WO1998030576A1 - Nouveaux polypeptides derives d'une proteine herisson - Google Patents

Nouveaux polypeptides derives d'une proteine herisson Download PDF

Info

Publication number
WO1998030576A1
WO1998030576A1 PCT/US1997/015753 US9715753W WO9830576A1 WO 1998030576 A1 WO1998030576 A1 WO 1998030576A1 US 9715753 W US9715753 W US 9715753W WO 9830576 A1 WO9830576 A1 WO 9830576A1
Authority
WO
WIPO (PCT)
Prior art keywords
hedgehog
polypeptide
protein
amino acid
expression
Prior art date
Application number
PCT/US1997/015753
Other languages
English (en)
Inventor
Philip A. Beachy
Jeffrey A. Porter
Original Assignee
The Johns Hopkins University School Of Medicine
Priority date (The priority date is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the date listed.)
Filing date
Publication date
Priority claimed from US08/729,743 external-priority patent/US6214794B1/en
Application filed by The Johns Hopkins University School Of Medicine filed Critical The Johns Hopkins University School Of Medicine
Priority to JP52494798A priority Critical patent/JP2002503088A/ja
Priority to AU48006/97A priority patent/AU728541B2/en
Priority to EP97910705A priority patent/EP0966478A4/fr
Priority to CA002267106A priority patent/CA2267106A1/fr
Publication of WO1998030576A1 publication Critical patent/WO1998030576A1/fr
Priority to IL129295A priority patent/IL129295A/en

Links

Classifications

    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
    • G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
    • G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
    • G01N33/5005—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving human or animal cells
    • G01N33/5008—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving human or animal cells for testing or evaluating the effect of chemical or biological compounds, e.g. drugs, cosmetics
    • G01N33/5044—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving human or animal cells for testing or evaluating the effect of chemical or biological compounds, e.g. drugs, cosmetics involving specific cell types
    • G01N33/5058—Neurological cells
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K14/00—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
    • C07K14/435—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans
    • C07K14/43504—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans from invertebrates
    • C07K14/43563—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans from invertebrates from insects
    • C07K14/43577—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans from invertebrates from insects from flies
    • C07K14/43581—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans from invertebrates from insects from flies from Drosophila
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K14/00—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
    • C07K14/435—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans
    • C07K14/46—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans from vertebrates
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K14/00—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
    • C07K14/435—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans
    • C07K14/46—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans from vertebrates
    • C07K14/47—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans from vertebrates from mammals
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
    • G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
    • G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
    • G01N33/5005—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving human or animal cells
    • G01N33/5008—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving human or animal cells for testing or evaluating the effect of chemical or biological compounds, e.g. drugs, cosmetics
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
    • G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
    • G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
    • G01N33/5005—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving human or animal cells
    • G01N33/5008—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving human or animal cells for testing or evaluating the effect of chemical or biological compounds, e.g. drugs, cosmetics
    • G01N33/5011—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving human or animal cells for testing or evaluating the effect of chemical or biological compounds, e.g. drugs, cosmetics for testing antineoplastic activity
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
    • G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
    • G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
    • G01N33/5005—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving human or animal cells
    • G01N33/5008—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving human or animal cells for testing or evaluating the effect of chemical or biological compounds, e.g. drugs, cosmetics
    • G01N33/502—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving human or animal cells for testing or evaluating the effect of chemical or biological compounds, e.g. drugs, cosmetics for testing non-proliferative effects
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
    • G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
    • G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
    • G01N33/5005—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving human or animal cells
    • G01N33/5091—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving human or animal cells for testing the pathological state of an organism
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
    • G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
    • G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
    • G01N33/68—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving proteins, peptides or amino acids
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K38/00—Medicinal preparations containing peptides
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K2319/00—Fusion polypeptide
    • C07K2319/20—Fusion polypeptide containing a tag with affinity for a non-protein ligand
    • C07K2319/21—Fusion polypeptide containing a tag with affinity for a non-protein ligand containing a His-tag
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K2319/00—Fusion polypeptide
    • C07K2319/20—Fusion polypeptide containing a tag with affinity for a non-protein ligand
    • C07K2319/23—Fusion polypeptide containing a tag with affinity for a non-protein ligand containing a GST-tag
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K2319/00—Fusion polypeptide
    • C07K2319/50—Fusion polypeptide containing protease site
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N2333/00—Assays involving biological materials from specific organisms or of a specific nature
    • G01N2333/435—Assays involving biological materials from specific organisms or of a specific nature from animals; from humans
    • G01N2333/43504—Assays involving biological materials from specific organisms or of a specific nature from animals; from humans from invertebrates
    • G01N2333/43552—Assays involving biological materials from specific organisms or of a specific nature from animals; from humans from invertebrates from insects
    • G01N2333/43569—Assays involving biological materials from specific organisms or of a specific nature from animals; from humans from invertebrates from insects from flies
    • G01N2333/43573—Assays involving biological materials from specific organisms or of a specific nature from animals; from humans from invertebrates from insects from flies from Drosophila
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N2333/00—Assays involving biological materials from specific organisms or of a specific nature
    • G01N2333/435—Assays involving biological materials from specific organisms or of a specific nature from animals; from humans
    • G01N2333/46—Assays involving biological materials from specific organisms or of a specific nature from animals; from humans from vertebrates
    • G01N2333/4603—Assays involving biological materials from specific organisms or of a specific nature from animals; from humans from vertebrates from fish
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N2333/00—Assays involving biological materials from specific organisms or of a specific nature
    • G01N2333/435—Assays involving biological materials from specific organisms or of a specific nature from animals; from humans
    • G01N2333/46—Assays involving biological materials from specific organisms or of a specific nature from animals; from humans from vertebrates
    • G01N2333/4606—Assays involving biological materials from specific organisms or of a specific nature from animals; from humans from vertebrates from amphibians
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N2333/00—Assays involving biological materials from specific organisms or of a specific nature
    • G01N2333/435—Assays involving biological materials from specific organisms or of a specific nature from animals; from humans
    • G01N2333/46—Assays involving biological materials from specific organisms or of a specific nature from animals; from humans from vertebrates
    • G01N2333/465—Assays involving biological materials from specific organisms or of a specific nature from animals; from humans from vertebrates from birds
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N2333/00—Assays involving biological materials from specific organisms or of a specific nature
    • G01N2333/435—Assays involving biological materials from specific organisms or of a specific nature from animals; from humans
    • G01N2333/46—Assays involving biological materials from specific organisms or of a specific nature from animals; from humans from vertebrates
    • G01N2333/47—Assays involving proteins of known structure or function as defined in the subgroups
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N2500/00—Screening for compounds of potential therapeutic value
    • G01N2500/10—Screening for compounds of potential therapeutic value involving cells

