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Binding Protein Domains
Involved in Activation and Transglutaminase-dependent
Cross-Linking of Latent Transforming Growth Factor-
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-
(TGF-) is secreted by many cell types as part of a large latent complex composed of three subunits: TGF-, the TGF-
propeptide, and the latent TGF- binding protein (LTBP). To interact with its cell surface receptors,
TGF- must be released from the latent complex by
disrupting noncovalent interactions between mature
TGF- and its propeptide. Previously, we identified
LTBP-1 and transglutaminase, a cross-linking enzyme, as reactants involved in the formation of TGF-. 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- by transglutaminase.
Antibodies to the amino and carboxyl regions of
LTBP-1S abrogate TGF- 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 (
N293) or 441 (N441) amino acids were expressed transiently in
CHO cells. Analysis of the LTBP-1S content in matrices of transfected CHO cultures revealed that N293
LTBP-1S was matrix associated via a transglutaminasedependent reaction, whereas N441 LTBP-1S was not.
This suggests that residues 294-441 are critical to the transglutaminase reactivity of LTBP-1S.
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