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• W2030943228 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. 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. The hedgehog proteins are a family of extracellular signaling proteins that regulate various aspects of embryonic development both in vertebrates and in invertebrates (for reviews, see Refs. 1Perrimon N. Cell. 1995; 80: 517-520Abstract Full Text PDF PubMed Scopus (144) Google Scholar and 2Johnson R.L. Tabin C. Cell. 1995; 81: 313-316Abstract Full Text PDF PubMed Scopus (112) Google Scholar). The most extensively characterized mammalian homolog is Sonic hedgehog (Shh), 1The 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. which is involved in diverse embryonic induction events, including the induction of floor plate and establishment of ventral polarity within the central nervous system as well as proper anterior-posterior patterning of the developing limb (3Riddle R.D. Johnson R.L. Laufer E. Tabin C. Cell. 1993; 75: 1401-1416Abstract Full Text PDF PubMed Scopus (1967) Google Scholar, 4Echelard Y. Epstein D.J. St-Jacques B. Shen L. Mohler J. McMahon J.A. McMahon A.P. Cell. 1993; 75: 1417-1430Abstract Full Text PDF PubMed Scopus (1813) Google Scholar, 5Roelink H. Augsberger A. Heemskerk J. Korzh V. Norlin S. Ruiz I. Altaba A. Tanabe Y. Placzek M. Edlund T. Jessell T.M. Dodd J. Cell. 1994; 76: 761-775Abstract Full Text PDF PubMed Scopus (756) Google Scholar, 6Roelink H. Porter J.A. Chiang C. Tanabe Y. Chang D.T. Beachy P.A. Jessell T.M. Cell. 1995; 81: 445-455Abstract Full Text PDF PubMed Scopus (773) Google Scholar). In mediating these effects, Shh is believed to act both as a short range, contact-dependent inducer and as a long range, diffusible morphogen. Shh is expressed in the embryonic notochord, and induces floor plate formation at the ventral midline of the neural tube in a contact-dependent manner (3Riddle R.D. Johnson R.L. Laufer E. Tabin C. Cell. 1993; 75: 1401-1416Abstract Full Text PDF PubMed Scopus (1967) Google Scholar, 5Roelink H. Augsberger A. Heemskerk J. Korzh V. Norlin S. Ruiz I. Altaba A. Tanabe Y. Placzek M. Edlund T. Jessell T.M. Dodd J. Cell. 1994; 76: 761-775Abstract Full Text PDF PubMed Scopus (756) Google Scholar, 6Roelink H. Porter J.A. Chiang C. Tanabe Y. Chang D.T. Beachy P.A. Jessell T.M. Cell. 1995; 81: 445-455Abstract Full Text PDF PubMed Scopus (773) Google Scholar). Data suggest Shh can also act as a long range, diffusible morphogen, to promote subsequent differentiation of ventral neurons in a region-specific manner; e.g. dopaminergic neurons in the midbrain (7Wang M.Z. Jin P. Bumcrot D.A. Marigo V. McMahon A.P. Wang E. Woolf T. Pang K. Nature Med. 1995; 1: 1184-1188Crossref PubMed Scopus (147) Google Scholar) and motor neurons in the spinal cord (6Roelink H. Porter J.A. Chiang C. Tanabe Y. Chang D.T. Beachy P.A. Jessell T.M. Cell. 1995; 81: 445-455Abstract Full Text PDF PubMed Scopus (773) Google Scholar). It is presently unclear whether the same molecular species of Shh mediates both of these effects. While the mechanism of action of hedgehog proteins is not fully understood, biochemical and genetic data suggest that the hedgehog receptor is the product of the tumor suppressor genepatched (8Marigo V. Davey R.A. Zuo Y. Cunningham J.M. Tabin C.J. Nature. 1996; 384: 176-179Crossref PubMed Scopus (719) Google Scholar, 9Stone D.M. Hynes M. Armanini M. Swanson T.A. Gu Q. Johnson R.L. Scott M.P. Pennica D. Goddard A. Phillips H. Noll M. Hooper J.E. de Sauvage F. Rosenthal A. Nature. 1996; 384: 129-134Crossref PubMed Scopus (971) Google Scholar, 10Motoyama J. Takabatake T. Takeshima K. Hui C-C. Nat. Genet. 1998; 18: 104-106Crossref PubMed Scopus (175) Google Scholar), and that other proteins, includingsmoothened (9Stone D.M. Hynes M. Armanini M. Swanson T.A. Gu Q. Johnson R.L. Scott M.P. Pennica D. Goddard A. Phillips H. Noll M. Hooper J.E. de Sauvage F. Rosenthal A. Nature. 