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-beta1.
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.
Gleizes
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PE
Department of Cell Biology and Kaplan Cancer Center and the Raymond and Beverly Sackler Foundation Laboratory, New York University Medical Center, New York, New York 10016, USA.
Beavis
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RC
Mazzieri
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R
Shen
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Rifkin
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DB
D000818:Animals; D016466:CHO Cells; D002352:Carrier Proteins / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism; D002460:Cell Line; D006224:Cricetinae; D003545:Cysteine / Q000737:chemistry*; D006801:Humans; D047908:Intracellular Signaling Peptides and Proteins; D053779:Latent TGF-beta Binding Proteins; D016296:Mutagenesis; D017924:Nucleopolyhedroviruses / Q000235:genetics; D011485:Protein Binding; D011994:Recombinant Proteins / Q000737:chemistry / Q000378:metabolism; D017384:Sequence Deletion; D019032:Spectrometry, Mass, Matrix-Assisted Laser Desorption-Ionization; D018411:Spodoptera; D016212:Transforming Growth Factor beta / Q000378:metabolism*
D002352:Carrier Proteins; D047908:Intracellular Signaling Peptides and Proteins; C509032:LTBP1 protein, human; D053779:Latent TGF-beta Binding Proteins; D011994:Recombinant Proteins; D016212:Transforming Growth Factor beta; D003545:Cysteine