Betaglycan has Two Independent Domains Required for High Affinity TGF-β Binding: Proteolytic Cleavage Separates the Domains and Inactivates the Neutralizing Activity of the Soluble Receptor

• Published in: Biochemistry (Easton) Vol. 48; no. 49; pp. 11755 - 11765
• Main Authors: Mendoza, Valentín, Vilchis-Landeros, M. Magdalena, Mendoza-Hernández, Guillermo, Huang, Tao, Villarreal, Maria M, Hinck, Andrew P, López-Casillas, Fernando, Montiel, Jose-Luis
• Format: Journal Article
• Language: English
• Published: United States American Chemical Society 15.12.2009
• Subjects: Amino Acid Sequence; Animals; Base Sequence; Cercopithecus aethiops; COS Cells; Extracellular Space - chemistry; Extracellular Space - metabolism; Fibrinolysin - metabolism; Humans; Molecular Sequence Data; Neutralization Tests; Peptide Fragments - chemistry; Peptide Fragments - genetics; Peptide Fragments - metabolism; Protein Binding; Protein Structure, Secondary; Protein Structure, Tertiary - genetics; Proteoglycans - antagonists & inhibitors; Proteoglycans - chemistry; Proteoglycans - genetics; Proteoglycans - metabolism; Receptors, Transforming Growth Factor beta - antagonists & inhibitors; Receptors, Transforming Growth Factor beta - chemistry; Receptors, Transforming Growth Factor beta - genetics; Receptors, Transforming Growth Factor beta - metabolism; Recombinant Proteins - antagonists & inhibitors; Recombinant Proteins - chemistry; Recombinant Proteins - metabolism; Solubility; Structure-Activity Relationship; Transforming Growth Factor beta - chemistry; Transforming Growth Factor beta - metabolism
• ISSN: 0006-2960; 1520-4995
• DOI: 10.1021/bi901528w

Betaglycan is a coreceptor for members of the transforming growth factor β (TGF-β) superfamily. Mutagenesis has identified two ligand binding regions, one at the membrane-distal and the other at the membrane-proximal half of the betaglycan ectodomain. Here we show that partial plasmin digestion of soluble betaglycan produces two proteolysis-resistant fragments of 45 and 55 kDa, consistent with the predicted secondary structure, which indicates an intervening nonstructured linker region separating the highly structured N- and C-terminal domains. Amino terminal sequencing indicates that the 45 and 55 kDa fragments correspond, respectively, to the membrane-distal and -proximal regions. Plasmin treatment of membrane betaglycan results in the production of equivalent proteolysis-resistant fragments. The 45 and 55 kDa fragments, as well as their recombinant soluble counterparts, Sol Δ10 and Sol Δ11, bind TGF-β, but nonetheless, compared to intact soluble betaglycan, have a severely diminished ability to block TGF-β activity. Surface plasmon resonance (SPR) analysis indicates that soluble betaglycan has K d's in the low nanomolar range for the three TGF-β isoforms, while those for Sol Δ10 and Sol Δ11 are 1−2 orders of magnitude higher. SPR analysis further shows that the K d's of Sol Δ11 are not changed in the presence of Sol Δ10, indicating that the high affinity of soluble betaglycan is a consequence of tethering the domains together. Overall, these results suggest that betaglycan ectodomain exhibits a bilobular structure in which each lobule folds independently and binds TGF-β through distinct nonoverlapping interfaces and that linker modification may be an approach to improve soluble betaglycan’s TGF-β neutralizing activity.