Mutational analysis of structure--activity relationships in human tumor necrosis factor-alpha.
To determine the region of human tumor necrosis factor-alpha (TNF-alpha), essential for cytotoxic activity against mouse L-M cells, single amino-acid-substituted TNF-alpha mutant proteins (muteins) were produced in Escherichia coli by protein engineering techniques. An expression plasmid for TNF-alpha was mutagenized by passage through an E. coli mutD5 mutator strain and by oligonucleotide-directed mutagenesis. Approximately 100 single amino-acid-substituted TNF-alpha muteins were produced and assayed for cytotoxic activity. The cytotoxic activities of purified TNF-alpha muteins, e.g. TNF-31T, -32Y, -82D, -85H, -115L, -141Y, -144K and -146E, were less than 1% of that of parent TNF-alpha. These results indicate that the integrity of at least four distinct regions of the TNF-alpha molecule is required for full biological activity. These regions are designated as follows: region I, from position 30 to 32; region II, from position 82 to 89; region III, from position 115 to 117; region IV, from position 141 to 146. In addition, TNF-141Y could not completely compete with parent TNF-alpha for binding to the receptor. This demonstrates that region IV, and at least aspartic acid at position 141, must be involved in the TNF receptor binding site.
Yamagishi
J
J
Research Laboratories, Dainippon Pharmaceutical Co. Ltd, Osaka, Japan.
Kawashima
H
H
Matsuo
N
N
Ohue
M
M
Yamayoshi
M
M
Fukui
T
T
Kotani
H
H
Furuta
R
R
Nakano
K
K
Yamada
M
M
D000595:Amino Acid Sequence; D000818:Animals; D001483:Base Sequence; D002460:Cell Line; D002470:Cell Survival / Q000187:drug effects; D004926:Escherichia coli; D015964:Gene Expression Regulation, Bacterial; D005796:Genes; D006801:Humans; D006863:Hydrogen-Ion Concentration; D051379:Mice; D008969:Molecular Sequence Data; D016297:Mutagenesis, Site-Directed; D009154:Mutation; D015202:Protein Engineering; D012689:Sequence Homology, Nucleic Acid; D013329:Structure-Activity Relationship; D014409:Tumor Necrosis Factor-alpha / Q000235:genetics* / Q000378:metabolism / Q000633:toxicity
D014409:Tumor Necrosis Factor-alpha