A pre-aspartate-specific protease from human leukocytes that cleaves pro-interleukin-1 beta.
Interleukin-1 beta is a 17.4-kilodalton hormone derived from a 33-kilodalton inactive precursor produced by monocytes. We used the precursor as a substrate to detect proteolytic activities in peripheral blood mono-nuclear cell-conditioned medium that might be involved in interleukin-1 beta processing. We found that the conditioned medium, following passage through DEAE-Sephacel, generates a biologically active fragment from the precursor that runs slightly higher than the mature hormone in sodium dodecyl sulfate-polyacrylamide gel electrophoresis. The responsible activity behaved as a single protein in ion exchange chromatography. It was completely inhibited by metal ion chelators and not by inhibitors of serine, cysteine, or aspartate proteases, and it was dependent on both calcium (or magnesium) and zinc. The enzyme was not inhibited by three substrate-based metalloprotease inhibitors, phosphoramidon, benzyloxycarbonyl-Gly-Leu-NH2, and N-(2-carboxy-3-phenylpropionyl)-Leu. NH2-terminal sequence analysis showed that cleavage of the precursor occurred between a histidine and an aspartate residue, and digestion of synthetic peptides indicated that the protease is specific for pre-aspartate cleavages.
Black
R A
RA
Immunex Corporation, Seattle, Washington 98101.
Kronheim
S R
SR
Merriam
J E
JE
March
C J
CJ
Hopp
T P
TP
D000595:Amino Acid Sequence; D000626:Aminopeptidases / Q000097:blood* / Q000302:isolation & purification; D020170:Caspase 1; D002413:Cations, Divalent; D002478:Cells, Cultured; D002852:Chromatography, Ion Exchange; D003470:Culture Media; D006801:Humans; D007375:Interleukin-1 / Q000378:metabolism*; D007962:Leukocytes / Q000201:enzymology*; D008969:Molecular Sequence Data; D010446:Peptide Fragments / Q000032:analysis; D011480:Protease Inhibitors / Q000494:pharmacology; D011498:Protein Precursors / Q000378:metabolism*; D013379:Substrate Specificity
D002413:Cations, Divalent; D003470:Culture Media; D007375:Interleukin-1; D010446:Peptide Fragments; D011480:Protease Inhibitors; D011498:Protein Precursors; C043830:interleukin 1 precursor; D000626:Aminopeptidases; D020170:Caspase 1