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 (TGFbeta) 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 TGFbeta through disruption of the noncovalent interactions with its propeptide or latency associated protein (LAP). Complex formation or dissociation between LAP and TGFbeta plays a very important role in TGFbeta 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 TGFbeta1. The complex formation showed an equilibrium Kd 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 TGFbeta activation. The same approach was used to examine whether latent TGFbeta1 could interact with thrombospondins, previously described as activators of latent TGFbeta. Using this method, we could not detect any direct interaction of thrombospondins with either LAP alone, TGFbeta1 alone, or the small latent TGFbeta1 complex. This suggests that activation of latent TGFbeta1 complex by thrombospondins is through an indirect mechanism.
Bailly
S
S
INSERM U244, DBMS/BRCE, Commissariat à l'Energie Atomique Grenoble, 17 rue des martyrs, 38054 Grenoble Cedex 9, France. S.Bailly@geant.ceng.cea.fr
Brand
C
C
Chambaz
E M
EM
Feige
J J
JJ
D006801:Humans; D008562:Membrane Glycoproteins / Q000378:metabolism*; D010446:Peptide Fragments / Q000378:metabolism; D019699:Thrombospondins; D016212:Transforming Growth Factor beta / Q000378:metabolism*
D008562:Membrane Glycoproteins; D010446:Peptide Fragments; D019699:Thrombospondins; D016212:Transforming Growth Factor beta