In vitro reconstitution of vertebrate Sonic Hedgehog protein cholesterolysis

Extracellular secretion of the oncogenic sonic hedgehog signaling ligand is contingent on its release from a precursor protein through peptide bond cholesterolysis, mediated by the hedgehog C-terminal domain, SHhC. In this work, we describe the in vitro reconstitution of cholesterolysis activity for SHhC domains from vertebrate model organisms, Xenopus laevis (Xla) and Danio rerio (Dre). Cholesterolysis is assayed continuously in multi-well plates by monitoring changes in fluorescence resonance energy transfer (FRET) from an engineered precursor construct, expressed in E. coli and purified in soluble form. Using this FRET assay, we found that Xla and Dre SHhC exhibit high substrate stereospecificity, accepting cholesterol, (KM, 1-2 {micro}M, cholesterolysis t1/2 of [~]11 min) while rejecting the 3-alpha epimer, epi-cholesterol (KM > 100 {micro}M, t1/2 > 10 hr). By screening a 96-member detergent/surfactant library for compatibility with SHhC activity, we identify cationic detergents that inhibit cholesterolysis and find a shared preference for the zwitterionic n-dodecyl-phosphocholine (DPC, Fos-choline-12), which supported the fastest reaction kinetics. Lastly, we report that alanine point mutation at a conserved aspartate residue (D46A) in Xla SHhC and Dre SHhC blocks cholesterolysis; however, activity could be chemically rescued with rationally designed hyper-nucleophilic sterols. Of those sterols, 2-beta carboxy cholestanol was active as a substrate with D46A variants only; the remaining sterols were accepted by both D46A and wild-type SHhC. In summary, we have established the first in vitro kinetic assay to continuously monitor enzymatic activity of wild-type and mutant vertebrate SHhC domains in multi-well plates, a key step toward pharmacological manipulation of Sonic hedgehog protein biosynthesis in vivo.