1996; 384: 129-134Crossref PubMed Scopus (971) Google Scholar, 11Alcedo J. Ayzenzon M. von Ohlen T. Noll M. Hooper J.E. Cell. 1996; 86: 221-232Abstract Full Text Full Text PDF PubMed Scopus (489) Google Scholar), Cubitus interruptus or its mammalian counterpart gli (12Dominguez M. Brunner M. Hafen E. Basler K. Science. 1996; 272: 1621-1625Crossref PubMed Scopus (275) Google Scholar, 13Alexandre C. Jacinto A. Ingham P.W. Genes & Dev. 1996; 10: 2003-2013Crossref PubMed Scopus (342) Google Scholar), and fused(14Therond P.P. Knight J.D. Kornberg T.B. Bishop J.M. Proc. Natl. Acad. Sci. U. S. A. 1996; 93: 4224-4228Crossref PubMed Scopus (102) Google Scholar), are involved in the hedgehog signaling pathway. Shh is synthesized as a 45-kDa precursor protein that is cleaved autocatalytically to yield a 20-kDa NH2-terminal fragment that is responsible for all known hedgehog biological activity (residues 24–197 in the human gene sequence) and a 25-kDa COOH-terminal fragment that contains the autoprocessing machinery (15Lee J.J. Ekker S.C. von Kessler D.P. Porter J.A. Sun B.I. Beachy P.A. Science. 1994; 266: 1528-1536Crossref PubMed Scopus (453) Google Scholar, 16Bumcrot D.A. Takada R. McMahon A.P. Mol. Cell Biol. 1995; 15: 2294-2303Crossref PubMed Scopus (260) Google Scholar, 17Porter J.A. von Kessler D.P. Ekker S.C. Young K.E. Lee J.J. Moses K. Beachy P.A. Nature. 1995; 374: 363-366Crossref PubMed Scopus (439) Google Scholar). The NH2-terminal fragment remains membrane-associated through the addition of a lipid tether at its COOH terminus (18Porter J.A. Young K.E. Beachy P.A. Science. 1996; 274: 255-258Crossref PubMed Scopus (1114) Google Scholar, 19Porter J.A. Ekker S.C. Park W-J. von Kessler D.P. Young K.E. Chen C-H. Ma Y. Woods A.S. Cotter R.J. Koonin E.V. Beachy P.A. Cell. 1996; 86: 21-34Abstract Full Text Full Text PDF PubMed Scopus (434) Google Scholar). This tether is critical for restricting the tissue localization of the hedgehog signal. Recent biochemical data have identified the lipid tether as a cholesterol (18Porter J.A. Young K.E. Beachy P.A. Science. 1996; 274: 255-258Crossref PubMed Scopus (1114) Google Scholar), the addition of which is catalyzed by the COOH-terminal domain during the autoprocessing step. Here we expressed full-length human Shh in a variety of systems and in characterizing the proposed NH2-terminal fragment, determined that cholesterol alone could not account for the increased mass of the membrane-tethered form of the protein. We identified a second lipid modification, a palmitoyl group, attached at its NH2 terminus. Along with the cholesterol, the palmitic acid modification is likely to have evolved as part of the mechanism for regulating short range-long range signaling by hedgehog. The cDNA for full-length human Shh was provided as a 1.6-kilobase EcoRI fragment subcloned into pBluescript SK+ (20Marigo V. Roberts D.J. Lee S.M.K. Tsukurov O. Levi T. Gastier J.M. Epstein D.J. Gilbert D.J. Copeland N.G. Seidman C.E. Jenkins N.A. Seidman J.G. McMahon A.P. Tabin C. Genomics. 1995; 28: 44-51Crossref PubMed Scopus (172) Google Scholar) (a gift of David Bumcrot from Ontogeny). 5′ and 3′ NotI sites immediately flanking the Shh open reading frame were added by unique site elimination mutagenesis using a Pharmacia kit following the manufacturer's recommended protocol. The 1.4-kilobase NotI fragment carrying the full-length Shh cDNA was then subcloned into the insect expression vector, pFastBac (Life Technologies, Inc.). Recombinant baculovirus was generated by using the procedures supplied by Life Technologies, Inc. The resulting virus was used to create a high-titer virus stock. Methods used for production and purification of Shh are described below. The presence of membrane-associated Shh