Mechanism of activation of latent recombinant transforming growth factor beta 1 by plasmin.

Abstract

Medium conditioned by Chinese hamster ovary (CHO) cells transfected with the simian pre-pro-TGF.beta.1 cDNA contains high levels of latent TGF.beta.1. The amino-terminal region of the TGF.beta.1 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 TGF.beta.1 contains both the cleaved amino-terminal glycopeptide and mature TGF.beta.1 polypeptide in a noncovalent association and that this association confers latency. The data presented here do not support the involvement of a unique TGF.beta. binding protein(s) in latent recombinant TGF.beta.1. Plasmin treatment of CHO-conditioned medium resulted in the appearance of TGF.beta. competing activity. In addition, immunoblot analysis of plasmin-treated CHO-conditioned medium indicates that the amino-terminal glycopeptide is partially degraded and that mature TGF.beta.1 is released. Thus, activation of latent TGF.beta.1 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 TGF.beta.1. Acid activation of latent TGF.beta., in comparison, appears to be due to association of the amino-terminal glycopeptide from the mature polypeptide.