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

摘要

Betaglycan is a coreceptor formembers of the transforming growth factor β (TGF-β) superfamily.nMutagenesis has identified two ligand binding regions, one at the membrane-distal and the other at thenmembrane-proximal half of the betaglycan ectodomain. Here we show that partial plasmin digestion ofnsoluble betaglycan produces two proteolysis-resistant fragments of 45 and 55 kDa, consistent with thenpredicted secondary structure, which indicates an intervening nonstructured linker region separating thenhighly structured N- and C-terminal domains. Amino terminal sequencing indicates that the 45 and 55 kDanfragments correspond, respectively, to the membrane-distal and -proximal regions. Plasmin treatment ofnmembrane betaglycan results in the production of equivalent proteolysis-resistant fragments. The 45 andn55 kDa fragments, as well as their recombinant soluble counterparts, Sol Δ10 and Sol Δ11, bind TGF-β, butnnonetheless, compared to intact soluble betaglycan, have a severely diminished ability to blocknTGF-β activity. Surface plasmon resonance (SPR) analysis indicates that soluble betaglycan has Kd’s innthe low nanomolar range for the three TGF-β isoforms, while those for Sol Δ10 and Sol Δ11 are 1-2 orders ofnmagnitude higher. SPR analysis further shows that the Kd’s of Sol Δ11 are not changed in the presence of SolnΔ10, indicating that the high affinity of soluble betaglycan is a consequence of tethering the domains together.nOverall, these results suggest that betaglycan ectodomain exhibits a bilobular structure in which each lobulenfolds independently and binds TGF-β through distinct nonoverlapping interfaces and that linker modifica-ntion may be an approach to improve soluble betaglycan’s TGF-β neutralizing activity.