was examined by FACS and Western blot analysis. Peak expression occurred 48 h post-infection. For Western blot analysis, supernatants and cell lysates from Shh-infected or uninfected cells were subjected to SDS-PAGE on a 10–20% gradient gel under reducing conditions, transferred electrophoretically to Immobilon-P (Millipore), and the Shh detected with a rabbit polyclonal antiserum raised against an NH2-terminal Shh 15-mer peptide-keyhole limpet hemocyanin conjugate. The cell lysates were made by incubating the cells for 5 min at 25 °C in 20 mm Na2HPO4, pH 6.5, 1% Nonidet P-40, 150 mm NaCl or in 20 mmTris-HCl, pH 8.0, 50 mm NaCl, 0.5% Nonidet P-40, 0.5% sodium deoxycholate, and then pelleting particulates at 13,000 rpm for 10 min at 4 °C in an Eppendorf centrifuge. For expression of full-length Shh in mammalian cells, the 1.4-kilobaseNotI fragment containing full-length Shh was cloned into a derivative of the pCEP4 (Invitrogen) vector, CH269 (21Sanicola M. Hession C. Worley D. Walus L. Carmillo P. Ehrenfels C. Robinson S. Jarworski G. Wei H. Tizard R. Whitty A. Pepinsky R.B. Cate R. Proc. Natl. Acad. Sci. U. S. A. 1997; 94: 6238-6243Crossref PubMed Scopus (278) Google Scholar). The construct was transfected into EBNA-293 cells using LipofectAMINE (Life Technologies, Inc.) and the cells were harvested 48 h post-transfection. The expression of surface Shh was verified by FACS and Western blot analysis. A chimeric gene product in which the NH2-terminal fragment (residues 24–197) of human Shh had been fused to the hinge and CH2CH3 regions of human IgG1 was expressed in Chinese hamster ovary cells. Other constructs encoding soluble human Shh (residues 24–197 without the lipid modification), a mutant version of soluble human Shh containing a Cys-24 to Ser point mutation (C24S), and soluble human Indian hedgehog (Ihh, residues 28–202 in the human gene sequence) were expressed in Escherichia coli as His-tagged fusion proteins with an enterokinase cleavage site immediately adjacent to the start of the mature sequence. All of the constructs were confirmed by DNA sequencing. The fidelity of the final products after removal of the His tag with enterokinase was confirmed by NH2-terminal sequencing and electrospray ionization-mass spectrometry (ESI-MS). The cDNA encoding the NH2-terminal fragment of rat Sonic hedgehog (residues 25–198 in the rat gene sequence) was cloned into the baculovirus expression vector pBluebac III (Invitrogen) and expressed in insect cells essentially as described for the human full-length construct. The NH2-terminal fragment of rat Sonic hedgehog starts at Cys-25 and not at Cys-24 as in the human protein, since the signal sequence of the rat protein contains an additional amino acid residue. The rat sequence (residues 25–198) differs from the human sequence (residues 24–197) by only two residues; Ser-67 and Gly-196 in the human protein are replaced in the rat protein by threonine and aspartic acid, respectively. The membrane-tethered form of Shh was produced in High FiveTMinsect cells (Invitrogen) using the recombinant baculovirus encoding full-length Shh discussed above. High Five cells were grown at 28 °C in Sf-900 II serum-free medium (Life Technologies, Inc.) in a 10-liter bioreactor controlled for oxygen. The cells were infected in late log phase at about 2 × 106 cells/ml with virus at a multiplicity of infection of 3 and harvested 48 h post-infection (cell viability at the time of harvest was >50%). The cells were collected by centrifugation and washed in 10 mmNa2HPO4, pH 6.5, 150 mm NaCl, containing 0.5 mm phenylmethylsulfonyl fluoride. The resulting cell pellet (150 g wet weight) was suspended in 1.2 liters of 10 mm Na2HPO4, pH 6.5, 150 mm NaCl, 0.5 mm phenylmethylsulfonyl fluoride, 5 μm pepstatin A, 10 μg/ml leupeptin, 2 μg/ml E64, and 120 ml of a 10% solution of Triton