Disulfide assignments in recombinant mouse and human interleukin 4.
The disulfide pairings of mouse and human interleukin 4 (IL-4) proteins have been determined. The purified proteins, synthesized by recombinant DNA technology, are fully active as judged by their ability to stimulate an appropriate biological response in a variety of functional assays. Peptide maps were produced by digesting the proteins with pepsin and separating the resulting fragments by reverse-phase HPLC using linear acetonitrile-TFA gradients. Cystine-containing peptides were identified by determining which reverse-phase peaks showed an altered elution pattern after reduction. These peptides were purified further and defined by composition and sequence analysis. Three sets of disulfide-linked peptides were consistently identified for each protein. For mouse IL-4, the first and fifth, second and fourth, and third and sixth cysteines are joined. The disulfide bonds in human IL-4 are between the first and sixth, second and fourth, and third and fifth cysteines. A large double-loop region within the central three-fifths of each protein is stabilized by these bonds. Sequence analysis of the peptides containing the third and fifth cysteines of human IL-4 also demonstrated that only one of the potential N-glycosylation sites is used by C127 mammary tumor cells. Complete alkylation of mouse IL-4 under mild conditions completely destroyed its biological activity in a hematopoietic precursor cell proliferation assay.
Carr
C
C
Department of Biological Sciences, Monsanto Company, St. Louis, Missouri 63198.
Aykent
S
S
Kimack
N M
NM
Levine
A D
AD
D000595:Amino Acid Sequence; D000818:Animals; D002455:Cell Division / Q000187:drug effects; D002460:Cell Line; D003001:Cloning, Molecular; D004220:Disulfides / Q000032:analysis*; D004229:Dithiothreitol / Q000494:pharmacology; D004926:Escherichia coli / Q000235:genetics; D006801:Humans; D015847:Interleukin-4 / Q000737:chemistry* / Q000235:genetics / Q000302:isolation & purification / Q000494:pharmacology; D051379:Mice; D008969:Molecular Sequence Data; D011487:Protein Conformation; D011994:Recombinant Proteins / Q000235:genetics / Q000302:isolation & purification / Q000494:pharmacology; D012689:Sequence Homology, Nucleic Acid
D004220:Disulfides; D011994:Recombinant Proteins; D015847:Interleukin-4; D004229:Dithiothreitol