A ROLE OF THE LATENT TGF-BETA-1-BINDING PROTEIN IN THE ASSEMBLY AND SECRETION OF TGF-BETA-1

Transforming growth factor-beta-1 (TGF-beta-1) is synthesized as latent complexes with high molecular weights. The large latent complex of TGF-beta-1 in platelets is composed of three components, i.e. the mature TGF-beta-1, which is non-covalently associated with a disulphide-bonded complex of the N-terminal remnant of the TGF-beta-1 precursor (TGF-beta-1-latency associated peptide) and the latent TGF-beta-1 binding protein (LTBP). The TGF-beta-1-latency associated peptide is sufficient for the latency of TGF-beta-1, whereas the functions of LTBP remain to be elucidated. In a human erythroleukemia cell line, HEL, the production of the latent form of TGF-beta-1 was induced more than 100-fold by phorbol 12-myristate 13-acetate. Analysis by Northern blotting revealed that both the TGF-beta-1 precursor and LTBP were induced in a coordinated fashion. Analysis by immunoprecipitation using antibodies against LTBP and the TGF-beta-1 precursor dimer revealed that LTBP has a molecular size of 205 kd under reducing conditions in this cell type, i.e. similar to that from cells transfected with cDNA for LTBP, but larger than the platelet form (125-160 kd). Limited tryptic digestion of LTBP in HEL cells and analysis by SDS-PAGE showed protein bands of similar sizes to those of platelet LTBP, suggesting that the difference in molecular sizes of LTBP involves cell-specific processing. The biosynthesis of the latent TGF-beta-1 was studied by pulse-chase analysis. LTBP became covalently associated with the TGF-beta-1 precursor within 15 min after synthesis in this cell line. Secretion of the large latent TGF-beta-1 complex was observed as early at 30 min after the synthesis of LTBP; at the same time, a free form of LTBP not bound to the TGF-beta-1 precursor was seen. In contrast, the TGF-beta-1 precursor remained inside the cells in an unprocessed form for a longer time period and the TGF-beta-1 precursor dimer without LTBP was secreted only very slowly. Furthermore, the results of partial tryptic digestion of this molecule suggested that it contained improper disulphide bonding. These results suggest that LTBP plays a critical role in the assembly and secretion of the latent TGF-beta-1.