X-100 was added. After a 30-min incubation on ice, particulates were removed by centrifugation (1500 × g, 10 min). All subsequent steps were performed at 4–6 °C. The pH of the supernatant was adjusted to 5.0 with a stock solution of 0.5 m MES, pH 5.0 (50 mm final), and loaded onto a 150-ml SP-Sepharose FF column (Pharmacia). The column was washed with 300 ml of 5 mmNa2HPO4, pH 5.5, 150 mm NaCl, 0.5 mm phenylmethylsulfonyl fluoride, 0.1% Nonidet P-40, then with 200 ml of 5 mm Na2HPO4, pH 5.5, 300 mm NaCl, 0.1% Nonidet P-40, and bound hedgehog eluted with 5 mm Na2HPO4, pH 5.5, 800 mm NaCl, 0.1% Nonidet P-40. The Shh was next subjected to immunoaffinity chromatography on a mAb 5E1-Sepharose resin that was prepared by conjugating 4 mg of the anti-hedgehog antibody (27Ericson J. Morton S. Kawakami A. Roelink H. Jessell T.M. Cell. 1996; 87: 661-673Abstract Full Text Full Text PDF PubMed Scopus (757) Google Scholar) per ml of CNBr-activated Sepharose 4B resin. The SP-Sepharose elution pool was diluted with 2 volumes of 50 mm HEPES pH 7.5, and batch loaded onto the 5E1 resin (1 h). The resin was collected in a column, washed with 10 column volumes of phosphate-buffered saline, 0.1% hydrogenated Triton X-100 (Calbiochem), and eluted with 25 mm NaH2PO4, pH 3.0, 200 mm NaCl, 0.1% hydrogenated Triton X-100. The elution fractions were neutralized with 0.1 volumes of 1 m HEPES pH 7.5, and analyzed for total protein content from absorbance measurements at 240–340 nm, and for purity by SDS-PAGE. Fractions were stored at −70 °C. Peak fractions from three affinity steps were pooled, diluted with 1.3 volumes of 50 mm HEPES pH 7.5, 0.2% hydrogenated Triton X-100 and again batch loaded onto the 5E1 resin. The resin was collected in a column, washed with 3 column volumes of phosphate-buffered saline, pH 7.2, 1% octylglucoside (U. S. Biochemical Corp.), and eluted with 25 mmNaH2PO4, pH 3.0, 200 mm NaCl, 1% octylglucoside. The elution fractions were neutralized and analyzed as described above, pooled, filtered through a 0.2-micron filter, aliquoted, and stored at −70 °C. For metabolic labeling studies, High Five cells were seeded into T-75 flasks and infected with recombinant baculovirus encoding full-length Shh or with a control virus. 28 h post-infection the culture medium was replaced with 10 ml/flask of fresh medium supplemented with 250 μCi/ml [9,10-3H]palmitic acid (50 Ci/mmol; NEN Life Science Products) and the cells were further incubated for 12 h at 28 °C. The cells were removed from the flasks by scraping, washed with phosphate-buffered saline, and lysed in 1 ml of 20 mmHEPES pH 7.5, 100 mm NaCl, 1% Triton X-100 plus protease inhibitors following the same protocol described above. Half of each lysate was treated for 1 h at 4 °C with 25 μl of 5E1-Sepharose and the other half with a control mAb-Sepharose of the same isotype. The resins were collected, washed three times with lysis buffer containing 0.2% Triton X-100, and treated with electrophoresis sample buffer. The samples were subjected to SDS-PAGE on a 10–20% gradient gel, and visualized by fluorography (3-day exposure). Aliquots of Shh were subjected to reverse phase HPLC on a C4 column (Vydac, catalog number 214TP104, column dimensions 0.46-cm inner diameter by 25 cm) at ambient temperature. Bound components were eluted with a 30-min 0–70% gradient of acetonitrile in 0.1% trifluoroacetic acid at a flow rate of 1.4 ml/min. The column effluent was monitored at 280 nm and 0.5-min fractions were collected. 