N-linked glycosylation of IL-13R alpha2 is essential for optimal IL-13 inhibitory activity.
A high-affinity receptor for interleukin (IL)-13 (interleukin-13R alpha 2) is over-expressed in disease-related fibroblasts and neoplastic cells and is involved in cancer, allergic, and inflammatory diseases. The extracellular domain of IL-13R alpha2 (ECD alpha2) could be cleaved, which serves as a decoy receptor. We have expressed and purified ECD alpha2 in both Escherichia coli (E. coli) and mammalian systems as a soluble fragment and studied its biological activities. Although both products of ECD alpha2 showed IL-13 inhibitory activities, mammalian cell-derived ECD alpha2 appeared to be superior compared with purified protein from E. coli. When expressed in E. coli, ECD alpha2 appeared to be a monomer of 42 but a 60 kDa protein when purified from mammalian cells due to heavy glycosylation. The purified glycosylated ECD alpha2 efficiently inhibited IL-13-induced STAT6 phosphorylation in immune and Hodgkin's lymphoma cell lines, IL-13 binding, and cytotoxicity of IL-13 cytotoxin in various cancer cell lines. The improved potency of mammalian cell-derived ECD alpha2 was shown over ECD alpha2/Fc fusion protein. The N-linked glycosylation of ECD alpha2 was found to be essential for optimal IL-13 inhibitory activity as deglycosylation by PNGase F showed lower activity. ECD alpha2 did not inhibit IL-4-induced STAT6 phosphorylation, indicating that inhibitory effects of ECD alpha2 are receptor specific. These results indicate that glycosylated ECD alpha2 can serve as a potent inhibitor of IL-13 in a variety of conditions in which IL-13 is a key mediator, e.g., pulmonary, allergic, fibrotic, and neoplastic diseases.
Kioi
Mitomu
M
Tumor Vaccines and Biotechnology Branch, Division of Cellular and Gene Therapies, Food and Drug Administration, Center for Biologics Evaluation and Research, Bethesda, MD 20892, USA.
Seetharam
Saraswathy
S
Puri
Raj K
RK
D000818:Animals; D001483:Base Sequence; D019556:COS Cells; D002455:Cell Division; D002460:Cell Line; D002470:Cell Survival / Q000187:drug effects; D002522:Chlorocebus aethiops; D003001:Cloning, Molecular; D017931:DNA Primers; D006031:Glycosylation; D006801:Humans; D018793:Interleukin-13 / Q000502:physiology*; D053661:Interleukin-13 Receptor alpha1 Subunit / Q000235:genetics / Q000502:physiology*; D051379:Mice; D010957:Plasmids; D011994:Recombinant Proteins / Q000494:pharmacology; D012190:Retroviridae
D017931:DNA Primers; C508181:IL13RA1 protein, human; D018793:Interleukin-13; D053661:Interleukin-13 Receptor alpha1 Subunit; D011994:Recombinant Proteins