Latent transforming growth factor-beta binding protein domains involved in activation and transglutaminase-dependent cross-linking of latent transforming growth factor-beta.
Transforming growth factor-beta (TGF-beta) is secreted by many cell types as part of a large latent complex composed of three subunits: TGF-beta, the TGF-beta propeptide, and the latent TGF-beta binding protein (LTBP). To interact with its cell surface receptors, TGF-beta must be released from the latent complex by disrupting noncovalent interactions between mature TGF-beta and its propeptide. Previously, we identified LTBP-1 and transglutaminase, a cross-linking enzyme, as reactants involved in the formation of TGF-beta. In this study, we demonstrate that LTBP-1 and large latent complex are substrates for transglutaminase. Furthermore, we show that the covalent association between LTBP-1 and the extracellular matrix is transglutaminase dependent, as little LTBP-1 is recovered from matrix digests prepared from cultures treated with transglutaminase inhibitors. Three polyclonal antisera to glutathione S-transferase fusion proteins containing amino, middle, or carboxyl regions of LTBP-1S were used to identify domains of LTBP-1 involved in cross-linking and formation of TGF-beta by transglutaminase. Antibodies to the amino and carboxyl regions of LTBP-1S abrogate TGF-beta generation by vascular cell cocultures or macrophages. However, only antibodies to the amino-terminal region of LTBP-1 block transglutaminase-dependent cross-linking of large latent complex or LTBP-1. To further identify transglutaminase-reactive domains within the amino-terminal region of LTBP-1S, mutants of LTBP-1S with deletions of either the amino-terminal 293 (deltaN293) or 441 (deltaN441) amino acids were expressed transiently in CHO cells. Analysis of the LTBP-1S content in matrices of transfected CHO cultures revealed that deltaN293 LTBP-1S was matrix associated via a transglutaminase-dependent reaction, whereas deltaN441 LTBP-1S was not. This suggests that residues 294-441 are critical to the transglutaminase reactivity of LTBP-1S.
Nunes
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Department of Cell Biology, Kaplan Cancer Center, New York University Medical Center, New York 10016, USA.
Gleizes
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PE
Metz
C N
CN
Rifkin
D B
DB
D000818:Animals; D016466:CHO Cells; D002103:Cadaverine / Q000031:analogs & derivatives / Q000494:pharmacology; D002352:Carrier Proteins / Q000235:genetics / Q000378:metabolism*; D002478:Cells, Cultured; D006224:Cricetinae; D003432:Cross-Linking Reagents; D004791:Enzyme Inhibitors / Q000494:pharmacology; D005109:Extracellular Matrix / Q000378:metabolism*; D006801:Humans; D047908:Intracellular Signaling Peptides and Proteins; D053779:Latent TGF-beta Binding Proteins; D011993:Recombinant Fusion Proteins; D013552:Swine; D016212:Transforming Growth Factor beta / Q000378:metabolism*; D011503:Transglutaminases / Q000037:antagonists & inhibitors / Q000494:pharmacology*
D002352:Carrier Proteins; D003432:Cross-Linking Reagents; D004791:Enzyme Inhibitors; D047908:Intracellular Signaling Peptides and Proteins; C509032:LTBP1 protein, human; D053779:Latent TGF-beta Binding Proteins; D011993:Recombinant Fusion Proteins; D016212:Transforming Growth Factor beta; D011503:Transglutaminases; C008542:monodansylcadaverine; D002103:Cadaverine