IDENTIFICATION AND ANALYSIS OF DISCRETE FUNCTIONAL DOMAINS IN THE PRO REGION OF PRE-PRO-TRANSFORMING GROWTH FACTOR-BETA-1

A series of site-specific insertion and deletion mutants was prepared in the pro domain of transforming growth factor beta-1 (TGF-beta-1) encoded by simian TGF-beta-1 cDNA. These mutants were transiently expressed in COS-1 cells and the ability of each to be properly processed, folded correctly, and secreted was determined by immunoblot analysis of cells and culture supernatants. Insertions in regions corresponding to amino acid residues 50, 154, and 170 blocked secretion; culture supernatants from COS-1 cells showed no immunologically reactive proteins, whereas intact cells contained high levels of the mutant polypeptides. Insertions in the middle portion of the pro domain at residues 81, 85, and 144 affected disulfide maturation of the mature TGF-beta-1. An insertion at residue 110, on the other hand, appeared to destabilize the mature TGF-beta-1 polypeptide, resulting in degraded growth factor. Relatively small (10 amino acids) to large (125 amino acids) deletion mutations in the pro domain of TGF-beta-1, when expressed as the full-length pre-pro-TGF-beta-1, appeared to block secretion. By contrast, if the pro domain (designated beta-1-latency-associated peptide [beta-1-LAP]) was expressed independently, deletion mutants in the region 40-110 were readily secreted by the COS-1 cells, whereas deletions in residues 110-210 either destabilized the structure of the protein or blocked its intracellular transport. Cross-linking assays employing radioiodinated TGF-beta-1 and biological assays indicate that residues 50-85 of beta-1-LAP are required for association with mature TGF-beta-1.