The Hedgehog (Hh) family of morphogenic proteins exhibits complex posttranslational processing involving cleavage of a precursor molecule, attachment of a C-terminal cholesterol moiety, attachment of an N-terminal palmitoyl group, and proteolysis by disintegrin and metalloprotease (ADAM) proteins. Inhibition of palmitoylation in cells or animals either with palmitoylation-deficient mutants or by deficiency in the palmitoyl transferases compromises Hh activity, yet recombinant Hh that is not lipidated retains activity. Ohlig et al. provide a mechanistic basis for the role of palmitoylation in the activity of the Hh family member Sonic hedgehog (Shh). Shh that had undergone all of the posttranslational processing steps (ShhNp) lost its palmitoylation when released from cells into the culture medium by proteolytic processing. Secreted ShhNp formed large multimers when analyzed by gel filtration. In contrast, recombinant that had never been modified with cholesterol or palmitate (ShhN) was monomeric. Monomeric forms of Shh retained activity in a cell-based assay, whereas medium containing a mutant that could not be palmitoylated (ShhNpC25S) showed substantially reduced activity. Using tagged ShhNpC25S (and wild-type ShhNp tagged in the same location), the authors showed that this palmitoylation-deficient mutant formed multimers and was cleaved at the C terminus, as was wild-type ShhNp, but that the mutant failed to undergo N-terminal cleavage when released from cells. Inspection of the published crystal structure of human ShhN led the authors to suggest a model in which the N-terminal region of one ShhN molecule interferes with the zinc coordination site of an adjacent molecule before proteolytic processing of the N-terminal domain. Because the zinc coordination site contributes to the interaction of Shh with its receptor Patched, the interaction between the N-terminal domain of ShhN and the zinc coordination site may be a mechanism for autoinhibition of ShhN. Immunoprecipitation experiments with the conformation-specific antibody 5E1 that recognizes the zinc coordination site showed that the binding of this antibody to ShhNpC25S was reduced compared to the interaction with wild-type ShhNp. Furthermore, cell-associated ShhN was not effectively recognized by 5E1, whereas ShhNp released into the medium was, suggesting that before release from the membrane, the zinc coordination site is blocked. Thus, the authors proposed that the multimeric palmitoylated and cholesterol-modified ShhN that is associated with cells is maintained in an inactive conformation due to the interaction of the N-terminal domain of the ShhN with the zinc coordination site and that, in the absence of palmitoylation, the released ShhN multimers persist in this autoinhibited conformation. Indeed, truncation of the N terminus by deletion mutation of the ShhNpC25S mutant restored the activity of this palmitoylation-deficient mutant in a cell-based assay.
S. Ohlig, P. Farshi, U. Pickhinke, J. van den Boom, S. Höing, S. Jakuschev, D. Hoffmann, R. Dreier, H. R. Schöler, T. Dierker, C. Bordych, K. Grobe, Sonic hedgehog shedding results in functional activation of the solubilized protein. Dev. Cell 20, 764–774 (2011). [PubMed]