Research: (Patho) biochemistry of heparan sulfate

Superimposition of Shh with two coordinated calcium ions (blue spheres, pdb: 3d1m) and without calcium coordination (pdb: 3m1n). Zinc as part of the receptor binding site is shown as a black sphere. Calcium coordination strongly repositions a flexible loop (shown in red, highlighted in light blue).

The group of Dr. Grobe addresses the important question of how vertebrate and invertebrate cells coordinate developmental behaviours with that of their neighbours. It is well established that one way to achieve this goal is that organizing cells secrete signaling molecules such as the Hedgehog (Hh) morphogens to instruct receiving cells in concentration-dependent manner. In vertebrates, the Hh family member Sonic Hh (Shh) is essential for patterning of the ventral neural tube, for specifying digit identities and for axon guidance. In the adult, Shh pathway activation has been implicated in maintaining the stem/cancer stem cell niche and in the progression of various cancers. Despite these important roles, various aspects of Hh solubilization, its transport and signaling function remain unclear.

 

In the past, we have established that Hhs are solubilized by proteolytic processing of their lipidated N- and C-terminal Hh peptides (called shedding) in vitro (Dierker et al., 2009; Ohlig et al., 2011; Ohlig et al., 2012) and in vivo (Kastl et al., 2018; Schurmann et al., 2018). We also found that Hh shedding depends on accessory glycoproteins called heparan sulfate proteoglycans (HSPGs) (Grobe et al., 2005; Jakobs et al., 2016; Ortmann et al., 2015). HSPGs are extracellular proteins linked to highly anionic heparan sulfate (HS) glycosaminoglycan chains that bind Hhs and factors required for their regulated release (called Scubes)(Jakobs et al., 2014; Jakobs et al., 2016; Jakobs et al., 2017). Still, the hierarchical assembly and molecular structure of such Hh assembly and release platforms as determinants of subsequent Hh spreading and signaling are only poorly understood. Therefore, by using advanced microscopy in conjunction with biochemistry, we characterize the molecular mechanisms that govern dynamic Hh platform assembly as well as Hh platform structure and its molecular composition at the cell surface (Manikowski, Ehring, Schulz, Kupich).

 

In a second line of research, we currently decipher Hh long-range transport in the Drosophila wing disc by site-directed mutagenesis of HS-binding or calcium-coordinating Hh amino acids (Manikowski, Froese, Schulz, Kupich). So far, removal of selected Hh residues was found to vastly increase its signalling range, resulting in striking patterning abnormalities such as mirror-image duplications of anterior wing tissue, while other residues have the opposite role. These findings provide a new mechanism to explain the essential role of HSPGs for accurate, defined and robust Hh signalling during development.

 

 

Dierker, T., Dreier, R., Petersen, A., Bordych, C., and Grobe, K. (2009). Heparan Sulfate-modulated, Metalloprotease-mediated Sonic Hedgehog Release from Producing Cells. J Biol Chem 284, 8013-8022.

Grobe, K., Inatani, M., Pallerla, S.R., Castagnola, J., Yamaguchi, Y., and Esko, J.D. (2005). Cerebral hypoplasia and craniofacial defects in mice lacking heparan sulfate Ndst1 gene function. Development 132, 3777-3786.

Jakobs, P., Exner, S., Schurmann, S., Pickhinke, U., Bandari, S., Ortmann, C., Kupich, S., Schulz, P., Hansen, U., Seidler, D.G., et al. (2014). Scube2 enhances proteolytic Shh processing from the surface of Shh-producing cells. J Cell Sci 127, 1726-1737.

Jakobs, P., Schulz, P., Ortmann, C., Schurmann, S., Exner, S., Rebollido-Rios, R., Dreier, R., Seidler, D.G., and Grobe, K. (2016). Bridging the gap: heparan sulfate and Scube2 assemble Sonic hedgehog release complexes at the surface of producing cells. Sci Rep 6, 26435.

Jakobs, P., Schulz, P., Schurmann, S., Niland, S., Exner, S., Rebollido-Rios, R., Manikowski, D., Hoffmann, D., Seidler, D.G., and Grobe, K. (2017). Calcium coordination controls sonic hedgehog structure and Scube2-cubulin domain regulated release. J Cell Sci 130, 3261-3271.

Kastl, P., Manikowski, D., Steffes, G., Schurmann, S., Bandari, S., Klambt, C., and Grobe, K. (2018). Disrupting Hedgehog Cardin-Weintraub sequence and positioning changes cellular differentiation and compartmentalization in vivo. Development 145.

Ohlig, S., Farshi, P., Pickhinke, U., van den Boom, J., Hoing, S., Jakuschev, S., Hoffmann, D., Dreier, R., Scholer, H.R., Dierker, T., et al. (2011). Sonic hedgehog shedding results in functional activation of the solubilized protein. Dev Cell 20, 764-774.

Ohlig, S., Pickhinke, U., Sirko, S., Bandari, S., Hoffmann, D., Dreier, R., Farshi, P., Gotz, M., and Grobe, K. (2012). An emerging role of sonic hedgehog shedding as a modulator of heparan sulfate interactions. J Biol Chem 287, 43708-43719.

Ortmann, C., Pickhinke, U., Exner, S., Ohlig, S., Lawrence, R., Jboor, H., Dreier, R., and Grobe, K. (2015). Sonic hedgehog processing and release are regulated by glypican heparan sulfate proteoglycans. J Cell Sci 128, 2374-2385.

 

Schurmann, S., Steffes, G., Manikowski, D., Kastl, P., Malkus, U., Bandari, S., Ohlig, S., Ortmann, C., Rebollido-Rios, R., Otto, M., et al. (2018). Proteolytic processing of palmitoylated Hedgehog peptides specifies the 3-4 intervein region of the Drosophila wing. Elife 7.