Inactivation of N-terminal signaling domain of Sonic hedgehog by forming adisulfide bond

The N-terminal domain of mouse Sonic hedgehog (Shh-N) expressed in mammalia n cells showed four-fold bands on nonreduced SDS-PAGE, though it was homoge neous under reduced conditions. It contains three cysteine residues, Cys-25 , Cys-103, and Cys-184, which may be concerned with this heterogeneity. The refore, we examined the formation of a disulfide bond in the recombinant Sh h-N and identified three kinds of disulfides with a combination of peptide mapping and NH2-terminal amino acid sequencing analysis. Among them, one ty pe of the Shh-N containing a disulfide bond of Cys-103/Cys-184 could be sep arated from the other Shh-Ns using reverse phase HPLC and had no activity o f alkaline phosphatase induction in C3H10T1/2 cells. This molecule could al so be made by denaturation of the purified Shh-N with guanidine-HCl under n onreduced conditions. On the other hand, the reduced Shh-N and the reduced S-methylated Shh-N at cysteine residues showed approximately 10-fold higher activity compared to the originally purified Shh-N. These results suggeste d that Shh-N was synthesized as an active form whose three cysteine residue s did not form disulfide and inactivated finally by forming a disulfide bon d between Cys-103 and Cys-184. (C) 2000 Elsevier Science B.V. All rights re served.