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

Abstract

SummaryBetaglycan is a co-receptor for members of the 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 non-structured 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, nonetheless, compared to intact soluble betaglycan, have 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 of the domains together. Overall, these results, suggest that betaglycan ectodomain exhibits a bi-lobular structure in which each lobule folds independently, binds TGF-β through distinct non-overlapping interfaces, and that linker modification may be an approach to improve soluble betaglycan's TGF-β neutralizing activity.Address correspondence to Jose-Luis Montiel, PhD, Av. Universidad 1001, Colonia Chamilpa, Cuernavaca, Morelos, 62209, México. Phone and fax: +(52) 77 73297000 ext. 3371. jlmontiel@uaem.mx. Supporting InformationThis manuscript presents biochemical evidence that betaglycan ectodomain is composed of two independently folded TGF-β binding domains. Determination of their TGF-β affinity binding constants indicate both of these domains are required for the high affinity TGF-β binding and the complete TGF-β neutralizing activity of the full-length soluble betaglycan. Supporting information can be found at http://pubs.acs.org. NIH Public Access Author ManuscriptBiochemistry. Author manuscript; available in PMC 2010 December 15. NIH-PA Author ManuscriptNIH-PA Author Manuscript NIH-PA Author ManuscriptBetaglycan (BG) is a membrane proteoglycan that serves as a co-receptor for diverse members of the TGF-β superfamily of autocrine and paracrine factors. These factors are involved in diverse biological functions that include, among others, embryonic development, cell differentiation and proliferation, control of the immune response and wound repair (1). BG binds TGF-β superfamily factors with a characteristic selectivity: TGF-β2 > TGF-β1 > inhibin A, and establishes ligand-dependent complexes with several type II receptors (2). Th...

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