Definitions

  • This invention relates generally to the field of protein processing and protein signalling pathways and specifically to two novel proteins having distinct activities, which are derived from a common hedgehog protein precursor.
  • Embryologists have long performed experimental manipulations that reveal the striking abilities of certain structures in vertebrate embryos to impose pattern upon surrounding tissues. Speculation on the mechanisms underlying these patterning effects usually centers on the secretion of signaling molecule that elicits an appropriate response from the tissues begin patterned. More recent work aimed at the identification of such signaling molecules implicates secreted proteins encoded by individual members of a small number of gene families. One such family of proteins which may have an influential effect upon patterning activities are those proteins encoded by the hedgehog gene family.
  • the hedgehog (hh) gene was initially identified based on its requirement for normal segmental patterning in Drosophila (N ⁇ sslein-Volhard, C. & Wieschaus, E. Nature 287:795-801, 1980). Its functions include local signaling to coordinate the identities of adjacent cells within early embryonic segments (Hooper, J.E., & Scott, M.P. Early Embryonic Development of Animals, pp.1-48, 1992) and a later function in cuticle patterning that extends across many cell diameters (Heernskerk, J. & DiNardo, S., Cell, 76:449-460, 1994).
  • the hh gene also functions in the patterning of imaginal precursors of adult structures, including the appendages and the eye (Mohler, J. Genetics, 120: 1061- 1072, 1988; Ma, et al, Cell, 75:927-938, 1993; Heberlein, et al, Cell, 75:913-926, 1993; Tabata, T. & Kornberg, T.D., Cell, 76:89-102, 1992; Basler. K. & Struhl, G.. Nature, 368:208-214, 1994).
  • the vertebrate ventral midbrain contains neurons whose degeneration or abnormal function are linked to a number of diseases, including Parkinson's disease and schizophrenia. It is known that motor neurons develop in close proximity to the floor plate in the ventral midbrain. Midbrain projections to the striatum are involved in the control of voluntary movement (Bjorklund and Lindvall, In: Handbook of Chemical Neuroanatomy, eds., Borklund, et al., Amsterdam: Elsevier, pp55-122, 1984) and loss of these neurons results in the motor disorders of Parkinson's disease (Hirsch, et al, Nature, 334:345, 1988). Midbrain dopaminergic neurons that innervate limbic structures and the cortex influence emotional and cognitive behavior, respectively, and abnormal function of these neurons has been associated with schizophrenia and drug addiction (Seeman, et al, Nature, 365:441, 1993).
  • TGF- ⁇ transforming growth factor- ⁇
  • hedgehog protein family Smith, J.C., Cell, 76:193, 1994
  • the invention shows that internal cleavage of hedgehog protein product is critical for full function, and that the two novel products of this auto-proteolytic cleavage display distinguishable activities, thus demonstrating that hh signaling activity is a composite effect of two separate signaling proteins that derive from a common hh protein precursor.
  • the invention provides the means for specific patterning and proliferation of desired neuronal cell types for addressing disorders which arise from neuronal degeneration or abnormal function.
  • the present invention is based on the seminal discovery that hedgehog proteins undergo auto-proteolytic cleavage which results in two separate proteins having distinct functional and structural characteristics.
  • the two polypeptides referred to as the "N" and “C” fragments of hedgehog, or N-terminal and C-terminal fragments, respectively, are produced after specific cleavage at a G'CF site recognized by the autoproteolytic domain in the native protein.
  • the "C” fragment functions as a cholesterol transferase during autoproteolysis thus allowing cholesterol modification of the "N" fragment.
  • the invention provides a substantially pure polypeptide characterized by having an amino acid sequence derived from amino terminal amino acids of a hedgehog protein and having at its carboxy terminus, a G I CF cleavage site specifically recognized by a proteolytic activity of the carboxy terminal fragment of the native hedgehog polypeptide.
  • the invention also provides a substantially pure polypeptide characterized by having an amino acid sequence of a hedgehog polypeptide or a fragment derived from amino terminal amino acids of a hedgehog polypeptide, wherein the polypeptide or fragment thereof comprises a sterol moiety. Fragments derived from a native hedgehog polypeptide are included and preferably include extracellular amino acid residues, such as those derived from the N fragment.
  • the sterol moiety is cholesterol.
  • the invention provides a substantially pure polypeptide characterized by having an amino acid sequence derived from carboxy terminal amino acids of a hedgehog protein and having at its amino terminus, a G I CF cleavage site specifically recognized by a proteolytic activity of the carboxy terminal fragment of the native hedgehog polypeptide.
  • the invention also provides a method for modulating proliferation or differentiation of neuronal cells, comprising contacting the cells with a hedgehog polypeptide.
  • the native hedgehog polypeptide, the N, or the C fragment, or functional fragments derived therefrom, are most useful for the induction of proliferation or differentiation of neuronal cells substantially derived from floor plate neuronal cells.
  • the invention provides a method for identifying a compound which affects hedgehog activity comprising incubating the compound with hedgehog polypeptide, or with biologically active fragments thereof, or with a recombinant cell expressing hedgehog, under conditions sufficient to allow the components to interact; and determining the effect of the compound on hedgehog activity or expression.
  • cholesterol level e.g., biosynthesis or transport
  • the method provides a means for affecting cholesterol biosynthesis or transport in a cell comprising contacting a cell with an effective amount of a compound that affects hedgehog, thereby affecting cholesterol biosynthesis or transport. The effect may be inhibition or stimulation of cholesterol biosynthesis or transport.
  • FIGURE 1 shows processing of the hh protein by immunoblots (A,C) with antibodies against amino (AM) and carboxy-terminal (Ab2) epitopes.
  • FIGURE IB and D are blots of samples immunoprecipitated with Abl (B, lanes 7-9), Ab2 (D, lanes 19-21), or pre- immune serum (B, lanes 10-12, and D, lanes 22-24).
  • FIGURES IE and IF show a schematic illustration of the hedgehog cleavage mechanism.
  • FIGURE 2 shows sequence similarity between hh proteins and serine proteases, hh protein sequences are aligned to residues 323 to 329 of the D. melanogaster protein and numbered as positions 1 to 7 (group A).
  • the catalytic histidines of mammalian serine proteinases (group B) are aligned to the invariant histidine at position 7 in hh proteins.
  • FIGURE 3 shows autoproteolysis of the hh protein.
  • 3 A shows a coomasie blue stained polyacrylamide gel showing production and purification of His 6 -U and His -l ⁇ 329A proteins from E. coli.
  • Samples were molecular weight markers (lanes 1 and 2); lysates of E. coli cells carrying the His 6 -U expression construct without (lane 3) and with (lane 4) induction by IPTG; purified His 6 -U protein (lane 5); lysates of E. coli cells that carry the His 6 -U H329A expression construct without (lane 6) and with (lane 7) induction by IPTG; purified His 6 -U H329A protein (lane 8).
  • FIGURE 3(B) is an immunoblot detected with Ab2 showing transfected S2 cells induced to express hh (lane 1); His 6 -U and His 6 -U H329A proteins incubated in cleavage reaction buffer for 0 hours (lanes 2 and 5), for 20 hours (lanes 3 and 6), and for 20 hours in the presence of 20 mM TAME (a serine protease inhibitor) (lanes 4 and 7).
  • TAME a serine protease inhibitor
  • FIGURE 4 shows autoproteolytic functions ⁇ Drosophila (4A-C) and zebrafish (D) hh proteins map to the carboxy terminal fragments by in vitro translations of wild-type and mutant hh proteins. The locations of mutations and cleavage sites (arrows) in these proteins are illustrated schematically in 4E.
  • FIGURE 5 shows immunoblots showing heat shock induced expression of wild type and H329A mutant hh proteins in Drosophilia embryos (A) and (B) are immunoblots developed using Abl and Ab2 antibodies, respectively.
  • Lanes 1 and 6 induced untransfected S2 cells; lanes 2 and 7, transfected S2 cells induced to express hh; lanes 3 and 8, heat shocked wild-type embryos; lanes 4 and 9, heat shocked hshh embryos; lanes 5 and 10, heat shocked hshh H329A embryos.
  • FIGURE 6 shows in situ hybridization showing the embryonic effects of ubiquitously expressed wild type and H329A hh proteins.
  • FIGURE 6 shows the embryonic distribution of wingless (wg) RNA as revealed by in situ hybridization is shown in (A) wild-type (homozygous 1 w 1 " 8 ), (B) hshh, and (C) hshh H329A embryos that were exposed to two 10 minute heat shocks separated by a 90-minute recovery period (33). Wild-type embryos showed little change in wg expression, whereas the wild-type protein and, to a lesser extent, the H329A protein each induced ectopic wg expression (Table 1).
  • Panels (D), (E), and (F) show the dorsal surfaces of v' w 1 " 8 , hshh, and hshh H329A larvae, respectively, at the level of the fourth abdominal segment. These larvae were shocked for 30 minutes as embryos and allowed to complete embryogenesis. Cuticle cell types (1 °, 2°, 3 °, and 4°) are labeled as described (J. Heemskerk and S. DiNardo, Cell 76, 449, 1994).
  • FIGURE 7 shows X-gal staining to show imaginal disc effects of ubiquitous wild type and H329 hh proteins.
  • X-gal staining was used to follow expression of wg (A-C) or dpp (D-O) in imaginal discs of late third-instar larvae that carry wg-lacZ or dpp-lacZ reporter genes.
  • Leg (A-F), wing (G-I) and eye-antennal discs (J-L) from control larvae (A, D, G, J), larvae carrying the hshh transgene (B, E, H, K) and larvae carrying the hshh H329A transgene (C, F, I, L) are displayed. In all panels anterior is to the left.
  • FIGURE 8 are immunoblots of cell pellets (lane 1 ) or supernatants (lane 2) from transfected S2 cell cultures expressing HH protein, developed with Ab 1 (A) and Ab2 (B). Samples in each lane were from the same volume of resuspended total culture. Whereas N remained mostly associated with the cell pellet (compare lanes 1 and 2 in A), C was nearly quantitatively released into the supernatant (compare lanes 1 and 2 in B). U displayed partitioning properties in between those of N and C (A and B). (C) demonstrates the heparin binding activity of various HH protein species generated by in vitro translations with microsomes (38).
  • Samples were: total translation mix (lane 1); supernatant after incubation with heparin agarose or agarose (control) beads (lanes 2 and 4); and material eluted from heparin agarose or agarose beads after washing (lanes 3 and 5).
  • F, U, N ss and N fragments are depleted from reactions incubated with heparin agarose but not agarose beads (compare lanes 2 and 4 to 1), and the same species subsequently can be eluted from the heparin agarose but not the agarose beads (compare lanes 3 and 5 with lane 1).
  • FIGURE 9 shows the differential localizations of N and C in embryos by in situ localization of the hh transcript.
  • Fig. 9 (A) is shown in comparison to the distribution of N and C epitopes detected with Abl and Ab2 in panels (B) and (C), respectively. Note that the distribution of N and C epitopes span approximately one-third and one-half of each segmental unit respectively, while the transcript is limited to approximately one-quarter of each unit.
  • D the localization of C epitopes in embryos homozygous for the z 2 13E allele is detected with the use of Ab2.
  • C epitopes in this mutant which displays impaired auto-proteolytic activity (see text), are more restricted, and resemble the wild-type localization of N. Homozygous hh UE embryos were identified by loss of a marked balancer from a heterozygous parent stock. All embryos are at mid to late stage 9 (extended germ-band).
  • FIGURE 10 shows a signal relay versus dual function models for hh protein action.
  • the long-range effects of hh signaling are achieved indirectly through short- range induction of a second signaling molecule (X).
  • X second signaling molecule
  • N is presumed to represent the active short-range signal while the role of C would be limited to supplying the catalytic machinery required for biogenesis of N.
  • the long- and short-range signaling functions of hh are supplied by the N and C proteins derived by internal auto-proteolysis of the U precursor.
  • N is implicated in short-range signaling by retention near its cellular site of synthesis, while C is less restricted in its distribution and would execute long-range signaling functions. In both models, auto-proteolysis is required to generate fully active signaling proteins.
  • FIGURES 10 C and D show an immunoblot of the N fragment synthesized from a wild type construct (C) or a consruct lacking the C domain (D).
  • FIGURES 11 A and B show the nucleotide and deduced amino acid sequences for partial human hh clones.
  • FIGURE 12 A and B show in vitro cleavage reactions of a Drosophila hh protein produced in E. coli and purified to homogeneity.
  • FIGURE 12 Panel A shows a time course of cleavage after initiation by addition of DTT.
  • Panel B shows incubations of concentrations ranging over three order of magnitude for a fixed time period (four hours), with no difference in the extent of conversion to the cleaved form.
  • Panel C shows the sequence around the cleavage site as determined by amino-terminal sequence of the cleaved fragment C. The cleavage site is denoted by the arrow, and the actual residues sequenced by Edman degradation of the C fragment are underlined.
  • Panel C also shows an alignment of all published vertebrate hh sequences plus some of unpublished sequences from fish and Xenopus.
  • the sequences shown correspond to the region of Drosophila hh where the cleavage occurs, and demonstrates the absolute conservation of the Gly-Cys-Phe sequence at the site of cleavage.
  • Panel D shows a SDS-PAGE gel loaded with in vitro transcription/translation reactions as described in the previous Examples, using various hh genes as templates, dhh is Drosophila, twhh and zfshh are the twiggy-winkle and sonic hh genes of the zebrafish, and mshh is the shl ⁇ /Hgh-l/vhh-1 gene of the mouse.
  • Panel E shows that Edman degradation of the C fragments releases 35 S counts on the first but not subsequent rounds for all these proteins, indicating that the site of autoproteolytic cleavage for all of these hh proteins is the amide bond to the amino-terminal side of the Cys residue that forms the center of the conserved Gly-Cys- Phe sequence highlighted in panel C.
  • FIGURE 13 shows the predicted amino acid sequences are shown in single letter code.
  • 13(a) shows sequences common to five distinct hh-h e genes are shown with a dot indicating identity with the corresponding residue of zebrafish twiggy-winkle.
  • 13(b) shows amino acid sequences of twhh and shh are aligned to those of the soniclvhh-l class from chick and mouse. The amino-terminal hydrophobic stretch common to all four hh genes is shaded.
  • the asterisk (*) denotes invariant amino acid residues associated with the proteolytic domain of C fragment from various species.
  • 13(c) shows percent identity of residues carboxy-terminal to the hydrophobic region.
  • FIGURES 14A-S show a comparative expression of twhh, shh, and pax-2 during zebrafish embryogenesis.
  • FIGURE 15, panels 15A-15I show the effects of ectopic hh on zebrafish development.
  • Wild type zebrafish, Danio rerio, Ekkwill Waterlife Resources were maintained at 28.5°C, some embryos were then cultured overnight at RT.
  • Zebrafish embryos were injected at the 1-8 cell stage with twhh, shh, or / ⁇ cZRNA and examined at 28 h of development, (a-c) Dorsal view of the midbrain-hindbrain region; anterior is left, (a) lacZ. (b) twhh. (c) shh. (d-f) Frontal optical section of the forebrain region; anterior is up. (d) lacZ. (h) twhh. (f) shh. (g-1) Lateral view of the eye region; anterior is left, (g) lacZ. (h) twhh. (i) twhh.
  • FIGURE 16 is a table showing the effects of ectopic expression of shh, twhh and twhh mutants on zebrafish embryonic development.
  • FIGURE 17 shows zebrafish twiggy-winkle hedgehog derivatives.
  • FIGURE 17A shows cartoons of various twhh open reading frames.
  • SS (shaded) is the predicted N-terminal signal sequence for secretion of these proteins and encompasses the first 27 amino acids of each open reading frame.
  • the arrow indicates the predicted internal site of auto- proteolytic cleavage. Amino acid residue numbers are according to Figure 13b.
  • the filled triangle denotes the normal termination codon for the twhh open reading frame.
  • Construct U HA contains a mutation that blocks auto-proteolysis (the histidine at residue 273 is changed to an alanine; see Lee, J.J., et al, supra.).
  • Construct U356 H ⁇ contains a stop codon in place of amino acid residue 357 as well as the H273A mutation in U HA .
  • Construct N encodes just the first 200 amino acids of twhh.
  • Construct C has had the codons for residues 31-197 deleted.
  • FIGURE 17B shows in vitro translation of the expression constructs shown schematically in part a. Constructs were translated in vitro in the presence of 35 S methionine and analyzed by autoradiography after SDS-PAGE.
  • FIGURES 18A and 18B show Northern blot analysis of the effect of hedgehog on expression of various neural markers.
  • FIGURES 19A and 19B show hh synergy with naturally occurring neural markers or agents (e.g., XAG-1, XANF-2, Otx-A, En-2, Krox-20, Xlh box-6, NCAM, and EF-l ⁇ ).
  • naturally occurring neural markers or agents e.g., XAG-1, XANF-2, Otx-A, En-2, Krox-20, Xlh box-6, NCAM, and EF-l ⁇ .
  • FIGURE 20 A shows hh constructs including delta N-C.
  • FIGURE 20B shows a Northern blot analysis of the effect of hedgehog N or C on various neural markers.
  • FIGURE 21 shows ⁇ N-C interferes with X-bhh and N-activity in animal cap explants as shown by RT-PCR analysis.
  • FIGURE 22A is an illustration of lipid stimulation of hedgehog autoprocessing.
  • FIGURE 22B shows a Coomassie blue stained SDS-PAGE of autocleavage reactions in bacterially expressed His 6 Hh-C protein.
  • FIGURE 23A is a thin layer chromatography (TLC) plate coated with silica gel G (Merck) showing the fractionation of bulk S2 cell lipids using a heptane:ether:formic acid solvent (80:20:2).
  • TLC thin layer chromatography
  • FIGURE 23B is a Coomassie blue-stained SDS-polyacrylamide gel showing in vitro autocleavage reactions of the bacterially expressed His6Hh-C protein incubated with 1 mMDTT plus either unfractionated S2 cell lipids (lane 1 ), or spots A through F (lanes 2- 7, respectively).
  • FIGURE 23 C is TLC of S2 cell lipids (lane 1) along with selected lipid standards: phosphatidylcholine (lane 2), a diacylglycerol (lane 3), cholesterol (lane 4), stearic acid (lane 5), a triacylglycerol (lane 6), and cholesteryl ester (lane 7).
  • Lipid spot B comigrates with cholesterol, as also demonstrated by mixing radio-labeled cholesterol with S2 lipids before TLC fractionation.
  • FIGURE 23D is a Coomassie blue stained SDS-polyacrylamide gel showing that relative to 1 mMDTT alone (lane 1) cholesterol (0.35 mM) + 1 mMDTT (lane 2) stimulates His2Hh-C autocleavage in vitro.
  • FIGURE 23E is an autoradiogram of electrophoretically-resolved products of His6Hh-C autocleavage reactions driven by 20 mMDTT (lane 1) or 1 mMDTT+0.35 mM cholesterol (lane 2).
  • FIGURE 24A shows Coomassie stained gels of His6Hh-C autocleavage reactions carried out in the presence of 20 mMDTT (lane 1), or 1 mMDTT+0.35 mM cholesterol (lane 2).
  • Lane 3 contains a mixture of the samples loaded in lanes 1 and 2.
  • FIGURE 24B is Coomassie stained gels showing protein products of His6Hh-C a- utocleavage reactions carried out in the presence of 1 mM DTT+0.35 mM cholesterol (lanes 1 and 2) or with 20 mM DTT (lane 3).
  • FIGURE 24C is an autoradiogram of immunoblotted Hh amino-terminal domains purified from cultured S2 cells.
  • FIGURE 25 A is an audioradiogram of a gel loaded with total cell proteins from S2 cells containing a stably integrated Cu++-inducible hedgehog gene.
  • FIGURE 25B is an HPLC profile of sterols separated on a C18 column by isocratic elution with a solvent containing methanol:ethanol:water (86:10:4).
  • FIGURE 25C shows HPLC analysis as in (B) of the adduct released by base treatment of Hh-Np metabolically labeled with [3 H] cholesterol (A).
  • FIGURE 25D shows metabolic labeling of vertebrate Sonic hedgehog protein with [3H]cholesterol.
  • FIGURE 26A is a schematic drawing of a two-step mechanism for Hh autoprocessing. Aided by deprotonation by either solvent or a base (Bl), the thiol group of Cys-258 initiates a nucleophilic attack on the carbonyl carbon of the preceding residue, Gly-257. This attack results in replacement of the peptide bond between Gly-257 and Cys-258 by a thioester linkage (step 1). The emerging ⁇ -amino group of Cys-258 likely becomes protonated, and an acid (A) is shown donating a proton.
  • the Thioester is subject to a second nucleophilic attack from the 3 ⁇ -hydroxyl group of a cholesterol molecule, shown here facilitated by a second base (B2), resulting in a cholesterol-modified amino-terminal domain and a free carboxy-terminal domain.
  • B2 second base
  • In vitro cleavage reactions may also be stimulated by addition of small nucleophiles including DTT, glutathione, and hydroxyl- amine.
  • Figure 26B is a schematic drawing of a mechanism for intein self-splicing.
  • a base (B 1 ') or solvent deprotonates a cysteine or serine residue at the N-extein/intein junction (shown here as a cysteine residue) for attack on the carbonyl group of the preceding amino-acid residue resulting in the formation of a thioester/ester intermediate.
  • An acid (A') may protonate the ⁇ -amino group of the cysteine/serine residue promoting its release.
  • the thioester/ester is then subject to a second nucleophilic attack from a cysteine, serine, or threonine residue at the intein/C-extein junction (shown here as a cysteine residue).
  • a second base (B2') is shown facilitating deprotonation of the second nucleophile, although this function may also be carried out by Bl'. This reaction produces a branched protein intermediate that ultimately resolves to a free intein and ligated exteins.
  • Figure 27 is a Coomassie Brilliant Blue-stained SDS-polyacrylamide gel showing in vitro autocleavage reactions of bacterially-expressed His 6 Hh-C 25 (lanes 1-3) and Hi% Hh-C, 7 (lanes 4-6) proteins. Proteins were incubated with 1 mM DTT (lanes 1 and 4), 50 mM DTT (lanes 2 and 5) or 350 ⁇ M cholesterol/1 mM DTT (lanes 3 and 6). The uncleaved His 6 Hh-C 25 protein migrates as a ⁇ 29-kDa species, and the carboxy-terminal cleavage product of this protein migrates as a ⁇ 25-kDa species (Porter et al., 1996).
  • the uncleaved His 6 Hh-C ⁇ protein migrates as a -21 -kDa species, and the carboxy-terminal product of this truncated protein migrates as a ⁇ 14-kDa species.
  • the amino-terminal product of the His 6 Hh-C 25 and His 6 Hh-C 17 proteins migrates as a ⁇ 7-kDa species when DTT-modified or as a ⁇ 5-kDa species when cholesterol-modified.
  • His 6 Hh-C 17 was also incubated with 46 ⁇ M [ 3 H] cholesterol/ 1 mM DTT, and no cholesterol-modified product was detected by autoradiography. A cholesterol-transfer activity 1% of wildtype could have been detected by this radioassay.
  • Figure 28A is a ribbon diagram of Hh-C l7 .
  • the amino- (N) and carboxy- (C) termini are labeled.
  • This panel was prepared with MOLSCRIPT (Kraulis, 1991).
  • Figure 28B is a topology diagram of Hh-C 17 .
  • Residues in ⁇ strands are in boxes with amino-acid type and number indicated.
  • Residues in turns of 3 I0 helix are ovals with amino-acid type and number indicated.
  • Other residues in the structure are in boxes with amino-acid number indicated.
  • Hydrogen bonds between ⁇ strands are indicated with arrows.
  • a pseudo two-fold axis of symmetry is indicated with a diamond. This panel was prepared using the output of the program PROMOTIF.
  • FIGURE 29A is a pseudo two-fold symmetry in Hh-C 17 .
  • a stereodiagram of a trace of the ⁇ -carbon backbone of residues 258-393 of Hh-C17 viewed along the pseudo-twofold symmetry axis is shown. Equivalent loops are colored identically. Residues 258-276 and 324-347 are colored yellow, residues 276-301 and 347-373 are colored magenta, and residues 312-320 and 381-389 are colored cyan.
  • the pseudo two-fold axis is indicated with a closed circle.
  • FIGURE 29B is a stereodiagram of a backbone trace of Hh-C17 is shown with residues 258-323 colored green and residues 324-395 colored yellow.
  • the extended loops that make up the Hh-C 17 structure are labeled in the order in which they appear in the amino- acid sequence, A1-A2-A3-B1-B2-B3.
  • Two structurally cohesive subdomains are apparent, one comprising loops Al, A2, and B3 and another comprising loops Bl, B2, and A3.
  • Hh-C 17 appears to have arisen from a tandem duplication of a primordial gene to produce the 'A' and 'B' sequence regions coupled with exchange of the homologous A3 (residues 310-323) and B3 (residues 379-395) loops to form structural subdomains that are hybrids of 'A' and 'B' sequences.
  • a pivot about which exchange 9of these loops appears to have occurred is indicated by an arrow.
  • FIGURE 29C is a stereodiagram of backbone traces of the regions of Hh-C17 corresponding to the sequence duplication (residues 259-320 colored green and residues 325-389 colored yellow) following superposition is shown.
  • FIGURE 29D is a structure-based alignment of the amino-acid sequences of the two subdomains of Hh-C 17 . conserveed amino acids are highlighted with yellow. Active site residues are in red. ⁇ strands are indicated with arrows, ⁇ lb and ⁇ 2b are slightly longer than ⁇ la and ⁇ 2a, respectively, and are indicated with lighter green coloring.
  • Fractional solvent accessibility (FSA) is shown in blue for each residue in the Hh-C, 7 structure.
  • the FSA is the ratio of the solvent accessible surface area of residue X in a Gly-X-Gly tripeptide vs. in the Hh-C17 structure. A value of 0 represents a value from 0.00 to 0.09, 1 represents 0.10 to 0.19, and so on.
  • Type I ⁇ turns are conserved at homologous positions in both Hh-C I7 subdomains at residues 260-263 (homologous to residues 326- 329) and residues 317-320 (homologous to residues 386-389).
  • a type II ⁇ turn is conserved between both subdomains at residues 279-282 (homologous to residues 350- 353), and a type IV ⁇ turn is conserved between both subdomains at residues 288-291 (homologous to residues 359-362).
  • ⁇ bulges are found at homologous positions in both Hh-C 17 subdomains at residues 282 (homologous to residue 353) and 300 (homologous to 372).
  • FIGURE 30A is a stereodiagram of the nucleophilic residue, Cys-258, and nearby residues. Distances (A) between atoms are indicated.
  • FIGURE 30B is a ribbon diagram of Hh-C 17 with the side chains of Cys-258 and other putative active site residues indicated. Panels A and B were prepared with MOLSCRIPT (Kraulis, 1991, supra).
  • FIGURE 30C shows a Coomassie Brilliant Blue-stained SDS-polyacrylamide gel showing in vitro autocleavage reactions of bacterially-expressed His 6 HhC wildtype (lanes 1-3) and mutant proteins, H329A (lanes 4-6), T326A (lanes 7-9), and D303A (lanes 10-12).
  • Proteins were incubated with 1 mM DTT (lanes 1, 4, 7, and 10), 50 mM DTT (lanes 2, 5, 8, and 11) or 350 ⁇ M cholesterol/1 mM DTT (lanes 3, 6, 9, and 12).
  • the uncleaved protein migrates as a ⁇ 29 kDa species.
  • the carboxy-terminal cleavage product migrates as a ⁇ 25-kDa species and the amino-terminal product migrates as a ⁇ 7- kDa species when DTT-modified or as a ⁇ 5-kDa species when cholesterol-modified.
  • D303A The significant level of apparent cleavage seen with the D303A protein with 1 mM DTT results from preexisting cleavage products in the preparation; however, addition of 50 mM DTT greatly increases the amount of cleavage products and addition of cholesterol does not produce a cholesterol-modified product ( ⁇ 5-kDa species).
  • D303A was also incubated with 46 ⁇ M [ 3 H]cholesterol/l mM DTT, and no cholesterol-modified product was detected by autoradiography (data not shown). A cholesterol-transfer activity 1% of wildtype could have been detected by this radioassay.
  • FIGURE 31 A is an alignment of the Hh-C 17 amino-acid sequence (residues 258-402) with other Hh sequences, with nematode sequences homologous to Hh-C, and intein sequences.
  • the alignment was constructed by superimposing the Hh-C and intein alignments produced by the CLUSTALW program using the results of the PSI-BLAST analysis as a guide (Thompson et al., 1994). Additionally, the alignment was verified by analyzing a subset of the sequences containing fifteen diverse intein sequences and three Hh-C sequences with the MACAW program (Schuler et al., 1991, supra).
  • a second inserted domain in the PI-Scel/YEAST intein thought to be involved in DNA recognition (DRR) is located between ⁇ lb and ⁇ 2b.
  • Three inteins, GYRA/MYCXE, DNAB/PORPU, and KLBA METJA, contain a short insert replacing the endonuclease domain.
  • the yeast HO endonuclease does not undergo self-splicing, but contains a vestigial, inactive intein domain.
  • the KLBA/METJA intein homologue in which the amino-terminal nucleophile is replaced by alanine is likely inactive as well.
  • a consensus sequence is shown above the aligned sequences and shows amino acid residues conserved in at least one half of the sequences in each of the two aligned sets.
  • 'U' indicates a bulky hydrophobic residue (I, L, V, M, F, Y, W), and "-" indicates a negatively-charged residue (Dor E).
  • Catalytic site residues are highlighted with red; hydrophobic residues are highlighted with yellow; other residues that conform with the consensus are highlighted with blue.
  • the secondary structure elements and for Hh-C 17 are shown. Every tenth residue in the Hh-C l7 sequence is indicated with a dot.
  • the leftmost column shows abbreviated protein and species names, and the second column shows the gene identification number in the NCBI protein database.
  • CAEEL Caenorhabditis elegans
  • DANRE Danio rerio
  • XENLA - Xenopus laevis
  • Cynpy - Cynops pyrrhogaster DROHY - Drosophila hydei
  • DROME Drosophila melanogaster
  • CANTR Candida tropicalis
  • MYCLE Mycobacterium leprae
  • MYCXE - Mycobacterium xenopi MYCTU - Mycobacterium tuberculosis
  • PORPU Porphyra purpurea
  • SYNSP Synechocystis sp
  • CHLEU Chlamydomonas
  • METJA Methanococcus jannaschii
  • PYRFU Pyrococcus furiosus
  • PYRSP Pyrococcus sp.
  • THELI Thermococcus lit
  • FIGURE 3 IB is a stereo ribbon diagram of Hh-C 17 , showing where the endonuclease domain and additional DNA recognition region of Pl-Scel are inserted.
  • the loop where the endonuclease domain is inserted is colored red and the loop where the additional DNA recognition region ("the arm of the self-splicing domain" is inserted is colored blue.
  • the orientation of the Hh-C l7 in this view is the same as the orientation of the PI- Scel intein in Figure 2 of Duan, et al., 1997, supra. This panel was prepared with MOLSCRIPT.
  • FIGURE 32 is a schematic drawing illustrating the duplication and insertion events that appear to have occurred during the evolution of Hh proteins and inteins.
  • the insertion of the intein into a host protein is not shown.
  • the order of some of these events is speculative. For example, dimerization through loop swapping may have preceded the gene duplication that produced an Hh-C 17 -like protein.
  • FIGURE 33 shows inhibition of cholesterol biosynthesis by the plant steroidal alkaloid, jervine.
  • the present invention provides two novel polypeptides originally derived from a single precursor protein, both of which have distinct structural and functional characteristics.
  • the proteins are derived from a hedgehog protein and can be naturally produced by auto- proteolytic cleavage of the full-length hedgehog protein. Based on evidence provided herein, which indicates that hedgehog precursor protein and the auto-proteolytic products of hedgehog precursor protein are expressed in the floo ⁇ late of the ventral midline of the neural tube and notochord, the invention now provides a method for the induction of proliferation or differentiation of neuronal cells associated with or in close proximity to the floo ⁇ late and notochord.
  • the invention also provides cholesterol modified hedgehog polypeptides and function fragments thereof.
  • the invention provides a substantially pure polypeptide characterized by having an amino acid sequence derived from amino terminal amino acids of a hedgehog protein and having at its carboxy terminus, a glycine-cysteine- phenylalanine (G 1 CF) cleavage site specifically recognized by a proteolytic activity of the carboxy terminal fragment of the native hedgehog polypeptide.
  • G 1 CF glycine-cysteine- phenylalanine
  • the N fragment includes amino acids 1-257 of hedgehog protein, wherein amino acids 85-257 have a molecular weight of about 19 kD by non- reducing SDS-PAGE (Amino acid residue numbers 1-257 include non-structural features such as signal sequences.).
  • the G 1 CF cleavage site in Drosophila hedgehog precursor protein occurs at amino acid residues 257-259. Those of skill in the art will be able to identify the G1CF cleavage site in other hedgehog genes, as the amino acid location will be similar and the site will be specifically recognized by the autoproteolytic activity of the corresponding C fragment.
  • the N-terminal polypeptide is also characterized by being cell-associated in cells expressing the polypeptide in vitro, and being specifically localized in vertebrate or Drosophila cells or embryos, for example. In other words, this N-terminal fragment of hedgehog, remains close to the site of cellular synthesis.
