Identification and characterization of an eight-cysteine repeat of the latent transforming growth factor- beta binding protein-1 that mediates bonding to the latent transforming growth factor- beta 1
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 w...
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Veröffentlicht in: | The Journal of biological chemistry 1996-11, Vol.271 (47), p.29891-29896 |
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Format: | Artikel |
Sprache: | eng |
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Zusammenfassung: | 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- beta 1. Coexpression in Sf9 cells of small latent TGF- beta 1 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- beta 1. 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- beta 1 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 . |
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ISSN: | 0021-9258 |
DOI: | 10.1074/jbc.271.47.29891 |