Recombinant soluble betaglycan is a potent and isoform-selective transforming growth factor-β neutralizing agent

Abstract

Betaglycan is an accessory receptor of members of the transforming growth factor-β (TGF-β) superfamily, which regulates their actions through ligand-dependent interactions with type II receptors. A natural soluble form of betaglycan is found in serum and extracellular matrices. Soluble betaglycan, prepared as a recombinant protein using the baculoviral expression system, inhibits the actions of TGF-β. Because of its potential use as an anti-TGF-β therapeutic agent, we have purified and characterized baculoviral recombinant soluble betaglycan. Baculoviral soluble betaglycan is a homodimer formed by two 110kDa monomers associated by non-covalent interactions. This protein is devoid of glycosaminoglycan chains, although it contains the serine residues, which, in vertebrate cells, are modified by these carbohydrates. On the other hand, mannose-rich carbohydrates account for approximately 20kDa of the mass of the monomer. End-terminal sequence analysis of the soluble betaglycan showed that Gly24 is the first residue of the mature protein. Similarly to the natural soluble betaglycan, baculoviral soluble betaglycan has an equilibrium dissociation constant (Kd) of 3.5nM for TGF-β1. Ligand competition assays indicate that the relative affinities of recombinant soluble betaglycan for the TGF-β isoforms are TGF-β2>TGF-β3>TGF-β1. The anti-TGF-β potency of recombinant soluble betaglycan invitro is 10-fold higher for TGF-β2 than for TGF-β1. Compared with a commercial pan-specific anti-TGF-β neutralizing antibody, recombinant soluble betaglycan is more potent against TGF-β2 and similar against TGF-β1. These results indicate that baculoviral soluble betaglycan has the biochemical and functional properties that would make it a suitable agent for the treatment of the diseases in which excess TGF-β plays a central physiopathological role.