Analysis and characterisation of IL-1beta processing in rainbow trout, Oncorhynchus mykiss.
Mammalian IL-1beta 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-1beta is poorly understood. In the present study, the native IL-1beta 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-1beta (rmIL-1beta) 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-1beta precursor molecule tagged with 6 histidines at its C terminus. Only the recombinant mature 24 kDa) IL-1beta/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-1beta molecule is processed in trout macrophages in an analogous way to the situation with mammalian IL-1beta despite the lack of a clear ICE cut site.
Hong
Suhee
S
Scottish Fish Immunology Research Centre, University of Aberdeen, Zoology Building, Tillydrone Avenue, Aberdeen, Scotland AB24 2TZ, UK.
Zou
Jun
J
Collet
Bertrand
B
Bols
Niels C
NC
Secombes
Chris J
CJ
D000818:Animals; D015153:Blotting, Western; D002460:Cell Line; D017931:DNA Primers; D018076:DNA, Complementary / Q000235:genetics; D004591:Electrophoresis, Polyacrylamide Gel; D007375:Interleukin-1 / Q000096:biosynthesis* / Q000235:genetics; D008264:Macrophages / Q000276:immunology / Q000378:metabolism*; D017686:Oncorhynchus mykiss / Q000276:immunology*; D010957:Plasmids / Q000378:metabolism; D011498:Protein Precursors / Q000378:metabolism*; D011499:Protein Processing, Post-Translational; D014162:Transfection
D017931:DNA Primers; D018076:DNA, Complementary; D007375:Interleukin-1; D011498:Protein Precursors