Creation of interferon-alpha8 mutants with amino acid substitutions against interferon-alpha receptor-2 binding sites using phage display system and evaluation of their biologic properties.
In this study, we describe the creation of three interferon-alpha (IFN-alpha)8 mutants with markedly higher antiviral and antiproliferative activities in comparison with those of the wild-type (wt)IFN-alpha8, wtIFN-alpha2, and IFN-con1 using a phage display system. Sequence analysis showed that three out of the six hot-spot amino acid residues of wtIFN-alpha8 known to be important for the interaction with the IFN-alpha receptor-2 (IFNAR-2)-binding sites were substituted to other amino acids and the others remained. Although affinity analysis revealed that the dissociation constant (K(D)) of IFN-alpha8 mutants was almost the same with that of wtIFN-alpha8, furthermore, the rates of association (k(a)) and dissociation (k(d)) were relatively lower. These results suggest that changes in the surface electronic charge of amino acid residues lead to changes in binding affinity and kinetics (prolonged dissociation time) toward the IFNAR-2, resulting in the modification of the biological activity. Moreover, our results demonstrate that the molecular engineering of the IFN-alpha8 provides important insight into action of IFN and also it would be useful in the development of therapeutically prominent IFN preparations than those used in clinical practice.
Yamamoto
Kouzo
K
Biomedical Institute, Research Center, Hayashibara Biochemical Laboratories, Inc., Fujisaki, Okayama, Japan.
Taniai
Madoka
M
Torigoe
Kakuji
K
Yamamoto
Shigeto
S
Arai
Norie
N
Suemoto
Yasuo
Y
Yoshida
Kenshi
K
Okura
Takanori
T
Mori
Tetsuya
T
Fujioka
Noboru
N
Tanimoto
Tadao
T
Miyata
Manabu
M
Ariyasu
Harumi
H
Ushio
Chie
C
Fujii
Mitsukiyo
M
Ariyasu
Toshio
T
Ikeda
Masao
M
Ohta
Tsunetaka
T
Kurimoto
Masashi
M
Fukuda
Shigeharu
S
D019943:Amino Acid Substitution; D000998:Antiviral Agents / Q000737:chemistry / Q000494:pharmacology; D001665:Binding Sites / Q000235:genetics; D001667:Binding, Competitive; D045744:Cell Line, Tumor; D049109:Cell Proliferation / Q000187:drug effects; D006131:Growth Inhibitors / Q000737:chemistry / Q000494:pharmacology; D056945:Hep G2 Cells; D006801:Humans; D007370:Interferon Type I / Q000235:genetics / Q000378:metabolism / Q000494:pharmacology; D016898:Interferon-alpha / Q000235:genetics* / Q000378:metabolism* / Q000494:pharmacology; D007700:Kinetics; D008958:Models, Molecular; D009154:Mutation; D019151:Peptide Library; D011485:Protein Binding; D011487:Protein Conformation; D053633:Receptor, Interferon alpha-beta / Q000378:metabolism*; D011994:Recombinant Proteins; D012845:Sindbis Virus / Q000187:drug effects; D020349:Surface Plasmon Resonance; D020298:U937 Cells; D014721:Vesicular stomatitis Indiana virus / Q000187:drug effects
D000998:Antiviral Agents; D006131:Growth Inhibitors; C507964:IFNAR1 protein, human; C508033:IFNAR2 protein, human; D007370:Interferon Type I; D016898:Interferon-alpha; D019151:Peptide Library; D011994:Recombinant Proteins; C056556:methionyl human consensus alpha interferon; D053633:Receptor, Interferon alpha-beta