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© The Rockefeller University Press, 0021-9525/1997//1151 $5.00
The Journal of Cell Biology, Volume 136, Number 5, , 1997 1151-1163

Article

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



Irene Nunes, Pierre-Emmanuel Gleizes, Christine N. Metz, and Daniel B Rifkin

Department of Cell Biology, Kaplan Cancer Center, and the Raymond and Beverly Sackler Foundation Laboratory, New York University Medical Center, New York 10016

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 ({Delta}N293) or 441 ({Delta}N441) amino acids were expressed transiently in CHO cells. Analysis of the LTBP-1S content in matrices of transfected CHO cultures revealed that {Delta}N293 LTBP-1S was matrix associated via a transglutaminasedependent reaction, whereas {Delta}N441 LTBP-1S was not. This suggests that residues 294–441 are critical to the transglutaminase reactivity of LTBP-1S.

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 TGF-β; binding protein-1; MDC, monodansylcadaverine; MLEC, mink lung epithelial cells; PAI-1, plasminogen activator inhibitor-1; TGF-β, transforming growth factor-β; WT, wild type.

This research was supported by grants CA 2753 (D.B Rifkin), CA 34282 (D.B Rifkin), EY 06537 (I. Nunes), T32GM 07238 (I. Nunes), and CA 09161 (C.N. Metz) from the National Institutes of Health. P.-E. Gleizes was the recipient of a fellowship from the Association pour la Recherche Contre le Cancer.

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.


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