Analysis of small latent transforming growth factor-beta complex formation and dissociation by surface plasmon resonance - Absence of direct interaction with thrombospondins

Transforming growth factor-beta (TGF beta) is a pluripotent regulator of cell growth and differentiation. The growth factor is expressed as a latent complex that must be converted to an active form before interacting with its ubiquitous high affinity receptors. This conversion involves the release of the mature TGF beta through disruption of the noncovalent interactions with its propeptide or latency associated protein (LAP). Complex formation or dissociation between LAP and TGF beta plays a very important role in TGF beta biological activity at different steps. To further characterize the kinetic parameters of this interaction, we have employed surface plasmon resonance biosensor methodology. Using this technique, we observed real time association of LAP with mature TGF beta 1. The complex formation showed an equilibrium K-d around 3-7 nM. Furthermore, we observed dissociation of the complex in the presence of extreme pH, chaotropic agents, or plasmin, confirming their effects on TGF beta activation, The same approach was used to examine whether latent TGF beta 1 could interact with thrombospondins, previously described as activators of latent TGF beta. Using this method, we could not detect any direct interaction of thrombospondins with either LAP alone, TGF beta 1 alone, or the small latent TGF beta 1 complex. This suggests that activation of latent TGF beta 1 complex by thrombospondins is through an indirect mechanism.