  • the association of N with the cell is a result of the processing event which involves lipophilic modification of the amino terminal domain. (See Figure IE and Example 19) This modification is initiated by the action of the carboxy terminal domain, generating a thioester intermediate; the carboxy-terminal domain thus does not act simply as a protease, although cleavage of a peptide bond does ultimately result from its action.
  • the lipid modification is a cholesterol moiety.
  • the N fragment binds to heparin agarose in vitro.
  • the N polypeptide of the invention is characterized by having an amino acid sequence derived from amino terminal amino acids of hedgehog protein, e.g., 1-257 in Drosophila, wherein amino acids 1-257 have a molecular weight of about 19 kD by non-reducing SDS-PAGE.
  • the N polypeptide includes smaller fragments which retain the functional characteristics of full length N, e.g., bind to heparin.
  • the hedgehog protein from which N is derived includes, but is not limited to Drosophila, Xenopus, chicken, zebrafish, mouse, and human. Crystallographic analysis shows the structure of SHH-N includes the presence of a zinc ion.
  • Zinc hydrolases include proteases such as carboxypeptidase A and thermolysin, Upases such as phospholipase C, and other enzymes such as carbonic anhydrase.
  • Alterations in the zinc hydrolase site of the amino terminal signaling domain may be useful for modulating the range of diffusion of a hedgehog protein or to alter the signaling characteristics of the amino terminal signaling domain.
  • a mutation in the zinc hydrolase site may result in a tethered protein where ordinarily the protein is secreted at a distance. The result would be induction of a cell type not typically induced.
  • Alteration in the zinc site may result in a molecule capable of inducing motor neurons and not floor plate, and vice versa.
  • the identification of a cell-surface, or extracellular matrix localization of N and its expression in notochord and floor plate-associated cells provides a means for isolation or specific selection of cells expressing N, e.g., to isolate a notochord sample or to isolate floor plate cells.
  • antibodies directed to N are useful for histological analysis of tissues suspected of expressing N protein.
  • the invention also provides a substantially pure polypeptide characterized by having an amino acid sequence derived from carboxy terminal amino acids of a hedgehog protein and having at its amino terminus a GICF cleavage site specifically recognized by a proteolytic activity of the carboxy terminal fragment of the native hedgehog polypeptide.
  • This fragment is denoted the C-terminal fragment or polypeptide or "C", herein.
  • this "C" polypeptide derives from the C-terminal domain of hedgehog precursor protein beginning at amino acid residue 258, wherein the full length C-terminal domain has a molecular weight of about 25 kD by non-reducing SDS-PAGE, a histidine residue at position 72, and has protease activity.
  • the GICF cleavage site specifically recognized by the proteolytic activity of the carboxy terminal fragment of the native hedgehog polypeptide is located at amino acid residues 257-259.
  • the present invention has shown the precise cleavage recognition site for the autoproteolytic domain of hedgehog, those of skill in the art can readily discern the cleavage site in other hedgehog proteins thereby allowing the ready identification of any N or C polypeptide of any hedgehog precursor protein.
  • the "C” polypeptide of the invention is derived from the C-terminus of a hedgehog precursor protein, beginning at the autoproteolytic cleavage site identified at the GCF amino acid sequence, which in Drosophila corresponds to amino acids 257-259.
  • the histidine residue found invariably at amino acid residue 329 of the native hedgehog protein, and at amino acid residue 72 of the C polypeptide is essential for auto-proteolytic cleavage between amino acids 257 and 258 (G and C).
  • Corresponding C-polypeptides of the invention will likewise contain a similarly located histidine residue which can be readily identified, such as by comparison to the Drosophila C-polypeptide.
  • the proteolytic domain can be characterized by the amino acid sequence -XTXXHLXX-.
  • the C polypeptide of the invention unlike N, does not significantly bind to heparin agarose.
  • C is characterized by being released into the culture supernatant of cells expressing C polypeptide in vitro and by being localized diffusely in cells and embryos. Because C polypeptide diffuses freely, it would be detectable in various body fluids and tissues in a subject. Identification of C polypeptide expression near the midline of the neural tube, as described herein, provides a useful assay for neural tube closure in an embryo/fetus, for example. The presence of C polypeptide in amniotic fluid would be diagnostic of a disorder in which the neural tube may be malformed.
  • Altered levels of C polypeptide in cerebrospinal fluid may be indicative of neuro- degenerative disorders, for example. Because C polypeptide is released from the cell after synthesis and autoproteolysis of native hedgehog precursor polypeptide, tumors synthesizing and releasing high levels of C polypeptide would be detectable without prior knowledge of the exact location of the tumor.
  • C fragment is effective in inducing genes of the pituitary and anterior brain as well.
  • induction is increased by the addition of a member of the TGF- ⁇ family of growth factors.
  • human activin in combination with C fragment may be effective in enhancing pituitary cell growth and activity or development.
  • C fragment possesses cholesterol transferase activity thereby effecting precursor cleavage and transfer of a cholesterol moiety to N fragment, resulting in a biologically active N fragment.
  • the invention includes a polypeptide deleting amino acid residues 28-194 of X-bhh. (Autoproteolysis gives a C domain of 198-409 as well as a seven amino acid peptide, representing aa 24-27 and 195-197). This polypeptide blocks the activity of X-bhh and N in explants and reduces dorsoanterior structures in embryos. Also included are polynucleotide sequences encoding ⁇ N-C.
  • ⁇ N-C is useful for increasing expression of posterior neural markers (e.g., En-2, Krox-20, Xlttbox-6) and decreasing expression of anterior neural markers (e.g., XANF-2, XAG-1, Otx-A) when desirable to do so to modulate neural patterning.
  • posterior neural markers e.g., En-2, Krox-20, Xlttbox-6
  • anterior neural markers e.g., XANF-2, XAG-1, Otx-A
  • substantially pure refers to hedgehog N or C polypeptide which is substantially free of other proteins, lipids, carbohydrates, nucleic acids or other materials with which it is naturally associated.
  • One skilled in the art can purify hedgehog N or C polypeptide using standard techniques for protein purification.
  • the substantially pure polypeptide will yield a single major band on a non-reducing polyacrylamide gel.
  • the purity of the hedgehog N or C polypeptide can also be determined by amino- terminal amino acid sequence analysis.
  • the invention includes a functional N or C polypeptide, and functional fragments thereof.
  • the term "functional polypeptide” or “functional fragment” refers to a polypeptide which possesses a biological function or activity which is identified through a defined functional assay and which is associated with a particular biologic, mo ⁇ hologic, or phenotypic alteration in the cell.
  • Functional fragments of the hedgehog N or C polypeptide include fragments of N or C polypeptide as long as the activity, e.g., proteolytic activity or cholesterol transferase activity of C polypeptide remains. Smaller peptides containing the biological activity of N or C polypeptide are therefore included in the invention.
  • the biological function can vary from a polypeptide fragment as small as an epitope to which an antibody molecule can bind to a large polypeptide which is capable of participating in the characteristic induction or programming of phenotypic changes within a cell.
  • a "functional polynucleotide” denotes a polynucleotide which encodes a functional polypeptide as described herein.
  • Biologically active or functional fragments of hedgehog are included in the invention and can be identified as such by functional assays.
  • fragments of hedgehog are identified as inducing differentiation of neuronal cells; regulating differentiationof chondrocytes; able to complement a loss of function mutation of hedgehog, for example in a transgenic Drosophila; binding to Patched (Ptc); or having cholesterol transferase activity (e.g., C fragment).
  • Fragments of the invention may be from about 30 to 450 amino acids in length; from about 50 to 300 amino acids in length; from about 75 to 250 amino acids in length; or from about 100 to 200 amino acids in length, as long as a biological activity of hedgehog is retained therein.
  • N or C polypeptide primary amino acid sequence may result in polypeptides which have substantially equivalent activity as compared to the N or C polypeptide described herein. Such modifications may be deliberate, as by site-directed mutagenesis, or may be spontaneous. All of the polypeptides produced by these modifications are included herein as long as the proteolytic activity of C polypeptide, for example, is present. Further, deletion of one or more amino acids can also result in a modification of the structure of the resultant molecule without significantly altering its activity. This can lead to the development of a smaller active molecule which would have broader utility. For example, it is possible to remove amino or carboxy terminal amino acids which may not be required for N or C polypeptide activity.
  • the N or C polypeptide of the invention also includes conservative variations of the polypeptide sequence.
  • conservative variation denotes the replacement of an amino acid residue by another biologically similar residue. Examples of conservative variations include the substitution of one hydrophobic residue such as isoleucine, valine, leucine or methionine for another, or the substitution of one polar residue for another, such as the substitution of arginine for lysine, glutamic for aspartic acids, or glutamine for asparagine, and the like.
  • conservative variation also includes the use of a substituted amino acid in place of an unsubstituted parent amino acid provided that antibodies raised to the substituted polypeptide also immunoreact with the unsubstituted polypeptide.
  • the N fragment of the invention includes both the active form of the polypeptide and the N fragment including the uncleaved signal sequence.
  • the signal sequence is internal (at about amino acids 60-80)
  • the entire uncleaved N fragment beginning at the initiating methionine is included in the invention.
  • Those of skill in the art can readily ascertain the nature and location of the signal sequence by using, for example, the algorithm described in von Heijne, G., Nucl. Acids Res. 14:4683, (1986).
  • Hedgehog polypeptides of the invention include polypeptides having at least about 50%- 100% homology with the hedgehog polypeptides provided herein, for example 52%, 64%, 68%, 70%, 75%, 80%, 85%, 90%, 95% and up to 100% homology.
  • homologous polypeptides are derived from vertebrate species, most preferably mammalian species, such as humans.
  • the invention also provides an isolated polynucleotide sequence encoding a polypeptide having the amino acid sequence of N or C polypeptide of the invention.
  • isolated includes polynucleotides substantially free of other nucleic acids, proteins, lipids, carbohydrates or other materials with which it is naturally associated.
  • Polynucleotide sequences of the invention include DNA, cDNA and RNA sequences which encode N or C polypeptide. It is understood that all polynucleotides encoding all or a portion of N or C polypeptide are also included herein, as long as they encode a polypeptide with N or C polypeptide activity.
  • polynucleotides include naturally occurring, synthetic, and intentionally manipulated polynucleotides.
  • N or C polypeptide polynucleotide may be subjected to site-directed mutagenesis.
  • the polynucleotide sequence for N or C polypeptide also includes antisense sequences.
  • the polynucleotides of the invention include sequences that are degenerate as a result of the genetic code. There are 20 natural amino acids, most of which are specified by more than one codon. Therefore, all degenerate nucleotide sequences are included in the invention as long as the amino acid sequence of N or C polypeptide polypeptide encoded by the nucleotide sequence is functionally unchanged.
  • the invention also includes a polynucleotide consisting essentially of a polynucleotide sequence encoding a polypeptide having an amino acid sequence of N or C and having at least one epitope for an antibody immunoreactive with N or C polypeptide.
  • the polynucleotide encoding N or C polypeptide includes the entire polypeptide or fragments thereof, as well as nucleic acid sequences complementary to that sequence.
  • a complementary sequence may include an antisense nucleotide.
  • the sequence is RNA
  • the deoxynucleotides A, G, C, and T are replaced by ribonucleotides A, G, C, and U, respectively.
  • fragments of the above-described nucleic acid sequences that are at least 15 bases in length, which is sufficient to permit the fragment to selectively hybridize to DNA that encodes the protein under physiologi- cal conditions.
  • Hedgehog encoding polynucleotides of the invention include nucleic acid sequences identified by hybridization to a hedgehog nucleic acid described herein.
  • the conditions used to achieve a particular level of stringency will vary, depending on the nature of the nucleic acids being hybridized. For example, the length, degree of complementarity, nucleotide sequence composition (e.g., GC v. AT content), and nucleic acid type (e.g., RNA v. DNA) of the hybridizing regions of the nucleic acids can be considered in selecting hybridization conditions.
  • An additional consideration is whether one of the nucleic acids is immobilized, for example, on a filter.
  • An example of progressively higher stringency conditions is as follows: 2 x SSC/0.1% SDS at about room temperature (hybridization conditions); 0.2 x SSC/0.1 % SDS at about room temperature (low stringency conditions); 0.2 x SSC/0.1% SDS at about 42°C (moderate stringency conditions); and 0.1 x SSC at about 68 °C (high stringency conditions). Washing can be carried out using only one of these conditions, e.g., high stringency conditions, or each of the conditions can be used, e.g., for 10-15 minutes each, in the order listed above, repeating any or all . of the steps listed. However, as mentioned above, optimal conditions will vary, depending on the particular hybridization reaction involved, and can be determined empirically.
  • DNA sequences of the invention can be obtained by several methods.
  • the DNA can be isolated using hybridization techniques which are well known in the art. These include, but are not limited to: 1) hybridization of genomic or cDNA libraries with probes to detect homologous nucleotide sequences; 2) antibody screening of expression libraries to detect cloned DNA fragments with shared structural features; and 3) PCR amplification of a desired nucleotide sequence using oligonucleotide primers.
  • the hedgehog, N, or C polynucleotide of the invention is derived from a vertebrate organism, and most preferably from human. Screening procedures which rely on nucleic acid hybridization make it possible to isolate any gene sequence from any organism, provided the appropriate probe is available. Oligonucleotide probes, which correspond to a part of the sequence encoding the protein in question, can be synthesized chemically. This requires that short, oligopeptide stretches of amino acid sequence must be known. The DNA sequence encoding the protein can be deduced from the genetic code, however, the degeneracy of the code must be taken into account. It is possible to perform a mixed addition reaction when the sequence is degenerate.
  • hybridization is preferably performed on either single-stranded DNA or denatured double-stranded DNA.
  • Hybridization is particularly useful in the detection of cDNA clones derived from sources where an extremely low amount of mRNA sequences relating to the polypeptide of interest are present.
  • stringent hybridization conditions directed to avoid non-specific binding, it is possible, for example, to allow the autoradiographic visualization of a specific cDNA clone by the hybridization of the target DNA to that single probe in the mixture which is its complete complement (Wallace, et al, Nucl. Acid Res., 9:879, 1981).
  • DNA sequences encoding hedgehog can also be obtained by: 1) isolation of double-stranded DNA sequences from the genomic DNA; 2) chemical manufacture of a DNA sequence to provide the necessary codons for the polypeptide of interest; and 3) in vitro synthesis of a double-stranded DNA sequence by reverse transcription of mRNA isolated from a eukaryotic donor cell. In the latter case, a double- stranded DNA complement of mRNA is eventually formed which is generally referred to as cDNA.
  • genomic DNA isolates are the least common. This is especially true when it is desirable to obtain the microbial expression of mammalian polypeptides due to the presence of introns.
  • DNA sequences are frequently the method of choice when the entire sequence of amino acid residues of the desired polypeptide product is known.
  • the direct synthesis of DNA sequences is not possible and the method of choice is the synthesis of cDNA sequences.
  • the standard procedures for isolating cDNA sequences of interest is the formation of plasmid- or phage-carrying cDNA libraries which are derived from reverse transcription of mRNA which is abundant in donor cells that have a high level of genetic expression. When used in combination with polymerase chain reaction technology, even rare expression products can be cloned.
  • the production of labeled single or double-stranded DNA or RNA probe sequences duplicating a sequence putatively present in the target cDNA may be employed in DNA/DNA hybridization procedures which are carried out on cloned copies of the cDNA which have been denatured into a single-stranded form (Jay, et al, Nucl. Acid Res., ⁇ :2325, 1983).
  • a preferred method for obtaining genomic DNA is Polymerase Chain Reaction (PCR), which relies on an in vitro method of nucleic acid synthesis by which a particular segment of DNA is specifically replicated.
  • PCR Polymerase Chain Reaction
  • Two oligonucleotide primers that flank the DNA fragment to be amplified are utilized in repeated cycles of heat denaturation of the DNA, annealing of the primers to their complementary sequences, and extension of the annealed primers with DNA polymerase. These primers hybridize to opposite strands of the target sequence and are oriented so that DNA synthesis by the polymerase proceeds across the region between the primers. Since the extension products themselves are also complementary to and capable of binding primers, successive cycles of amplification essentially double the amount of the target DNA synthesized in the previous cycle.
  • a cDNA expression library such as ⁇ gtl 1
  • ⁇ gtl 1 can be screened indirectly for hedgehog, N, or C polypeptides having at least one epitope, using antibodies specific for hedgehog, N, or C.
  • Such antibodies can be either polyclonally or monoclonally derived and used to detect expression product indicative of the presence of the desired hedgehog cDNA.
  • the polynucleotide sequence for hedgehog, N, or C also includes sequences complementary to the polynucleotide encoding hedgehog, N or C (antisense sequences).
  • Antisense nucleic acids are DNA or RNA molecules that are complementary to at least a portion of a specific mRNA molecule (Weintraub, Scientific American, 262:40, 1990).
  • the invention embraces all antisense polynucleotides capable of inhibiting production of hedgehog, N, or C polypeptide.
  • the antisense nucleic acids hybridize to the corresponding mRNA, forming a double-stranded molecule.
  • antisense nucleic acids interfere with the translation of the mRNA since the cell will not translate a mRNA that is double-stranded.
  • Antisense oligomers of about 15 nucleotides are preferred, since they are easily synthesized and are less likely to cause problems than larger molecules when introduced into the target hedgehog, N, or C-producing cell.
  • the use of antisense methods to inhibit the translation of genes is well known in the art (Marcus-Sakura, Anal. Biochem., 172:289, 1988). Inhibition of target nucleotide would be desirable, for example, in inhibiting cell-proliferative disorders, such as certain tumors, which are mediated by hedgehog, N or C.
  • Ribozyme nucleotide sequences for hedgehog, N or C are included in the invention.
  • Ribozymes are RNA molecules possessing the ability to specifically cleave other single-stranded RNA in a manner analogous to DNA restriction endonucleases. Through the modification of nucleotide sequences which encode these RNAs, it is possible to engineer molecules that recognize specific nucleotide sequences in an RNA molecule and cleave it (Cech, J. Amer. Med. Assn., 260:3030, 1988). A major advantage of this approach is that, because they are sequence-specific, only mRNAs with particular sequences are inactivated.
  • ribozymes There are two basic types of ribozymes namely, tetrahymena-type (Hasselhoff, Nature, 334:585, 1988) and "hammerhead"-type. Tetrahymena-type ribozymes recognize sequences which are four bases in length, while “hammerhead”-type ribozymes recognize base sequences 1 1-18 bases in length. The longer the recognition sequence, the greater the likelihood that sequence will occur exclusively in the target mRNA species. Consequently, hammerhead-type ribozymes are preferable to tetrahymena-type ribozymes for inactivating a specific mRNA species and 18-based recognition sequences are preferable to shorter recognition sequences.
  • DNA sequences encoding hedgehog, N or C can be expressed in vitro by DNA transfer into a suitable host cell.
  • "Host cells” are cells in which a vector can be propagated and its DNA expressed.
  • the term also includes any progeny of the subject host cell. It is understood that all progeny may not be identical to the parental cell since there may be mutations that occur during replication. However, such progeny are included when the term "host cell” is used. Methods of stable transfer, meaning that the foreign DNA is continuously maintained in the host, are known in the art.
  • the hedgehog, N or C polynucleotide sequences may be inserted into a recombinant expression vector.
  • recombinant expression vector refers to a plasmid, virus or other vehicle known in the art that has been manipulated by insertion or inco ⁇ oration of the hedgehog, N or C genetic sequences.
  • Such expression vectors contain a promoter sequence which facilitates the efficient transcription of the inserted genetic sequence of the host.
  • the expression vector typically contains an origin of replication, a promoter, as well as specific genes which allow phenotypic selection of the transformed cells.
  • Vectors suitable for use in the present invention include, but are not limited to the T7-based expression vector for expression in bacteria (Rosenberg, et al, Gene, 56:125, 1987), the pMSXND expression vector for expression in mammalian cells (Lee andNathans, J Biol. Chem., 263:3521, 1988) and baculovirus-derived vectors for expression in insect cells.
  • the DNA segment can be present in the vector operably linked to regulatory elements, for example, a promoter (e.g., T7, metallothionein I, or polyhedrin promoters).
  • Polynucleotide sequences encoding hedgehog, N or C can be expressed in either prokaryotes or eukaryotes, although post-translational modification of eukaryotically derived polypeptides, such as carboxylation, would occur in a eukaryotic host.
  • Hosts can include microbial, yeast, insect and mammalian organisms. Methods of expressing DNA sequences having eukaryotic or viral sequences in prokaryotes are well known in the art. Biologically functional viral and plasmid DNA vectors capable of expression and replication in a host are known in the art. Such vectors are used to inco ⁇ orate DNA sequences of the invention.
  • a variety of host-expression vector systems may be utilized to express the hedgehog, N or C coding sequence. These include but are not limited to microorganisms such as bacteria transformed with recombinant bacteriophage DNA, plasmid DNA or cosmid DNA expression vectors containing the hedgehog, N or C coding sequence; yeast transformed with recombinant yeast expression vectors containing the hedgehog, N or C coding sequence; plant cell systems infected with recombinant virus expression vectors (e.g., cauliflower mosaic virus, CaMV; tobacco mosaic virus, TMV) or transformed with recombinant plasmid expression vectors (e.g., Ti plasmid) containing the Hedgehog, N or C coding sequence; insect cell systems infected with recombinant virus expression vectors (e.g., baculovirus) containing the hedgehog, N or C coding sequence; or animal cell systems infected with recombinant virus expression vectors (e.g., retroviruses, adeno
  • any of a number of suitable transcription and translation elements including constitutive and inducible promoters, transcription enhancer elements, transcription terminators, etc., may be used in the expression vector (see e.g., Bitter, etal, 1987, Methods in Enzymology, 153:516-544).
  • inducible promoters such as pL of bacteriophage ⁇ , plac, pt ⁇ , ptac (pt ⁇ -lac hybrid promoter) and the like may be used.
  • promoters derived from the genome of mammalian cells e.g., metallothionein promoter
  • mammalian viruses e.g., the retrovirus long terminal repeat; the adenovirus late promoter; the vaccinia virus 7.5K promoter
  • Promoters produced by recombinant DNA or synthetic techniques may also be used to provide for transcription of the inserted hedgehog, N or C coding sequence.
  • vectors may be advantageously selected depending upon the use intended for the expressed. For example, when large quantities of hedgehog, N or C are to be produced, vectors which direct the expression of high levels of fusion protein products that are readily purified may be desirable. Those which are engineered to contain a cleavage site to aid in recovering are preferred.
  • Such vectors include but are not limited to the E coli expression vector pUR278 (Ruther, et al..
  • yeast a number of vectors containing constitutive or inducible promoters may be used.
  • Current Protocols in Molecular Biology Vol. 2, 1988, Ed. Ausubel, et al, Greene Publish. Assoc. & Wiley Interscience, Ch. 13; Grant, et al, 1987, Expression and Secretion Vectors for Yeast, in Methods in Enzymology, Eds. Wu and Grossman, 31987, Acad. Press, N.Y., Vol. 153, pp.516-544; Glover, 1986, DNA Cloning, Vol. II, IRL Press, Wash., D.C., Ch.
  • yeast promoter such as ADH or LEU2 or an inducible promoter such as GAL may be used (Cloning in Yeast, Ch. 3, R. Rothstein In: DNA Cloning Vol.l l, A Practical Approach, Ed. DM Glover, 1986, IRL Press, Wash., D.C.).
  • vectors may be used which promote integration of foreign DNA sequences into the yeast chromosome.
  • the expression of the hedgehog, N or C coding sequence may be driven by any of a number of promoters.
  • viral promoters such as the 35S RNA and 19S RNA promoters of CaMV (Brisso et al, Nature, ___:511, 1984), or the coat protein promoter to TMV (Takamatsu, et al, EMBO J., 6:307, 1987) may be used; alternatively, plant promoters such as the small subunit of RUBISCO (Coruzzi, et al, EMBO J, 3:1671-1680, 1984; Broglie, et al, Science, 224:838, 1984); or heat shock promoters, e.g., soybean hspl7.5-E or hspl7.3-B (Gurley, et al., Mol.
  • An alternative expression system which could be used to express is an insect system.
  • Autographa californica nuclear polyhedrosis virus (AcNPV) is used as a vector to express foreign genes.
  • the virus grows in Spodoptera frugiperda cells.
  • the hedgehog, N or C coding sequence may be cloned into non-essential regions (for example the polyhedrin gene) of the virus and placed under control of an AcNPV promoter (for example the polyhedrin promoter).
  • Successful insertion of the hedgehog, N or C coding sequence will result in inactivation of the polyhedrin gene and production of non-occluded recombinant virus (i.e., virus lacking the proteinaceous coat coded for by the polyhedrin gene).
  • Eukaryotic systems and preferably mammalian expression systems, allow for proper post-translational modifications of expressed mammalian proteins to occur.
  • Eukaryotic cells which possess the cellular machinery for proper processing of the primary transcript, glycosylation, phosphorylation, and advantageously, secretion of the gene product may be used as host cells for the expression of hedgehog, N or C.
  • Mammalian cell lines may be preferable. Such host cell lines may include but are not limited to CHO, VERO, BHK, HeLa, COS, MDCK, -293, and WI38.
  • Mammalian cell systems which utilize recombinant viruses or viral elements to direct expression may be engineered.
  • the hedgehog, N or C coding sequence may be ligated to an adenovirus transcription/- translation control complex, e.g., the late promoter and tripartite leader sequence.
  • This chimeric gene may then be inserted in the adenovirus genome by in vitro or in vivo recombination. Insertion in a non-essential region of the viral genome (e.g., region El or E3) will result in a recombinant virus that is viable and capable of expressing the protein in infected hosts (e.g., see Logan and Shenk, Proc. Natl.
  • the vaccinia virus 7.5K promoter may be used, (e.g., see, Mackett, etal, Proc. Natl. Acad. Sci. USA, 79:7415, 1982; Mackett, et al, J. Virol, 49: 857, 1984; Panicali, et al., Proc. Natl. Acad. Sci. USA, 79:4927, 1982).
  • vectors based on bovine papilloma virus which have the ability to replicate as extrachromosomal elements (Sarver, et al., Mol Cell. Biol, 1:486, 1981).
  • the plasmid Shortly after entry of this DNA into mouse cells, the plasmid replicates to about 100 to 200 copies per cell. Transcription of the inserted cDNA does not require integration of the plasmid into the host's chromosome, thereby yielding a high level of expression.
  • These vectors can be used for stable expression by including a selectable marker in the plasmid, such as, for example, the neo gene.
  • the retroviral genome can be modified for use as a vector capable of introducing and directing the expression of the hedgehog, N or C gene in host cells (Cone and Mulligan, Proc. Natl. Acad. Sci. USA, 81:6349, 1984). High level expression may also be achieved using inducible promoters, including, but not limited to, the metallothionine IIA promoter and heat shock promoters.
  • host cells can be transformed with the hedgehog, N or C cDNA controlled by appropriate expression control elements (e.g., promoter, enhancer, sequences, transcription terminators, polyadenylation sites, etc.), and a selectable marker.
  • expression control elements e.g., promoter, enhancer, sequences, transcription terminators, polyadenylation sites, etc.
  • the selectable marker in the recombinant plasmid confers resistance to the selection and allows cells to stably integrate the plasmid into their chromosomes and grow to form foci which in turn can be cloned and expanded into cell lines.
  • engineered cells may be allowed to grow for 1-2 days in an enriched media, and then are switched to a selective media.
  • a number of selection systems may be used, including but not limited to the he ⁇ es simplex virus thymidine kinase (Wigler, et al, Cell, __: 223, 1977), hypoxanthine-guanine phosphoribosyltransferase (Szybalska and Szybalski, Proc. Nat Acad. Sci.
  • adenine phosphoribosyltransferase genes can be employed in tk “ , hgprf or aprt " cells respectively.
  • antimetabo- lite resistance can be used as the basis of selection for dhfr, which confers resistance to methotrexate (Wigler, et al, Natl. Acad. Sci. USA, 77: 3567, 1980; O'Hare. et al, Proc. Natl Acad. Sci. USA, 78: 1527, 1981); gpt, which confers resistance to mycophenolic acid (Mulligan and Berg, Proc.
  • ODC ornithine decarboxylase
  • Transformation of a host cell with recombinant DNA may be carried out by conventional techniques as are well known to those skilled in the art.
  • the host is prokaryotic, such as E. coli
  • competent cells which are capable of DNA uptake can be prepared from cells harvested after exponential growth phase and subsequently treated by the CaCl 2 method using procedures well known in the art.
  • MgCl 2 or RbCl can be used. Transformation can also be performed after forming a protoplast of the host cell if desired.
  • Eukaryotic cells can also be cotransformed with DNA sequences encoding the hedgehog, N or C of the invention. and a second foreign DNA molecule encoding a selectable phenotype, such as the he ⁇ es simplex thymidine kinase gene.
  • Another method is to use a eukaryotic viral vector, such as simian virus 40 (SV40) or bovine papilloma virus, to transiently infect or transform eukaryotic cells and express the protein.
  • a eukaryotic viral vector such as simian virus 40 (SV40) or bovine papilloma virus
  • SV40 simian virus 40
  • bovine papilloma virus bovine papilloma virus
  • Isolation and purification of microbial expressed polypeptide, or fragments thereof, provided by the invention may be carried out by conventional means including preparative chromatography and immunological separations involving monoclonal or polyclonal antibodies.
  • the invention includes antibodies immunoreactive with or which bind to hedgehog, N or C polypeptide or functional fragments thereof.
  • Antibody which consists essentially of pooled monoclonal antibodies with different epitopic specificities, as well as distinct monoclonal antibody preparations are provided.
  • Monoclonal antibodies are made from antigen containing fragments of the protein by methods well known to those skilled in the art (Kohler, et al, Nature, 256:495. 1975).
