|Budget Amount *help
¥3,700,000 (Direct Cost : ¥3,700,000)
Fiscal Year 1999 : ¥1,600,000 (Direct Cost : ¥1,600,000)
Fiscal Year 1998 : ¥2,100,000 (Direct Cost : ¥2,100,000)
To determine the minimum requirement in the 76-residue leader sequence of pro-tumor necrosis factor (TNF) for membrane translocation across the endoplasmic reticulum (ER) and for the maturation of pro-TNF, we have constructed pro-TNF mutants in which a part of the transmembrane domain of pro-TNF was directly linked to the N-terminus of the mature domain, and evaluated their translocational behavior across the ER-membrane and their secretion from the transfected cells.
The in vitro translation/translocation assay using a canine pancreatic microsomal membrane system with a mutant, Δ-75--47,-32--1, revealed that the N-terminal half of the transmembrane domain of pro-TNF consisting of 14 residues functioned as a cleavable signal sequence ; it generated a cleaved form of TNF having a molecular mass similar to that of mature TNF. Analysis of the cleavage site by site-directed mutagenesis indicated that the site was inside of the leader sequence of this mutant. When the mutant Δ-75--47, -32--1 was expressed in COS-1 cells, efficient secretion of a biologically active soluble TNF was observed. Further deletion of the hydrophobic domain from this mutant inhibited the translocation, indicating that some extent of hydrophobicity is indispensable for the membrane translocation of the mature domain of TNF. Thus, the N-terminal half of the transmembrane domain of pro-TNF could function as a cleavable signal sequence when linked to the mature domain of TNF, and secretion of biologically active secretory form of TNF could be achieved with this 14-residue hydrophobic segment.
In intact pro-TNF, however, this 14-residue sequence could not function as a cleavable signal sequence during intracellular processing, indicating that the remainder of the 76-residue leader sequence of pro-TNF inhibits the signal peptide cleavage and enables the leader sequence to function as a type II signal-anchor sequence that generates a transmembrane form of TNF.