Analysis and characterisation of IL-1 β processing in rainbow trout, Oncorhynchus mykiss

Abstract

Mammalian IL-1 β is produced as a biologically inactive 31 kDa precursor, which is converted to the active 18 kDa form by proteolytic processing. Synthesis and processing of native piscine IL-1 β is poorly understood. In the present study, the native IL-1 β precursor or mature peptides were detected at sizes of approx. 29 kDa and 24 kDa in cell lysates of a rainbow trout macrophage cell line RTS-11, with or without LPS stimulation, by Western blot analysis using a polyclonal antibody against the putative trout mature IL-1 β (rmIL-1 β) produced in Escherichia coli. Processing of the 29 kDa precursor into a 24 kDa mature peptide was confirmed by analysis of such proteins using a monoclonal conjugate (Ni-NTA-HRP) against 6 histidines in lysates of the RTS-11 cells transfected with an expression plasmid containing the IL-1 β precursor molecule tagged with 6 histidines at its C terminus. Only the recombinant mature 24 kDa) IL-1 β/HIS protein was purified from the culture supernatants of the transfected cells, indicating the molecule is cleaved to be secreted. These findings strongly suggest that the trout IL-1 β molecule is processed in trout macrophages in an analogous way to the situation with mammalian IL-1 β despite the lack of a clear ICE cut site.