In the present work we have functionally characterized these ligand binding regions. Similar to the wild type receptor, both regions bind TGF-␤2 with higher affinity than TGF-␤1. However, only the endoglin-related region increases the TGF-␤2 labeling of the TGF-␤ type II receptor, the so-called "TGF-␤ -presentation" function of the wild type receptor. Despite this preference, both regions as well as the wild type receptor mediate the TGF-␤2-dependent Smad2 phosphorylation, indicating that they can function indistinguishably as TGF-␤-enhancing coreceptors. On the other hand, we found that the recently described ability of the wild type betaglycan to bind inhibin A is a property of the core protein that resides in the uromodulin-related region. Binding competition experiments indicate that this region binds inhibin and TGF-␤ with the following relative affinities: TGF-␤2 > inhibin A > TGF-␤1. All together, the present results suggest that betaglycan ectodomain is endowed with two bona fide independent ligand binding domains that can perform specialized functions as co-receptors of distinct members of the TGF-␤ superfamily. Transforming growth factor-␤ (TGF-␤)1 is the prototype of a superfamily of growth factors involved in the regulation of cell proliferation, differentiation, and development (1, 2). TGF-␤ signals through a complex of transmembrane serine/threonine kinase receptors, the TGF-␤ type I and type II receptors. Ligand binding promotes the association between the type I and II receptors. In this complex, phosphorylation of the type I receptor kinase by the constitutively active type II receptor kinase results in its activation. Active type I receptor phosphorylates members of a novel family of transcriptional regulators, the Smads, which transduce the TGF-␤ signal into the cell nucleus (3, 4).TGF-␤ has two known co-receptors, betaglycan and endoglin, which are transmembrane glycoproteins with large extracellular regions that bind TGF-␤ and small cytoplasmatic regions without any clearly identifiable signaling motif (5-8). Betaglycan is a membrane proteoglycan containing heparan and chondroitin sulfate chains whose core protein binds all three TGF-␤ isoforms (9 -11). Betaglycan is capable of fine tuning the availability of TGF-␤ to the signaling receptors, thereby determining the outcome of the TGF-␤ stimulation (12, 13). This regulation is both positive and negative. Although the membrane-bound form of betaglycan increases the binding of TGF-␤ to the signaling complex, the soluble form of betaglycan prevents this binding and therefore blocks the actions of TGF-␤ (14). These effects are more dramatic for TGF-␤2, the isoform for which betaglycan has higher affinity (15-17). Expression of membrane betaglycan in cells that normally do not express this co-receptor increases their binding to TGF-␤2 and corrects for their low sensitivity to this TGF-␤ isoform (18,19). Presumably, this effect is mediated by a TGF-␤-induced "presentation complex" formed between membrane-bound betaglycan and the TGF-␤ type II rece...

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.

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...

An important step of innate immune response is the recruitment of polymorphonuclear leukocytes (PMN) to injured tissues through chemotactic molecules. Galectins, a family of endogenous lectins, participate in numerous functions such as lymphoid cell migration, homing, cell-cell and cell-matrix interactions. Particularly, galectin-3 (Gal-3) and -9 have been implicated in the modulation of acute and chronic inflammation by inducing the directional migration of monocytes/macrophages and eosinophils, whereas Gal-1 is considered to function as an anti-inflammatory molecule, capable of inhibiting the influx of PMN to the site of injury. In this study, we assessed the effect of Gal-1 on neutrophil recruitment, in the absence of additional inflammatory insults. Contrasting with its capacity to inhibit cell trafficking and modulate the release of mediators described in models of acute inflammation and autoimmunity, we evidenced that Gal-1 has the capacity to induce neutrophil migration both in vitro and in vivo. This effect is not mediated through a G-protein-coupled receptor but potentially through the sialoglycoprotein CD43, via carbohydrate binding and through the p38 mitogen-activated protein kinase pathway. These results suggest a novel biological function for CD43 on neutrophils and highlight that depending on the environment, Gal-1 can act either as chemoattractant or, as a molecule that negatively regulates migration under acute inflammatory conditions, underscoring the potential of Gal-1 as a target for innovative drug development.

Betaglycan is an accessory receptor of members of the transforming growth factor-beta (TGF-beta) 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-beta. Because of its potential use as an anti-TGF-beta therapeutic agent, we have purified and characterized baculoviral recombinant soluble betaglycan. Baculoviral soluble betaglycan is a homodimer formed by two 110 kDa 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 20 kDa of the mass of the monomer. End-terminal sequence analysis of the soluble betaglycan showed that Gly(24) is the first residue of the mature protein. Similarly to the natural soluble betaglycan, baculoviral soluble betaglycan has an equilibrium dissociation constant (K(d)) of 3.5 nM for TGF-beta1. Ligand competition assays indicate that the relative affinities of recombinant soluble betaglycan for the TGF-beta isoforms are TGF-beta2>TGF-beta3>TGF-beta1. The anti-TGF-beta potency of recombinant soluble betaglycan in vitro is 10-fold higher for TGF-beta2 than for TGF-beta1. Compared with a commercial pan-specific anti-TGF-beta neutralizing antibody, recombinant soluble betaglycan is more potent against TGF-beta2 and similar against TGF-beta1. 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-beta plays a central physiopathological role.