  • the term antibody as used in this invention is meant to include intact molecules as well as fragments thereof, such as Fab and F(ab') 2 , which are capable of binding an epitopic determinant on hedgehog, N or C.
  • the antibodies of the invention include antibodies which bind to the N or C polypeptide and which bind with immunoreactive fragments N or C.
  • antibody as used in this invention includes intact molecules as well as fragments thereof, such as Fab, F(ab') 2 , and Fv which are capable of binding the epitopic determinant. These antibody fragments retain some ability to selectively bind with its antigen or receptor and are defined as follows:
  • Fab the fragment which contains a monovalent antigen-binding fragment of an antibody molecule can be produced by digestion of whole antibody with the enzyme papain to yield an intact light chain and a portion of one heavy chain;
  • Fab' the fragment of an antibody molecule can be obtained by treating whole antibody with pepsin, followed by reduction, to yield an intact light chain and a portion of the heavy chain; two Fab' fragments are obtained per antibody molecule;
  • Fv defined as a genetically engineered fragment containing the variable genetically fused single chain molecule.
  • epitopic determinants means any antigenic determinant on an antigen to which the paratope of an antibody binds.
  • Epitopic determinants usually consist of chemically active surface groupings of molecules such as amino acids or sugar side chains and usually have specific three dimensional structural characteristics, as well as specific charge characteristics.
  • Antibodies which bind to the hedgehog, N or C polypeptide of the invention can be prepared using an intact polypeptide or fragments containing small peptides of interest as the immunizing antigen.
  • the polypeptide such as N or C, or fragments thereof used to immunize an animal can be derived from translated cDNA or chemical synthesis which can be conjugated to a carrier protein, if desired.
  • carrier protein e.g., a carrier protein
  • Such commonly used carriers which are chemically coupled to the peptide include keyhole limpet hemocyanin (KLH), thyroglobulin, bovine serum albumin (BSA), and tetanus toxoid.
  • KLH keyhole limpet hemocyanin
  • BSA bovine serum albumin
  • the coupled peptide is then used to immunize the animal (e.g., a mouse, a rat, or a rabbit).
  • polyclonal or monoclonal antibodies can be further purified, for example, by binding to and elution from a matrix to which the polypeptide or a peptide to which the antibodies were raised is bound.
  • a matrix to which the polypeptide or a peptide to which the antibodies were raised is bound.
  • monoclonal antibodies See for example, Coligan, et al, Unit 9, Current Protocols in Immunology, Wiley Interscience, 1991, inco ⁇ orated by reference).
  • an anti-idiotypic monoclonal antibody made to a first monoclonal antibody will have a binding domain in the hypervariable region which is the "image" of the epitope bound by the first monoclonal antibody.
  • Antibodies as described herein as having specificity for N polypeptide are useful for specific identification of cells or tissues expressing the N fragment of hedgehog.
  • antibodies described herein as having specificity for C polypeptide e.g., Ab2 (residues 300-391), are useful for specific identification of cells or tissues expressing the C fragment of hedgehog. Both antibodies, naturally, will also detect native hedgehog polypeptide.
  • N and C-specific antibodies of the invention are useful for purification of N and C polypeptide, respectively, especially using the antibodies immobilized on solid phase.
  • N and native hedgehog polypeptides By contacting a sample with anti-N antibody, both N and native hedgehog polypeptides can be isolated.
  • anti-N antibodies By next contacting the sample removed by anti-N antibodies, with anti- C antibodies, the native hedgehog polypeptide is removed, thus allowing purification of N polypeptide.
  • C polypeptide can be antibody purified from a sample.
  • Monoclonal antibodies of the invention are suited for use, for example, in immunoassays in which they can be utilized in liquid phase or bound to a solid phase carrier.
  • the monoclonal antibodies in these immunoassays can be detectably labeled in various ways.
  • Examples of types of immunoassays which can utilize monoclonal antibodies of the invention are competitive and non-competitive immunoassays in either a direct or indirect format. Examples of such immunoassays are the radioimmunoassay (RIA) and the sandwich (immunometric) assay.
  • Detection of the antigens using the monoclonal antibodies of the invention can be done utilizing immunoassays which are run in either the forward, reverse, or simultaneous modes, including immunohistochemi- cal assays on physiological samples. Those of skill in the art will know, or can readily discern, other immunoassay formats without undue experimentation.
  • immunometric assay or "sandwich immunoassay” includes simultaneous sandwich, forward sandwich and reverse sandwich immunoassays. These terms are well understood by those skilled in the art. Those of skill will also appreciate that antibodies according to the present invention will be useful in other variations and forms of assays which are presently known or which may be developed in the future. These are intended to be included within the scope of the present invention.
  • Monoclonal antibodies can be bound to many different carriers and used to detect the presence of N or C polypeptide.
  • carriers include glass, polystyrene, polypropylene, polyethylene, dextran, nylon, amylases, natural and modified celluloses, polyacrylamides, agaroses and magnetite.
  • the nature of the carrier can be either soluble or insoluble for pu ⁇ oses of the invention. Those skilled in the art will know of other suitable carriers for binding monoclonal antibodies, or will be able to ascertain such using routine experimentation.
  • N or C polypeptide may be detected by the monoclonal antibodies when present in biological fluids and tissues.
  • Any sample containing a detectable amount of N or C can be used.
  • a sample can be a liquid such as urine, saliva, cerebrospinal fluid, blood, serum and the like, or a solid or semi-solid such as tissues, feces. and the like, or, alternatively, a solid tissue such as those commonly used in histological diagnosis.
  • C polypeptide in particular is detectable in biological samples, since it tends to diffuse more readily than N polypeptide. In performing the assays it may be desirable to include certain "blockers" in the incubation medium (usually added with the labeled soluble antibody).
  • blockingers are added to assure that non-specific proteins, proteases, or anti-heterophilic immuno- globulins to anti-C or N immunoglobulins present in the experimental sample do not cross-link or destroy the antibodies on the solid phase support, or the radiolabeled indicator antibody, to yield false positive or false negative results.
  • the selection of "blockers” therefore may add substantially to the specificity of the assays described in the present invention.
  • the invention also provides a method for modulating proliferation or differentiation of neuronal cells comprising contacting the cells with a hedgehog polypeptide.
  • the hedgehog polypeptide may be a native hedgehog polypeptide, or a N or C polypeptide, or functional fragments thereof.
  • the modulation is induction of proliferation or differentiation of a particular cell type. This can involve either synergistic positive induction of neuronal cells by N, or negative modulation by delta N-C for example (Lai, etal, Development 121:2349, 1995). Delta N-C enhances expression of posterier relative to anterior neural genes and does so through inhibition of N (see EXAMPLE 18 and Figure 18D).
  • a TGF- ⁇ factor may also be utilized in the method of the invention.
  • the term "substantially derived” refers to those cells from the floor plate or proximate to the floor plate.
  • such cells include motor neurons and dopaminergic neurons.
  • Those of skill in the art will be able to identify other neuronal cells substantially derived from the floor plate.
  • the cells are vertebrate cells and most preferably, human cells.
  • hedgehog and particularly C fragment, induces the expression of pituitary genes.
  • Hedgehog is also effective in inducing anterior brain gene expression as exemplified by the OTX-A marker.
  • TGF- ⁇ family member for example activin
  • Other TGF- ⁇ family members will be known to those of skill in the art. This apparent synergy of hh fragments with TGF- ⁇ family members occurs through the TGF- ⁇ protein inducing expression of neural inducers such as noggin and follistatin. The hh fragment then synergizes with these inducers to pattern neural gene expression.
  • hh fragments may also be useful as nerve-sparing agents or in restoring or promoting appropriate patterning during the healing of major limb trauma.
  • the N and C fragments may be useful in the area of genetic counseling. Specifically, familial midline defects such as cyclopia, polydactyly or neural tube defects may be diagnosed by mapping close to hh. Since autoproteolytic defects may be responsible for the disorders, N or C therapy could be provided.
  • the invention also provides an autoproteolytic fusion protein comprising a first polypeptide including the proteolytic domain of the C polypeptide of the invention, a cleavage site recognized by the first polypeptide, and a second polypeptide. (It is understood that the first and second polypeptides can be reversed.)
  • the auto-proteolytic activity of the native hedgehog protein is found entirely within the C polypeptide, therefore, the C polypeptide is useful for producing a fusion polypeptide which can then be cleaved at the junction of the C polypeptide and the second polypeptide.
  • the fusion protein may optionally have a purification tag, such as a poly-histidine tag for isolation on a nickel column, or an antibody epitope tag, preferably on the C fragment.
  • the cleavage site includes the sequence "GCF", which is recognized by the proteolytic domain of the C polypeptide and is utilized to cleave the second polypeptide from the C fragment.
  • GCF sequence of the C polypeptide
  • the invention also provides a method for producing an autoproteolytic fusion protein comprising operably linking a first polynucleotide, wherein the first polynucleotide encodes a first polypeptide including the proteolytic domain of the C polypeptide of the invention and the cleavage site recognized by the proteolytic domain, and a second polynucleotide encoding a second polypeptide.
  • the fusion protein may also include a carcier peptide and/or a purification tag.
  • the C polypeptide or functional fragment thereof is useful as a fusion partner to cause lipophilic modification and tethering of other proteins in vivo or in vitro.
  • Such fusion proteins may be desirable for factors whose activity is required in a localized manner, either by targeting DNA constructs to specific cells or by introducing cells transfected with specific DNA constructs, for example. It may be desirable to lipid-modify a normally secreted protein in order to produce a cell-associated protein. For example, it may be desirable to produce a viral antigen that remains cell associated.
  • cholesterol is covalently attached to the N-terminal protein during autoprocessing and the C polypeptide acts as an intramolecular cholesterol transferase.
  • the C polypeptide or functional fragments thereof can be used as a fusion partner with a protein of interest (e.g.. Protein X fused to hh-C domain).
  • a protein of interest e.g.. Protein X fused to hh-C domain.
  • Such fusions form thioesters at the junction between Protein X and hh-C (via an S to N shift).
  • the thioesters are then available as substrates for a peptide ligation reaction in which any peptide or protein having an amino terminal cysteine (Peptide Y) is added and undergoes spontaneous rearrangement (S to N shift) that generates a stable peptide bond between Protein X and Peptide Y (Protein X-peptide bond-Peptide Y).
  • a protein that is toxic when produced in vivo could be produced in vitro using the hh-C domain fusion protein method.
  • the fusion polypeptide may also include an optional carrier peptide.
  • the "carrier peptide", or signal sequence is located at the amino terminal end of the fusion peptide sequence. In the case of eukaryotes, the canier peptide is believed to function to transport the fusion polypeptide across the endoplasmic reticulum.
  • the secretory protein is then transported through the Golgi apparatus, into secretory vesicles and into the extracellular space or, preferably, the external environment.
  • Carrier peptides which can be utilized according to the invention include pre-pro peptides which contain a proteolytic enzyme recognition site.
  • Acceptable carrier peptides include the amino terminal pro-region of calcitonin or other hormones, which undergo cleavage at the flanking dibasic sites.
  • the invention is not limited to the use of any particular peptide as a carrier.
  • Other carrier peptides are known to those skilled in the art or can be readily ascertained without undue experimentation.
  • a carrier peptide which is a signal sequence is included in the expression vector, specifically located adjacent to the N-terminal end of the fusion polypeptide.
  • This signal sequence allows the fusion protein to be directed toward the endoplasmic reticulum.
  • the signal sequence consists of a leader of from about 16 to about 29 amino acids, starting with two or three polar residues and continuing with a high content of hydrophobic amino acids; there is otherwise no detectable conservation of sequence known.
  • signal sequences are known to those of skill in the art, and include the naturally occurring signal sequence derived from a hedgehog protein.
  • the fusion polypeptide of the invention includes a polypeptide encoded by a structural gene, preferably at the amino-terminus of the fusion polypeptide. Any structural gene is expressed in conjunction with the C-polypeptide (polynucleotide) and optionally a carrier peptide. The structural gene is operably linked with the carrier in an expression vector so that the fusion polypeptide is expressed as a single unit.
  • the identification of the autoproteolysis of hedgehog into the N and C domains is useful in a screening method to identify compounds or compositions which affect this processing activity.
  • the invention provides a method for identifying a composition which affects hh processing, which can be determined by activity or gene expression, comprising incubating the components, which include the composition to be tested (e.g., a drug, a small molecule, a protein) and a hh polypeptide or a recombinant cell expressing hedgehog or a gene encoding a C domain or functional fragment thereof operably linked to an N domain or functional fragment thereof, under conditions sufficient to allow the components to interact, then subsequently measuring the effect the composition has on hedgehog activity or expression.
  • the composition to be tested e.g., a drug, a small molecule, a protein
  • a hh polypeptide or a recombinant cell expressing hedgehog or a gene encoding a C domain or functional fragment thereof operably linked to an N domain or functional fragment thereof under conditions sufficient to allow the components to interact
  • Fragments of hedgehog polypeptide or polynucleotide can be used in the method of the invention as long as autoproteolytic activity remains (e.g., the construct exemplified in Figure 12a and 12b, Example 10).
  • the observed effect on hh may be either inhibitory or stimulatory. For example, one can determine whether the N domain is associated with the cell, or whether the N domain is secreted into the medium, in other words, whether incomplete processing has occurred.
  • Such methods for determining the effect of the compound or composition on hh processing include those described herein (see Example 10, Figure 12a and 12b) such as time course of autoproteolytic cleavage or course of cleavage based on concentration ranges.
  • the effect of the composition on hh can be determined by the expression of anterior or posterior neural markers.
  • Other methods for determining the effect of a composition on processing of N and C will be known to those of skill in the art.
  • Various labels can be used to detect the N and C domains, for example, a radioisotope, a fluorescent compound, a bioluminescent compound, a c- hemiluminescent compound, a metal chelator or an enzyme could be used.
  • a radioisotope for example, a fluorescent compound, a bioluminescent compound, a c- hemiluminescent compound, a metal chelator or an enzyme could be used.
  • Those of ordinary skill in the art will know of other suitable labels or will be able to ascertain such, using routine experimentation.
  • the identification of the lipid modification of the N domain of hedgehog by the C domain, resulting in a biologically active N domain is useful in a screening method to identify compounds or compositions which affect the cholesterol transferase/processing activity of hedgehog.
  • the modification may be a general sterol or lipid modification, and not limited to cholesterol.
  • the invention provides a method for identifying a composition which affects hh biological activity, which can be determined by activity or lipid modification (e.g., cholesterol), comprising incubating the components, which include the composition to be tested (e.g., a drug, a small molecule, a protein) and a hh polypeptide or a recombinant cell expressing hedgehog or a gene encoding a C domain or functional fragment thereof operably linked to an N domain or functional fragment thereof, under conditions sufficient to allow the components to interact, then subsequently measuring the effect the composition has on hedgehog activity.
  • the composition to be tested e.g., a drug, a small molecule, a protein
  • a hh polypeptide or a recombinant cell expressing hedgehog or a gene encoding a C domain or functional fragment thereof operably linked to an N domain or functional fragment thereof under conditions sufficient to allow the components to interact
  • Fragments of hedgehog polypeptide or polynucleotide can be used in the method of the invention as long as cholesterol transferase activity remains, for example.
  • the effect on hh may be either inhibitory or stimulatory. For example, one can determine whether the N domain is associated with the cell, or whether the N domain is secreted into the medium, in other words, whether incomplete processing and modification has occurred.
  • Such methods for determining the effect of the compound or composition on hh processing include those described herein (see Example 10, Figure 12a and 12b) such as time course of autoproteolytic cleavage or course of cleavage based on concentration ranges.
  • the effect of the composition on hh can be determined by the level of cholesterol modification as determined by thin layer chromatography (e.g., Example 19, Figure 23) or inco ⁇ oration of labeled cholesterol into hh protein (e.g., Example 19, Figure 25) or into a fragment appended to the transferase (c) domain.
  • Other methods for determining the effect of a composition on processing and cholesterol modification of N and C will be known to those of skill in the art.
  • Various labels can be used to detect the N and C domains, for example, a radioisotope, a fluorescent compound, a bioluminescent compound, a c- hemiluminescent compound, a metal chelator or an enzyme could be used.
  • hh activity refers preferably to autoproteolytic activity.
  • screening assay to identify a composition having an affect on other hh activities, for example, zinc hydrolase activity or cholesterol transferase activity; or inductionor regulatoin of differentiation of neuronal cells or chondrocytes. Appropriate assays for determining the effect on such activities will be known to those of skill in the art.
  • Example 19 provides lipophilic modification assays useful in the described screening methods above.
  • SLOS Lemli-Optiz syndrome
  • Another aspect of the present invention concerns three-dimensional molecular models of the subject hedgehog proteins, and their use as templates for the design of agents able to inhibit or potentiate at least one biological activity of the hedgehog, particularly the autoproteolytic.
  • An integral step to our approach to designing inhibitors of the subject hegehog proteins involves construction of computer graphics models of the hegehog protein which can be used to design pharmacophores by rational drug design.
  • an inhibitor to interact optimally with the subject proteolytic domain of hedgehog it will generally be desirable that it have a shape which is at least partly complimentary to that of a particular binding site of the enzyme, as for example those portions of the human hegehog protein which are involved in the autoproteolytic activity.
  • other factors including electrostatic interactions, hydrogen bonding, hydrophobic interactions, desolvation effects, and cooperative motions of ligand and enzyme, all influence the binding effect and should be taken into account in attempts to design bioactive inhibitors.
  • a computer-generated molecular model of the subject hedgehog proteins can be created.
  • at least the C ⁇ -carbon positions of the hedgehog sequence of interest are mapped to a particular coordinate pattern, such as the coordinates for hedgehog determined by x-ray crystallography, by homology modeling, and the structure of the protein and velocities of each atom are calculated at a simulation temperature (T 0 ) at which the docking simulation is to be determined.
  • T 0 simulation temperature
  • such a protocol involves primarily the prediction of side-chain conformations in the modeled protein, while assuming a main-chain trace taken from a tertiary structure such as provided in x- crystallographic model described herein.
  • Computer programs for performing energy minimization routines are commonly used to generate molecular models.
  • Common features of such molecular modeling methods include: provisions for handling hydrogen bonds and other constraint forces; the use of periodic boundary conditions; and provisions for occasionally adjusting positions, velocities, or other parameters in order to maintain or change temperature, pressure, volume, forces of constraint, or other externally controlled conditions.
  • energy minimization methods can be carried out for a given temperature, T which may be different than the docking simulation temperature, T 0 .
  • T which may be different than the docking simulation temperature, T 0 .
  • T j coordinates and velocities of all the atoms in the system are computed.
  • the normal modes of the system are calculated. It will be appreciated by those skilled in the art that each normal mode is a collective, periodic motion, with all parts of the system moving in phase with each other, and that the motion of the molecule is the supe ⁇ osition of all normal modes.
  • the mean square amplitude of motion in a particular mode is inversely proportional to the effective force constant for that mode, so that the motion of the molecule will often be dominated by the low frequency vibrations.
  • the system is "heated” or "cooled” to the simulation temperature, T 0 , by carrying out an equilibration run where the velocities of the atoms are scaled in a step-wise manner until the desired temperature, T Q , is reached.
  • the system is further equilibrated for a specified period of time until certain properties of the system, such as average kinetic energy, remain constant.
  • the coordinates and velocities of each atom are then obtained from the equilibrated system.
  • a second class of methods involves calculating approximate solutions to the constrained EOM for the protein. These methods use an iterative approach to solve for the Lagrange multipliers and, typically, only need a few iterations if the corrections required are small.
  • SHAKE Rivaert et al. (1977) J Comput Phys 23:327; and Van Gunsteren et al. (1977) Mol Phys 34: 131 1
  • RATTLE is based on the velocity version of the Verlet algorithm.
  • SHAKE Rivaert et al. (1977) J Comput Phys 23:327; and Van Gunsteren et al. (1977) Mol Phys 34: 131
  • RATTLE is based on the velocity version of the Verlet algorithm. Like SHAKE, RATTLE is an iterative algorithm and can be used to energy minimize the model of the subject hedgehog protein.
  • Directed methods generally fall into two categories: (1) design by analogy in which 3-D structures of known molecules (such as from a crystallographic database) are docked to the enzyme structure and scored for goodness-of-fit; and (2) de novo design, in which the ligand model is constructed piece-wise in the enzyme.
  • design by analogy in which 3-D structures of known molecules (such as from a crystallographic database) are docked to the enzyme structure and scored for goodness-of-fit
  • de novo design in which the ligand model is constructed piece-wise in the enzyme.
  • the latter approach in particular, can facilitate the development of novel molecules, uniquely designed to bind to, and, e.g., inhibit the proteolytic activity of a hegehog protein.
  • the design of potential hedgehog inhibitors begins from the general perspective of shape complimentary for the active site and substrate specificity subsites of the enzyme, and a search algorithm is employed which is capable of scanning a database of small molecules of known three-dimensional structure for candidates which fit geometrically into the target protein site. It is not expected that the molecules found in the shape search will necessarily be leads themselves, since no evaluation of chemical interaction necessarily be made during the initial search. Rather, it is anticipated that such candidates might act as the framework for further design, providing molecular skeletons to which appropriate atomic replacements can be made. Of course, the chemical complimentary of these molecules can be evaluated, but it is expected that atom types will be changed to maximize the electrostatic, hydrogen bonding, and hydrophobic interactions with the enzyme.
  • the program can also search a database of small molecules for templates whose shapes are complementary to particular binding sites of the enzyme (DesJarlais et al. (1988) J Med Chem 31 : 722- 729). These templates normally require modification to achieve good chemical and electrostatic interactions (DesJarlais et al. (1989) ACS Symp Ser 413: 60-69). However, the program has been shown to position accurately known cofactors for inhibitors based on shape constraints alone.
  • orientations are evaluated for goodness-of-fit and the best are kept for further examination using molecular mechanics programs, such as AMBER or CHARMM.
  • molecular mechanics programs such as AMBER or CHARMM.
  • Such algorithms have previously proven successful in finding a variety of molecules that are complementary in shape to a given binding site of a receptor-enzyme, and have been shown to have several attractive features.
  • First, such algorithms can retrieve a remarkable diversity of molecular architectures.
  • the best structures have, in previous applications to other proteins, demonstrated impressive shape complementarity over an extended surface area.
  • GRID computer program
  • Yet a further embodiment of the present invention utilizes a computer algorithm such as CLLX which searches such databases as CCDB for small molecules which can be oriented in the receptor binding site in a way that is both sterically acceptable and has a high likelihood of achieving favorable chemical interactions between the candidate molecule and the surrounding amino acid residues.
  • the method is based on characterizing the receptor site in terms of an ensemble of favorable binding positions for different chemical groups and then searching for orientations of the candidate molecules that cause maximum spatial coincidence of individual candidate chemical groups with members of the ensemble.
  • the current availability of computer power dictates that a computer-based search for novel ligands follows a breadth-first strategy.
  • a breadth-first strategy aims to reduce progressively the size of the potential candidate search space by the application of increasingly stringent criteria, as opposed to a depth-first strategy wherein a maximally detailed analysis of one candidate is performed before proceeding to the next.
  • CLIX conforms to this strategy in that its analysis of binding is rudimentary -it seeks to satisfy the necessary conditions of steric fit and of having individual groups in "corcect" places for bonding, without imposing the sufficient condition that favorable bonding interactions actually occur.
  • a ranked "shortlist" of molecules, in their favored orientations, is produced which can then be examined on a molecule-by-molecule basis, using computer graphics and more sophisticated molecular modeling techniques.
  • CLIX is also capable of suggesting changes to the substituent chemical groups of the candidate molecules that might enhance binding.
  • the algorithmic details of CLLX is described in Lawerence et al. (1992) Proteins 12:31- 41, and the CLLX algorithm can be summarized as follows.
  • the GRID program is used to determine discrete favorable interaction positions (termed target sites) in the binding site of the protein for a wide variety of representative chemical groups. For each candidate ligand in the CCDB an exhaustive attempt is made to make coincident, in a spatial sense in the binding site of the protein, a pair of the candidate's substituent chemical groups with a pair of conesponding favorable interaction sites proposed by GRID. All possible combinations of pairs of ligand groups with pairs of GRID sites are considered during this procedure.
  • the program Upon locating such coincidence, the program rotates the candidate ligand about the two pairs of groups and checks for steric hindrance and coincidence of other candidate atomic groups with appropriate target sites. Particular candidate/orientation combinations that are good geometric fits in the binding site and show sufficient coincidence of atomic groups with GRID sites are retained.
  • Yet another embodiment of a computer-assisted molecular design method for identifying inhibitors of the subject hegehog protein comprises the de novo synthesis of potential inhibitors by algorithmic connection of small molecular fragments that will exhibit the desired structural and electrostatic complementarity with the active site of the enzyme.
  • the methodology employs a large template set of small molecules with are iteratively pieced together in a model of the hedgehog active site.
  • Each stage of ligand growth is evaluated according to a molecular mechanics-based energy function, which considers van der Waals and coulombic interactions, internal strain energy of the lengthening ligand, and desolvation of both ligand and enzyme.
  • the search space can be managed by use of a data tree which is kept under control by pruning according to the binding criteria.
  • the search space is limited to consider only amino acids and amino acid analogs as the molecular building blocks.
  • Such a methodology generally employs a large template set of amino acid conformations, though need not be restricted to just the 20 natural amino acids, as it can easily be extended to include other related fragments of interest to the medicinal chemist, e.g. amino acid analogs.
  • the putative ligands that result from this construction method are peptides and peptide-like compounds rather than the small organic molecules that are typically the goal of drug design research.
  • peptide building approach is not that peptides are preferable to organics as potential pharmaceutical agents, but rather that: (1) they can be generated relatively rapidly de novo; (2) their energetics can be studied by well- parameterized force field methods; (3) they are much easier to synthesize than are most organics; and (4) they can be used in a variety of ways, for peptidomimetic inhibitor design, protein-protein binding studies, and even as shape templates in the more commonly used 3D organic database search approach described above.
  • GROW a software package called GROW (Moon et al. (1991) Proteins 11 :314-328).
  • GROW a software package
  • standard interactive graphical modeling methods are employed to define the structural environment in which GROW is to operate.
  • environment could be the active site cleft of hedgehog, or it could be a set of features on the protein's surface to which the user wishes to bind a peptide-like molecule, a peptide sequence based on the cleavage site of hedgehog itself (e.g., to represent the autoproteolytic event).
  • the GROW program then operates to generate a set of potential ligand molecules.
  • GROW operates on an atomic coordinate file generated by the user in the interactive modeling session, such as the coordinates provided in the crystaliographic- derived models, plus a small fragment (e.g., an acetyl group) positioned in the active site to provide a starting point for peptide growth. These are referred to as "site” atoms and “seed” atoms, respectively.
  • a second file provided by the user contains a number of control parameters to guide the peptide growth (Moon et al. (1991) Proteins 11 :314-328).
  • GROW proceeds in an iterative fashion, to systematically attach to the seed fragment each amino acid template in a large preconstructed library of amino acid conformations.
  • a template When a template has been attached, it is scored for goodness-of-fit to the receptor site, and then the next template in the library is attached to the seed. After all the templates have been tested, only the highest scoring ones are retained for the next level of growth.
  • This procedure is repeated for the second growth level; each library template is attached in turn to each of the bonded seed/amino acid molecules that were retained from the first step, and is then scored. Again, only the best of the bonded seed/dipeptide molecules that result are retained for the third level of growth.