25-μl aliquots of fractions containing protein were dried in a Speed Vac concentrator (Savant), dissolved in electrophoresis sample buffer, and analyzed by SDS-PAGE. Fractions containing hedgehog were pooled, concentrated 4-fold in a Speed Vac concentrator, and the protein content calculated from the absorbance at 280 nm using a molar extinction coefficient of 26,030 liter mol−1 cm−1 that was calculated from the tyrosine and tryptophan content of Shh. Samples were subjected to ESI-MS on a Micromass Quattro II triple quadrupole mass spectrometer, equipped with an electrospray ion source. A volume of 6 μl (1 pmol/μl) of HPLC-purified hedgehog was infused directly into the ion source at a rate of 10 μl/min using 50% water, 50% acetonitrile, 0.1% formic acid as the solvent in the syringe pump. Scans were acquired throughout the sample infusion. All electrospray mass spectral data were acquired and stored in profile mode and were processed using the Micromass MassLynx data system. Peptides from an endoproteinase Lys-C digest of pyridylethylated Shh were analyzed by reverse phase HPLC on-line with a Micromass Quattro II triple quadrupole mass spectrometer. The digest was separated on a Reliasil C18 column using a Michrom Ultrafast Microprotein Analyzer system at a flow rate of 50 μl/min with a 25-min 5–85% acetonitrile gradient in 0.05% trifluoroacetic acid. Scans were acquired from m/z 400 to 2000 throughout the run and processed as described above. Sequencing of the tethered Shh was performed by post-source decay (PSD) measurement (22Spengler B. Hirsch D. Kaufmann R. Rapid. Commun. Mass Spectrom. 1992; 6: 105-108Crossref PubMed Scopus (287) Google Scholar, 23Spengler B. Hirsch D. Kaufmann R. J. Phys. Chem. 1992; 96: 9678-9684Crossref Scopus (146) Google Scholar) on a Voyager-DETM STR (PerSeptive Biosystems, Framingham, MA) matrix-assisted laser desorption/ionization time-of-flight (MALDI-TOF) mass spectrometer using α-cyano-4-hydroxycinnamic acid as the matrix (24Beavis R.C. Chaudhary T. Chait B.T. Org. Mass. Spectrom. 1992; 27: 156-158Crossref Scopus (438) Google Scholar). 0.5 μl (1 pmol) of HPLC-purified endoproteinase Lys-C peptide was mixed with 0.5 μl of matrix (10 mg/ml in 50% acetonitrile) on the target plate. To cover the entire spectrum of fragment ions, the mirror voltages were decreased from 20 to 1.2 kv in 11 steps. Soluble human Shh was labeled with [3H]palmitic acid in a cell-free system by using a modified version of a published procedure (25Caron J.M. Mol. Biol. Cell. 1997; 8: 621-636Crossref PubMed Scopus (61) Google Scholar). A crude microsomal fraction from rat liver was prepared by subjecting a liver homogenate to sequential centrifugation at 3,000 × g for 10 min, 9,000 × g for 20 min, and 100,000 × gfor 30 min. The 100,000 × g pellet was suspended in 10 mm HEPES pH 7.4, 10% sucrose and again pelleted at 100,000 × g for 20 min. The final pellet (derived from 10 g of liver) was suspended in 3 ml of 20 mmTris-HCl, pH 7.4, 150 mm NaCl, 1 mm EDTA, 10 μg/ml leupeptin, 0.15% Triton X-100, aliquoted, and stored at −70 °C. Reactions containing 3 μg of Shh, 1 μl of rat microsomes, 50 ng/ml coenzyme A (Sigma), 0.3 mm ATP, 20 mm Tris-HCl, pH 7.4, 150 mm NaCl, 1 mm EDTA, 10 μg/ml leupeptin, and 0.5 μCi of [9,10-3H]palmitic acid (50 Ci/mmol; NEN Life Science Products) were performed at room temperature for 1 h. Reactions were stopped with reducing electrophoresis sample buffer, subjected to SDS-PAGE on a 10–20% gradient gel, and visualized by fluorography. Shh was tested for function in a cell-based assay measuring alkaline phosphatase induction in C3H10T1/2 cells (26Kinto N. Iwamoto M. Enomoto-Iwamoto M. Noji S. Ohnuchi H. Yoshioka H. KaKaoka H. Wado Y. Yuhao G. Takahashi H.E. Yoshiki S. Yamaguchi A. FEBS Lett. 1997; 404: 319-323Crossref PubMed Scopus (109) Google Scholar) with a 5-day readout. The assay was performed in a 96-well format. Samples were run in duplicate. For tethered Shh (100 μg/ml), the samples were first diluted 200-fold with normal growth medium then subjected to serial 2-fold dilutions down the plates. Wells were normalized for potential effects of the added octylglucoside by including 0.005% octylglucoside in the culture