title: Immunologic Function and Molecular Insight of Recombinant Interleukin-18.
abstract: In recent years, cytokine-mediated therapy has emerged as further advance alternative in cancer therapy. Interleukin-18 (IL-18) has exhibited interesting anti-cancer properties especially when combined with IL-12. We engineered IL-18 in order to improve its activity using single point mutagenesis. IL-18 mutants were constructed according to binding residues and polarity which we tried to increase polarity in M33Q and M60Q, enhanced cationicity in E6K, and flexibility in T63A. All IL-18 proteins were expressed in Pichia pastoris, purified, and then measured the activity by treating with the NK-92MI cell line to evaluate interferon-γ (IFN-γ) stimulation. The E6K and T63A mutant forms showed higher activity with respect to native proteins at the concentration of 200 ng mL-1 by inducing the expression of IFN-γ, about factors of 9 and 4, respectively. Meanwhile, M33Q and M60Q had no significant activity to induce IFN-γ. Interestingly, the combination of E6K and T63A mutations could synergize the induction activity of IL-18 to be 16 times at 200 ng mL-1. Furthermore, molecular dynamics studies have elucidated the effect due to mutation on conformation of the binding site of IL-18. The results turn out that E6K provides structural perseverance against mutation, while M33Q and M60Q promote vivid overall change in protein conformation, especially at the binding site. For T63A, mutation yields small difference in structure but clearly increases structural flexibility. However, a small structural change was observed when T63A was combined with E6K. Our research resulted in a novel version of IL-18 which could be a new key candidate for cytokine-mediated therapy.
authors.affiliation: Department of Biomedical Sciences, Faculty of Medicine, Prince of Songkla University, Songkhla, 90110, Thailand.|Graduate School, Prince of Songkla University, Songkhla, 90110, Thailand.
authors.forename: Jirakrit
authors.initials: J
authors.lastname: Saetang
authors.affiliation: Microbial Cell Factory Laboratory, Bioresources Technology Unit, National Center for Genetic Engineering and Biotechnology, National Science and Technology Development Agency, 113 Thailand Science Park, Phahonyothin Road, Khlong Nueng, Khlong Luang, Pathum Thani, 12120, Thailand.
authors.forename: Aekkachai
authors.initials: A
authors.lastname: Puseenam
authors.affiliation: Microbial Cell Factory Laboratory, Bioresources Technology Unit, National Center for Genetic Engineering and Biotechnology, National Science and Technology Development Agency, 113 Thailand Science Park, Phahonyothin Road, Khlong Nueng, Khlong Luang, Pathum Thani, 12120, Thailand.
authors.forename: Niran
authors.initials: N
authors.lastname: Roongsawang
authors.affiliation: Department of Microbiology and Natural Product Research Center of Excellence, Faculty of Science, Prince of Songkla University, Songkhla, 90110, Thailand.
authors.forename: Supayang Piyawan
authors.initials: SP
authors.lastname: Voravuthikunchai
authors.affiliation: Department of Biomedical Sciences, Faculty of Medicine, Prince of Songkla University, Songkhla, 90110, Thailand.|Department of Surgery, Faculty of Medicine, Prince of Songkla University, Songkhla, 90110, Thailand.
authors.forename: Surasak
authors.initials: S
authors.lastname: Sangkhathat
authors.affiliation: Department of Biomedical Sciences, Faculty of Medicine, Prince of Songkla University, Songkhla, 90110, Thailand.
authors.forename: Varomyalin
authors.identifier: http://orcid.org/0000-0001-6017-7519
authors.initials: V
authors.lastname: Tipmanee
mesh_terms: D000595:Amino Acid Sequence; D001665:Binding Sites; D045744:Cell Line, Tumor; D003001:Cloning, Molecular; D015870:Gene Expression; D006801:Humans; D007371:Interferon-gamma / Q000096:biosynthesis* / Q000378:metabolism; D020382:Interleukin-18 / Q000737:chemistry* / Q000235:genetics / Q000276:immunology / Q000494:pharmacology; D007694:Killer Cells, Natural / Q000166:cytology / Q000187:drug effects* / Q000276:immunology; D007700:Kinetics; D008213:Lymphocyte Activation / Q000187:drug effects*; D008958:Models, Molecular; D008970:Molecular Weight; D010843:Pichia / Q000235:genetics / Q000378:metabolism; D011485:Protein Binding; D000072756:Protein Conformation, alpha-Helical; D000072757:Protein Conformation, beta-Strand; D015202:Protein Engineering; D054730:Protein Interaction Domains and Motifs; D053724:Receptors, Interleukin-18 / Q000737:chemistry* / Q000235:genetics / Q000276:immunology; D011994:Recombinant Proteins / Q000737:chemistry / Q000235:genetics / Q000276:immunology; D016415:Sequence Alignment; D013329:Structure-Activity Relationship; D013379:Substrate Specificity
chemical_list: D020382:Interleukin-18; D053724:Receptors, Interleukin-18; D011994:Recombinant Proteins; D007371:Interferon-gamma