Identification of critical regions in mouse granulocyte-macrophage colony-stimulating factor by scanning-deletion analysis.
Structure-function relationships for mouse granulocyte-macrophage colony-stimulating factor were examined by generating a series of small deletions scanning the entire length of the molecule. Deletions of three amino acids were introduced at intervals of five amino acids by site-directed mutagenesis of the mature mouse granulocyte-macrophage colony-stimulating factor gene. The mutant proteins were expressed in Escherichia coli and assayed for biological activity. This procedure identified four regions critical to activity. These critical regions were further delineated by additional three-amino acid deletion mutants. Larger deletions at each terminus were also made, as well as changes of specific amino acid residues. The four critical regions span amino acid residues 18-22, 34-41, 52-61, and 94-115. The disulfide bridge between Cys-51 and Cys-93 was also shown to be essential for activity, whereas that between Cys-85 and Cys-118 could be removed without loss of activity. The possible structural and/or functional roles of the critical regions are discussed.
Shanafelt
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Department of Molecular Biology, DNAX Research Institute of Molecular and Cellular Biology, Palo Alto, CA 94304-1104.
Kastelein
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RA
D000595:Amino Acid Sequence; D000818:Animals; D002455:Cell Division / Q000187:drug effects; D002460:Cell Line; D002872:Chromosome Deletion; D003001:Cloning, Molecular; D003115:Colony-Stimulating Factors / Q000235:genetics* / Q000302:isolation & purification / Q000494:pharmacology; D004274:DNA, Recombinant / Q000378:metabolism; D004926:Escherichia coli / Q000235:genetics; D005796:Genes; D016178:Granulocyte-Macrophage Colony-Stimulating Factor; D006133:Growth Substances / Q000235:genetics* / Q000302:isolation & purification / Q000494:pharmacology; D051379:Mice; D008969:Molecular Sequence Data; D009154:Mutation; D011994:Recombinant Proteins / Q000302:isolation & purification / Q000494:pharmacology; D013329:Structure-Activity Relationship
D003115:Colony-Stimulating Factors; D004274:DNA, Recombinant; D006133:Growth Substances; D011994:Recombinant Proteins; D016178:Granulocyte-Macrophage Colony-Stimulating Factor