Structure-function studies of human granulocyte-macrophage colony-stimulating factor. Identification of residues required for activity.
Human granulocyte-macrophage colony stimulating factor (hGM CSF), a protein containing 127 amino acids, was chemically synthesized by using automated stepwise solid-phase methods. The unpurified synthetic hGM-CSF had the same range of actions on hemopoietic cells as the purified recombinant protein. The structural requirements for the activities of synthetic hGM-CSF were examined by the design and synthesis of fragments and analogs. The synthetic fragment, hGM-CSF (54-127), containing all four of the cysteine residues found in the intact protein, lacked detectable activity. Assays of fragments shortened at the N terminus showed that the residues 1-13 were not required for activity, but that the integrity of residues 14-25, particularly residues 16, 17, and 18, was critical for biologic activity. The 14-25 region is predicted to form the first alpha-helix in hGM-CSF. Synthetic peptides within the N-terminal 53 residue region lacked detectable activity. The synthetic analog hGM-CSF (1-121), which lacks the C-terminal 6 residues, had similar activity to hGM-CSF (1-127) indicating that residues 122-127 are not required for activity. An analog, [Ala88] hGM-CSF (14-96), which lacks the hydrophobic C-terminal region and 2 cysteine residues, had low but readily detectable activity suggesting that residues 14-96 are sufficient for detectable synthetic hGM-CSF activity, although the presence of residues 97-121 are required for full activity. No dissociation of the multiple biological activities of hGM-CSF was detected.
Clark-Lewis
I
I
California Institute of Technology, Division of Biology, Pasadena.
Lopez
A F
AF
To
L B
LB
Vadas
M A
MA
Schrader
J W
JW
Hood
L E
LE
Kent
S B
SB
D000595:Amino Acid Sequence; D000596:Amino Acids / Q000502:physiology; D002455:Cell Division / Q000187:drug effects; D003115:Colony-Stimulating Factors / Q000031:analogs & derivatives / Q000138:chemical synthesis / Q000502:physiology; D016178:Granulocyte-Macrophage Colony-Stimulating Factor; D006098:Granulocytes / Q000378:metabolism; D006133:Growth Substances / Q000138:chemical synthesis / Q000502:physiology; D006801:Humans; D008969:Molecular Sequence Data; D010101:Oxygen Consumption / Q000187:drug effects; D010446:Peptide Fragments / Q000138:chemical synthesis / Q000502:physiology; D013329:Structure-Activity Relationship; D014407:Tumor Cells, Cultured
D000596:Amino Acids; D003115:Colony-Stimulating Factors; D006133:Growth Substances; D010446:Peptide Fragments; D016178:Granulocyte-Macrophage Colony-Stimulating Factor