medium. Blocking studies using the neutralizing mouse monoclonal antibody 5E1 (27Ericson J. Morton S. Kawakami A. Roelink H. Jessell T.M. Cell. 1996; 87: 661-673Abstract Full Text Full Text PDF PubMed Scopus (757) Google Scholar) were performed by mixing Shh with serial dilutions of the antibody for 30 min at ambient temperature in culture medium prior to adding the test samples to the plates. When full-length human Shh was expressed in High Five insect cells, over 95% of the NH2-terminal fragment was in a cell-associated form. The Shh was purified from a detergent lysate of the cells by a combination of SP-Sepharose chromatography and immunoaffinity chromatography on a 5E1-Sepharose column. By SDS-PAGE, the purified protein migrated as a single sharp band with apparent mass of 20 kDa (Fig. 1, lane c). The protein migrated faster by about 0.5 kDa than a soluble version of the protein that had been produced in E. coli (Fig. 1,lanes b-d), consistent with previously published data (19Porter J.A. Ekker S.C. Park W-J. von Kessler D.P. Young K.E. Chen C-H. Ma Y. Woods A.S. Cotter R.J. Koonin E.V. Beachy P.A. Cell. 1996; 86: 21-34Abstract Full Text Full Text PDF PubMed Scopus (434) Google Scholar). Similarly, as described (19Porter J.A. Ekker S.C. Park W-J. von Kessler D.P. Young K.E. Chen C-H. Ma Y. Woods A.S. Cotter R.J. Koonin E.V. Beachy P.A. Cell. 1996; 86: 21-34Abstract Full Text Full Text PDF PubMed Scopus (434) Google Scholar), the soluble and membrane-bound Shh proteins were also readily distinguishable by reverse phase HPLC, where the tethered form eluted later in the acetonitrile gradient. The concentration of acetonitrile needed for elution of the membrane-bound form was 60 versus 45% for the soluble form, indicating a significant increase in the hydrophobicity of the protein. ESI-MS data for the soluble and membrane-bound forms of Shh showed primary species with masses of 19,560 and 20,167 Da, respectively (Fig.2). The measured mass of 19,560 Da matches the predicted mass for Shh starting with Cys-24 and terminating with Gly-197 (calculated mass of 19,560.02 Da). By contrast, the 20,167 Da mass did not match the predicted mass for residues 24–197 plus cholesterol (calculated mass of 19,928.64 Da), nor could the difference in the masses of the tethered and soluble forms, 607 Da, be accounted for by any known modification or by aberrant proteolytic processing. Since Porter et al. (18Porter J.A. Young K.E. Beachy P.A. Science. 1996; 274: 255-258Crossref PubMed Scopus (1114) Google Scholar) had previously demonstrated thatDrosophila hedgehog contained a cholesterol moiety, it was possible that the mass difference in the human Shh was due, at least in part, to cholesterol (calculated average mass for esterified cholesterol is 368.65 Da). Indeed, the presence of a minor component in the mass spectrum of tethered Shh at 19,796 Da (371 Da smaller than the primary ion) supported this notion (Fig. 2 B). Further evidence for cholesterol modification was obtained by treating the tethered Shh with a mild alkali under conditions that can hydrolyze the cholesterol linkage without disrupting peptide bonds (18Porter J.A. Young K.E. Beachy P.A. Science. 1996; 274: 255-258Crossref PubMed Scopus (1114) Google Scholar), and then reanalyzing the reaction products by MS. Base treatment caused a 387-Da shift in the observed mass of the tethered Shh, which is consistent with the loss of cholesterol plus water (see TableI), while the mass of soluble Shh was not affected by the treatment. Together, these observations suggested that the membrane-tethered human Shh contained two modifications with total mass of 607 Da, a cholesterol (368.65 Da), and a second moiety with mass of 238 Da. The similarity in mass between this value and the mass of a palmitoyl group (calculated mass of 238.47 Da) suggested that the protein might be palmitoylated, which was verified through more rigorous biochemical studies (see below). While the mass data clearly support the notion that Shh is cholesterol modified and have extended this observation to human Shh, the loss of water following base treatment is inconsistent with the proposed model in which the cholesterol is attached to the COOH-terminal carboxyl group through an ester linkage (18Porter J.A. Young K.E. Beachy P.A. Science. 