Identification of key residues in the amino-terminal third of human interleukin-1 alpha.
Two mutational approaches were used to perform a thorough structure-function analysis of the first 53 residues of the 159-residue cytokine human interleukin-1 alpha (hIL-1 alpha). In this study, a total of 26 deletions, 97 multiple amino acid substitutions, and 46 single amino acid substitutions were examined. A synthetic hIL-1 alpha gene with many unique restriction sites was constructed to facilitate the molecular manipulations that were performed. The mutational methods employed include: Bal-31 exonuclease-generated deletions at unique restriction sites and combinatorial cassette mutagenesis via segment replacement with synthetic DNA. The mutant hIL-1 alpha proteins were expressed at high levels in Escherichia coli and were assayed for biological activity in a mouse T cell proliferation assay. We observed that the activity of hIL-1 alpha was extraordinarily sensitive to deletion mutations. Most internal deletions of as few as 1 or 2 residues substantially reduced biological activity. Combinatorial cassette mutagenesis on residues 13-53 of hIL-1 alpha identified 15 important residue positions. Of these, 8 displayed strong preferences for residues with hydrophilic side chains, and the remainder preferred hydrophobic side chains. We found that functional hIL-1 alpha had an absolute requirement for a basic residue (Arg, Lys, or His) at either position 15 or 16, and that Leu was preferred at position 40.
Yanofsky
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DNAX Research Institute of Molecular and Cellular Biology, Palo Alto, California 94304.
Zurawski
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G
D000595:Amino Acid Sequence; D000818:Animals; D001483:Base Sequence; D002872:Chromosome Deletion; D004926:Escherichia coli / Q000235:genetics; D005813:Genes, Synthetic; D005822:Genetic Vectors; D006801:Humans; D015151:Immunoblotting; D007375:Interleukin-1 / Q000235:genetics*; D008969:Molecular Sequence Data; D009154:Mutation; D010957:Plasmids; D012689:Sequence Homology, Nucleic Acid
D007375:Interleukin-1