All Hedgehog (Hh) proteins are released from producing cells despite being synthesized as N- and C-terminally lipidated, membrane-tethered molecules. Thus, a cellular mechanism is needed for Hh solubilization. We previously suggested that a disintegrin and metalloprotease (ADAM)-mediated shedding of Sonic hedgehog (ShhNp) from its lipidated N and C termini results in protein solubilization. This finding, however, seemed at odds with the established role of N-terminal palmitoylation for ShhNp signaling activity. We now resolve this paradox by showing that N-palmitoylation of ShhNp N-terminal peptides is required for their proteolytic removal during solubilization. These peptides otherwise block ShhNp zinc coordination sites required for ShhNp binding to its receptor Patched (Ptc), explaining the essential yet indirect role of N-palmitoylation for ShhNp function. We suggest a functional model in which membrane-tethered multimeric ShhNp is at least partially autoinhibited in trans but is processed into fully active, soluble multimers upon palmitoylation-dependent cleavage of inhibitory N-terminal peptides.

The ectodomains of numerous proteins are released from cells by matrix metalloproteases to yield soluble intercellular regulators. A disintegrin and metalloprotease (ADAM) family members have often been found to be the responsible "sheddases," ADAM17/tumor necrosis factor-␣-converting enzyme being its best characterized member. In this work, we show that ShhNp (lipidated and membrane-tethered Sonic hedgehog) is released from Bosc23 cells by metalloprotease-mediated ectodomain shedding, resulting in a soluble and biologically active morphogen. ShhNp shedding is increased by ADAM17 coexpression and cholesterol depletion of cells with methyl-␤-cyclodextrin and is reduced by metalloprotease inhibitors as well as ADAM17 RNA interference. We also show that the amount of shed ShhNp is modulated by extracellular heparan sulfate (HS) and that ShhNp shedding depends on specific HS sulfations. Based on those data, we suggest new roles for metalloproteases, including but not restricted to ADAM17, and for HS-proteoglycans in Hedgehog signaling.The proteins of the Hedgehog (Hh) 3 family are powerful morphogens that control growth and patterning during development. Establishing the molecular mechanisms that generate the Hh gradient is essential for our understanding of how the Hh signal elicits multiple responses in a temporally and spatially specific manner. The Hh spreading mechanism is especially intriguing, because all Hhs are released from the producing cells despite being synthesized as dually lipidated molecules, whereas lipid-modified peptides normally appear firmly tethered to membranes. Both in vertebrates and in Drosophila melanogaster, the Hhs are synthesized as precursor proteins consisting of the N-terminal signaling domain and a C-terminal cholesterol transferase domain. The precursor first undergoes internal cleavage between residues Gly 198 and Cys 199 (of murine Sonic hedgehog (Shh)) linked to the addition of a cholesteryl moiety to Gly 198 of the N-terminal cleavage product (1). This reaction is catalyzed by the C-terminal cholesterol transferase domain through a nucleophilic substitution resembling intein-mediated protein splicing. Inteins, also called protein introns, are parts of protein sequences that are post-translationally excised, their flanking regions (exteins) being spliced together to yield an additional protein product in a self-catalyzed manner. The second lipid adduct that modifies the Hh proteins is palmitic acid, which attaches to the N-terminal cysteine residue exposed after signal peptide cleavage (2), resulting in the formation of the processed (HhNp) protein. The molecular mechanisms through which the lipid-modified HhNp morphogen is able to diffuse long distances are currently under intense debate, and its ability to do so has been linked to oligomer formation or co-transport with lipoprotein particles (3, 4). HhNp gradient formation also depends on the presence of heparan sulfate proteoglycans (HSPGs).Heparan sulfate (HS) is produced by most cell types in vertebrates and invertebrates....

The fly morphogen Hedgehog (Hh) and its mammalian orthologs, Sonic, Indian, and Desert hedgehog, are secreted signaling molecules that mediate tissue patterning during embryogenesis and function in tissue homeostasis and regeneration in the adult. The function of all Hh family members is regulated at the levels of morphogen multimerization on the surface of producing cells, multimer release, multimer diffusion to target cells, and signal reception. These mechanisms are all known to depend on interactions of positively charged Hh amino acids (the Cardin-Weintraub (CW) motif) with negatively charged heparan sulfate (HS) glycosaminoglycan chains. However, a precise mechanistic understanding of these interactions is still lacking. The proteins of the Hh family are powerful morphogens that control growth and patterning of developing embryos. Current models for Hh activity suggest that the morphogen disperses from a localized source and forms a gradient that patterns fields of responsive cells expressing the Hh receptor Patched (Ptc).Genetic evidence suggests that this process critically depends on heparan sulfate proteoglycan (HSPG) 2 expression. Upon secretion to the cell surface, Drosophila Hh forms nanoscale oligomers on the cell surface that co-localize with HSPGs (1). HS binds to the Cardin-Weintraub (CW) motif found on all known Hhs and regulates their function in flies (2, 3) and mice (4). In Drosophila (5) as well as in mammalian cell culture (6, 7), Hhs are always released from producing cells in multimeric form, as demonstrated by gel filtration analysis of the soluble morphogen. Release of the multimeric morphogen (the processed Hh N-terminal signaling domain, designated HhNp) from the cell surface depends on the expression of Dispatched (8) and A disintegrin and metalloprotease (ADAM) family members that mediate ectodomain shedding from transfected Bosc23 cells (9). HS is involved in the formation of the HhNp extracellular gradient, which, in the fly, depends on the expression of the Drosophila Exostosin (Ext) family of proteins, encoded by the genes tout velu (ttv), brother of tout velu, and sister of tout velu and the glycosylphosphatidylinositollinked HSPGs Dally and Dally-like, corresponding to vertebrate glypicans (2, 3, 10). HS expression and Dally-like/glypican expression are also essential for signal reception and modulation on Ptc-expressing receiving cells (10 -14) and participate in HhNp-Ihog interaction (15). However, the essential role of direct morphogen-HSPG interactions in embryonic patterning was recently challenged (16). In that report, transgenic mice made deficient in two ShhNp CW amino acid residues implicated in HS binding (17) lacked an Shh-related phenotype, suggesting that direct morphogen-HS interactions were not essential for normal development. However, in that report as well as in others (16 -18), CW-dependent HS interactions were characterized using a recombinant, non-physiological monomeric morphogen termed ShhN, whereas embryogenesis depends entirely on the activity of morp...

