Abstract
In a prior study, we found that the processed form of human interleukin-1 beta (mature IL-1 beta) is secreted to a significantly greater extent than the precursor form of the protein, indicating that the precursor domain acts in some manner to reduce the secretory potential of the protein. In view of this observation, we sought to define the sequence(s) in the IL-1 beta precursor that limit the secretion of the protein as well as the sequences in the mature protein that promote secretion. The P388D1 murine macrophage cell line and the Jurkat human T-cell line were transiently transfected with cDNA expression vectors encoding truncated forms of human precursor IL-1 beta proteins, lacking either the first 76, 94, 99, or 104 amino acids. The removal of increasing numbers of precursor amino acid residues resulted in a graded increase in the secretion of the truncated precursor IL-1 beta proteins from both cell lines. The minimal region of the precursor sequence required to inhibit the optimal secretion of IL-1 beta occurs between amino acids 100 and 104 for P388D1 cells and 95-99 for Jurkat cells. Deletion of the amino acids within these regions increased the secretion level of the truncated proteins to that of mature IL-1 beta. Mutagenesis of the mature IL-1 beta sequence revealed that a region of basic amino acids may play an important role in the optimal secretion of mature IL-1 beta in P388D1 cells, but not in Jurkat cells. Based on the differences in the structural requirements for IL-1 beta secretion in P388D1 and Jurkat cell lines, it is likely that the secretion of IL-1 beta may be subject to multiple levels of regulation that are differentially operative in different cell types.
Highlights
In support of this hypothesis, we have found that mature IL-1(3 proteins with conformation-altering deletions at the carboxyl or amino termini are poorly secreted in transiently transfected P388Dl cells, a murine macrophage cell line [6]
Secretion of the Protein by P388D1 Cells-Previous experiments demonstrated that precursor IL-l{3 is poorly secreted from P388Dl cells, whereas the mature IL-l{3 is secreted in a rapid and efficient manner following the addition of the calcium ionophore, ionomycin [6]
This conclusion is based upon the assumption that the mechanism of IL-l secretion in the two cell types is fundamentally the same, a view that is supported by the following common features ofIL-l secretion in the two cell lines: 1) mature IL-l is secreted to a greater extent than the precursor; 2) the secretion of mature IL-l is enhanced by calcium ionophores; 3) precursor domains truncated to amino acid 95 impair secretion; and 4) the secretion of improperly folded proteins such as the F162D mutant are reduced relative to the wild-type mature protein
Summary
In a recent study [6], we reported that the mature forms of IL-la and -(3 are secreted to a markedly greater extent than the precursor proteins, indicating that the optimal secretion of IL-1a and -(3 may be dependent upon a change in the conformation of the proteins that enhances their interaction with one or more components of the secretory pathway In support of this hypothesis, we have found that mature IL-1(3 proteins with conformation-altering deletions at the carboxyl or amino termini are poorly secreted in transiently transfected P388Dl cells, a murine macrophage cell line [6]. The results of these experiments are consistent with the hypothesis that IL-1 secretion is a multistep process that is subject to regulation in a cell-type specific manner
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