title: Inactivation of N-terminal signaling domain of Sonic hedgehog by forming a disulfide bond.
abstract: The N-terminal domain of mouse Sonic hedgehog (Shh-N) expressed in mammalian cells showed four-fold bands on non-reduced SDS-PAGE, though it was homogeneous under reduced conditions. It contains three cysteine residues, Cys-25, Cys-103, and Cys-184, which may be concerned with this heterogeneity. Therefore, we examined the formation of a disulfide bond in the recombinant Shh-N and identified three kinds of disulfides with a combination of peptide mapping and NH(2)-terminal amino acid sequencing analysis. Among them, one type of the Shh-N containing a disulfide bond of Cys-103/Cys-184 could be separated from the other Shh-Ns using reverse phase HPLC and had no activity of alkaline phosphatase induction in C3H10T1/2 cells. This molecule could also be made by denaturation of the purified Shh-N with guanidine-HCl under non-reduced 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 suggested that Shh-N was synthesized as an active form whose three cysteine residues did not form disulfide and inactivated finally by forming a disulfide bond between Cys-103 and Cys-184.
authors.affiliation: Discovery Research Laboratories, Research and Development Division, Hoechst Marion Roussel Ltd., 1-3-2, Minamidai, Kawagoe, Saitama, Japan. ksaeki@lsi.kurume-u.ac.jp
authors.forename: K
authors.initials: K
authors.lastname: Saeki
authors.forename: M
authors.initials: M
authors.lastname: Katsuura
authors.forename: S
authors.initials: S
authors.lastname: Yanagisawa
authors.forename: R
authors.initials: R
authors.lastname: Suzuki
authors.forename: M
authors.initials: M
authors.lastname: Okazaki
authors.forename: M
authors.initials: M
authors.lastname: Kimura
mesh_terms: D000818:Animals; D004220:Disulfides; D053823:Hedgehog Proteins; D051379:Mice; D011487:Protein Conformation; D011506:Proteins / Q000737:chemistry* / Q000378:metabolism; D011994:Recombinant Proteins / Q000737:chemistry / Q000378:metabolism; D015534:Trans-Activators
chemical_list: D004220:Disulfides; D053823:Hedgehog Proteins; D011506:Proteins; D011994:Recombinant Proteins; D015534:Trans-Activators