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.
DNAX Research Institute of Molecular and Cellular Biology, Palo Alto, California 94304.
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Yanofsky
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Zurawski
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