Identification of the High Affinity Binding Site in Transforming Growth Factor-β Involved in Complex Formation with α2-Macroglobulin

The biological activities of transforming growth factor-β isoforms (TGF-β1,2) are known to be modulated by α2-macroglobulin (α2M). α2M forms complexes with numerous growth factors, cytokines, and hormones, including TGF-β. Identification of the binding sites in TGF-β isoforms responsible for high affinity interaction with α2M many unravel the molecular basis of the complex formation. Here we demonstrate that among nine synthetic pentacosapeptides with overlapping amino acid sequences spanning the entire TGF-β1 molecule, the peptide (residues 41–65) containing Trp-52 exhibited the most potent activity in inhibiting the formation of complexes between 125I-TGF-β1and activated α2M (α2M*) as determined by nondenaturing polyacrylamide gel electrophoresis and by plasma clearance in mice. TGF-β2 peptide containing the homologous sequence and Trp-52 was as active as the TGF-β1 peptide, whereas the corresponding TGF-β3 peptide lacking Trp-52, was inactive. The replacement of the Trp-52 with alanine abolished the inhibitory activities of these peptides. 125I-TGF-β3, which lacks Trp-52, bound to α2M* with an affinity lower than that of 125I-TGF-β1. Furthermore, unlabeled TGF-β3 and the mutant TGF-β1W52A, in which Trp-52 was replaced with alanine, were less potent than unlabeled TGF-β1 in blocking I125-TGF-β1 binding to α2M*. TGF-β1 and TGF-β2 peptides containing Trp-52 were also effective in inhibiting I125-nerve growth factor binding to α2M*. Τhese results suggest that Trp-52 is involved in high affinity binding of TGF-β to α2M*. They also imply that TGF-β and other growth factors/cytokines/hormones may form complexes with α2M* via a common mechanism involving the interactions between topologically exposed Trp and/or other hydrophobic residues and a hydrophobic region in α2M*.