  • the growth of peptides can proceed in the N-to-C direction only, the reverse direction only, or in alternating directions, depending on the initial control specifications supplied by the user. Successive growth levels therefore generate peptides that are lengthened by one residue.
  • the procedure terminates when the user-defined peptide length has been reached, at which point the user can select from the constructed peptides those to be studied further.
  • the resulting data provided by the GROW procedure include not only residue sequences and scores, but also atomic coordinates of the peptides, related directly to the coordinate system of the receptor site atoms.
  • potential pharmacophoric compounds can be determined using a method based on an energy minimization-quenched molecular dynamics algorithm for determining energetically favorable positions of functional groups in the binding sites of the subject hegehog protein.
  • the method can aid in the design of molecules that inco ⁇ orate such functional groups by modification of known ligands or de novo construction.
  • the multiple copy simultaneous search method described by Miranker et al. (1991) Proteins 11 : 29-34.
  • MCSS multiple copy simultaneous search method
  • To determine and characterize a local minima of a functional group in the forcefield of the protein multiple copies of selected functional groups are first distributed in a binding site of interest on the hedgehog protein. Energy minimization of these copies by molecular mechanics or quenched dynamics yields the distinct local minima. The neighborhood of these minima can then be explored by a grid search or by constrained minimization.
  • the MCSS method uses the classical time dependent Hartee (TDH) approximation to simultaneously minimize or quench many identical groups in the forcefield of the protein.
  • TDH time dependent Hartee
  • Implementation of the MCSS algorithm requires a choice of functional groups and a molecular mechanics model for each of them.
  • Groups must be simple enough to be easily characterized and manipulated (3-6 atoms, few or no dihedral degrees of freedom), yet complex enough to approximate the steric and electrostatic interactions that the functional group would have in binding to the site of interest in the hedgehog protein.
  • a preferred set is, for example, one in which most organic molecules can be described as a collection of such groups (Patai's Guide to the Chemistry of Functional Groups, ed. S. Patai (New York: John Wiley, and Sons, (1989)). This includes fragments such as acetonitrile, methanol, acetate, methyl ammonium, dimethyl ether, methane, and acetaldehyde.
  • Determination of the local energy minima in the binding site requires that many starting positions be sampled. This can be achieved by distributing, for example, 1,000-5,000 groups at random inside a sphere centered on the binding site; only the space not occupied by the protein needs to be considered. If the interaction energy of a particular group at a certain location with the protein is more positive than a given cut-off (e.g. 5.0 kcal/mole) the group is discarded from that site. Given the set of starting positions, all the fragments are minimized simultaneously by use of the TDH approximation (Elber et al. (1990) J Am Chem Soc 1 12: 9161-9175). In this method, the forces on each fragment consist of its intemal forces and those due to the protein. The essential element of this method is that the interactions between the fragments are omitted and the forces on the protein are normalized to those due to a single fragment. In this way simultaneous minimization or dynamics of any number of functional groups in the field of a single protein can be performed.
  • Minimization is performed successively on subsets of, e.g. 100, of the randomly placed groups. After a certain number of step intervals, such as 1 ,000 intervals, the results can be examined to eliminate groups converging to the same minimum. This process is repeated until minimization is complete (e.g. RMS gradient of 0.01 kcal/mole/ A).
  • minimization e.g. RMS gradient of 0.01 kcal/mole/ A.
  • the next step then is to connect the pharmacophoric pieces with spacers assembled from small chemical entities (atoms, chains, or ring moieties).
  • each of the disconnected can be linked in space to generate a single molecule using such computer programs as, for example, NEWLEAD (Tschinke et al. ( 1993) J Med Chem 36 : 3863,3870).
  • the procedure adopted by NEWLEAD executes the following sequence of commands (1) connect two isolated moieties, (2) retain the intermediate solutions for further processing, (3) repeat the above steps for each of the intermediate solutions until no disconnected units are found, and (4) output the final solutions, each of which is single molecule.
  • Such a program can use for example, three types of spacers: library spacers, single-atom spacers, and fuse-ring spacers.
  • library spacers are optimized structures of small molecules such as ethylene, benzene and methylamide.
  • the output produced by programs such as NEWLEAD consist of a set of molecules containing the original fragments now connected by spacers. The atoms belonging to the input fragments maintain their original orientations in space.
  • the molecules are chemically plausible because of the simple makeup of the spacers and functional groups, and energetically acceptable because of the rejection of solutions with van-der Waals radii violations.
  • the three-dimensional stmcture of hedgehog is useful to aid in screening and development of diagnostic and therapeutic protein fragments as in rational drug design, to search for structural analogs of known protein stmctures, or to aid in an analysis of biological function and activity. Also, the method may be used to predict protein secondary stmctures and protein subsecondary stmctures from amino acid sequences alone, and to predict those regions of a protein molecule that are on the outside and those that are on the inside.
  • Compounds can also be prepared using the three-dimensional stmcture provided herein and tested using assays known to those of skill in the art. For example, compounds can be synthesized and screened for hedgehog autoproteolytic activity by cleavage assays (see for example, Porter et al., Cell 86:21, 1996; W096/17924, herein inco ⁇ orated by reference).
  • peptidomimetics are synthetic compounds having a three-dimensinal stmcture (i.e., a "peptide motif) based upon the three-dimensional stmcture of a selected peptide.
  • the peptide motif provides the peptidomimetic compound with Hedgehog agonist or antagonist activity that is substantially the same as, or greater than, the Hedgehog agonist or antagonist activity of the peptide from which the peptidomimetic was derived.
  • Peptidomimetic compounds can have additional characteristics that enhance their therapeutic application, e.g., enhanced cell permeability, increased receptor or polypeptide binding affinity and/or avidity, and prolonged biological half-life.
  • the design of peptidomimetic compounds having agonist or antagonist activity can be aided through computer modeling techniques well known in the art. Other methods for the design, as well as the preparation of, p- eptidomimemtic compounds are well known in the art.
  • Atomic coordinates and stmcture factors have been deposited in the Brookhaven Protein Data Bank. Applicant assures complete access and disclosure of these coordinates and factors upon issuance of a patent.
  • FIGURES 1 (A) and (C) are immunoblots with antibodies against amino- (Abl) and carboxy-terminal (Ab2) epitopes.
  • GST fusion proteins containing either residues 83 to 160 or 300 to 391 from HH protein were expressed in Escherichia coli, purified as recommended [F. M. Ausubel, et al, Current Protocols in Molecular Biology (Greene and Wiley-Interscience, New York, 1991)], and used to immunize rabbits by standard methods.
  • the antibodies were affinity purified on a column of His 6 -U protein [E. Harlow and D.
  • Biotinylated hh antibodies were prepared by purifying the rabbit antisera over a protein A column, followed by biotinylation with the use of the Immunoprobe biotinylation kit (Sigma).
  • Immunoprecipitations were performed as described [Harlow and Lane] with the use of cold RIPA lysis buffer containing 0.25 mM phenylmethylsulfonyl fluoride (PMSF) and 5 mM EDTA for tissue homogenization. Lysates were precleared twice with pre-immune rabbit serum plus protein A beads (Gibco-BRL). Affinity-purified antibodies or preimmune semm was then added, and the immunoprecipitation was performed with protein A beads, with the use of NP-40 lysis buffer for the washes.
  • PMSF phenylmethylsulfonyl fluoride
  • Immunoblots were performed with affinity purified Abl or Ab2 by either of two chemiluminescence based protocols.
  • samples were resolved on 15 percent or 12 percent SDS-polyacrylamide gels (F. M. Ausubel et al., supra) and transferred to Magnagraph nylon membranes (MSI) by electroblotting.
  • MSI Magnagraph nylon membranes
  • Lanes contain protein from induced untransfected S2 cells (lanes 1 and 13), transfected S2 cells induced to express hh (lanes 2 and 14), imaginal discs (lanes 3 and 15), wild type embryos (lanes 6 and 18), and in vitro translations of synthetic h mRNA both in the presence (lanes 5 and 17) and absence of microsomes (lanes 4 and 16).
  • cDNAs encoding various hh protein species were cloned into the pMK33 vector, which allows for inducible expression under metallothionein promoter control (M. R. Koelle et al., Cell 67:59,1991).
  • Stable S2 cell lines were made by transfection of the hh/pMK33 plasmids with constant selection for hygromycin resistance. Proteins were expressed by plating a log phase culture of cells diluted to 0.1 A 595 units, waiting 48 hours, inducing with CuS0 4 at 0.2 mM final concentration, and harvesting the cells and/or supematant 24 hours later.
  • FIGURES 1 (B) and (D) are blots of samples immunoprecipitated with Abl (B, lanes 7-9), Ab2 (D, lanes 19-21), or pre-immune semm (B, lanes 10-12 and D, lanes 22-24). Detection was with biotinylated derivatives of Abl (B) and Ab2 (D). Samples used were: induced untransfected S2 cells, lanes 7, 10, 19 and 22; transfected S2 cells induced to express hh, lanes 8, 11, 20 and 23; and embryos, lanes 9, 12, 21 and 24. For either antibody, hh protein fragments were specifically immunoprecipitated from hh expressing cells and embryos, but not from untransfected cells.
  • cleavage sites are denoted by arcows.
  • the cleavage site marked by the asterisk is infened by identification of only one cleavage product and may therefore occur at another location within the C fragment.
  • the first two columns to the right of the diagram indicate the reactivity of Abl and Ab2 to each hh fragment.
  • the other columns indicate the presence (+) or absence (-) of each hh fragment in the various samples. Parentheses around F and N ss indicate that these species are detected in in vitro translation reactions but not in vivo.
  • the 46kD species was detected from in vitro translation extracts by Abl and Ab2 (FIGURE 1 , lanes 4 and 16), and was partially converted to a species of 39 kD (U) when translation occuned in the presence of microsomes (FIGURE 1, lanes 5 and 17).
  • a 39 kD species co-migrating with U is also present in extracts from all in vivo sources, but none of these extracts contain detectable levels of F.
  • U represents the signal-cleaved form of F; signal cleavage thus appears to be relatively inefficient in vitro, as reported previously, (J. J. Lee, etal, Cell, 21:33, 1992), but is highly efficient in vivo.
  • the 19 kD species hereafter is refened to as N (N-terminal fragment), the 25 kD species as C (C-terminal fragment) and the 16 kD species as C * ; these species represent the major forms of endogenous hh protein present in vivo.
  • the proposed cleavages by which these species arise are shown schematically in the bottom portion of FIGURE 1.
  • the N and C species are uniquely detected by Abl and Ab2, respectively, and the sum of the relative masses of the two smaller species is roughly equivalent to the relative mass of U.
  • the electrophoretic mobilities of the F and U species are somewhat at variance with their predicted relative masses (52.1 kD and 43.3 kD, respectively).
  • the identities of these species were confirmed by in vitro translation of a variety of hedgehog open reading frames modified to contain different extents of sequence at the NH 2 - or COOH- terminus, and by insertion of epitope tags.
  • the migration anomalies appear to be associated with protein species in which sequences from both the NH,- and COOH-terminal fragments are simultaneously present.
  • the mobilities of the NH 2 - and COOH-terminal fragments in contrast, conespond to relative masses (19 kD and 25 kD, respectively) that sum to yield 44 kD, roughly equivalent to the expected relative mass of U.
  • Processing of the hh protein when translated in vitro also yields a 25 kD species (C; lanes 16 and 17) and either a 29 kD or 19 kD (N) species (lanes 4 and 5).
  • the 19 kD species comigrates with N, and its formation depends upon the presence of microsomes, consistent with the proposal that N derives from F by signal cleavage and a further intemal cleavage.
  • the overall pathway for formation of the predominant forms of hh protein observed in vivo thus appears to involve signal cleavage of F to generate U.
  • FIGURE 2 shows limited sequence similarity between hh proteins and serine proteinases.
  • hh protein sequences are aligned to residues 323 to 329 of the D. melanogaster protein and numbered as positions 1 to 7 (group A). conserveed hh residues are in bold letters.
  • the catalytic histidines A. J. Banett, in Proteinase inhibitors A. J. Barrett, G. Salvesen, Eds. (Elsevier, Amsterdam, 1986) pp.
  • bovine trypsin ELA, porcine elastase
  • UKH human urokinase
  • C1R human complement factor 1R
  • CIS human complement factor IS
  • MCP rat mast cell protease
  • FAX human blood clotting factor X
  • TPA human tissue plasminogen activator.
  • Figure 2 shows that a seven residue region of hh coding sequence (residues 323 to 329 in the Drosophila protein) displays some similarity to the sequences of serine proteases. This region lies approximately two thirds of the distance from the signal cleavage site to the carboxy-terminus, and includes Thr and His, residues (positions 4 and 7 in FIGURE 2) that are invariant among all hh sequences from all species. In the serine proteases, this conserved sequence contains an invariant His that acts as a general base in catalysis (A. J. Barrett, in Proteinase inhibitors A. J. Banett, G. Salvesen, Eds. Elsevier. Amsterdam, 1986, pp. 3-22).
  • FIGURE 3(A) is a coomasie blue stained polyacrylamide gel that shows the production and purification of His 6 -U and His 6 -U H329A proteins from E. coli.
  • Samples were molecular weight markers (lanes 1 and 2); lysates of E. coli cells carrying the His 6 -U expression construct without (lane 3) and with (lane 4) induction by IPTG; purified His 6 -U protein (lane 5); lysates of E. coli cells that carry the His 6 -U H329A expression constmct without (lane 6) and with (lane 7) induction by IPTG; purified His 6 -U H329A protein (lane 8).
  • FIGURE 3 (B) is an immunoblot detected with Ab2 showing transfected S2 cells induced to express hh (lane 1); His 6 -U and Hi% -L, 329A proteins incubated in cleavage reaction buffer for 0 hours (lanes 2 and 5), for 20 hours (lanes 3 and 6), and for 20 hours in the presence of 20 mM TAME (a serine protease inhibitor) (lanes 4 and 7).
  • TAME serine protease inhibitor
  • soybean trypsin inhibitor, a, anti-trypsin, aprotitin, leupeptin, and E-64 do not block auto-proteolysis of translated Hh protein
  • TAME partially inhibits auto-proteolysis of purified His 6 -U protein (FIGURE 3, panel B).
  • the wild type protein but not the H329A mutant protein released a 25 kD species detectable by Ab2 and identical in mobility with the C species produced from in vitro translations and various in vivo sources. This cleavage was also observed when the wild type protein was purified and renatured by other protocols and cleaved under distinct conditions. Plasmids encoding the His 6 -U and His6-U H329A proteins were generated by inserting sequences conespond- ing to residues 83 to 471 from the wild-type or hh H329A ORF into the pRSETB expression vector (Invitrogen).
  • Proteins were induced in BL21(DE3)/pLysS E. coli cells as described (F. M. Ausubel et al., supra). The basic purification was performed on Ni-NTA agarose beads (Qiagen) by a denaturing protocol with the use of 6 M guanidinium HCl and 8 M urea essentially as recommended (a detailed protocol of exact conditions used is available upon request). Washes contained 0.2 percent Tween 20 and 5 mM b-mercaptoethanol. The final wash buffer was: 6 M urea, 100 mM Tris, 500 mM NaCl, 20 percent glycerol, (pH 7.4). Elutions were with the final wash buffer containing 250 mM imidazole.
  • In vitro cleavage reactions were performed by incubating the purified protein (diluted 1 :30 in the final mix) in cleavage buffer [50 mM Tris, 500 mM NaCl, 5 percent glycerol, 0.2% Triton X-100, 50 mM DTT, (pH 7.4)]. To isolate soluble full-length His 6 -U protein free from denaturants or detergents, additional steps were taken (this refers to the other renaturation protocols mentioned in the text). Full-length protein from the eluate described above was further purified from breakdown products by precipitation, by urea removal through dialysis.
  • the precipitate was then re-solubilized in a buffer containing guanidinium HCl and loaded onto another Ni-NTA agarose column. After washing as described, the protein was re-folded (while attached to the beads) by gradual dilution of urea (from 6M to 0.5M) with dilution buffer [(100 mM Tris, 500 mM NaCl, 20 percent glycerol, (pH 7.4)] over an 8 hour period at 4° C. The protein was eluted with dilution buffer containing 250 mM imidazole and 0.5M urea. The eluate was dialyzed in 100 mM Tris, 150 mM NaCl, 10 percent glycerol, (pH 7.4) at 4° C and stored at -70° C.
  • H329A/flu227 double mutant is not cleaved by wild-type protein in a mixing experiment (lane 1 1), suggesting an intramolecular mechanism for auto-proteolysis.
  • Hh proteins encoded by the zebrafish genes twhh and shh display a pattem of processing (D) similar to that of the Drosophila protein although the NH 2 -terminal fragment of each zebrafish protein (23 kD for twhh and 22 kD for shh) has a lower apparent mass than the COOH-terminal fragment (25 kD for twhh and shh). This is the result of a shorter stretch of residues that precedes the signal sequences as compared to the Drosophila protein.
  • the flu408 and flu227 mutations were generated by inserting a trimer of the influenza hemagglutinin antigen (42 residues for flu408 and 43 residues for flu227) into the AlwN I and Bgl I sites present in the hh ORE (nucleotide positions 1604 and 1058 respectively) (J. J. Lee, et al, supra).
  • the ⁇ 89-254 mutation was generated by removing sequences between the EcoN I site (644) and the Pml I site (1145).
  • the 294 tmnc mutation was generated by removing sequences between the Ace I site (1265) and the Xcm I site (1792).
  • the 410 tmnc mutation was previously generated and identified as Hh 4]0 (J. J. Lee, et al, supra).
  • DNA isolated from hh E /TM3 was used to seed PCR reactions generating regions of the hh ORF and flanking sequences, which were subcloned into Bluescript KSM (Stratagene). Six clones each, derived from two different PCR amplifications were sequenced.
  • this constmct generates a full length species of a mobility conesponding to the expected relative mass of 33 kD, and two cleaved products whose apparent relative masses (25 and 9 kD) sum to give the relative mass of the larger species.
  • the smaller of the cleaved products will occasionally migrate as two bands as seen in Fig 4A.
  • the 456 trunc hh protein is like that encoded by the EMS-induced hh £ mutant allele, which results in the loss of 15 residues from the carboxy-terminus of the protein.
  • This protein undergoes auto-proteolysis, as demonstrated by the appearance of a 24 kD band in place of C, but the efficiency of the reaction is much impaired in vitro (FIGURE 4B).
  • Autoproteolysis of the hh protein relies mainly upon residues within C; deletion or alteration of residues within this domain is associated with reduced efficiency of processing, and one such deletion appears to be the cause of the hh UE mutation.
  • sequence homology and auto-proteolytic function of the full length hh protein suggested the possibility that F or the C fragment is a sequence-specific protease.
  • an influenza vims epitope tag was introduced into the N-terminus of a hh open reading frame that also canied a H329A mutation.
  • FIGURE 4C shows that the insertion of the epitope tag alone does not interfere with auto-proteolysis (lane 9), and yields a normal C fragment and an N fragment of increased relative mass (compare to wild type in lane 12).
  • the hh gene has been broadly conserved in evolution, with single homologues unidentified in a wide variety of invertebrate species and multiple distinct homologues in each of several vertebrate species (Y. Echelard et al., Cell 7_5: 1417, 1993; S. Krauss, et al, Cell 75: 1431, 1993; H. Roelink et al., Cell, supra). As seen in FIGURE 2, all of these coding sequences contain an invariant histidine and other conserved residues at a position conesponding to H329 in the Drosophila protein.
  • the protein encoded by at least one of the mouse genes appears to be processed in vivo to yield two smaller species in a manner resembling the in vivo processing of the Drosophila protein.
  • auto-proteolysis may also play a role in vertebrates we examined the behavior of proteins encoded by two distinct hh homologues from the zebrafish, twhh and shh.
  • FIGURE 4D demonstrates that when these sequences are translated in vitro, smaller species are generated whose relative masses sum to yield approximately the relative mass of the full length protein (lanes 1 and 3).
  • the H329A mutant gene under control of the hsp 70 promoter was introduced by P element-mediated transformation into the Drosophila germline.
  • the hshh H329A constmct was made identically to the hshh constmct with the use of a hh ORF fragment containing the H329A mutation.
  • Transgenic flies were generated from ay 1 w 1 " 8 parental strain using standard methods of P element mediated transformation (A. C. Spradling and G. M. Rubin, Science 218: 341 1982).
  • a line, HA3, carrying the hshh H329A P element on the second chromosome was maintained as a homozygous stock.
  • Embryos from a hh l3E /TM3 ft ⁇ -lacZ (the balancer chromosome was from the Bloomington Stock Center, strain 3218) stock homozygous for the hh E allele were identified by the lack of staining with an anti b-galactosidase antibody (Promega) in a double stain with Ab2 (FIGURE 9, panel D). Staining in FIGURE 9, panels B and C were performed formaldehyde fixed Canton-S embryos with the use of an AP conjugated anti-rabbit IgG secondary. Although standard formaldehyde fixation was generally used, heat and acid-formaldehyde fixation also gave similar results.
  • GST fusion proteins containing either residues 83 to 160 or 300 to 391 from the Hh protein were expressed in E. coli, purified as recommended (F. M. Ausubel et al., supra), and used to immunize rabbits by standard methods.
  • the antibodies were affinity purified on a column of His 6 -U protein (Harlow and Lane, supra) linked to Affi-Gel 10 beads (Bio-Rad). The purification was performed as described (Harlow and Lane, supra) except that the acid and base elutions contained 10 percent dioxane.
  • Biotinylated hh antibodies were prepared by purifying the rabbit antisera over a protein A column, followed by biotinylation with the use of the Immunoprobe biotinylation kit (Sigma). Immunoprecipitations were performed as described (Harlow and Lane, supra) with the use of cold RIPA lysis buffer containing 0.25 mM PMSF and 5 mM EDTA for tissue homogenization. Lysates were precleared twice with pre-immune rabbit semm plus protein A beads (Gibco BRL). Affinity purified antibodies or pre-immune serum was then added, and the immunoprecipitation was performed with protein A beads, with the use of NP-40 lysis buffer for the washes.
  • FIGURE 5 (A) and (B) are immunoblots developed with the use of Abl and Ab2 antibodies respectively.
  • heat shocked hshh embryos the wild-type Hh protein is both induced and properly processed to generate the U, N C and C* species seen in other expression contexts.
  • FIGURE 5 shows that heat shock induction results in the formation of an abundant species that conesponds to U based on its mobility and its interaction with Abl and Ab2 (lanes 5 and 10).
  • induction of wild type hh protein using a similar contmct resulted in similar levels of the N and C processed products (lanes 4 and 9), with very little uncleaved U.
  • the H329A mutation in embryos appears to greatly reduce the efficiency of auto-proteolytic cleavage of the hh protein.
  • FIGURE 6 the embryonic distribution of wingless (wg) RNA as revealed by in situ hybridization is shown in FIGURE 6 (A) wild-type (homozygous 1 w" 18 ), (B) hshh, and (C) hshh H329A embryos that were exposed to two 10 minute heat shocks separated by a 90-minute recovery period. Wild-type embryos showed little change in wg expression, whereas the wild-type protein and, to a lesser extent, the H329A protein each induced ectopic wg expression (Table 1).
  • Panels (D), (E), and (F) show the dorsal surfaces ofy 1 w 1118 , hshh, and hshh H329A larvae, respectively, at the level of the fourth abdominal segment. These larvae were shocked for 30 minutes as embryos and allowed to complete embryogenesis. Cuticle cell types (1 °, 2°, 3 °, and 4°) are labeled as described (J. Heemskerk and S. DiNardo, Cell 76: 449, 1994).
  • Hh protein has a requirement for maintenance of an adjacent stripe of wingless (wg) gene expression in each embryonic segment (A. Martinez Arias, etal, Development 103: 157, 1988; and S. DiNardo, et al, Nature 332: 604, 1988).
  • This requirement is deduced from the loss of wg expression when hh function is absent; in addition, the ubiquitous expression of wild-type Hh protein induces expansion of the domain of w gene expression (P. W. Ingham, Nature 366: 560, 1993).
  • the effects of the H329A mutation upon wg expansion were examined by heat shocking embryos carrying the H329A mutant constmct in parallel with embryos containing the wild-type constmct.
  • the H329A mutant protein is able to induce some expansion of the wg domain, the efficiency of this activity is impaired relative to that of the wild-type protein (FIGURE 6, B and C; Table 1). The difference in efficiency ranges nearly as high as threefold depending upon the heat shock regime, and these results suggest that a uto-proteolysis of the Hh protein is important for optimal activity in embryonic signaling to induce wg expression.
  • Hh protein The effects of Hh protein on the patteming of cuticular stmctures are most clearly visible on the dorsal surface of the larva, where four distinctive cell types can be identified in each parasegment. These cell types have been designated 1 °, 2,° 3 °, and 4°, from anterior to posterior, with hh transcription occurring in precursors of the 1 ° cells (J. Heemskerk and S. DiNardo, supra). Differentiation of the first three cell types was shown to be dependent upon hh gene function, and it has been proposed that the fates of these cells are determined by the concentration of Hh protein, with highest concentra- tions producing the 1 ° fate, intermediate concentrations producing the 2° fate, and the lowest concentrations producing the 3 ° fate (J.
  • H329A mutant protein were extended to the function to the patteming of adult structures and signaling within imaginal discs.
  • imaginal disc hh function is required for appropriate development of pattem (J. Mohler, Genetics L20: 1061, 1988; J. J. Lee, supra; and J. Mohler and K. Vani, supra) and more recently has been shown to control progression of a wave of differentiation via induction of decapentaplegic (dpp) gene expression in the mo ⁇ hogenetic funow of the eye (U. Heberlein, et al, Cell 7_5_: 913, 1993; and C. Ma, et al, Cell _5: 927, 1993).
  • dpp decapentaplegic
  • ectopic expression of hh has also been shown to yield pattem duplications and defects and is associated with induction of ectopic expression of other signaling molecules normally expressed in a zone along the anterior/posterior compartment boundary (T. Tabata and T. B. Komberg, Cell 16: 89, 1994; and K. Basler and G. Stmhl, Nature 368 : 208. 1994).
  • a thermal cycler was utilized to subject larvae carrying heat shock-inducible hh constmcts to successive rounds of heat shock and recovery.
  • AJTOWS highlight the following features: an ectopic patch of dpp expression in the anterior compartment of wing discs in hshh H329A larvae (I); and an ectopic band of dpp expression in eye portion of the eye-antennal disc anterior to the mo ⁇ hogenetic furrow (marked by the other band of dpp expression more posteriorly) in hshh larvae (K).
  • M-O The eye phenotypes of adult control (M), hshh (N) and hshh H329A (O) flies that were shocked during larval development in a manner similar to that of the imaginal disc experiments above. Duplicated eye stmctures were observed in hshh flies, but never in hshh H329A flies. The anow in (N) points to a thin strip of cuticle between the two eye stmctures. Other deformities were also seen in hshh flies (for example, compare the thorax in N to M) .
  • Progeny were grown at 25 °C in aerated 0.5-ml microcentrifuge tubes containing yeast paste until the late second instar or early third instar stage of larval development. The larvae were then cycled continuously at 37° C for 30 minutes followed by 25° C for 90 minutes in a Perkin-Elmer thermal cycler until they reached the late third instar stage. They were subsequently dissected and stained with X-gal as described (M. Ashbumer, supra) or allowed to grow to adulthood for phenotypic analysis.
  • wg expression normally occurs in a ventral sector of the leg disc along the anterior/posterior compartment boundary while dpp is expressed in the dorsal portion of the disc along this boundary (FIGURE 7D).
  • thermal cycling of larvae carrying the wild-type hh gene produced abnormal leg disc mo ⁇ hology and extensive ectopic expression of both target genes, as previously reported for ectopic hh expression (FIGURE 7B and E), the H329A constmct produced little if any detectable difference in these patterns of expression (FIGURE 7, C and F).
  • Ectopic hh expression in the wing disc also leads to mo ⁇ hological changes and expanded expression of dpp (compare FIGURE 7, G and H), but the H329A constmct produced only an occasional small patch of anterior ectopic expression (FIGURE 71).
  • FIGURE 7N An apparently duplicated eye stmcture such as that in FIGURE 7N can be observed, with two eye stmctures separated by a thin strip of cuticle (arrow).
  • the H329A mutant protein did not induce expansion of dpp expression in either portion of the eye-antennal disc (FIGURE 7L), and does not induce eye duplications or cuticle defects in the adult (FIGURE 70).
  • the experiments described thus far comprise multiple series of larvae subjected to two days of thermal cycling followed by immediate dissection for analysis of imaginal structures or further incubation at constant temperature for analysis of adult stmctures.
  • the H329A protein appeared to have little activity in these experiments, the small patch of ectopic dpp expression induced in the wing disc (FIGURE 71, anow) suggested that some residual activity remained.
  • This suggestion was bome out in a similar experiment involving three days of cycling prior to dissection: the H329A protein clearly displayed some p-inducing activity in this experiment, presumably as a result of the higher amounts of protein that accumulated during the longer cycling period.
  • the H329A protein Although its potency is greatly reduced relative to wild-type, the H329A protein retained at least some activity in early embryonic and imaginal disc induction of wg and dpp expression; in contrast, even under heat shock conditions far more severe than those required for effects by the wild-type protein, the H329A mutant remained completely inert with respect to the re-specification of cell fates in the dorsal cuticle of the larva.
  • FIGURES 8 (A) and (B) are immunoblots of cell pellets (lane 1) or supematants (lane 2) from transfected S2 cell cultures expressing Hh protein, developed with Abl (A) and Ab2 (B). Samples in each lane were from the same volume of resuspended total culture.
  • N remained mostly associated with the cell pellet (compare lanes 1 and 2 in A)
  • C was nearly quantitatively released into the supematant (compare lanes land 2 in B).
  • U displayed partitioning properties in between those of N and C (A and B).
  • (8C) demonstrates the heparin binding activity of various Hh protein species generated by in vitro translations with microsomes. Samples were: total translation mix (lane 1); supematant after incubation with heparin agarose or agarose (control) beads (lanes 2 and 4); and material eluted from heparin agarose or agarose beads after washing (lanes 3 and 5).
  • FIGURES 8, A and B indeed show that these proteins behave differently, with most of the N fragment remaining cell-associated and all, or nearly all, of C being released into the culture supematant.
  • N but not C was retained upon the heparin agarose beads upon extensive washing with a solution that contains 0.1% Triton X-100 and 150 mM NaCl; in contrast, neither fragment was retained by unmodified agarose.
  • N, but not C binds tightly to heparin, and this behavior suggests that the low concentration of N released into culture supematants may be the result of binding to the extracellular matrix.
  • Another mechanism that might contribute to the differential release of N and C into culture supematant would be the expression in S2 cells of a receptor for N but not for C. Our cunent data can not distinguish these possibilities.
  • FIGURE 9 shows the differential localizations of N and C in embryos by in situ localization of the hh transcript.
