Latent Transforming Growth Factor-β Binding Protein Domains Involved in Activation and Transglutaminase-dependent Cross-Linking of Latent Transforming Growth Factor-β

Abstract

Transforming growth factor-β (TGF-β) is secreted by many cell types as part of a large latent complex composed of three subunits: TGF-β, the TGF-β propeptide, and the latent TGF-β binding protein (LTBP). To interact with its cell surface receptors, TGF-β must be released from the latent complex by disrupting noncovalent interactions between mature TGF-β and its propeptide. Previously, we identified LTBP-1 and transglutaminase, a cross-linking enzyme, as reactants involved in the formation of TGF-β. 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 crosslinking and formation of TGF-β by transglutaminase. Antibodies to the amino and carboxyl regions of LTBP-1S abrogate TGF-β 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 (ΔN293) or 441 (ΔN441) amino acids were expressed transiently in CHO cells. Analysis of the LTBP-1S content in matrices of transfected CHO cultures revealed that ΔN293 LTBP-1S was matrix associated via a transglutaminasedependent reaction, whereas ΔN441 LTBP-1S was not. This suggests that residues 294–441 are critical to the transglutaminase reactivity of LTBP-1S.

Analysis of the expression pattern of the latent transforming growth factor ? binding protein isoforms in normal and diseased human liver reveals a new splice variant missing the proteinase-sensitive hinge region

“…It is discussed that the availability of TGF-␤ seems to be modulated by the association of the small latent TGF-␤ complex to the ECM via LTBP by transglutaminasedependent cross-linking. 45,46 The activation of TGF-␤ by various proteases like thrombin, plasmin, mast cell chymase, or leukocyte elastase occurs most probably via proteolytic cleavage of LTBP-1, although the activation and the release from matrix seem to be controlled by different mechanisms. 17 The release of latent TGF-␤ from ECM is most likely the prerequisite for the activation of TGF-␤ on cell surfaces.…”

Localization of Tissue Transglutaminase in Human Carotid and Coronary Artery Atherosclerosis: Implications for Plaque Stability and Progression

“…TGF-␤1 is secreted in an inactive complex comprised of latent TGF-␤1 and the latency associated peptide. TG-mediated activation of TGF-␤1 requires cross-linking of latency associated peptide to components of the ECM (Kojima et al, 1993;Nunes et al, 1995Nunes et al, , 1997. Once activated, TGF-␤1 enhances the expression of a variety of ECM components and induces inhibitors of matrix degradation (plasminogen activator inhibitor type 1 [PAI-1] and TIMP).…”

Roles of transglutaminases in cardiac and vascular diseases

“…The upregulation of TG2 by TGF-beta 1 leads to another positive feedback loop. TG2 activates TGF-beta by cross linking the latent TGF-beta binding protein to the extracellular matrix (72). TGF-beta expression down regulates inflammatory and autoimmune responses (73).…”