MECHANISM OF ACTIVATION OF LATENT RECOMBINANT TRANSFORMING GROWTH FACTOR-BETA-1 BY PLASMIN

Medium conditioned by Chinese hamster ovary (CHO) cells transfected with the simian pre-pro-TGFβ1 cDNA contains high levels of latent TGFβ1. The amino-terminal region of the TGFβ1 precursor is secreted and can be detected in the conditioned medium by immunoblotting using peptide antibodies specific for amino-terminal peptides. Chemical cross-linking of CHO-conditioned medium using bis-(sulfosuccinimidyl)-suberate (BS3) followed by immunoblot analyses indicates that latent recombinant TGFβ1 contains both the cleaved amino-terminal glycopeptide and mature TGFβ1 polypeptide in a noncovalent association and that this association confers latency. The data presented here do not support the involvement of a unique TGFβ binding protein(s) in latent recombinant TGFβ1. Plasmin treatment of CHO-conditioned medium resulted in the appearance of TGFβ competing activity. In addition, immunoblot analysis of plasmin-treated CHO-conditioned medium indicates that the amino-terminal glycopeptide is partially degraded and that mature TGFβ1 is released. Thus, activation of latent TGFβ1 may occur by proteolytic nicking within the amino-terminal glycopeptide thereby causing a disruption of tertiary structure and noncovalent bonds, which results in the release of active, mature TGFβ1. Acid activation of latent TGFβ, in comparison, appears to be due to dissociation of the amino-terminal glycopeptide from the mature polypeptide.