Creation of lysine-deficient mutant lymphotoxin-alpha with receptor selectivity by using a phage display system.
The cytokine lymphotoxin-alpha (LT alpha) activates various biological functions through its three receptor subtypes, tumor necrosis factor receptor 1 (TNFR1), TNFR2 and herpes virus entry mediator (HVEM), but the relative contribution of each receptor to each function is unclear. Therefore it is important to create mutant LT alpha with receptor selectivity for optimized cancer therapy and the analysis of receptor function. Here, we attempted to create a lysine-deficient mutant LT alpha with TNFR1-selective bioactivity using a phage display technique. We obtained the TNFR1-selective mutant LT alpha R1selLT, which contained the mutations K19N, K28Q, K39S, K84Q, K89V, and K119H. Compared with wild-type LT alpha (wtLT alpha), R1selLT showed several-fold higher bioactivity via TNFR1 but 40-fold lower bioactivity via TNFR2. Kinetic association-dissociation parameters of R1selLT with TNFR2 were higher than those of wtLT alpha, whereas these parameters of R1selLT with TNFR1 were lower than those of wtLT alpha, suggesting that destabilization of the R1selLT-TNFR2 complex causes the decreased bioactivity of R1selLT on TNFR2. We also showed that the K84Q mutation contributed to the enhanced activity via TNFR1, and K39S lowered activity via TNFR2. R1selLT likely will be useful in cancer therapy and in analysis of the LT alpha structure-function relationship.
Yoshioka
Yasuo
Y
The Center for Advanced Medical Engineering and Informatics, Osaka University, 1-6, Yamadaoka, Suita, Osaka, 565-0871, Japan. yasuo@phs.osaka-u.ac.jp
Watanabe
Hikaru
H
Morishige
Tomohiro
T
Yao
Xinglei
X
Ikemizu
Shinji
S
Nagao
Chioko
C
Ahmad
Shandar
S
Mizuguchi
Kenji
K
Tsunoda
Shin-ichi
S
Tsutsumi
Yasuo
Y
Mukai
Yohei
Y
Okada
Naoki
N
Nakagawa
Shinsaku
S
D000595:Amino Acid Sequence; D000818:Animals; D016923:Cell Death; D045744:Cell Line, Tumor; D006801:Humans; D007526:Isoelectric Point; D007700:Kinetics; D008233:Lymphotoxin-alpha / Q000737:chemistry / Q000378:metabolism*; D008239:Lysine / Q000172:deficiency*; D051379:Mice; D008958:Models, Molecular; D008969:Molecular Sequence Data; D050505:Mutant Proteins / Q000378:metabolism*; D019151:Peptide Library; D017354:Point Mutation / Q000235:genetics; D011485:Protein Binding; D011956:Receptors, Cell Surface / Q000378:metabolism*
D008233:Lymphotoxin-alpha; D050505:Mutant Proteins; D019151:Peptide Library; D011956:Receptors, Cell Surface; D008239:Lysine