:Molecular Sequence Data; D046912:Multiprotein Complexes / Q000737:chemistry* / Q000378:metabolism; D015202:Protein Engineering / Q000379:methods; D058849:Protein Refolding; D017433:Protein Structure, Secondary; D011994:Recombinant Proteins / Q000096:biosynthesis / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism; D017550:Spectroscopy, Fourier Transform Infrared
D007376:Interleukin-2; D046912:Multiprotein Complexes; D011994:Recombinant Proteins