Human IL-1 beta processing and secretion in recombinant baculovirus-infected Sf9 cells is blocked by the cowpox virus serpin crmA.
Biologically active, mature IL-1 beta (mIL-1 beta) is released from activated monocytes after proteolytic processing from an inactive precursor (pIL-1 beta). IL-1 beta converting enzyme (ICE), the first member of a newly discovered family of cysteine proteinases, is required for this processing event. The cleaved cytokine is released from monocytes by an unknown mechanism which does not employ a standard hydrophobic signal sequence. As in mammalian fibroblasts, insect Sf9 cells do not normally process or secrete human IL-1 beta. The expression of active ICE enables Sf9 cells to process 31-kDa pIL-1 beta correctly at Asp27 and Asp116, and to export 17.5-kDa mIL-1 beta. The recombinant heterodimeric human enzyme purified from Sf9 cells possesses a sp. act. of 2.9 +/- 0.5 x 10(6) U/mg and is indistinguishable from native ICE with regard to its subunit composition and catalytic properties. In this system, co-expression of the cowpox virus crmA gene, an extremely potent serpin inhibitor of ICE (Ki < 7 pM), inhibits ICE activation completely and blocks pIL-1 beta processing and mIL-1 beta secretion by approximately 95%. The results indicate that ICE, in addition to its processing function, facilitates the transport of IL-1 beta across the plasma membrane.
Howard
A D
AD
Department of Immunology, Merck Research Laboratories, Rahway, NJ 07065.
Palyha
O C
OC
Griffin
P R
PR
Peterson
E P
EP
Lenny
A B
AB
Ding
G J
GJ
Pickup
D J
DJ
Thornberry
N A
NA
Schmidt
J A
JA
Tocci
M J
MJ
D000595:Amino Acid Sequence; D000818:Animals; D016367:Baculoviridae / Q000235:genetics; D001665:Binding Sites / Q000235:genetics; D020170:Caspase 1; D002460:Cell Line; D015608:Cowpox virus / Q000235:genetics; D003546:Cysteine Endopeptidases / Q000235:genetics / Q000378:metabolism; D006801:Humans; D066298:In Vitro Techniques; D007375:Interleukin-1 / Q000378:metabolism*; D007700:Kinetics; D008969:Molecular Sequence Data; D009842:Oligopeptides / Q000235:genetics; D011499:Protein Processing, Post-Translational / Q000187:drug effects; D011994:Recombinant Proteins / Q000235:genetics / Q000378:metabolism; D015843:Serpins / Q000235:genetics / Q000494:pharmacology*; D018411:Spodoptera; D013379:Substrate Specificity; D014764:Viral Proteins
D007375:Interleukin-1; D009842:Oligopeptides; D011994:Recombinant Proteins; D015843:Serpins; D014764:Viral Proteins; C074641:interleukin-1beta-converting enzyme inhibitor; D003546:Cysteine Endopeptidases; D020170:Caspase 1