1996; 274: 255-258Crossref PubMed Scopus (1114) Google Scholar, 19Porter J.A. Ekker S.C. Park W-J. von Kessler D.P. Young K.E. Chen C-H. Ma Y. Woods A.S. Cotter R.J. Koonin E.V. Beachy P.A. Cell. 1996; 86: 21-34Abstract Full Text Full Text PDF PubMed Scopus (434) Google Scholar). Base hydrolysis of an ester linkage would not result in the loss of water. More extensive studies are needed to address this issue.Table ICharacterization of tethered human Shh by MSProteinMass (Da)CalculatedMeasureda. KOH-treated Shh No tether (−treatment)19,560.0219,560 No tether (+treatment)19,560.0219,561 Tethered (−treatment)20,167.1420,167 Tethered (+treatment)19,798.4919,780b. NH2-terminal endoproteinase Lys-C peptide (MH+)1-aAll mass values presented in the paper for peptides are for their protonated masses. No tether983.49983.50 Tethered1,221.721,221.79Insect cell-derived Shh was treated with 50 mm KOH, 95% methanol for 1 h at ambient temperature and then analyzed by ESI-MS or digested with endoproteinase Lys-C and subjected to LC-MS on the Michromass Quattro II triple quadrupole mass spectrometer. For samples subjected to LC-MS, the proteins were first treated with 4-vinyl pyridine. Calculated mass values were determined using average residue masses in part a, and protonated monoisotopic masses in part b.1-a All mass values presented in the paper for peptides are for their protonated masses. Open table in a new tab Insect cell-derived Shh was treated with 50 mm KOH, 95% methanol for 1 h at ambient temperature and then analyzed by ESI-MS or digested with endoproteinase Lys-C and subjected to LC-MS on the Michromass Quattro II triple quadrupole mass spectrometer. For samples subjected to LC-MS, the proteins were first treated with 4-vinyl pyridine. Calculated mass values were determined using average residue masses in part a, and protonated monoisotopic masses in part b. The distinctive chromatographic properties of the lipid-modified forms of Shh on reverse phase HPLC was used as an assay to quantify the extent of modification (see Fig.3 B). In this assay, the unmodified Shh elutes first (peak 1), followed by cholesterol-modified Shh (peak 2), and finally the Shh containing both the cholesterol and palmitic acid modifications (peak 3). Over 80% of the tethered Shh recovered from High Five cells contained both cholesterol and the palmitoyl moiety. The shoulder on peak 3 was caused by a modified form of the palmitoyl moiety, containing an unsaturated bond, that was identified through peptide mapping and sequence analysis by MALDI PSD measurement (data not shown). The mass of this variant was 2 Da smaller than that of the main peak. Direct evidence for palmitic acid modification was obtained using an in vitro modification reaction in which Shh was treated with [3H]palmitic acid under conditions that promote protein palmitoylation. As shown in Fig. 1 (lane e), Shh is readily labeled with the radioactive tracer. None of the approximately 100 other proteins in the reaction mixture were labeled (see the corresponding Coomassie Blue-stained gel profile inlane j), indicating a high degree of specificity of the palmitoylation reaction. As further evidence for the specificity of the palmitoylation reaction, we tested two Shh variants in which the site of palmitoylation (Cys-24, see below) had been elimin" @default.
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