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

Transforming growth factorb (TGFb ) is secreted by many cell types as part of a large latent complex composed of three subunits: TGFb , the TGFb propeptide, and the latent TGFb binding protein (LTBP). To interact with its cell surface receptors, TGFb must be released from the latent complex by disrupting noncovalent interactions between mature TGFb and its propeptide. Previously, we identified LTBP-1 and transglutaminase, a cross-linking enzyme, as reactants involved in the formation of TGFb . 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 TGFb by transglutaminase. Antibodies to the amino and carboxyl regions of LTBP-1S abrogate TGFb 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 ( D N293) or 441 ( D N441) amino acids were expressed transiently in CHO cells. Analysis of the LTBP-1S content in matrices of transfected CHO cultures revealed that D N293 LTBP-1S was matrix associated via a transglutaminasedependent reaction, whereas D N441 LTBP-1S was not. This suggests that residues 294–441 are critical to the transglutaminase reactivity of LTBP-1S. M ost cell types secrete transforming growth factorb 1 (TGFb ) 1 in a biologically inactive form (42). Mature TGFb is a homodimer composed of two 12.5-kD polypeptides joined by a disulfide bond at cysteine 77 (14). The monomeric subunits are produced by intercellular cleavage of a higher mol wt precursor at a dibasic site immediately preceding Ala-279 (17, 21). However, after secretion the propeptides remain associated with TGFb through noncovalent interactions, rendering TGFb inactive (20). TGFb with its propeptide, also known as the latency associated peptide (LAP), is referred to as the small latent complex. Both in vitro and in vivo, latent TGFb is secreted as part of a large latent complex in which a second gene product, the latent TGFb binding protein (LTBP), is disulfide-linked to LAP (42). The dissociation of TGFb from LAP is required for TGFb to bind to its receptors and exert its effects on cell proliferation, extracellular matrix (ECM) deposition, cell migration, and differentiation (34, 38, 58). Latent TGFb is activated by heat, acid or alkaline treatment, binding to thrombospondin, deglycosylation, proteolysis, or irradiation (5, 9, 34, 35, 56). The most extensively studied process for activating large latent complex is a plasmin-dependent mechanism observed in several tissue culture systems including bovine aortic endothelial (BAE) cells treated with retinoids, cocultures of endothelial cells and either smooth Address all correspondence to Irene Nunes, Department of Cell Biology, MSB 650, NYU Medical Center, 550 First Ave., New York, NY 10016. Tel.: (212) 263-5327. Fax: (212) 263-8139. Christine Metz’s current address is The Picower Institute for Medical Research, 350 Community Drive, Manhasset, NY 11030. 1. Abbreviations used in this paper : BAE, bovine aortic endothelial; BSM, bovine smooth muscle; CM, conditioned medium; ECM, extracellular matrix; GST, glutathione S–transferase; LAP, latency associated peptide; LPS, lipopolysaccharide; LTBP-1, latent TGFb binding protein-1; MDC, monodansylcadaverine; MLEC, mink lung epithelial cells; PAI-1, plasminogen activator inhibitor-1; TGFb , transforming growth factorb ; WT, wild type. on M ay 1, 2017 D ow nladed fom Published March 10, 1997