Transforming growth factor‐β1 is a heparin‐binding protein: Identification of putative heparin‐binding regions and isolation of heparins with varying affinity for TGF‐β1

Abstract

Previous studies indicated that a major factor in heparin's ability to suppress the proliferation of vascular smooth muscle cells is an interaction with transforming growth factor-beta 1 (TGF-beta 1). Heparin appeared to bind directly to TGF-beta 1 and to prevent the association of TGF-beta 1 with alpha 2-macroglobulin (alpha 2-M). The present studies indicate that 20-70% of iodinated TGF-beta 1 binds to heparin-Sepharose and the retained fraction is eluted with approximately 0.37 M NaCl. Native, unlabelled platelet TGF-beta 1, however, is completely retained by heparin-Sepharose and eluted with 0.9-1.2 M NaCl. Using synthetic peptides, the regions of TGF-beta 1 that might be involved in the binding of heparin and other polyanions were examined. Sequence analysis of TGF-beta 1 indicated three regions with a high concentration of basic residues. Two of these regions had the basic residues arranged in a pattern homologous to reported consensus heparin-binding regions of other proteins. The third constituted a structurally novel pattern of basic residues. Synthetic peptides homologous to these three regions, but not to other regions of TGF-beta 1, were found to bind to heparin-Sepharose and were eluted with 0.15 M-0.30 M NaCl. Only two of these regions were capable of blocking the binding of heparin to 125I-TGF-beta. Immobilization of these peptides, followed by affinity purification of heparin, indicated that one peptide was capable of isolating subspecies of heparin with high and low affinity for authentic TGF-beta 1. The ability of TGF-beta 1 to bind to heparin or related proteoglycans under physiological conditions may be useful in understanding the biology of this pluripotent growth and metabolic signal. Conversely, a subspecies of heparin molecules with high affinity for TGF-beta 1 may be a factor in some of the diverse biological actions of heparin.

The Interaction of the Transforming Growth Factor-βs with Heparin/Heparan Sulfate Is Isoform-specific

“…1A), confirming the original observation of McCaffrey et al (6). The bound fraction was predominantly released by a 0.5 M NaCl step, although a proportion required a higher ionic strength for elution.…”

“…McCaffrey et al (6) identified three potential heparin-binding sites in TGF-␤1, corresponding to residues 23-32, 30 -41, and 92-99. Residues 23-32 were considered most likely to constitute an in vivo binding site, being most able, as a free peptide, to inhibit the binding of TGF-␤1 to immobilized heparin (6).…”

“…Residues 23-32 were considered most likely to constitute an in vivo binding site, being most able, as a free peptide, to inhibit the binding of TGF-␤1 to immobilized heparin (6). In light of the extensive sequence homologies between TGF-␤s, but the specific absence of a heparin/HS-binding site in TGF-␤3, we thought it possible that information on the binding site may be indirectly obtained from the conservation of basic amino acids (important for interaction with polyanionic GAGs) across the isoforms (Fig.…”

“…They are believed to have an important role in natural repair processes, but overexpression or dysregulation can lead to the development of various fibrotic disorders. TGF-␤1 has been demonstrated to possess strong heparin binding activity in vitro (6). Such interaction protects TGF-␤1 from proteolytic degradation in vitro (7), and also prevents the formation of inactive complexes with ␣ 2 -macroglobulin (␣ 2 M) (8).…”

Proteolysis of Latent Transforming Growth Factor-β (TGF-β)-binding Protein-1 by Osteoclasts

“…For instance, many growth factors can bind to matrix via heparan sulfate-containing proteoglycans and other members of the proteoglycan family. Mature TGF-␤ can bind to heparin (44) and our own unpublished studies indicate that LTBP1 itself binds to heparin. 3 Studies by Tiedemann et al (45) have demonstrated the importance of heparan sulfatecontaining proteoglycans in assembly of fibrillin-1 into the ECM.…”