Characterization of a low molecular weight isoform of IL-1 receptor antagonist

Abstract

IL-1R antagonist (IL-1Ra) exists in two well-characterized forms, 17-kDa secretory IL-1Ra (sIL-1Ra) and 18-kDa intracellular IL-1Ra (icIL-1Ra), that arise by alternative transcription of the same IL-1Ra gene. A third, lower molecular mass form (approximately 16 kDa) was detected by immunoblot within lysates of a variety of cells, including human monocytes and myelomonocytic cell lines. The 16-kDa isoform was designated icIL-1RaII, and the previously established 18-kDa form was designated icIL-1RaI. Intracellular IL-1RaII bound type I IL-1R up to fivefold less avidly than did sIL-1Ra and icIL-1RaI. Microsequencing of cyanogen bromide fragments of purified icIL-1RaII provided evidence consistent with initiation of protein translation at the second start site in either IL-1Ra mRNA. The results of site-directed mutation experiments established that icIL-1RaII could be derived by alternative translation initiation. In vitro transcription and translation of intact sIL-1Ra cDNA in rabbit reticulocyte lysates led to both pro-sIL-1Ra and icIL-1RaII proteins, whereas transcription and translation of icIL-1RaI cDNA produced both icIL-1RaI and icIL-1RaII proteins. Mutation of the first 5' ATG in sIL-1Ra cDNA led to translation of only icIL-1RaII, while only sIL-1Ra was observed after mutation of the second ATG. These results indicate that icIL-1RaII is a third member of the IL-1Ra family and is a 16-kDa, 143-amino acid intracellular protein derived by alternative translation initiation from either sIL-1Ra mRNA or icIL-1Ra mRNA. The role in biology of either intracellular form of IL-1Ra remains unknown.