  • FIGURE 9 (A) is shown in comparison to the distribution of N and C epitopes detected with Abl and Ab2 in panels (9B) and (9C), respectively. Note that the distribution of N and C epitopes span approximately one-third and one-half of each segmental unit respectively, while the transcript is limited to approximately one-quarter of each unit.
  • N epitopes at later stages accumulate in large punctate stmctures.
  • Our analysis here concentrates on the earlier stage, when antibody staining is weaker but before formation of the invaginations and grooves that later crease the epidermis and thereby complicate the inte ⁇ retation.
  • Ab2 was also utilized to detect C-specific epitopes with a variety of fixation and staining procedures. Although detection of C epitopes above background is more difficult than for N, we consistently observed a segmentally modulated pattem, albeit with a broader distribution than N (FIGURE 9C). This localization is also distinctive in that C epitopes at early or late stages are not found in the punctate stmctures characteristic of N.
  • the hh UE mutation encodes a prematurely tmncated protein that is missing 15 residues normally present at the COOH-terminus. Because this protein displays a much reduced efficiency in auto-proteolysis the distribution of C in this mutant background was examined.
  • FIGURE 9D shows that C epitopes in a homozygous hh ] E embryo (identified by absence of a marked balancer) are distributed in a much tighter segmental pattem than in wild-type. This localization resembles that of N, and we thus conclude that the broad distribution of C epitopes normally seen is altered in hh E by retention of the uncleaved precursor near the site of synthesis.
  • the hh protein undergoes auto-proteolysis at an intemal site to generate the predominant protein species observed in vivo. All or most of the amino acid residues required for this auto-proteolysis function map to C, the carboxy-terminal product of this intemal cleavage.
  • H329A single residue mutation that blocks auto-proteolysis of the hh protein in vitro and demonstrated that both processing and function of this protein is impaired in vivo.
  • the H329A Hh protein appears to retain weak activity in embryonic signaling to induce ectopic wg expression and, to a lesser degree, can function in imaginal disc signaling for induction of ectopic wg and dpp expression.
  • the H329 protein is completely inert when assayed for the ability to reprogram cell fates in the dorsal cuticle of the larva.
  • the assays in which the H329A protein is active or partially active involve short-range signaling that normally occurs across one or at most several cell diameters; in contrast, the H329A protein fails to exert any effect upon patteming of the dorsal cuticle, a long-range activity that normally operates across most of the segment.
  • Previous proposals to account for long-range patteming activities have suggested that hh expression induces other signaling molecules which are then responsible for executing the patterning functions (the signal relay model; see FIGURE 10A).
  • FIGURE 10 shows a signal relay versus dual function models for hh protein action.
  • the long-range effects of hh signaling are achieved indirectly through short-range induction of a second signaling molecule (X).
  • N is presumed to represent the active short-range signal while the role of C would be limited to supplying the catalytic machinery required for biogenesis of N.
  • the long- and short-range signaling functions of hh are supplied by the N and C proteins derived by intemal auto-proteolysis of the U precursor.
  • N is implicated in short-range signaling by retention near its cellular site of synthesis, while C is less restricted in its distribution and would execute long-range signaling functions.
  • auto-proteolysis is required to generate fully active signaling proteins. See text for further discussion.
  • the uncleaved H329A protein thus would carry all the residues that normally interact with a presumed receptor for N, although there might be some effect on the affinity of the interaction due to the presence of carboxy-terminal sequences, thus accounting for the decreased potency of the H329A protein.
  • the partial function of H329A protein may derive from an extremely small fraction of protein that appears to be cleaved, a very faint band with identical mobility to C appears in in vitro translations with the H329A protein (FIGURE 4, lane 3).
  • Execution of long-range functions by C is also consistent with our observations because long-range signaling might require the release of the C fragment or otherwise require the H329 residue for some function other than for cleavage.
  • N When N is synthesized from a native constmct (wild type hh), it remains primarily cell- associated (FIGURE 1 OC), however, N generated from a tmncated constmct in cultured cells predominantly enters the culture medium (FIGURE 10D) (For constmcts, see Porter, et al, Nature, 374:363, 1995). These results further confirm that autoprocessing by fragment C may regulate the degree of N association with the cell surface and therefore its range of action.
  • PCR polymerase chain reaction
  • Amplification was as follows: 94°C 5 min, addition of Taq polymerase at 75°C, followed by 94°C 1 min, 52°C 1.5 min and 72°C 1 min for 30 cycles and a final extension of 72 °C for 5 min. All PCR products were cloned into pBluescript (Stratagene) prior to sequence determination.
  • Mouse clones obtained in this manner contained 144 bases of sequence between the primer ends and were labelled with [ ⁇ "32 P]dATP and used for high stringency screens of mouse cDNA libraries made from whole 8.5 dpc embryonic RNA and from 14.5 dpc embryonic brain in the ⁇ ZAP vector (a gift from A. Lanahan).
  • Several clones conesponding to Hhg- ⁇ were isolated and the largest, 2629 bp in length (pDTC8.0), was chosen for sequence analysis using dideoxy chain termination (Sanger, et al. 1977) and Sequenase v2.0 (US Biochemicals). Compressions were resolved by using 7-deaza- guanosine (US Biochemicals). Sequence analysis made use of the Geneworks 2.0 (IntelliGenetics) and MacVector 3.5 (IB I) software packages.
  • Hhg- ⁇ The largest methionine-initiated open reading frame within this cDNA encompasses 437 codons, and is preceded by one in frame upstream stop codon. Sequence comparisons indicate that the protein encoded by Hhg- ⁇ is identical to the independently characterized mouse Shh (Echelard, et al, Cell, 75:1417-1430, 1993) except for an arginine to lysine difference at residue 122. Hhg-l also conesponds closely to the rat vhh-l gene (97% amino acid identity; Roelink, et al, Cell, 16:761-115, 1994), the chicken Sonic hedgehog
  • the overall level of amino acid identity between Hhg-l and hh carboxy-terminal to the signal sequences is 46%.
  • a closer examination shows that the amino terminal portion, from residues 25 to 187, displays 69% identity, while remaining residues in the carboxy- terminal portion display a much lower 31% identity.
  • the Hhg-l coding sequence is divided into three exons, and the boundaries of these exons are at the same positions within coding sequence as those of the three Drosophila hh exons. Curiously, the boundary between coding sequences of the second and third exons occurs near the transition from high to low levels of overall sequence conservation. The coincidence of these two boundaries suggests a possible demarcation of functional domains within these proteins. This location within Hhg-l coding sequence also coincides approximately with the site of a presumed proteolytic cleavage.
  • Partial sequence for two human hh genes has been obtained by DNA sequencing of clones derived by PCR amplification from genomic DNA with degenerate primers as outlined in Chang, et al, (Development, 120:3339, 1994) and EXAMPLE 9
  • FIGURE 11 A and B More extensive screening by the same approach, either with the same primers or with other primers from the hh coding region or with the human hh fragments seen in FIGURES 11A and B, is expected to yield at the least a third gene, and possibly more, since at least three genes are found in the mouse.
  • These segments of human hh genes can be used to obtain full coding sequences for human proteins by the following cloning method commonly used by those of skill in the art and which are extensively described in the literature.
  • ready-made cDNA libraries or RNAs from a variety of human sources including various fetal stages and organs (from abortuses) and specific infant or adult organs (from pathological or autopsy specimens), are being tested for the presence of hh sequences by PCR or RT-PCR using the primers described in Chang, et al, supra, and other primers derived directly from the sequence of the human fragments.
  • Ready-made libraries containing hh sequences are being screened directly and, where necessary, new libraries are being constmcted by standard methods from RNA sources containing hh sequences. The probe for these screens is a mixture of all the distinct human hh fragments. Sequences of cDNA clones can then be determined.
  • clones containing the probe sequences which are located in the N region, will also include a full C coding region since standard methods of library constmction result in cDNA clones that are most complete at their 3' ends. All full length /2/2-coding sequences obtained previously in vertebrates and invertebrates contain N and C sequences encoded in a single RNA. Screening is continued until complete open reading frames that conespond to all of the fragments of human hh genes are obtained. Specifically, 1.2 x 10 6 clones from a human fetal brain library (Stratagene, La Jolla, CA) was screened using a mixture of the two human hh fragments (FIGURE 11A and B) as probes. Twenty-nine clones were identified as specifically hybridizing with these probes.
  • RNA sources identified as containing hh sequences can be used as templates from anchored PCR (also refened to in the literature as RACE, for rapid amplification of cDNA ends). Briefly, this method provides a means to isolate further mRNA sequence in either the 5' or 3' direction provided that sequence is known from an intemal starting point. Anchored PCR can also be used to isolate sequences from cDNA library.
  • genomic libraries can be screened with the probes described in the first technique.
  • human hh exons and coding sequences are being identified by hybridization to previously isolated human and mouse coding sequences by sequence determination, and by exon-trapping methods to identify all hh coding sequences within genomic clones; these coding sequences can be "stitched" together by standard recombinant DNA methods to generate complete hh open reading frames.
  • FIGURE 12 A and B show in vitro cleavage reactions of a Drosophila hh protein produced in E. coli and purified to homogeneity. This protein has residues 89-254 deleted, rendering it more soluble and easier to purify. It also contains a His 6 purification tag appended to the N-terminus. Autoproteolysis of this protein is triggered by the addition of reducing agents (DTT), and the resulting product conesponds to the C fragment identified in vivo.
  • DTT reducing agents
  • Panel A shows a time course of cleavage after initiation by addition of DTT.
  • Panel B shows incubations of concentrations ranging over three order of magnitude for a fixed time period (four hours), with no difference in the extent of conversion to the cleaved form.
  • Panel C shows the sequence around the cleavage site as determined by amino-terminal sequence of the cleaved fragment C.
  • the cleavage site is denoted by the anow, and the actual residues sequenced by Edman degradation of the C fragment are underlined.
  • Panel C also shows an alignment of all published vertebrate hh sequences plus some of unpublished sequences from fish and Xenopus. The sequences shown conespond to the region of Drosophila hh where the cleavage occurs, and demonstrates the absolute conservation of the Gly-Cys-Phe sequence at the site of cleavage.
  • Panel D shows a SDS-PAGE gel loaded with in vitro transcription/translation reactions as described in the previous Examples, using various hh genes as templates, dhh is Drosophila, twhh and zfshh are the twiggy-winkle and sonic hh genes of the zebrafish, and shh is the shh/Hgh-l/vhh-1 gene of the mouse.
  • the translation mix included 35 S-labelled cysteine, used to visualize the resulting products by autoradiography. Note that each gene give a larger product (the precursor or U) and two smaller products of cleavage (N and C).
  • the larger species is C for each of the vertebrate genes, whereas the Drosophila N is larger than C due to the presence of -60 residues occuning amino-terminal to the signal sequence that are present in the vertebrate open reading frame.
  • This panel shows that vertebrate hh proteins are processed similarly to the Drosophila protein.
  • Panel E shows that Edman degradation of the C fragments releases 35 S counts on the first but not subsequent rounds for all these proteins, indicating that the site of autoproteolytic cleavage for all of these hh proteins is the amide bond to the amino-terminal side of the Cys residue that forms the center of the conserved Gly-Cys-Phe sequence highlighted in panel C. This is a generalizable approach to establish the composition of protein fragments from any other hh family members.
  • Partial sequences corcesponding to five distinct zebrafish M-like genes were isolated and the complete coding sequences for two of these genes were obtained from an embryonic cDNA library.
  • One of these two sequences is identical to that of the zebrafish nhh-I gene (Roelink, et al, Cell, 76:761, 1994), and appears to conespond to the shh gene reported by Krauss, et al, (Cell, 75:1431, 1993) (See FIGURE 13 description); the other gene, tiggy-winkle (Potter, B., The Tale of Mrs. Tiggy- Winkle, The Penguin Group, London, 1905), represents a novel vertebrate hh.
  • Coding sequences for both are shown in alignment to mouse and chicken sequences of the sonic/vhh-I class (FIGURE 13b).
  • the twhh and shh proteins contain an amino-terminal stretch of hydrophobic residues. These residues function as signal sequences since cleavage is observed when coding sequences are translated in the presence of micro- somoses; vertebrate hh genes thus appear to encode secreted proteins, as previously reported for Drosophila hh (Kimmel C.B. & Warga, R.M., Developmental Biology, 124:269-280, 1987; Warge, R.M., & Kimmel, C.B., Development, 108:569-580, 1990).
  • the first four sequences were isolated from zebrafish genomic DNA (a gift from J. Pellegrino) using degenerate primers in polymerase chain reactions as described (Chang, et al, supra), twhh and shh clones were isolated from a 20-28 hour cDNA library (a gift from R. Riggleman, K. Helde, D. Gmnwald and J. Pellegrino) using the first three sequences as probes.
  • the translational reading frames for twhh and shh were closed 12 and 16 codons, respectively, upstream of the putative initiating methionine.
  • Figure 13 shows the predicted amino acid sequences are shown in single letter code.
  • 13(a) shows sequences common to five distinct hh-like genes are shown with a dot indicating identity with the conesponding residue of zebrafish twiggy-winkle (twhh; Potter 1905; supra), hh[zfB] and hh[zfC] is more diverged and appears to represent a novel class.
  • 13(b) shows amino acid sequences of twhh and shh are aligned to those of the soniclvhh-1 class from chick and mouse (Riddle, et al, Cell, 75_: 1401-1416, 1993; Chang, D.T., et al, Development, supra; Echelard, Y., et al, Cell, 25:1431-1444, 1993).
  • Zebrafish sonic hedgehog (shh) is identical in sequence to z-vhh-I reported by Roelink, et al, Cell, 76761-115, 1994.
  • FIG. 14 shows a comparative expression of twhh, shh, and pax-2 during zebrafish embryogenesis.
  • Whole mount in situ hybridizations on 0-36 hour embryos were performed using a modification of the procedure of Tautz and Pfeifle, Chronosoma, 98:81-85, 1989, with antisense probes. Transcript localization is revealed by the pu ⁇ le product of an alkaline phosphatase enzymatic reaction.
  • shh is also expressed strongly in the protuberance
  • shh is also expressed strongly in the protuberance
  • shh is also expressed strongly in the protuberance
  • shh is expressed in cells that will form both floor plate and notochord.
  • k, 1, m Localization of pax-2 during early optic vesicle formation;
  • m also shows twhh expression,
  • k 12 hour (4-5 somites) embryo.
  • Expression of pax-2 in the developing optic vesicle is in a gradient away from the protuberance.
  • pax-2 (asterisk) at the future midbrain-hindbrain border, (m) twhh (anow) and pax-2 expression in a 6-7 somite (13 hour) stage embryo.
  • twhh in ventral neural keel (conesponding to neural tube in other vertebrates), (n-s) Embryos at end of somitogenesis (22-24 hours), (n, o, p) Localization of twhh. (n, o) Developing brain.
  • the floor plate expression is contiguous caudally along the axis, (n) Lateral view, (o) Dorsal view, (p) Lateral view of tail. Expression is restricted to the floor plate, (q, r, s) Localization of shh. (q, r) Developing brain (q) Lateral view, pax-2 expression in the otic vesicle is indicated, (r) Dorsal view. Expression in the protuberance (anowhead) and in the neural keel, (s) Lateral view of tail. Expression is strongest in the floor plate, but contrary to the report of Krauss, et al, supra., is still also in the notochord.
  • this band of twhh expression shortens along the equatorial plane and extends along the incipient embryonic axis until, by the end of gastmlation, expression occurs throughout the entire axis
  • FIGURE 14c,d Early in somitogenesis, twhh RNA is found restricted to presumptive ventral neural tissue along the entire body (FIGURE 14e, f, g), the only exception being cells in and near the tailbud (FIGURE 14g). In contrast to the neural restriction of twhh, shh is localized both to presumptive neural and notochordal cells (FIGURE 14j).
  • twhh and shh are strongly expressed in the floor plate (FIGURES 14p, s), although shh transcripts remain detectable in the notochord at this stage and at 36 hours of development (FIGURES 14s; later stage not shown).
  • FIGURES 14s later stage not shown.
  • twhh transcripts are also found in a small cluster of cells within the first gill arch (not shown), as also reported for shh at 33 hours of development (Krauss, et al, supra).
  • Embryos injected with synthetic twhh or shh mRNA exhibited numerous yet highly reproducible abnormalities in comparison to control embryos injected with lacZ mRNA. These abnormalities, discussed below, are primarily defects in the brain and eyes. Although the effects of ectopic twhh and shh expression were qualitatively similar, the incidence and severity were greater with twhh RNA (see text below, FIGURE 15 and FIGURE 16). The proteins encoded by these two genes have qualitatively similar biological activities, but apparent differences in potency.
  • FIGURE 15 shows the effects of ectopic hh on zebrafish development.
  • Wild type zebrafish, Danio rerio, Ekkwill Waterlife Resources were maintained at 28.5°C, some embryos were then cultured overnight at RT.
  • Zebrafish embryos were injected at the 1-8 cell stage with twhh, shh, or ⁇ cZRNA and examined at 28 h of development, (a-c) Dorsal view of the midbrain-hindbrain region; anterior is left, (a) lacZ. (b) twhh. (c) shh. (d-f) Frontal optical section of the forebrain region; anterior is up. (d) lacZ. (h) twhh. (f) shh.
  • pax-2 Krauss, et al, Nature, 353_:267-270, 1991; Krauss, et al, Nature, 360:87-89, 1992
  • pax2 expression at this boundary is not dismpted by hh RNA injection, however, indicating that this phenotype does not result from dismption of rostra-caudal information.
  • the eye pheno types caused by hh RNA injection resemble those produced by treatment of zebrafish and Xenopus laevis embryos with retinoic acid.
  • phenotypes range from reduction of the eye and absence of the lends to eyes with retinal folds (resembling duplicated dyes) and multiple small lenses (Manns, M. & Fritzsch, B., Neurosci. Lett., 127:150-154, 1991).
  • zebrafish exposure to retinoic acid during gastmlation interferes with the formation of the eye (Holder, N.
  • pax-2 is expressed in a gradient, with highest RNA levels in the anterior and ventral regions of the optic vesicle (Krauss, et al, supra; FIGURE 14k, 1, m).
  • the protuberance Schoss, et al, supra
  • FIGURES 14e, f, h, i, m both twhh and shh but not pax-2 are strongly expressed
  • the concentra- tion gradient of pax-2 expression in the eptic vesicle thus appears to incline downward from its maximum at a location adjacent to the site of twhh and shh expression in the protuberance.
  • Supe ⁇ osition of developmental fate within the optic vesicle suggests that the gradient of pax-2 RNA prefigures the future proximal/distal axis of the eye.
  • Ectopic hh alters the expression of pax-2, pax-6, and F-spondin.
  • Zebrafish embryos were injected at the 1-8 cell stage with twhh or shh RNA and the pattem of pax-2, pax-6, or F-spondin expression was examined by whole mount in situ hybridization. Control embryos injected with lacZ RNA were performed in every case and displayed wild-type expression patterns.
  • the anterior-posterior axis of the optic vesicle corresponds to the future proximal-distal axis of the eye.
  • the posterior edge of the optic vesicle will separate from the diencephalon (Schmitt and Dowling, Comp. Neur., 344:532-542, 1994).
  • hh RNA Injection of either hh RNA causes uniform initiation of pax-2 expression along both the proximal-distal and dorsal-ventral axes of the optic vesicle as it begins to evaginate.
  • the ectopic pax-2 expression appears at the same time as normal pax-2 expression is initiated in the eye, and in some cases, is also seen in the diencephalon between the optic vesicles.
  • pax-2 RNA in hh injected embryos is detected in all but the most distal portion of the optic vesicle.
  • pax-6 which encodes a transcription factor critical for eye development was also studied.
  • pax-6 is normally expressed in the lens and in most of the distal part of the optic cup (Krauss, et al, supra; Puschel, et al, Development, 1 14:643-651, 1992).
  • pax-6 is repressed in the optic vesicle, although many embryos retain pax-6 expression in the most distal cells.
  • pax-2 and pax-6 as markers of positional identity
  • hh expression in the optic vesicle can be characterized as inducing proximal fates and repressing distal fates.
  • the distal part of the optic vesicle is the most refractory to z z-induced changes in both pax-2 and pax-6 gene expression. Due to a later rotation, this distal portion of the optic vesicle will give rise to the dorsal portion of the mature eye (Schmitt, et ⁇ l, supra); interestingly, this is the portion of the eye that remains in 3-day old injected embryos with intermediate phenotypes (see above).
  • pax-6 Lesions in the pax-6 gene have been assigned as the basis for the Aniridia (Ton, et al, Cell, 67:1059-1074, 1991 ; Glaser, et al, Nat. Genetics, 2:232-239, 1992), Small eye (Hill, et al, Nature, 354:522-525, 1992), and eyeless mutations (Quiring, et al, Science 265:785-789, 1994), in humans, mice and Drosophila, respectively; pax-6 function thus appears to be critically required for eye development in Drosophila and mammals.
  • ventral neural stmctures fail to form and the developing eyes fuse at the midline,, yielding an embryo with a single eye (Hatta, et al, Nature, 350:339-341, 1991).
  • the missing ventral stmctures in cyclops mutants include the regions where we observe expression of twhh and shh, and we therefore examined the effects of the cyclops mutation on hh expression.
  • cyc bi6 Hatta, et al, Nature, 350:339-341, 1991
  • heterozygous adults a kind gift of R. Riggleman
  • twhh RNA is only expressed in the presumptive tailbud (caret) of eye embryos.
  • caret the presumptive tailbud
  • twhh RNA in cyclops embryos is found only in a small patch of cells at the presumptive tailbud and neural expression was not detected at any later stage examined. Neural expression of shh is also lost in eye mutants, although expression in the notochord is reunited (Krauss, et al, supra; data not shown).
  • this mutation can be used as a genetic tool to examine the requirement for hh function in eye development. Iiatta, et al; Hatta, et al, Proc. Natl Acad. Sci. USA, 9 2061-2065, 1994), recently demonstrated that pax-6 expression is fused at the midline due to loss of ventral midline cells that normally do not express pax-6 and, in addition, pax-2 expression in the fused eye of eye mutant embryos is reduced. We extended these observations to an earlier stage when the optic vesicles first form and found that pax-2 expression is weak and fails to extend within the vesicles in eye mutants.
  • twhh The ventralizing activities of twhh confirm and extend those previously reported for shh/vhh-1 class genes of chicken, zebrafish, and rat (Echelard, et al, supra; Krauss, et al, supra; Roelink et al, supra).
  • the early restriction of twhh to midline neural progenitors suggests that it may play a specific role in the homeogentic mechanisms of floor plate maintenance and expansion (Placzek, et al, Dev., 117:205- 218, 1993).
  • the floor plate retains auto-inductive potential long after the loss of floor plate inducing properties by the notochord, despite continued expression of shh/vhhl in the notochord (Roelink, et al, supra; Placzek, et al, supra; Yamada, et al, Cell, 7_3_:673-686, 1993).
  • twhh twhh class
  • expression of other hh homologues in pattems more like those of twhh might help explain these discrepancies.
  • Endogenous hh protein in Drosophila is fund predominantly as an amino- and a carboxy- terminal fragment (N and C, respectively) derived by an intemal auto-proteolytic cleavage of a larger precursor (U for uncleaved), which also occurs in vivo but at lower levels (Lee, et al, supra).
  • Determinants within the amino-terminal domain appear not to be required for auto-proteolytic activity, whereas mutations affecting the carboxy- terminal domain can block auto-proteolysis and reduce activity in vivo (Lee. et al, supra).
  • the auto-proteolysis is blocked by a substitution of alanine for the histidine normally present at position 329. This histidine is absolutely invariant in alignments of all known hh genes, and its sequence context suggests a catalytic role in auto-proteolysis (Lee, et al, supra).
  • FIGURE 17 shows zebrafish twiggy-winkle hedgehog derivatives.
  • 17(a) Cartoons of various twhh open reading frames.
  • SS (shaded) is the predicted N-terminal signal sequence for secretion of these proteins and encompasses the first 27 amino acids of each open reading frame.
  • the anow indicates the predicted intemal site of auto-proteolytic cleavage.
  • Amino acid residue numbers are according to Figure 13b.
  • the filled triangle denotes the normal termination codon for the twhh open reading frame.
  • Constmct U HA contains a mutation that blocks auto-proteolysis (the histidine at residue 273 is changed to an alanine; see Lee, J.J., et al, supra).
  • Constmct U356 HA contains a stop codon in place of amino acid residue 357 as well as the H273A mutation in U HA .
  • Constmct N encodes just the first 200 amino acids of twhh.
  • Constmct C has had the codons for residues 31-197 deleted.
  • 17(b) shows in vitro translation of the expression constmcts shown schematically in part a. Constmcts were translated in vitro in the presence of 35 S methionine and analyzed by autoradiography after SDS-PAGE. The protein products are shown schematically to the left.
  • Lanes 1 and 6 Auto-proteolysis of the full-length (U ss ) protein creates two fragments, an N-terminal fragment (N ss ) and a C-terminal fragment (C).
  • Lane 2 Constmct U HA only makes an uncleaved form of twhh protein that comigrates with U ss twhh via auto-cleavage.
  • Lane 5 Constmct C encodes processed and unprocessed forms which are visible as two bands migrating closely together. The bottom band is the C protein made from auto-proteolysis of the U ss ( ⁇ 31-197). All constructs were made by in vitro mutagenesis of expression constmct T7TStwhh (see FIGURE 15) using the method of RPCR. The sequence of all constructs were made by in vitro mutagenesis of expression constmct T7TStwhh (see FIGURE 15) using the method of RPCR. The sequence of all
  • constmcts were confirmed by dideoxy sequencing. In vitro translations were performed according to manufacturer's instmctions (Promega).
  • the vertebrate hh proteins encoded by shh, twhh and mouse-shh/Hhg ⁇ l also undergo auto-proteolysis to yield two smaller species from a single larger precursor (Lee, et al, supra; Chang, et al, supra; see lanes 1 and 6 in FIGURE 17b).
  • the invariant histidine to alanine mutation to generate a constmct encoding a form of the twhh protein that is not auto-proteolytically cleaved (U HA ).
  • constmcts that produce either the amino- or the carboxy-terminal domains of twhh (N and C, respectively; see lanes 4 and 5 in FIGURE 17b); constmcts are schematically diagrammed in FIGURE 17a).
  • constmcts are schematically diagrammed in FIGURE 17a.
  • the uncleaved U HA protein is only somewhat less active than C in inducing pax-2, but it also was not able to repress pax ⁇ efficiently (FIGURE 16). The latter is particularly notable since the U HA protein ⁇ 35 ⁇ ; see FIGURE 17a, b) has activities not significantly different from N (FIGURE 16).
  • the C-terminus also contains a domain inhibitory to N-terminal function when in the context of the uncleaved hh protein.
  • the C-terminus can also inhibit N action by an intermolecular mechanism (Lai, et al, supra).
  • N and C derivatives of the Drosophila hh gene may offer some insight.
  • the Drosophila N derivative is retained close to its embryonic site of synthesis in a segmentally striped pattem (Tabata and Komberg, Cell, 26:89-102, 1994; Taylor, et al, Mech. Dev., 42 89-
  • Drosophila N and C may account for the short and long range nature of the functions associated with hh during Drosophila development.
  • tissue distributions of zebrafish N and C are not known, their activities in ectopic expression assays are also suggestive of short- and long-range functions when considered in the context of normal expression pattems of hh, pax-2 and pax-6.
  • the normal gradient of pax-2 expression in the optic vesicle extends a substantial distance from its maximum adjacent to the site of hh expression in the protuberance; the ability of ectopic C to activate pax-2 therefore suggests that, consistent with the distribution of C in Drosophila, zebrafish C may carry out a long-range function.
  • Repression of endogenous pax-6 expression in contrast, appears to be a short-range function since pax- 6 expression occurs close to endogenous hh expression.
  • Efficient repression of pax-6 is an attribute of constmcts producing N, and a short-range function for N would be consistent with the distribution of N in Drosophila.
  • Two types of /z/z-dependent activity have been reported for /z/z-transfected cultured cells.
  • One is the apparent contact-dependent induction of floor plate markers (Roelink, H., et al, Cell 76:761-775, 1994); the second induction of sclerotome markers in presomitic mesoderm, is diffusible and acts at long-range.
  • cDNAs encoding full-length Xenopus hedgehogs, or encoding amino terminal or carboxy terminal domains linked to secretory leader sequences were transcribed in vitro to yield translatable messenger RNA.
  • the synthetic messenger RNAs, and control mRNAs, were microinjected into the animal poles of cleavage stage Xenopus embryos, which were allowed to develop to the blastula stage, at which time the animal cap explants were prepared from the upper one fourth of the embryo. These blastula cap explants were then cultured in vitro in physiological saline in the presence or absence of the transforming growth factor beta family member, recombinant human activin A.
  • Explants were then extracted to yield mRNA by methods commonly used by those of skill in the art, which was used as template with reverse transcriptase to yield cDNA.
  • the cDNA was then used as template with various sets of primers for PCR for specific genes, reverse-transcriptase-polymerase chain reaction, or RT-PCR. This results in specific amplification of radioactive products which are diagnostic for the presence and level of the messenger RNAs which were present in the explants. Samples were separated on polyacrylamide gels, which were exposed to X-ray film to yield the bands shown in the figures. Thus, the darker bands conespond to a greater level of the specific mRNA.
  • FIGURE 18A and B demonstrate that hedgehog induces pituitary and anterior brain genes, and can cooperate with activin or with neural inducers such as noggin and follistatin which are induced by activin to elevate expression of these genes in explanted embryonic tissue. All odd numbered lanes lack reverse transcriptase in the RT-PCR reaction and are negative controls. All even numbered lanes have this enzyme, and thus give specific bands to mRNA.
  • Lanes 1-2 are control blastula caps
  • lanes 3-4 are Xenopus hedgehog-expressing blastula caps
  • lanes 5-6 are control blastula caps treated with activin
  • lanes 7-8 are hedgehog-expressing blastula caps treated with activin
  • 9-10 are prolactin-expressing blastula caps treated with activin to serve as a control for simply expressing a secreted protein in the blastula cap.
  • XAG 1 is a cement gland marker
  • XANF1B is a pituitary marker
  • otx-A is an anterior brain marker
  • en-2 is a midbrain- hindbrain boundary marker
  • krox 20 is a rhombomere-specific hindbrain marker
  • HIHbox 6 is a posterior hindbrain marker
  • NCAM is a general neural marker
  • activin is a control for mesoderm induction
  • elongation factor is a positive control to shown that all even numbered lanes did in fact have cDNA present.
  • the panel labelled XANF1B detects a pituitary gene.
  • Lane 4 panel A shows that hedgehog induces this pituitary marker, and thus likely pituitary cell types, in blastula cap explants (see also FIGURE 20, lane 6, for a stronger signal showing this), when compared to control explants in the absence of hedgehog (lane 2), which do not express this gene.
