Synthetic enzyme-substrate tethering obviates the Tolloid-ECM interaction during Drosophila BMP gradient formation

Abstract

Members of the Tolloid family of metalloproteinases liberate BMPs from inhibitory complexes to regulate BMP gradient formation during embryonic dorsal-ventral axis patterning. Here, we determine mechanistically how Tolloid activity is regulated by its non-catalytic CUB domains in the Drosophila embryo. We show that Tolloid, via its N-terminal CUB domains, interacts with Collagen IV, which enhances Tolloid activity towards its substrate Sog, and facilitates Tsg-dependent stimulation of cleavage. In contrast, the two most C-terminal Tld CUB domains mediate Sog interaction to facilitate its processing as, based on our structural data, Tolloid curvature positions bound Sog in proximity to the protease domain. Having ascribed functions to the Tolloid non-catalytic domains, we recapitulate embryonic BMP gradient formation in their absence, by artificially tethering the Tld protease domain to Sog. Our studies highlight how the bipartite function of Tolloid CUB domains, in substrate and ECM interactions, fine-tune protease activity to a particular developmental context.

DOI: http://dx.doi.org/10.7554/eLife.05508.001

eLife digest

The body of an animal is a highly organised structure of tissues and organs that contain cells with specialised roles. To achieve this level of organisation, it is important that the cells in the embryo know their location and receive the correct instructions on how to develop, when to divide or move. Many animals are roughly symmetrical about an imaginary line that runs from their head to their tail; a developing embryo can provide its cells with information about their position along this head-to-tail axis and the axis that runs from its front to its back.

Setting up the front-to-back axis in the embryo involves a family of proteins called the bone morphogenetic proteins (or BMPs). These proteins can bind to other proteins that act as signals to provide instructions to cells. However, many of the BMPs are unable to perform this job because they are trapped by inhibitory molecules that bind to them instead.

Enzymes belonging to the Tolloid family can break down these inhibitors to release the BMPs. Together, the inhibitors and Tolloid enzymes create a gradient of BMP activity across the embryo. The side of the embryo with the highest levels of active BMPs sets the position of the back of the body, while the opposite side—which has the lowest levels of active BMPs—becomes the front. However, it is not clear how Tolloid is controlled to create the BMP gradient.

Different parts of the Tolloid enzyme have different roles; one portion of the enzyme breaks down the inhibitory molecules, and there are also several so-called ‘non-catalytic domains’. Winstanley et al. used a combination of approaches to study how Tolloid is controlled in fruit fly embryos. The experiments show that two non-catalytic domains at one end of Tolloid help the enzyme to bind to the inhibitory molecules. At the other end of the Tolloid enzyme, another non-catalytic domain can bind to a structural protein called Collagen IV. This enhances the ability of the enzyme to break down the inhibitory molecules and release the BMPs.

These findings reveal how Tolloid's non-catalytic domains can fine-tune the activity of this enzyme to create the gradient of BMP activity that is needed to set the front-to-back direction in animal embryos. Future studies will focus on identifying other proteins that bind to the non-catalytic domains of Tolloid in order to further control its activity during development.

DOI: http://dx.doi.org/10.7554/eLife.05508.002