Purification and characterization of interleukin 1 receptor level antagonist proteins from THP-1 cells.
Culture medium conditioned by phorbol 12-myristate 13-acetate-differentiated THP-1 cells contained interleukin 1 (IL-1) antagonist activity as measured by inhibition of both IL-1 beta binding to receptors on YT cells and inhibition of IL-1/phytohemagglutinin-stimulated IL-2 synthesis by LBRM-33-1A5 T cells. Based on their ability to compete for 125I-IL-1 beta binding to receptors on YT cells, four distinct antagonist proteins were purified from THP-1 cell conditioned medium using a combination of ion-exchange, hydrophobic interaction, and size exclusion chromatographies. The four proteins had different isoelectric points with molecular masses in the range 22-26 kDa and had similar specific activities for inhibition of IL-1 beta binding to cell surface receptors (Ki values 0.33-0.64 nM) and for inhibition of IL-1/phytohemagglutinin-stimulated IL-2 synthesis by 1A5 cells (IC50 values 25-100 pM). Amino-terminal sequence analysis of the two major forms (25 kDa/pI 5.1 and 22 kDa/pI 5.8) revealed complete identity for the first 27 residues in both forms. Based on the results of peptide mapping, amino acid compositional analysis and immune blotting, all of the forms were deduced to be variants of a common protein. Deglycosylation of the antagonist proteins with N-glycanase converted them to a common form (22 kDa/pI 5.8), indicating that the four isoforms represent glycosylation variants of a common protein and that asparagine-linked oligosaccharides are responsible for the observed size and charge heterogeneity.
Bienkowski
M J
MJ
Department of Cell Biology, Upjohn Company, Kalamazoo, Michigan 49007.
Eessalu
T E
TE
Berger
A E
AE
Truesdell
S E
SE
Shelly
J A
JA
Laborde
A L
AL
Zurcher-Neely
H A
HA
Reardon
I M
IM
Heinrikson
R L
RL
Chosay
J G
JG
D000595:Amino Acid Sequence; D001667:Binding, Competitive; D002454:Cell Differentiation / Q000187:drug effects; D002460:Cell Line; D002850:Chromatography, Gel; D002852:Chromatography, Ion Exchange; D006801:Humans; D007375:Interleukin-1 / Q000037:antagonists & inhibitors* / Q000276:immunology; D008969:Molecular Sequence Data; D009000:Monocytes / Q000276:immunology*; D010449:Peptide Mapping; D011971:Receptors, Immunologic / Q000276:immunology / Q000378:metabolism*; D017472:Receptors, Interleukin-1; D013755:Tetradecanoylphorbol Acetate / Q000494:pharmacology
D007375:Interleukin-1; D011971:Receptors, Immunologic; D017472:Receptors, Interleukin-1; D013755:Tetradecanoylphorbol Acetate