  • Lane 6 shows that explants treated with activin, in the absence of hedgehog, also express the pituitary gene.
  • Lane 8 shows that explants treated with both hedgehog, and with activin, give highest levels of the pituitary gene.
  • Lane 10 proves that this effect of hedgehog is specific, since prolactin, another secreted protein, does not lead to this elevated level of pituitary gene.
  • the panel labelled OTX-A detects this anterior brain gene. Lane 4 (and 6 in Figure 20) shows that hedgehog can induce this neural-specific gene. Lane 8 shows that the level of this neural gene is highest in tissue treated with both activin and hedgehog, relative to hedgehog alone (lane 4), or activin along (lane 6), and control explants do not express this gene (lane 2). Again, this effect is specific to hedgehog, since prolactin (lane 10) did not lead to elevated expression of this gene.
  • the panel labelled XAG-1 detects a cement gland-specific gene, and lane 4 shows that hedgehog induces this gene at high level.
  • N behaves like X-bhh in that it induces elevated levels of XANF-2 and Otx-A (lane 6) relative to control activin-treated animal caps (lane 4). Moreover, N also leads to a decrease in the expression of more posterior markers, such as krox-20 and XlHbox-6, as observed following injection of X-bhh. In contrast to the activity of N (Fig. 4C, lane 6), ⁇ N-C decreases the expression of the anterior neural genes XANF-2 or Otx-A (Fig. 4C, lane 8) in activin-treated animal caps when compared to uninjected controls (lane 4). Moreover, ⁇ N-C also leads to an increase in the expression of more posterior markers, such as En-2 and Xlhbox-6.
  • FIGURE 19 shows X-bhh modifies the anteroposterior pattem of neural gene expression in explants under the influence of endogenous neural inducers.
  • XAG-1 is a cement gland marker
  • XANF-2 is an anterior pituitary marker
  • Otx-A is a forebrain marker
  • En-2 demarcates the midbrain-hindbrain boundary
  • XlHbox-6 is a spinal cord marker.
  • N-CAM is a general neural marker whose expression is not restricted along the anteroposterior axis. The EF- 1 ⁇ control demonstrates that a comparable amount of RNA was assayed in each set. Note that expression of XAG-1 and anterior neural markers is stimulated by X-bhh treatment, whereas expression of posterior neural markers is suppressed.
  • FIGURE 20 demonstration of differential activities of N and C domains of hedgehog proteins.
  • odd numbered lanes are negative control lanes, and positive numbered lanes show specific gene expression for the markers described above.
  • the N domain of hedgehog is encoded in the constmct called Xhhl208 (lane 8), and the C domain is encoded in the constmct called Xhhl delta 27-208 (lane 10).
  • the constmct Xhhl l-1270A (lane 12) is specifically mutated so that it is unable to undergo self- processing.
  • the ability of the N and C domains to induce the genes described above is compared to control blastula cap explants (lane 4), entire embryos as a positive control (lane 2), blastula cap explants expressing a mutated hedgehog as a negative control (lane 14), blastula caps expressing the entire hedgehog 1 (lane 6), and blastula cap explants treated with an independent neural inducer, noggin (lane 16) (discovered by Richard Harland at University of California at Berkeley).
  • FIGURE 18 The results in FIGURE 18 are novel insofar as they establish that the activity of hedgehog in inducing a pituitary gene, and an anterior brain gene, may be enhanced by the TGF ⁇ family of growth factors.
  • This enhancement likely applies to the N and C domains described in FIGURE 20, since the genes analyzed are the same.
  • This enhancement is due to hh synergizing with neural inducing factors which are themselves induced by TGF- ⁇ family members, including but not limited to such molecules as noggin and follistatin.
  • the data in FIGURE 20 makes several important points.
  • any clinical or diagnostic uses of hedgehog might be improved by use of the N or C domain, as one generally wishes to use the smallest protein which has an activity for clinical work, as it is less likely to evoke adverse immune responses, or other adverse side effects.
  • the data show that the C domain is better than the N domain in inducing pituitary gene expression and, since it has less induction of cement gland genes that intact hedgehog, or N domain, it suggests that the C domain might be useful in clinical situations where one wishes to enhance the development or expression of the pituitary as specifically as possible.
  • FIGURE 20 shows clearly that while both hedgehog and noggin can induce pituitary gene expression, hedgehog is more specific, since hedgehog does not induce the general neural marker NCAM, whereas noggin induces NCAM as well as pituitary.
  • FIGURE 21 shows ⁇ N-C interferes with X-bhh and N activity in animal cap explants.
  • Embryos were injected with various RNAs, animal cap explants were cultured until sibling embryos reached tailbud (stage 25), at which time RT-PCR was used to analyze the expression of the cement gland marker XAG-1 and the control RNA, EF-l ⁇ .
  • Lanes 1 , 2 control animal caps from uninjected embryos.
  • Lanes 3, 4 animal caps from embryos injected with both X-bhh and prolactin RNAs.
  • Lanes 5, 6 animal caps from embryos injected with box X-bhh and ⁇ N-C.
  • Lanes 7, 8 animal caps from embryos injected with both N and prolacting RNAs.
  • Lanes 9, 10 animal caps from embryos injected with both N and ⁇ N-C.
  • ⁇ N-C An intemal deletion of X-bhh ( ⁇ N-C) blocked the activity of X-bhh and N in explants and reduced dorsoanterior stmctures in embryos. As elevated hh activity increases the expression of anterior neural genes, and as ⁇ N-C reduces dorsoanterior stmctures, these complementary data support a role for hh in neural induction and anteroposterior patteming.
  • ⁇ N-C deletes amino acids 28-194 of X-bhh.
  • the primary translation product is predicted to undergo signal sequence cleavage removing amino acids 1-23, and to undergo autoproteolysis.
  • autoproteolysis Based on the cleavage site in Drosophila hh (Porter, et al, Nature, 324:363, 1995) autoproteolysis would generate a C domain of X-bhh amino acids 198- 409, as well as a predicted seven amino acid polypeptide, representing amino acids 24- 27, and 195-197 (Lai, et al, Development 121:2349, 1995).
  • Analysis of the effect of ⁇ N- C on neural markers was by standard methods including Northern blot analysis and in situ hybridization (Lai, et al, supra, inco ⁇ orated herein by reference).
  • ⁇ N-C does not induce the cement gland marker XAG-1, it decreases the expression of anterior ectodermal and neural markers in activin-treated animal caps. Thus, ⁇ N-C has the capacity to affect neural patteming. ⁇ N-C also promotes an increase in posterior neural markers in activin-treated animal caps. Mixing ⁇ N-C with N or full length X-bhh at a 1 :1 ratio led to a dramatic inhibition of the induction of cement gland in animal cap assays, supporting the hypothesis that ⁇ N-C interfered with X-hh.
  • Hh autoprocessing causes the covalent attachment of a lipophilic adduct to the COOH-terminus of Hh-N p (J.A.Porter et al., Cell 86, 21, 1996).
  • This modification is critical for the spatially restricted tissue localization of the Hh signal; in its absence, the signaling domain exerts an inappropriate influence beyond its site of expression (J.A.Porter et al., Cell 86, 21, 1996).
  • Physical and biochemical characterization of this lipophilic adduct indicates that it is not the glycosyl phosphatidyl inositol (GPI) anchor, the only other known lipophilic modification associated with secreted cell surface proteins in eukaryotes (S. Udenfriend and K. Kodukula, A nnu. Rev. Biochem. 64, 563, 1995; and P.J.Casey, Science 268, 221, 1995).
  • GPI glycosyl phosphatidyl inositol
  • Figure 22 shows lipid stimulation of Hh autoprocessing in vitro.
  • Panel A illustrates the mechanism of Hh processing. The reaction is initiated by formation of a thioester between the thiol side chain of cysteine 258 and the carbonyl carbon of glycine 257, and N to S shift. This activated intermediate then undergoes a nucleophilic attack by DTT in vitro or by a piophilic nucleophilic in vivo resulting in cleavage as well as a formation of a covalent adduct at the carboxy-terminus of the amino-terminal product, X denotes the attacking nucleophilic.
  • Panel B shows a coomassie blue stained SDS-polyacrylamide gel showing in vitro autocleavage reactions of the bacterially expressed His6Hh-C protein ( ⁇ 29kD) incubated for 3 hours at 30°C with no additions (lane 1), 50 mMDTT (lane 2), 1 mMDTT (lane 3), or 1 mMDTT plus bulk S2 cell lipids (lane 4).
  • the Hh-C product of the autoprocessing reaction migrates as an ⁇ 25kD species (lanes 2 and 4); the ⁇ 5kD NH2-terminal product is not resolved in this gel.
  • Figure 23 A is a thin layer chromatog- raphy (TLC) plate coated with silica gel G (Merck) showing the fractionation of bulk S2 cell lipids using a heptane:ether:formic acid solvent (80:20:2).
  • TLC thin layer chromatog- raphy
  • Figure 23B is a Coomassie blue-stained SDS-polyacrylamide gel showing in vitro autocleavage reactions of the bacterial expressed His 6 Hh-C protein incubated with 1 mMDTT plus either un- fractionated S2 cell lipids (lane 1), or spots A through F (lanes 2-7, respectively). Addition of lipid spot B but no other resulted in processing of His 6 Hh-C protein.
  • Figure 23 C is TLC of S2 cell lipids (lane 1) along with selected lipid standards: phospha- tidylcholine (lane 2), a diacylglycerol (lane 3), cholesterol (lane 4), stearic acid (lane 5), a triacylglycerol (lane 6), and cholesteryl ester (lane 7).
  • Lipid spot B comigrates with cholesterol, as also demonstrated by mixing radio-labeled cholesterol with S2 lipids before TLC fractionation.
  • Figure 23D is a Coomassie blue stained SDS-polyacrylamide gel showing that relative to 1 mMDTT alone (lane 1) cholesterol (0.35 mM) + 1 mMDTT (lane 2) stimulates His 2 Hh-C autocleavage in vitro.
  • Figure 23E is an autoradiogram of electrophoretically-resolved products of His 6 Hh-C autocleavage reactions driven by 20 mMDTT (lane 1) or 1 mMDTT+0.35 mM cholesterol (lane 2).
  • the active S2 cell-derived lipid displayed the same mobility as cholesterol in two other solvent systems and gave a positive color test when sprayed with a specific reagent that reacts with sterols;W.W. Christie, Lipid analysis (Pergamon, Oxford, ed.2nd, 1982); and R.R.Lowry, Journal of Lipid Research 9, 397, 1968).
  • W.W. Christie Lipid analysis (Pergamon, Oxford, ed.2nd, 1982); and R.R.Lowry, Journal of Lipid Research 9, 397, 1968).
  • the active lipid component is in the sterol fraction of the S2 lipids.
  • cholesterol which is the principal sterol in eukaryotic cell membranes (W.W. Christie, Lipid analysis (Pergamon.
  • the part of the sterol most likely to act as attacking is the 3 ⁇ hydroxyl. Such an attack would leave cholesterol as a covalent adduct in ester linkage to the carboxylate of the terminal residue of the NH2-terminal fragment (GLY 257).
  • Figure 24A shows Coomassie stained gels of His 6 Hh-C autocleavage reactions canied out in the presence of 20 mMDTT (lane 1). or 1 mMDTT+0.35 mM cholesterol (lane 2). Lane 3 contains a mixture of the samples loaded in lanes 1 and 2. The amino-terminal product of the cholesterol driven reaction migrates approximately 2 kD faster than the DTT-driven reaction fragment.
  • Figure 24B is Coomassie stained gels showing protein products of His 6 Hh-C autocleavage reactions canied out in the presence of 1 mM DTT+0.35 mM cholesterol (lanes 1 and 2) or with 20 mM DTT (lane 3).
  • FIG. 24C is an autoradiogram of immunoblotted Hh amino-terminal domains purified from cultured S2 cells.
  • Amino- terminal domains were derived either from a constmct tmncated after glycine 257 (Hh-N lanes 1,3,4,8, and 9) or from a constmct encoding wild-type Hh that produces the amino- terminal domain via the processing reaction (Hh-N p , lane 2,3,5,6,7,8, and 9). Proteins were either directly loaded (lanes 1 and 2) or base-treated (M.C. Field and A.K. Menon, in Lipid modification of proteins N.M. Hooper, A.J. Turner, Eds.
  • Lane 3 contains a mixture of the samples loaded in lanes 1 and 2
  • lane 8 contains a mixture of the samples loaded in lanes 7 and 4
  • lane 9 contains a mixture of the samples loaded in lanes 7 and 2.
  • Ester bonds are subject to hydrolysis in alkaline conditions and base treatment prior to electrophoresis indeed reduced the migration of the cholesterol-driven reaction product to a position coinciding with that of the dithiothreitol-driven reaction product. These results are consistent with stimulation of the in vitro processing reaction by direct nucleophilic attack of cholesterol on the thioester intermediate to form an ester-linked adduct. If processing of Hh also results in formation of an ester-linked cholesterol adduct in vivo, then the protein-lipid linkage should be subject to base hydrolysis with a concomitant shift in electrophoretic mobility of the protein (normally 18.5 kD).
  • the immunoblot in Fig 24C shows the base-induced appearance of a species of reduced mobility (19.5 kD), which increased in abundance from - 1/3 of the total after five minutes of treatment to most of the immunoreactive protein after one hour.
  • This novel species comigrated with tmncated, unprocessed Hh-N, which is not affected by base treatment.
  • FIG. 25A is an audioradiogram of a gel loaded with total cell proteins from S2 cells containing a stably integrated Cu++-inducible hedgehog gene. Prior to harvesting, these cells were grown in media supplemented with [ 3 H]cholesterol in the absence (lane 1) or presence (lane 2) of 1 mM CuS04. [ 3 H]cholesterol inco ⁇ oration is dependent upon Cu++ induction (lane 2) and is restricted to a single protein species migrating at a position conesponding to Hh-N p .
  • Figure 25B is an HPLC profile of sterols separated on a C18 column by isocratic elution with a solvent containing methan- ol:ethanol:water (86:10:4) (R.J. Rodriguez and L.W. Parks, Methods of Enzymology 11 1, 37, 1985). -5 ⁇ g of each sterol was mixed, loaded, and elution monitored by absorbance at 210 nM. The stmcture of cholesterol is shown above cholesterol peak.
  • sterols include: 1) 10 desmosterol, which contains one additional double bond between carbon 24 and 25; 2) 20 7-dehydrocholesterol, which contains one additional double bond between carbon 7 and 8; 3) campesterol which contains an additional methyl group on carbon 24; and 4) sitosterol, which contains an additional ethyl group on carbon 24.
  • Figure 25C shows HPLC analysis as in (B) of the adduct released by base treatment of Hh-N p metabolically labeled with [ 3 H] cholesterol (A). The radioactive species recovered from the metabolically labeled protein collates with cholesterol.
  • Figure 25D shows metabolic labeling of vertebrate Sonic hedgehog protein with [ 3 H] cholesterol.
  • COS-7 cells were incubated in culture medium supplemented with [ 3 H]cholesterol for 24 hours prior to and 36 hours after transfection (COS-7 cells grown at 37°C in DMEM supplemented with 10% fetal calf serum were plated at -35% confluence onto two 35 mm dishes in 1 ml of Optimem media (Gibco) containing 1.5% fetal bovine sera and 25 ⁇ Ci of [ 3 H]cholesterol, giving ⁇ 40 ⁇ g/ml as the final concentration of cholesterol with a specific activity of 2Ci/mmol (labeling medium).
  • Optimem media Gibco
  • the labeling medium was removed and the cells were transfected for 6 hours with Shh or Shh-N expression constmcts using lipofectamine (Gibco) and serum-free DMEM media. After transfection, 1 ml of fresh labeling medium was added to each dish and the cells were incubated for 36 hours at 37°C. The cells were then harvested without washing, lysed on the plate with Tris buffered saline plus 1% Triton X-100 and the total cell proteins were precipitated with acetone, washed and analyzed as described above for the S2 cell proteins). A strongly labeled species with the Shh but not Shh-N constmct. Several other less heavily labeled species are apparent in both lanes, and may represent other cholesterol-modified proteins.
  • the protein pellet was resuspended in 2% SDS in H 2 0 and reprecipitated with acetone several times to remove uninco ⁇ orated radioactivity prior to loading onto SDS polyacrylamide gels for analysis.
  • Initial labeling experiments in which 25 ⁇ Ci of cholesterol was added resulted in - 10 fold decrease in extent of label inco ⁇ orated into the inducible Hh-N p protein).
  • uninduced cells showed no inco ⁇ oration of [ 3 H] cholesterol into cellular proteins
  • cells induced to express Hh showed a single strong band with a mobility conesponding to that of Hh-N p (Fig 25 A).
  • the amount of radioactive cholesterol inco ⁇ orated is consistent with that expected if all of the Hh-N p synthesized upon induction received a cholesterol adduct (The specific activity of [ 3 H]cholesterol in the S2 cell labeling medium was -5 Ci/mmol. Assuming after a 24 hour doubling time that this concentration approximately represents that within the S2 cell membrane, then any protein subsequently expressed and receiving cholesterol as an adduct would also be labeled at the same specific activity.
  • Hh-N p As determined by standardized coomassie blue staining, -50-100 ng or 2.5 to 5 picomoles of Hh-N p is produced by one 35 mm dish of S2 cells containing the Cu ++ -inducible Hh constmct during 24 hours of induction with ImM CuS0 4 (13). This predicts - 12.5 to 25 nCi or 2.75 x 10 4 to 5.5 x 10* dpm of radioactivity would be inco ⁇ orated into Hh-N p protein produced in our labeling experiment assuming it is cholesterol modified.
  • Hh-N p be treated with CNBr/70% formic acid, i.e. full length Hh-N p could not be detected.
  • the mass discrepancy noted above could be accounted for by the net addition of formic acid (45 daltons) during CNBr digestion.
  • This reaction could involve the addition of H 2 0 across the 5,6 double bond of cholesterol, a common reaction of secondary alkenes in strong acids [R.T. Morrison, R.N. Boyd, Organic Chemistry (Allyn and Bacon, Boston, ed.3rd, 1973)], followed by esterification of formate via this newly formed alcohol [B.I. Cohen, G.S. Tint, T.Kuramoto, E.H.
  • Hh-C processing domain functions as a cholesterol transferase; as a result of this activity, a cholesterol adduct is attached via an ester linkage to the COOH-terminus of the NH 2 - terminal signaling domain of the Hh protein.
  • COS-7 cultured green monkey kidney cells
  • [ 3 H]cholesterol and transfected with expression constmcts containing (i) the full length murine Sonic hedgehog (Shh) open reading frame, leading to production of an autocatalytically processed signaling domain (Shh-N p ) or (ii) Shh coding sequences precisely tmncated at the site of cleavage, thus producing an unprocessed amino terminal signaling domain (Shh-N)
  • fetal calf semm were plated at -35% confluence onto two 35 mm dishes in 1 ml of Optimem media (Gibco) containing 1.5% fetal bovine sera and 25 ⁇ Ci of [ 3 H]cholesterol, giving ⁇ 40 ⁇ g/ml
  • the labeling medium was removed and the cells were transfected for 6 hours with Shh or Shh-N expression constmcts using lipofectamine (Gibco) and serum-free DMEM media. After transfection, 1 ml of fresh labeling medium was added to each dish and the cells were incubated for 36 hours at 37 °C. The cells were then harvested without washing, lysed on the plate with Tris buffered saline plus 1% Triton X-100 and the total cell proteins were precipitated with acetone, washed and analyzed as described above for the S2 cell proteins).
  • sterols were extracted and analyzed by HPLC from COS7 cells metabolically labelled with [ 3 H]-mevalonic acid in the presence or absence of jervine, a teratogenic plant steroidal alkaloid. In the presence of 28mM jervine, radiolabelled cholesterol levels were reduced and another radiolabelled sterol was found to accumulate. On the basis of its retention time in this reverse phase HPLC method, this abnormal sterol is tentatively identified as zymosterol, an intermediate in the cholesterol biosynthetic pathway.
  • Treated cells synthesized reduced levels of cholesterol and accumulated increased levels of another sterol that we have provisionally identified as the cholesterol precursor, zymosterol.
  • the natural product jervine at these concentrations thus inhibits cholesterol biosynthesis in cultured cells in much the same manner as the synthetic dmgs discussed above, although the specific enzyme(s) affected appear to differ. Given the similarities in their teratogenic effects, this inhibition seems likely to underlie the teratogenic effects of both the synthetic and natural compounds.
  • Procejn expression and purifjcnrjoji Drosophila melanogaster Hh pro t ein in which most of the amino-tcrr ⁇ i ⁇ al signaling domain and signal sequence have been replaced by a hexa-histidine ra ⁇ r (His 6 Hh-C 2 i) was expressed as previously described (Po ⁇ er e ⁇ al., 1 995). SeMet His 6 H' ⁇ -C-. ⁇ was prepared by expression in £ coli strain B334 (DE3 ) pLysS, a methionine auxotroph, and " growth in minimal media as previously described (Leahy et al., 1994).
  • This His ⁇ -ca ⁇ scd protein was purif i ed cn a Ni ⁇ -NTA agarose column and autocleavage stimula t ed by addition of 50 mM DTT. .A ter removal of the DTT by dialysis, the cleaved protein was passed over a N . --NTA agarose column and the Hh carboxy-terminal domain, Hh-C ;5 , collected in t he column ran through. Hh-C ⁇ was subjected to limited proteolysis by overnight incubation with 1 :500 ( :w) subtilisin (Bcehringer Mannheim) at 4°C.
  • a procease-siablc fragment of approximately 17 kDa, Hh-C 17 was identified by SDS-PAGE and purified by anion-exchangc chromatography utiiizins a Mono-Q column (Pharmacia).
  • 7 were determined to be Cys-258 and Ser-408, respectively, by mass spectral analysis of cyanogen bromide-cleaved fragments. Mass spectral analysis was performed as previously described (Po ⁇ er et l., 1996a).
  • Crystallization Crystals were grown from hanging drops by the method of vapor diffusion (Wlodawe et al., 1575). £ ⁇ l c f a 1 .4 mg/ml solution of Hh-C, 7 in 1 .4 mM ⁇ -mercapto ⁇ thanol were mixed with 2 ui cf a ] : 1 dilution cf resarvoi: solution (20% P ⁇ C 3350, 0 mM ammonium sulfate, and 10 mM sodium cacodyia.e, pH 5.2) with distilled water and equilibrated over the reservoir solution.
  • Data collection and p rocessing All data were collected from crystals soaked in mother liquor made 10% (w/v) ethylene glycol and flash frozen in a gaseous nitrogen stream at - 180°C. MAD data were collected at four wavelengths from a single SeMet crystal at beamli ⁇ e X-4A of the National Synchrotron Light Source at Brookhaven National Laboratory. Data were collec t ed using Fuji HR-V phosphor-imaging plates a ⁇ d digitized using a Fuji BA-3000 scanner. 2° oscillations at ⁇ and ⁇ +1S0° ere collected with no overlap for each oscillation range a t each wavelength.
  • MAD phase determinations were made with the program MLPH RE (Collaborative Computational Project, 1994; Ramakrishnan and Biou. 1997), and solvent-flattening and histogram-matching were per ormed wi:h the proeram DM (Collaborative Computational Project, 1994).
  • An atomic mode! consisting of Cys-258 to Tyr- 401 was readily built into electron density maps computed with MAD-derived phases for reflections in the range 20.0-2.0 A using the program "O" (Jones et al., 1991).
  • Drosophila mclann paster Hh in which the signal sequence and most cf the amino-terminal signaling domain have been replaced by a hexahistidine tag was expressed in £. coli as previously described (Porter et al., 1995). FoUowing purification ith N.--- ⁇ A agarose, this protein cleaves itself i vitro in the presence of either DTT or cholesterol tc liberate the 25 kDa Hh-C fragment (H -C ⁇ , residues Cys-258 to Asp--71 ). Hh-Ci prepared by this method was found to be poorly soluble in the absence of detergents and susceptible to further proteolytic breakdown when concentrated to 1 mg/ml or greater.
  • Hh-C protease-stable fragment of - 17 kDa molecular weight
  • Hh-C mass spectre-metric analysis
  • 7 showed it tc consist of residues Cys-258 to Ser-408 (data not shown). All residues absolutely conserved in Hh-C homclcgu ⁇ s (Potter et al., 1996a), including the nematode sequences, are contained in Hh- Ci .
  • 7 is capable of cleaving itseif in the presence of DTT but nc; cholesterol, indicating that Hi ⁇ ⁇ H -C ⁇ is able to form the thioester intermediate ( ⁇ ,cz Figure 1 A) but that some pc ⁇ icn nf the carbcxy- te ⁇ r ⁇ nal 63 residues of Hh-Cis (Leu-409 to Asp-471) is required for cholesterol transfer.
  • Hh-C i 7 possesses an all-fi structure that is roughly disk-shaped with a diameter of -35 A and width cf -20 A. The amino and carboxy termini emerge from the same surface cf Hh-C;; -6 A aDari. A ribbon drawing and topology diagram of the Hh-C 17 s true rare are shown ir. Figure 3. An unexpected feature of the Hh-C( 7 stmcture is the presence of two homologous subdomains related by a pseudo-twofold axis of symmetry ( Figures 4A and 43 ).
  • the subdomains adopt an irregular fold characterized by three extended ⁇ -hairpin loops and are intimately associated, burying 1372 A 1 of surface area at a hydrophobic interface such that a single hydrophobic cere exists for the entire Hh-C; 7 molecule.
  • the topology of the Hh-Ci subdomains matches that of snake toxins such as cardictcxin VII4 (Ree et al., 1990) and c-bungarotoxin (Lcv ⁇ and Stroud, 19S6), but the toxin and Hh-C; 7 structures do net superimpose well and these sir.icn.ires do not seem otherwise related.
  • the full Hh-C 17 fcld can be detected in the self-splicing region of inteins (Duar. st al., 1997), and the evidence for a divergent evolutionary relationship in this case is strong.
  • Eh- C1 7 The structurally cohesive subdomair.s of Eh- C1 7 are thus mosaics composed of elements from both units of the tandem sequence duplication.
  • the exchange of the third loop between subdomains can be represented as A1-A2-(A3-B l-32)-33 where the structurally distinct subdomains are composed of loops either inside or outside of the parentheses (see Figure 4B).
  • duplication coupled with an intcrdomain structural exchange such as appears to have occurred in Hh-Cp provides a mechanism to generate permutations in the order in which specific structural elements occur in the amino-acid sequence.
  • S ch permutauens have been noted in other systems including saposin homologues (Ponting and Russell, 1 95) and bacterial glucanascs (Hcinemarm and Hahn, 1995).
  • Hh-C 17 The amino-terminal residue of Hh-C 17 , Cys-258, is involved in both the thioester formation and cholesterol transfer steps of Hh autoprocessing (see Figure 1A). Amino-acid sice chains participating directly in Hh autoprocessing chemistry will most likely possess polar groups, and the only such residues near Cys-258 in the Hh-C
  • Kis-329 and Thr-326 arc absolutely conserved in all Hh-C homologues, and the side chains of both of these residues are within hydrogen bonding distance of the c-ami ⁇ o group of Cys-25S in the Hh-C 17 strucrure.
  • Asp-303 is invariably aspartic acid or histidine in Hh-C domains, and the sice chain cf Asp-303 is exposed to solvent 4.2-4.5 A away from the Cys-25S t hiol group. Significant structural rearrangements would appear necessary for additional residues in Hh-C 17 to participate directly in Hh autoprocessing.
  • Cys-258, Kis-329, and Asp-282 do not form a serir.e "Ctease- ⁇ ke catalytic triad as had been proposed (Lee et al., 1994; Porter et al.,
  • each of these residues was mutated to alanine within the context of the fall-length Hi sHh-C ; protein , and the mutant proteins were expressed and assayed for Hh autcprccessmg act:vi:y.
  • the autocleavi g activity of the mutant proteins in the presence of high cer.centraticns of DTT was usea as an assay for thioester fcrmaticn, ih ⁇ first step in the Hh autoprocessing reaction, while the auxocleaving activity in the presence of cholesterol was used to assay for cholesterol transfer, the second steu in the autoprocessing reaction.
  • Tne loss or dramatic reduction of autocleaving activity in the presence of both DTT and choles t erol for H329A and T325A implicates both His-329 and Thr-326 in formation of the in t ernal t hioester during Hh autoprocessing.
  • Possible roies for Kis-329 during t hioes t er formation include stabilization of negative charge on the carbonyl oxygen of Gly-257 , dona t ion of a proton to the free c- amino group of Cys-258, and maintenance cf an appropriate orien t a t ion of reaction components through polar interactions.
  • His-329 may also deprotonate the thiol group of Cys-258 prior to thioester formation, but if this is the case some rearrangement of Cys-258 relative to its position in the Hh-C 17 crystal structure would be required tc bring the thiol ⁇ rou D of Cys-25S into proximity with His-329.
  • a base may not be needed to catalyze thiol deprotonation.
  • Possible roles fc Thr-326 in t hioes t er formation seem more limited.
  • the high pK- of a threonine hydroxyl group (>15) makes Thr-326 an unlikely candidate for proton transfers, suggesting that this residue is needed to form oclar interactions that stabilize reactive conformations within the Hh protein.
  • the activity cf the D303A mutant in DTT- but not cholesterol-stimulated autoprocessing shows t ha t Asp-303 is not needed for thioester formation but is required for cholesterol transfer.
  • the ncsa ⁇ vely-chargcd aspartic acid residue seems unlikely to be involved in binding a hydrophobic cholesterol molecule.
  • a role in activating the cholesterol molecule for nucleophilic at t ack of the t hioester appears more plausible.
  • the 3 ⁇ -hydroxyl group must 'become deprctonated, and Asp-303 is a good candidate for the general base tha t ca t alyzes this deorotcr.aticn.
  • Substitution o ⁇ ' Asp-303 wit histidine in Hh-C hemoicgues is consistent with this hypothesis as histidine is also capabie of functioning as a genera! base.
  • the improved methods for database searching include s t a ⁇ s t icai analysis of gapped alignments and iterative database scanning with position- specific ma t rices derived from previous BLAST outputs (.Altschui ct al., in press).
  • a database search was initiated with any of the Hh-C sequences cr i ⁇ most of the intein sequences , members of t he respec t ive second protein family were the only additional sequences retrieved from the database at a statistically significant level.
  • Solution of the Hh-C 17 crystal structure showed the expanded region of Hh-C/intein sequence homology to terminate halfway through one of the subdomains in the tum region of an exposed loop between ⁇ strands 3b and 4b (sec Figure 3A).
  • This observation coupled with the presence cf characteristic endonuclease motifs in intein sequences shortly after the end of the detectable Hh-C/intcin homology suggested that the intein endonuclease domain had been inserted into ' the ⁇ 3b- ⁇ 4b loop of an Hh-Cp-li e structure.
  • the yeast HO endonuclease which lacks an amino- terminal serine or cysteine residue, does net a e self-sp ⁇ ci ⁇ g activity (psrier e: al.. 1997), and the only intein homologue in which this residue is replaced by a jar. ins ( L'b ⁇ protein homcicgue from Metkcr.ococziis j nncschii) is suspected to be inactive as well.
  • the onjy residue absolutely conserved between Hh-C homologues and inteins is a histidine corresponding to His-329 in Drosophila Hh-C.
  • His-329 in the active site of Drosophila Hh-C and the loss of thioester formation activity when His-329 is mutated strongly imply that this histidine is conserved because it performs a vital role in thioester formation and that it functions similarly in inteins and Hh-C homologues.
  • 34 sequences contain a threonine at a homologous position to Thr-326, while three inteins have serine, and one each have sparasine cr glutamic acid at this position.
  • Tne high level of conservation of threonine at this active site position and its substitution with similar amino acids suggests a conserved role for this threonine in inteins and Hh-C homologues.
  • 7 active site, is not found in intein sequences, consistent with its role in cholesrcrol activation rather than thioester formation.
  • the structures of the self-splicing region of the Pl-Scel intein (Duan et al., 1997) and Hh-C 1 7 arc clearly homologous.
  • the self-splicing region of Pl-Scel contains homologous subdomains related by pseudosymmetry.
  • the Pl-Scel subdomains are homologous to the Hh-C 17 subdomains and possess the same loop exchange observed in Hh-C 1 7.
  • these features are obscured by insertion of end ⁇ nucleasc-ass ⁇ ciated sequences.
  • the Pl-Scel intein contains an additional insertion of amino acids relative to the Hh-C 17 strucmre.
  • the site of this insertion occurs in the tum between ⁇ strands lb and 2b in the Hh-C 17 structure (see Figure 3A), and this inserted region is believed tc be involved in aiding DNA recognition by the PI-SccI intein (Duan et a!., 1997).
  • Figure 6B shows a stereodiagram of the Hh-C 17 structure depicted in the same orientation as the Pl-Scel intein structure in Duan, ct al. (1997) with the sites of the cnco ⁇ ciease-associated insertions indicated.
  • FIG. 7 shows a piausible evolutionary scenario for the development of the Hh-C and intein protein families from a primordial domain of nown r ⁇ c'.ion.
  • the Hh-C; module is sufficient ⁇ r.iy for the initial replacement of a peptide bond with a thioester or ester -in both the Hh autoprocessing and self-splicing reactions.
  • a protein catalylic framework with an N-terminal nucleophile is capable of self- activation. Nature 378, 16-419.
  • Seienomethionyl proteins produced fcr analysis by multiwavelength anomalous diffraction (MAD): a vehicle for direct determination of three-dimensional structure.
  • MAD multiwavelength anomalous diffraction
  • Hedgehog patterning activity Role of a lipophilic modification mediated by the carboxy-terrninal autoprocessing domain. Cell 86, 21-34.
  • MOLECULE TYPE protein
  • Gin Phe lie Pro Asn Val Ala Glu Lys Thr Leu Gly Ala Ser Gly Lys 50 55 60
  • Pro Asn Tyr Asn Pro Asp lie lie Phe Lys Asp Glu Glu Asn Thr Asn 85 90 95

