Abstract
Twisted gastrulation (Tsg) and chordin are secreted glycoproteins that function together as BMP (bone morphogenetic protein) antagonists to regulate BMP growth factor signalling. Chordin binds to BMPs, preventing them from interacting with their receptors and Tsg is known to strengthen this inhibitory complex. Tsg also acts as a BMP agonist by promoting cleavage of chordin by tolloid-family proteinases. Here we explore the structural mechanism through which Tsg exerts this dual activity. We have characterized the nanoscale structure of human Tsg using in-solution biomolecular analysis and show that Tsg is a globular monomer with a flattened cross shape. Tsg has a high proportion of N-linked glycans, in relation to its molecular weight, which supports a role in solubilising BMPs. Tsg binds with high affinity to the C-terminal region of chordin and was also able to inhibit BMP-7 signalling directly but did not have an effect on BMP-4 signalling. Although both Tsg and mammalian tolloid are involved in chordin cleavage, no interaction could be detected between them using surface plasmon resonance. Together these data suggest that Tsg functions as a BMP-agonist by inducing conformational change in chordin making it more susceptible to tolloid cleavage and as a BMP-antagonist either independently or via a chordin-mediated mechanism. Following single cleavage of chordin by tolloids, Tsg continues to strengthen the inhibitory complex, supporting a role for partially cleaved chordin in BMP regulation.
Highlights
Bone morphogenetic proteins (BMPs) are secreted growth factors of the transforming growth factor-β family
The multi-angle light scattering (MALS) profile (Fig. 1C) showed a single species that had a molecular mass of 32.3 kDa ±0.13%, which is larger than the predicted mass of a monomer (~23.8 kDa)
Using small angle X-ray scattering we show the nanoscale structure of Twisted gastrulation (Tsg), an elongated globular-like molecule and is not, as previously postulated a dimer [14,15] but rather a stable monomer in solution
Summary
Bone morphogenetic proteins (BMPs) are secreted growth factors of the transforming growth factor-β family They are essential for early embryonic patterning, most notably in forming the morphogen gradient controlling dorsal ventral patterning [1]. Chordin antagonizes signalling by binding to BMPs and preventing them from interacting with their cell surface receptors This regulation is modulated by the small secreted glycoprotein twisted gastrulation (Tsg) which can form a ternary complex with chordin and BMP [6,7,8]. We show that Tsg interacts with partially cleaved chordin to increase BMP inhibition and promote further cleavage by tolloids These findings support the physiological relevance of partially cleaved chordin as an important BMP regulator [19]. Our results point to a monomeric Tsg interacting directly with chordin or BMP and suggest that Tsg has a type-specific regulation of BMPs enabling finely controlled regulation of BMP signalling in conjunction with other regulators
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