Identification and Characterization of an Eight-cysteine Repeat of the Latent Transforming Growth Factor-β Binding Protein-1 that Mediates Bonding to the Latent Transforming Growth Factor-β1*
- From the Department of Cell Biology and Kaplan Cancer Center and the Raymond and Beverly Sackler Foundation Laboratory, New York University Medical Center, and
- § Department of Pharmacology and Skirball Institute of Biomedical Research, New York University Medical Center, New York, New York 10016, and Department of Chemistry, New York University, New York, New York 10003
- ‡ Recipient of a fellowship from the Association pour la Recherche contre le Cancer (ARC). To whom correspondence should be addressed: Dept. of Cell Biology, NYU Medical Center, 550 First Ave., New York, NY 10016. Tel.: 212-263-5327; Fax: 212-263-8139.
Abstract
Most cultured cell types secrete small latent transforming growth factor-β (TGF-β) as a disulfide-bonded complex with a member of the latent TGF-β binding protein (LTBP) family. Using the baculovirus expression system, we have mapped the domain of LTBP-1 mediating covalent association with small latent TGF-β1. Coexpression in Sf9 cells of small latent TGF-β1 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-β1. 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-β, 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-β1 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-β.
Footnotes
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↵* This research was supported in part by National Institutes of Health Grants CA 23753 (to D. B. R.) and T32 CA 09161 (to B. S.). The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked “advertisement” in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.
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↵1 The abbreviations used are:
- TGF-β
-
transforming growth factor-β
- LAP
-
latency-associated peptide
- LTBP
-
latent TGF-β binding protein
- EGF
-
epidermal growth factor
- PCR
-
polymerase chain reaction
- mAb
-
monoclonal antibody
- PAGE
-
polyacrylamide gel electrophoresis
- Ab
-
antibody.
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- Received May 6, 1996.
- Revision received September 10, 1996.
- © 1996 by The American Society for Biochemistry and Molecular Biology, Inc.