Landscapes

  • Health & Medical Sciences (AREA)
  • Life Sciences & Earth Sciences (AREA)
  • Engineering & Computer Science (AREA)
  • Chemical & Material Sciences (AREA)
  • Immunology (AREA)
  • Biomedical Technology (AREA)
  • Molecular Biology (AREA)
  • Urology & Nephrology (AREA)
  • Hematology (AREA)
  • Biochemistry (AREA)
  • General Health & Medical Sciences (AREA)
  • Medicinal Chemistry (AREA)
  • Toxicology (AREA)
  • Cell Biology (AREA)
  • Physics & Mathematics (AREA)
  • Analytical Chemistry (AREA)
  • Pathology (AREA)
  • General Physics & Mathematics (AREA)
  • Tropical Medicine & Parasitology (AREA)
  • Organic Chemistry (AREA)
  • Biotechnology (AREA)
  • Food Science & Technology (AREA)
  • Microbiology (AREA)
  • Zoology (AREA)
  • Bioinformatics & Cheminformatics (AREA)
  • Proteomics, Peptides & Aminoacids (AREA)
  • Genetics & Genomics (AREA)
  • Insects & Arthropods (AREA)
  • Gastroenterology & Hepatology (AREA)
  • Biophysics (AREA)
  • Neurology (AREA)
  • Neurosurgery (AREA)
  • Physiology (AREA)
  • Peptides Or Proteins (AREA)

Abstract

L'invention concerne deux nouveaux polypeptides, désignés par les termes de fragments hérissons N et C, ou de fragments N-terminal ou C-terminal, respectivement, qui sont obtenus par clivage spécifique au niveau du site G↓CF reconnu par le domaine auto-protéolytique de la protéine native. L'invention concerne également des polypeptides hérissons modifiés par un stérol, ainsi que leurs fragments fonctionnels. Elle concerne enfin des méthodes permettant d'identifier des compositions qui modifient l'activité hérisson en inhibant la modification par le cholestérol de la protéine hérisson.
PCT/US1997/015753 1996-10-07 1997-10-07 Nouveaux polypeptides derives d'une proteine herisson WO1998030576A1 (fr)

Priority Applications (5)

Application Number Priority Date Filing Date Title
JP52494798A JP2002503088A (ja) 1996-10-07 1997-10-07 新規ヘッジホッグ由来ポリペプチド
AU48006/97A AU728541B2 (en) 1996-10-07 1997-10-07 Novel hedgehog-derived polypeptides
EP97910705A EP0966478A4 (fr) 1996-10-07 1997-10-07 Nouveaux polypeptides derives d'une proteine herisson
CA002267106A CA2267106A1 (fr) 1996-10-07 1997-10-07 Nouveaux polypeptides derives d'une proteine herisson
IL129295A IL129295A (en) 1996-10-07 1999-03-31 A polypeptide containing a sequence of amino acids of the hedgehog polypeptide or a residue which is a consequence of the amino terminus of the hedgehog polypeptide, which contain a sterol group

Applications Claiming Priority (4)

Application Number Priority Date Filing Date Title
US08/729,743 US6214794B1 (en) 1994-12-02 1996-10-07 Method of using hedgehog polypeptides to regulate neuronal cell growth
US6132397P 1997-10-02 1997-10-02
US08/729,743 1997-10-02
US60/061,323 1997-10-02

Publications (1)

Publication Number Publication Date
WO1998030576A1 true WO1998030576A1 (fr) 1998-07-16

Family

ID=26740948

Family Applications (1)

Application Number Title Priority Date Filing Date
PCT/US1997/015753 WO1998030576A1 (fr) 1996-10-07 1997-10-07 Nouveaux polypeptides derives d'une proteine herisson

Country Status (6)

Country Link
EP (1) EP0966478A4 (fr)
JP (1) JP2002503088A (fr)
AU (1) AU728541B2 (fr)
CA (1) CA2267106A1 (fr)
IL (1) IL129295A0 (fr)
WO (1) WO1998030576A1 (fr)

Cited By (21)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO1999028343A2 (fr) * 1997-12-03 1999-06-10 Biogen, Inc. Compositions a base de proteines modifiees sur le plan hydrophobe et procedes d'elaboration
WO1999028876A2 (fr) * 1997-12-03 1999-06-10 Biogen, Inc. Compositions proteiniques modifiees de maniere hydrophobe et leurs procedes de fabrication
WO1999050385A2 (fr) * 1998-03-30 1999-10-07 President And Fellows Of Harvard College Regulation de la synthese de glycosaminoglycane, techniques apparentees et reactifs connexes
EP0953576A1 (fr) * 1998-04-30 1999-11-03 Boehringer Mannheim Gmbh Conjugé de protein hedgehog active, procedé pour sa production et utilisation
EP0953575A1 (fr) * 1998-04-30 1999-11-03 Boehringer Mannheim Gmbh Conjugué de proteine hedgehog active, procédé pour sa production et utilisation
EP1002547A1 (fr) * 1998-09-01 2000-05-24 F. Hoffmann-La Roche Ag Complex ionique hydrosoluble de la protein Hedgehog et son utilisation pharmaceutique
WO2000035948A1 (fr) * 1998-12-03 2000-06-22 Biogen, Inc. Procedes et compositions servant a traiter des troubles entrainant une excitotoxicite
WO2000051628A2 (fr) * 1999-03-03 2000-09-08 Biogen, Inc. Methodes de modulation du metabolisme et du stockage lipidiques
WO2000073337A1 (fr) * 1999-06-01 2000-12-07 Biogen, Inc. Conjugues polymeres de proteines hedgehog et leurs utilisations
WO2001026644A2 (fr) * 1999-10-14 2001-04-19 Curis, Inc. Mediateurs de voies de signalisation hedgehog, compositions et utilisations associees
WO2002024151A2 (fr) * 2000-09-22 2002-03-28 The Johns Hopkins University School Of Medicine Procede d'utilisation de proteine sonic hedgehog comme ligand pour patched
US6552016B1 (en) 1999-10-14 2003-04-22 Curis, Inc. Mediators of hedgehog signaling pathways, compositions and uses related thereto
WO2003072779A1 (fr) * 2002-02-27 2003-09-04 Japan Science And Technology Agency Methode d'utilisation de genes specifiques a l'hypophyse
US6639051B2 (en) 1997-10-20 2003-10-28 Curis, Inc. Regulation of epithelial tissue by hedgehog-like polypeptides, and formulations and uses related thereto
US6767888B1 (en) 1997-06-27 2004-07-27 Curis, Inc. Neuroprotective methods and reagents
US6884770B1 (en) 1998-11-06 2005-04-26 Curis, Inc. Methods and compositions for treating or preventing peripheral neuropathies
US6897297B1 (en) 1997-12-03 2005-05-24 Curis, Inc. Hydrophobically-modified protein compositions and methods
US6994990B1 (en) 1997-11-28 2006-02-07 Curis, Inc. Active modified hedgehog proteins
US7138492B2 (en) 1997-07-24 2006-11-21 Curis, Inc. Method of treating dopaminergic and GABA-nergic disorders
US9427431B2 (en) 1998-04-09 2016-08-30 Johns Hopkins University School Of Medicine Inhibitors of hedgehog signaling pathways, compositions and uses related thereto
US9440988B2 (en) 1998-04-09 2016-09-13 John Hopkins University School Of Medicine Regulators of the hedgehog pathway, compositions and uses related thereto

Families Citing this family (2)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
JP4668616B2 (ja) * 2002-08-19 2011-04-13 メルク パテント ゲゼルシャフト ミット ベシュレンクテル ハフツング オオアワガエリからの主要アレルゲンＰｈｌｐ１の変異体
AU2005271944B2 (en) * 2004-07-09 2012-02-16 Viacyte, Inc. Preprimitive streak and mesendoderm cells

Family Cites Families (2)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
NZ193564A (en) * 1979-05-02 1985-01-31 Aruba Pty Ltd Steroid alkaloids from solanum sodomeum and pharmaceutical compositions
US6281332B1 (en) * 1994-12-02 2001-08-28 The Johns Hopkins University School Of Medicine Hedgehog-derived polypeptides

Non-Patent Citations (5)

* Cited by examiner, † Cited by third party
Title
CLARKE S D, ARMSTRONG M K: "Cellular Lipid Binding Proteins: Expression, Function and Nutritional Regul ation", THE FASEB JOURNAL, FEDERATION OF AMERICAN SOCIETIES FOR EXPERIMENTAL BIOLOGY, US, vol. 3, 1 November 1989 (1989-11-01), US, pages 2480 - 2487, XP002964488, ISSN: 0892-6638 *
INGHAM P W: "Signalling by Hedgehog Family Proteins in Drosophila and Vertebrate Develop ment", CURRENT OPINION IN GENETICS & DEVELOPMENT., CURRENT BIOLOGY LTD., XX, vol. 5, 1 January 1995 (1995-01-01), XX, pages 492 - 498, XP002964489, ISSN: 0959-437X, DOI: 10.1016/0959-437X(95)90054-K *
LEE J J, ET AL.: "SECRETION AND LOCALIZED TRANSCRIPTION SUGGEST A ROLE IN POSITIONAL SIGNALING FOR PRODUCTS OF THE SEGMENTATION GENE HEDGEHOG", CELL, CELL PRESS, US, vol. 71, 2 October 1992 (1992-10-02), US, pages 33 - 50, XP001053397, ISSN: 0092-8674, DOI: 10.1016/0092-8674(92)90264-D *
MOHLER ET AL.: "Molecular organization and embryonic expression of the hedgehog gene involved in cell-cell communication in segmental patterning of Drosophila", DEVELOPMENT, THE COMPANY OF BIOLOGISTS LTD., GB, vol. 115., 1 January 1992 (1992-01-01), GB, pages 957 - 971., XP002082040, ISSN: 0950-1991 *
See also references of EP0966478A4 *

Cited By (39)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US6767888B1 (en) 1997-06-27 2004-07-27 Curis, Inc. Neuroprotective methods and reagents
US7138492B2 (en) 1997-07-24 2006-11-21 Curis, Inc. Method of treating dopaminergic and GABA-nergic disorders
US7144997B2 (en) 1997-07-24 2006-12-05 Curis, Inc. Vertebrate embryonic patterning-inducing proteins, compositions and uses related therto
US6639051B2 (en) 1997-10-20 2003-10-28 Curis, Inc. Regulation of epithelial tissue by hedgehog-like polypeptides, and formulations and uses related thereto
US6994990B1 (en) 1997-11-28 2006-02-07 Curis, Inc. Active modified hedgehog proteins
EP1577321A1 (fr) * 1997-12-03 2005-09-21 Biogen, Inc. Compositions à base de protéines modifiées sur le plan hydrophobe et procédés d'élaboration
WO1999028343A2 (fr) * 1997-12-03 1999-06-10 Biogen, Inc. Compositions a base de proteines modifiees sur le plan hydrophobe et procedes d'elaboration
US6897297B1 (en) 1997-12-03 2005-05-24 Curis, Inc. Hydrophobically-modified protein compositions and methods
WO1999028876A3 (fr) * 1997-12-03 1999-09-23 Biogen Inc Compositions proteiniques modifiees de maniere hydrophobe et leurs procedes de fabrication
WO1999028343A3 (fr) * 1997-12-03 1999-08-12 Biogen Inc Compositions a base de proteines modifiees sur le plan hydrophobe et procedes d'elaboration
US6444793B1 (en) 1997-12-03 2002-09-03 Curis, Inc. Hydrophobically-modified hedgehog protein compositions and methods
WO1999028876A2 (fr) * 1997-12-03 1999-06-10 Biogen, Inc. Compositions proteiniques modifiees de maniere hydrophobe et leurs procedes de fabrication
WO1999050385A2 (fr) * 1998-03-30 1999-10-07 President And Fellows Of Harvard College Regulation de la synthese de glycosaminoglycane, techniques apparentees et reactifs connexes
WO1999050385A3 (fr) * 1998-03-30 1999-11-18 Harvard College Regulation de la synthese de glycosaminoglycane, techniques apparentees et reactifs connexes
US9440988B2 (en) 1998-04-09 2016-09-13 John Hopkins University School Of Medicine Regulators of the hedgehog pathway, compositions and uses related thereto
US9427431B2 (en) 1998-04-09 2016-08-30 Johns Hopkins University School Of Medicine Inhibitors of hedgehog signaling pathways, compositions and uses related thereto
US7153836B2 (en) 1998-04-30 2006-12-26 Curis, Inc. Active hedgehog protein conjugate
US6468978B1 (en) 1998-04-30 2002-10-22 Curis, Inc. Active hedgehog protein conjugate
US6818623B2 (en) 1998-04-30 2004-11-16 Curis, Inc. Active hedgehog protein conjugate
EP0953575A1 (fr) * 1998-04-30 1999-11-03 Boehringer Mannheim Gmbh Conjugué de proteine hedgehog active, procédé pour sa production et utilisation
EP0953576A1 (fr) * 1998-04-30 1999-11-03 Boehringer Mannheim Gmbh Conjugé de protein hedgehog active, procedé pour sa production et utilisation
SG85670A1 (en) * 1998-09-01 2002-01-15 Hoffmann La Roche Water-soluble pharmaceutical composition in an ionic complex and the use thereof
US8519098B2 (en) 1998-09-01 2013-08-27 Hoffmann-La Roche Inc. Composition of a polypeptide and an amphiphilic compound in an ionic complex and the use thereof
EP1002547A1 (fr) * 1998-09-01 2000-05-24 F. Hoffmann-La Roche Ag Complex ionique hydrosoluble de la protein Hedgehog et son utilisation pharmaceutique
US6867182B2 (en) 1998-09-01 2005-03-15 Hoffmann-La Roche Inc. Composition of a polypeptide and an amphiphilic compound in an ionic complex and the use thereof
US6884770B1 (en) 1998-11-06 2005-04-26 Curis, Inc. Methods and compositions for treating or preventing peripheral neuropathies
WO2000035948A1 (fr) * 1998-12-03 2000-06-22 Biogen, Inc. Procedes et compositions servant a traiter des troubles entrainant une excitotoxicite
AU763032B2 (en) * 1998-12-03 2003-07-10 Curis, Inc. Methods and compositions for treating disorders involving excitotoxicity
WO2000051628A2 (fr) * 1999-03-03 2000-09-08 Biogen, Inc. Methodes de modulation du metabolisme et du stockage lipidiques
WO2000051628A3 (fr) * 1999-03-03 2001-04-26 Biogen Inc Methodes de modulation du metabolisme et du stockage lipidiques
WO2000073337A1 (fr) * 1999-06-01 2000-12-07 Biogen, Inc. Conjugues polymeres de proteines hedgehog et leurs utilisations
US7300929B2 (en) 1999-10-14 2007-11-27 Curis, Inc. Mediators of hedgehog signaling pathways, compositions and uses related thereto
US6552016B1 (en) 1999-10-14 2003-04-22 Curis, Inc. Mediators of hedgehog signaling pathways, compositions and uses related thereto
WO2001026644A3 (fr) * 1999-10-14 2002-04-18 Curis Inc Mediateurs de voies de signalisation hedgehog, compositions et utilisations associees
WO2001026644A2 (fr) * 1999-10-14 2001-04-19 Curis, Inc. Mediateurs de voies de signalisation hedgehog, compositions et utilisations associees
US6942988B2 (en) * 2000-09-22 2005-09-13 The Johns Hopkins University School Of Medicine Method of use of sonic hedgehog protein as a ligand for patched
WO2002024151A2 (fr) * 2000-09-22 2002-03-28 The Johns Hopkins University School Of Medicine Procede d'utilisation de proteine sonic hedgehog comme ligand pour patched
WO2002024151A3 (fr) * 2000-09-22 2004-09-23 Univ Johns Hopkins Med Procede d'utilisation de proteine sonic hedgehog comme ligand pour patched
WO2003072779A1 (fr) * 2002-02-27 2003-09-04 Japan Science And Technology Agency Methode d'utilisation de genes specifiques a l'hypophyse

Also Published As

Publication number Publication date
AU4800697A (en) 1998-08-03
EP0966478A4 (fr) 2002-08-21
IL129295A0 (en) 2000-02-17
EP0966478A1 (fr) 1999-12-29
AU728541B2 (en) 2001-01-11
CA2267106A1 (fr) 1998-07-16
JP2002503088A (ja) 2002-01-29

Similar Documents

Publication Publication Date Title
AU728541B2 (en) Novel hedgehog-derived polypeptides
CA2206509C (fr) Nouveau polypeptide extrait du herisson
US8362216B2 (en) Method of detection using antibodies that specifically bind hedgehog-derived polypeptides
Eddy et al. The spermatozoon
Beachy et al. Multiple roles of cholesterol in hedgehog protein biogenesis and signaling
Simon et al. Structure and activity of the sevenless protein: a protein tyrosine kinase receptor required for photoreceptor development in Drosophila.
US6057091A (en) Method of identifying compounds affecting hedgehog cholesterol transfer
Ruder et al. EBAG9 adds a new layer of control on large dense-core vesicle exocytosis via interaction with Snapin
JPH11502104A (ja) ゴナドトロピン放出ホルモン受容体活性の抑制および調節
US6214794B1 (en) Method of using hedgehog polypeptides to regulate neuronal cell growth
JP2004033211A (ja) 神経ペプチド受容体およびその使用
US20030207853A1 (en) Cholesterol and hedgehog signaling
IL129295A (en) A polypeptide containing a sequence of amino acids of the hedgehog polypeptide or a residue which is a consequence of the amino terminus of the hedgehog polypeptide, which contain a sterol group
US6225086B1 (en) Polynucleotides encoding ankyrin proteins
US7846683B2 (en) Method for identifying agents which modulate cell growth or survival
Chase A genetic analysis of the role of neurotransmitters in the development of the nervous system in Drosophila melanogaster
Manseau An immunochemical analysis of goldfish brain proteins using antisera raised against memberane fractions
Jin Constitutively active rhodopsin mutations and the molecular mechanisms of retinal diseases: A transgenic Xenopus laevis model
Khatkar Structural and phosphorylation studies of cardiac ATP-sensitive potassium channels
Müller et al. Biochemie der Gehirnentwicklung
Kelley Cloning, expression and characterization of bovine αB-crystallin
WO2000026241A2 (fr) Nouvelles compositions et nouveaux procedes pour identifier par criblage des modulateurs d'activation des lymphocytes t et des lymphocytes b

Legal Events

Date Code Title Description
AK Designated states

Kind code of ref document: A1

Designated state(s): AL AM AT AU AZ BA BB BG BR BY CA CH CN CU CZ DE DK EE ES FI GB GE HU ID IL IS JP KE KG KP KR KZ LC LK LR LS LT LU LV MD MG MK MN MW MX NO NZ PL PT RO RU SD SE SG SI SK SL TJ TM TR TT UA UG US UZ VN ZW AM AZ BY KG KZ MD RU TJ TM

AL Designated countries for regional patents

Kind code of ref document: A1

Designated state(s): GH KE LS MW SD SZ UG ZW AT BE CH DE DK ES FI FR GB GR IE IT LU MC NL PT

121 Ep: the epo has been informed by wipo that ep was designated in this application
ENP Entry into the national phase

Ref document number: 2267106

Country of ref document: CA

Ref country code: CA

Ref document number: 2267106

Kind code of ref document: A

Format of ref document f/p: F

Ref country code: JP

Ref document number: 1998 524947

Kind code of ref document: A

Format of ref document f/p: F

WWE Wipo information: entry into national phase

Ref document number: 1997910705

Country of ref document: EP

REG Reference to national code

Ref country code: DE

Ref legal event code: 8642

WWP Wipo information: published in national office

Ref document number: 1997910705

Country of ref document: EP

WWR Wipo information: refused in national office

Ref document number: 1997910705

Country of ref document: EP

WWW Wipo information: withdrawn in national office

Ref document number: 1997910705

Country of ref document: EP