Role of amino acid residue 90 in bioactivity and receptor binding capacity of tumor necrosis factor mutants.
We have previously produced two bioactive lysine-deficient mutants of TNF-alpha (mutTNF-K90R,-K90P) and found that these mutants have bioactivity superior to wild-type TNF (wtTNF). Because these mutants contained same amino acid except for amino acid 90, it is unclear which amino acid residue is optimal for showing bioactivity. We speculated that this amino acid position was exchangeable, and this amino acid substitution enabled the creation of lysine-deficient mutants with enhanced bioactivity. Therefore, we produced mutTNF-K90R variants (mutTNF-R90X), in which R90 was replaced with other amino acids, to assay their bioactivities and investigated the importance of amino acid position 90. As a result, mutTNF-R90X that replaced R90 with lysine, arginine and proline were bioactive, while other mutants were not bioactive. Moreover, these three mutants showed bioactivity as good as or better than wtTNF. R90 replaced with lysine or arginine had especially superior binding affinities. These results suggest that the amino acid position 90 in TNF-alpha is important for TNF-alpha bioactivity and could be altered to improve its bioactivity to generate a "super-agonist".
Shibata
Hiroko
H
National Institute of Biomedical Innovation, 7-6-8 Saito-Asagi, Ibaraki, Osaka 567-0085, Japan.
Kamada
Haruhiko
H
Kobayashi-Nishibata
Kyoko
K
Yoshioka
Yasuo
Y
Nishibata
Toshihide
T
Abe
Yasuhiro
Y
Nomura
Tetsuya
T
Nabeshi
Hiromi
H
Minowa
Kyoko
K
Mukai
Yohei
Y
Nakagawa
Shinsaku
S
Mayumi
Tadanori
T
Tsunoda
Shin-Ichi
S
Tsutsumi
Yasuo
Y
D019943:Amino Acid Substitution; D006801:Humans; D020125:Mutation, Missense; D011485:Protein Binding / Q000235:genetics / Q000276:immunology; D047888:Receptors, Tumor Necrosis Factor, Type I / Q000819:agonists* / Q000276:immunology; D047889:Receptors, Tumor Necrosis Factor, Type II / Q000819:agonists* / Q000276:immunology; D014409:Tumor Necrosis Factor-alpha / Q000235:genetics / Q000276:immunology*
D047888:Receptors, Tumor Necrosis Factor, Type I; D047889:Receptors, Tumor Necrosis Factor, Type II; D014409:Tumor Necrosis Factor-alpha