Structure-function analysis of murine interleukin 1: biologically active polypeptides are at least 127 amino acids long and are derived from the carboxyl terminus of a 270-amino acid precursor.

Murine interleukin 1 (IL-1) is initially synthesized as a 270-amino acid precursor protein. Guided by amino-terminal end sequence analyses of mouse macrophage-derived IL-1, it was shown that expression of the carboxyl-terminal 156 amino acids (i.e., amino acids 115-270) of this precursor in Escherichia coli yields biologically active recombinant IL-1 (rIL-1) protein. To answer questions about precursor processing and the size of the smallest biologically active IL-1 fragment, we have engineered deletions of the rIL-1 (115-270) gene to encode two amino-terminal deletion analogs, rIL-1 (131-270) and rIL-1 (144-270), and a carboxyl-terminal deletion analog, rIL-1 (131-257, 270). The analogs were produced in E. coli, purified to homogeneity, and assayed for biological activity on murine thymocytes, human rheumatoid synovial cells, and human dermal fibroblasts and for their ability to bind to IL-1 receptors on murine EL-4 thymoma cells. The amino-terminal deletion analog rIL-1 (131-270) possessed a specific activity in the murine thymocyte proliferation assay equivalent to that of the 115-270 parent protein and exhibited significant biological activity in stimulating the production of collagenase and prostaglandin E2 by synovial cells and fibroblasts. The more extensive amino-terminal deletion analog rIL-1 (144-270) was inactive in all biological assays and failed to compete in the receptor binding assay. The carboxyl-terminal deletion analog rIL-1 (131-257, 270) competed less efficiently (by a factor of 100) in the receptor binding assay, retained weak biological activity on synovial cells and fibroblasts, and only demonstrated full intrinsic activity in the thymocyte proliferation assay when 100-200 times more protein was assayed. These results suggest that biologically active murine IL-1 polypeptides are at least 127 amino acids long and are derived from the carboxyl terminus of the 270-amino acid precursor. Furthermore, it appears that the integrity of the carboxyl terminus of the 270-amino acid precursor is important for activity but that different amino termini can be utilized to generate molecules with equivalent specific activities. This amino-terminal end flexibility supports a processing model for IL-1 maturation that partially explains IL-1 polypeptide heterogeneity.