Identification of a Palmitic Acid-modified Form of Human Sonic hedgehog*
- R. Blake Pepinsky‡§,
- Chenhui Zeng‡,
- Dingyi Wen‡,
- Paul Rayhorn‡,
- Darren P. Baker‡,
- Kevin P. Williams‡,
- Sarah A. Bixler‡,
- Christine M. Ambrose‡,
- Ellen A. Garber‡,
- Konrad Miatkowski‡,
- Frederick R. Taylor‡,
- Elizabeth A. Wang¶ and
- Alphonse Galdes‡
- From ‡Biogen, Inc., Cambridge, Massachusetts 02142 and¶Ontogeny Inc., Cambridge, Massachusetts 02139
Abstract
During hedgehog biosynthesis, autocatalytic processing produces a lipid-modified amino-terminal fragment (residues 24–197 in the human Sonic hedgehog sequence) that is responsible for all known hedgehog signaling activity and that is highly conserved evolutionarily. Published in vitro biochemical studies using Drosophila hedgehog identified the membrane anchor as a cholesterol, and localized the site of attachment to the COOH terminus of the fragment. We have expressed full-length human Sonic hedgehog in insect and in mammalian cells and determined by mass spectrometry that, in addition to cholesterol, the human hedgehog protein is palmitoylated. Peptide mapping and sequencing data indicate that the palmitoyl group is attached to the NH2 terminus of the protein on the α-amino group of Cys-24. Cell-free palmitoylation studies demonstrate that radioactive palmitic acid is readily incorporated into wild type Sonic hedgehog, but not into variant forms lacking the Cys-24 attachment site. The lipid-tethered forms of hedgehog showed about a 30-fold increase in potency over unmodified soluble hedgehog in a cell- based (C3H10T1/2 alkaline phosphatase induction) assay, suggesting that the lipid tether plays an important role in hedgehog function. The observation that an extracellular protein such as Shh is palmitoylated is highly unusual and further adds to the complex nature of this protein.
Footnotes
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↵* The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked “advertisement” in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.
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↵§ To whom correspondence should be addressed: Dept. of Protein Chemistry, Biogen, Inc. Tel.: 617-679-3310; Fax: 617-679-2616.
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↵1 The abbreviations used are: Shh, Sonic hedgehog; FACS, fluorescence-activated cell sorter; PAGE, polyacrylamide gel electrophoresis; MES, 4-morpholineethanesulfonic acid; mAb, monoclonal antibody; HPLC, high performance liquid chromatography; ESI-MS, electrospray ionization-mass spectrometry; MALDI, matrix-assisted laser desorption/ionization; PSD, post-source decay; Ihh, Indian hedgehog.
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↵2 D. P. Baker, C. Zeng, and D. Wen, unpublished observations.
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↵3 E. A. Garber, S. A. Bixter, N. J. Sykes, and C. M. Ambrose, manuscript in preparation.
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↵4 R. B. Pepinsky, C. Zeng, D. Wen, D. P. Baker, K. P. Williams, and F. R. Taylor, unpublished observations.
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- Received December 22, 1997.
- Revision received March 24, 1998.
- The American Society for Biochemistry and Molecular Biology, Inc.
