@PhdThesis{duepublico_mods_00045208,
  author = 	{Bachvarova, Velina Dimitrova},
  title = 	{Role of Heparan sulfate in Indian Hedgehog signaling and Glycosaminoglycan composition},
  year = 	{2018},
  month = 	{Mar},
  day = 	{26},
  abstract = 	{Indian hedgehog (Ihh), which belongs to the conserved family of Hedgehog proteins, is one of the key regulators of embryonic bone development. Several lines of evidence indicate that the distribution and activity of Ihh in the cartilage tissue are dependent on the extracellular matrix (ECM) composition and, particularly, on the level and sulfation pattern of Heparan sulfate (HS) (Dierker et al., 2016; Koziel et al., 2004; Ratzka et al., 2008; Yasuda et al., 2010). This work investigates the properties of the HS-Ihh interaction and the role of HS in the multimerization of Ihh. Moreover, the changes in GAG composition of mice with an altered HS structure were analyzed. In the closely related Sonic Hedgehog (Shh), two conserved HS-binding sequences, called Cardin-Weintraub (CW) motifs, which support a direct interaction with HS, have been proposed. (Chang et al., 2011; Farshi et al., 2011; Ohlig et al., 2011; Whalen et al., 2013). The current work demonstrates that Ihh, like Shh, also contains two CW motifs (CW1 and CW2). Both domains are highly conserved, but the CW1 sequence is not identical between Shh and Ihh. Mutation analysis revealed that the binding of Ihh, expressed in HEK-293Ebna cells to HS is mediated by both motifs, which, however, interact to specifically sulfated HS in distinct manners. Moreover, analysis of the Ihh multimerization showed that the CW1 and CW2 domains are essential, but--correlating with their HS affinity--distinctly important for multimer formation. Surprisingly, analysis of the role of the HS modification pattern on the binding affinity of Shh and Ihh showed that loss of N-sulfation leads to an increased affinity of both proteins. 2-Osulfate deficient HS bound Ihh with similar, but Shh with higher affinity than wild type HS. To better understand how the differences between the Shh and Ihh CW1 sequences impact their HS binding, the affinities of the two paralogs to HS were compared. By exchanging the HS-interacting regions, this work demonstrates that these variations result in distinct affinities to HS and multimer size of the paralogous proteins. Although the interaction with HS seems to mediate the Ihh multimerization in HEK-293Ebna cells, no alterations in multimer size were observed for Ihh expressed in primary chondrocyte of mouse mutants with an altered HS structure. This indicates that another cartilage glycosaminoglycan partly compensates for the HS functions. In line with this, analysis of E16.5 cartilage by immunofluorescence staining indicated that in mice, deficient in HS synthesizing (Ext1gt/gt) or modifying enzymes (Ndst1-/-; Hs2st-/- and Sulf1gt/gt;Sulf2gt/gt), the altered HS was compensated by changes in the levels of Chondroitin sulfate (CS), the main sulfated glycosaminoglycans in chondrocytes. This finding was confirmed by disaccharide analysis of HS and CS purified from Ext1gt/gt and Hs2st-/- embryonic skeletons. Moreover, evaluating the chain length of GAGs, produced by primary chondrocytes revealed that the decreased HS levels in Ext1gt/gt mice resulted in more, but shorter CS chains, while Hs2st-/- cells compensated the altered HS sulfation pattern by an increase of cell surface-associated HS and an elongation of CS chains carried by secreted proteoglycans. Taken together, these data indicate that the interactions of Ihh with HS are determined by two motifs of positively charged residues. Each motif likely interacts with specifically sulfated HS, and possibly with CS. In addition, these data demonstrate for the first time that not only reduced levels, but also an altered HS structure leads to an increase in CS levels. This underlines the highly adaptable nature of ECM and strongly points to a functional overlap between HS and CS in regulating signaling molecules during bone development.},
  doi = 	{10.17185/duepublico/45208},
  url = 	{https://duepublico2.uni-due.de/receive/duepublico_mods_00045208},
  url = 	{https://doi.org/10.17185/duepublico/45208},
  file = 	{:https://duepublico2.uni-due.de/servlets/MCRFileNodeServlet/duepublico_derivate_00081251/Diss_Bachvarova.pdf:PDF},
  language = 	{en}
}