Sonic hedgehog (Shh) signaling plays major roles in embryonic development and has also been associated with the progression of certain cancers. Here, Shh family members act directly as long range morphogens, and their ability to do so has been linked to the formation of freely diffusible multimers from the lipidated, cell-tethered monomer (ShhNp). In this work we demonstrate that the multimeric morphogen secreted from endogenous sources, such as mouse embryos and primary chick chondrocytes, consists of oligomeric substructures that are "undisruptable" by boiling, denaturants, and reducing agents. Undisruptable (UD) morphogen oligomers vary in molecular weight and possess elevated biological activity if compared with recombinant Sonic hedgehog (ShhN). However, ShhN can also undergo UD oligomerization via a heparan sulfate (HS)-dependent mechanism in vitro, and HS isolated from different sources differs in its ability to mediate UD oligomer formation. Moreover, site-directed mutagenesis of conserved ShhN glutamine residues abolishes UD oligomerization, and inhibitors directed against transglutaminase (TG) activity strongly decrease the amount of chondrocyte-secreted UD oligomers. These findings reveal an unsuspected ability of the N-terminal hedgehog (Hh) signaling domain to form biologically active, covalently crosslinked oligomers and a novel HS function in this TG-catalyzed process. We suggest that in hypertrophic chondrocytes, HS-assisted, TG-mediated Hh oligomerization modulates signaling via enhanced protein signaling activity. Hedgehog (Hh)4 family members are involved in tissue patterning and progenitor cell proliferation by activation of distinct target genes in a concentration-dependent manner. In vertebrates, three closely related Hh morphogens (Sonic hedgehog (Shh), Indian hedgehog (Ihh), and Desert hedgehog) have been described, and a single ortholog is expressed in Drosophila melanogaster. Production of active morphogen begins with the cleavage of the signal sequence followed by autocatalytic cleavage of the 45-kDa precursor molecule to yield a 19-kDa N-terminal signaling domain. This domain becomes C-terminal-cholesterol-modified during processing and N-terminal-palmitoylated, resulting in a dual-lipidated molecule tightly bound to the surface of producing cells that constitutes the active morphogen (called ShhNp, if derived from the Shh precursor, or IhhNp, if derived from the Ihh precursor) (1). On the (Drosophila) cell surface, lipidated morphogen monomers are organized into suboptical oligomers that are further recruited to preexisting heparan sulfate proteoglycan (HSPG)-rich membrane subdomains to form large, visible multimeric clusters (2). Morphogen release from the cell surface then depends on the expression of Dispatched (3) and ADAM (a disintegrin and metalloprotease) family members. The latter mediate morphogen shedding in an HSregulated way, as has recently been shown for ShhNp in transfected Bosc23 cells, a HEK 293T-derived cell line (4).In addition to HS-regulated ShhNp shedding, HS is...

Analysis of heparan sulfate synthesized by HEK 293 cells overexpressing murine NDST1 and/or NDST2 demonstrated that the amount of heparan sulfate was increased in NDST2-but not in NDST1-overexpressing cells. Altered transcript expression of genes encoding other biosynthetic enzymes or proteoglycan core proteins could not account for the observed changes. However, the role of NDST2 in regulating the amount of heparan sulfate synthesized was confirmed by analyzing heparan sulfate content in tissues isolated from Ndst2 ؊/؊ mice, which contained reduced levels of the polysaccharide. Detailed disaccharide composition analysis showed no major structural difference between heparan sulfate from control and Ndst2 ؊/؊ tissues, with the exception of heparan sulfate from spleen where the relative amount of trisulfated disaccharides was lowered in the absence of NDST2. In vivo transcript expression levels of the heparan sulfate-polymerizing enzymes Ext1 and Ext2 were also largely unaffected by NDST2 levels, pointing to a mode of regulation other than increased gene transcription. Size estimation of heparan sulfate polysaccharide chains indicated that increased chain lengths in NDST2-overexpressing cells alone could explain the increased heparan sulfate content. A model is discussed where NDST2-specific substrate modification stimulates elongation resulting in increased heparan sulfate chain length.