Heparan Sulfate and Transglutaminase Activity Are Required for the Formation of Covalently Cross-linked Hedgehog Oligomers

Abstract

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...

An Emerging Role of Sonic Hedgehog Shedding as a Modulator of Heparan Sulfate Interactions

“…Consistent with previous observations, control ShhN eluted from HS at 0.6 M NaCl and from heparin at 1 M NaCl (30). In contrast, brainderived 5E1-reactive mShhNp (40) did not bind to HS but eluted from heparin at 0 -0.75 M NaCl. In this regard, endogenous mShhNp resembled ShhN ⌬CW , which also binds heparin but not HS (30,35).…”

“…In contrast to proteolysis-induced Wnt inactivation, however, removal of ShhNp N termini activates the oligomerized protein (40). This is supported by restored ShhNp C25S biofunction and 5E1 binding after deletion mutagenesis, as shown in this work.…”

Extracellular distribution of diffusible growth factors controlled by heparan sulfate proteoglycans during mammalian embryogenesis

“…B 369: 20130545 shifted protein. Consistent with these findings, in mammalian cell cultures, the release of soluble and oligomerized Shh from the membrane is considered to be mediated by HS-dependent mechanisms [78][79][80]. These data suggest that HSPGs can influence lipid-linked growth factor signalling-at least in part-by binding to lipoproteins.…”

Dual Roles of the Cardin-Weintraub Motif in Multimeric Sonic Hedgehog

“…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).…”