Abstract
Most cultured cell types secrete small latent transforming growth factor-beta (TGF-beta) as a disulfide-bonded complex with a member of the latent TGF-beta binding protein (LTBP) family. Using the baculovirus expression system, we have mapped the domain of LTBP-1 mediating covalent association with small latent TGF-beta1. Coexpression in Sf9 cells of small latent TGF-beta1 with deletion mutants of LTBP-1 showed that the third eight-cysteine repeat of LTBP-1 is necessary and sufficient for covalent interaction with small latent TGF-beta1. Analysis by mass spectrometry of this eight-cysteine repeat, produced as a recombinant peptide in Sf9 cells, confirmed that it was N-glycosylated, as expected from the primary sequence. No other post-translational modifications of this domain were detected. Alkylation of the recombinant peptide with vinyl pyridine failed to reveal any free cysteines, indicating that, in the absence of small latent TGF-beta, the eight cysteines of this domain are engaged in intramolecular bonds. These data demonstrate that the third LTBP-1 eight-cysteine repeat recognizes and associates covalently with small latent TGF-beta1 through a mechanism that does not require any specific post-translational modification of this domain. They also suggest that this domain adopts different conformations depending on whether it is free or bound to small latent TGF-beta.
Highlights
First identified as a transforming agent on cultured fibroblasts, transforming growth factor- (TGF-)1 is a potent, ubiquitous regulator of cell growth and differentiation and a modulator of the immune system [1,2,3]
In order to interact with specific cell surface serine/threonine kinase receptors, TGF- must dissociate from LAP, a process known as TGF- activation
The release of the large latent complex from the extracellular matrix mediated by cells has not been reported, this step is assumed to play a role in latent TGF- activation by allowing interaction of the complex with the cell surface [14]
Summary
First identified as a transforming agent on cultured fibroblasts, transforming growth factor- (TGF-)1 is a potent, ubiquitous regulator of cell growth and differentiation and a modulator of the immune system [1,2,3]. These data demonstrate that the third LTBP-1 eight-cysteine repeat recognizes and associates covalently with small latent TGF-1 through a mechanism that does not require any specific post-translational modification of this domain.
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