CHARACTERIZATION OF LATENT RECOMBINANT TGF-BETA-2 PRODUCED BY CHINESE-HAMSTER OVARY CELLS

Latent recombinant transforming growth factor-beta-2 (LrTGF-beta-2) complex has been purified from serum-free media conditioned by Chinese hamster ovary cells transfected with a plasmid encoding the TGF-beta-2 (414) precursor. Under neutral conditions, LrTGF-beta-2 had an apparent molecular weight of 130 kDa. The complex contained both mature and pro-region sequences. Acidification of LrTFG-beta-2 resulted in the release of mature 24 kDa TGF-beta-2 from the high molecular weight pro-region-containing complex, suggesting that TGF-beta-2 was non-covalently associated with this complex. These results were confirmed by crosslinking experiments performed on partially purified LrTGF-beta-2. Protein sequence analysis of the purified TGF-beta-2 pro-region indicated that signal peptide cleavage occurred between ser(20) and leu(21). The pro-region, which previously was found to contain mannose-6-phosphate, bound to the mannose-6-phosphate receptor. Proteolytic cleavage of mature TGF-beta-2 was inhibited by monensin and chloroquin suggesting that binding to this receptor and subsequent transport to acidic vesicles may be involved in the processing of rTGF-beta-2 precursor.