Cloning of candidate chicken prion
Chicken prion copurifies with ARIA
Post-translational processing of chicken prion
N-terminus essential for endocytosis
Copper binding to repeat region: I, II
Reported Chicken DNA and protein sequences
Molecular cloning of a candidate chicken prion proteinGabriel JM; Oesch B; Kretzschmar H; Scott M; Prusiner SB PNAS 89: 9097-101 (1992)Fractions enriched for acetylcholine receptor-inducing activity from chicken brain were found to contain a protein that was approximately 30% homologous with mammalian prion proteins [Harris, D. A., Falls, D. L., Johnson, F. A. & Fischbach, G. D. (1991) Proc. Natl. Acad. Sci. USA 88, 7664-7668]. To extend these observations, we recovered genomic clones encoding a putative chicken prion protein (PrP). Like mammalian PrP molecules, the candidate chicken PrP is encoded by a single-copy gene and the entire open reading frame is found within a single exon. All of the structural features of mammalian PrP were found in the chicken protein. When the N-terminal repeats of PrP were not considered, the chicken and mammalian proteins were approximately 55% homologous, allowing for conservative substitutions. Screening of a chicken genomic DNA library failed to identify a more closely related chicken PrP homologue. These findings argue that the protein which purifies with acetylcholine receptor-inducing activity is chicken PrP.
Chicken brain prion copurifies with acetylcholine receptor-inducing activityHarris DA; Falls DL; Johnson FA; Fischbach GD PNAS 88: 7664-8 (1991)The mammalian prion protein (PrPC) is a cellular protein of unknown function, an altered isoform of which (PrPSc) is a component of the infectious particle (prion) thought to be responsible for spongiform encephalopathies in humans and animals. We report here the isolation of a cDNA that encodes a chicken protein that is homologous to PrPC. This chicken prion-like protein (ch-PrLP) is identical to the mouse PrP at 33% of its amino acid positions, including an uninterrupted stretch of 24 identical residues, and it displays the same structural domains. In addition, ch-PrLP, like its mammalian counterpart, is attached to the cell surface by a glycosyl-phosphatidylinositol anchor. We find that ch-PrLP is the major protein in preparations of an acetylcholine receptor-inducing activity that has been purified greater than 10(6)-fold from brain on the basis of its ability to stimulate synthesis of nicotinic receptors by cultured myotubes. The ch-PrLP gene is expressed in the spinal cord and brain as early as embryonic day 6; and in the spinal cord, the protein appears to be concentrated in motor neurons. Our results therefore raise the possibility that prion proteins serve normally to regulate the chemoreceptor number at the neuromuscular junction and perhaps in the central nervous system as well.
Processing of a cellular prion protein: identification of N- and C-terminal cleavage sitesHarris DA; Huber MT; van Dijken P; Shyng SL; Chait BT; Wang R Biochemistry 32: 1009-16 (1993)ChPrP is the chicken homologue of PrPC, the cellular isoform of the mammalian prion protein. We have used sequence-specific antibodies to immunoprecipitate and immunoblot chPrP derived from stably transfected cultures of neuroblastoma cells, as well as from chicken brain and cerebrospinal fluid. We have also used mass spectrometry to characterize fragments of the protein purified from conditioned medium. The majority of chPrP protein present in neuroblastoma cells and on isolated brain membranes can be released by incubation with phosphatidylinositol-specific phospholipase C, indicating that these molecules are attached to the cell surface by a glycosylphosphatidylinositol anchor. Surprisingly, most of the surface-anchored molecules are truncated at their N-terminus distal to the proline/glycine-rich repeats.
The corresponding N-terminal fragments are found in medium conditioned by neuroblastoma cells, as well as in cerebrospinal fluid and a postmicrosomal supernatant of brain. One of these fragments extends from Lys25 to Phe116. 35-45-kDa forms of chPrP that can be metabolically labeled with [3H]ethanolamine can also be found in extracellular media. We propose that the chPrP molecule undergoes at least two cleavages as part of its normal metabolism: one within the glycosylphosphatidylinositol anchor and one within or just N-terminal to the central hydrophobic domain. The second cleavage lies within a region of 24 amino acids that is identical in chPrP and mammalian PrP, and represents a major processing event that may have physiological as well as pathological significance.
The N-terminal domain of GPI prion is essential for endocytosis via clathrin-coated pitsShyng SL; Moulder KL; Lesko A; Harris DA J Biol Chem 270: 14793-800 (1995)The cellular prion protein (PrPC) is a glycolipid-anchored protein that is involved in the pathogenesis of fatal spongiform encephalopathies. We have shown previously that, in contrast to several other glycolipid-anchored proteins, chPrP, the chicken homologue of mammalian PrPC, is endocytosed via clathrin-coated pits in cultured neuroblastoma cells, as well as in embryonic neurons and glia (Shyng, S.-L., Heuser, J. E., and Harris, D. A. (1994) J. Cell Biol. 125, 1239-1250).
In this study, we have determined that the N-terminal half of the chPrP polypeptide chain is essential for its endocytosis. Deletions within this region reduce the amount of chPrP internalized, as measured by surface iodination or biotinylation, and decrease its concentration in clathrin-coated pits, as determined by quantitative electron microscopic immunogold labeling. Mouse PrP, as well as two mouse PrP/chPrP chimeras, are internalized as efficiently as chPrP, suggesting that conserved features of secondary and tertiary structure are involved in interaction with the endocytic machinery. Our results indicate that the ectodomain of a protein can contain endocytic targeting information, and they strongly support a model in which the polypeptide chain of PrPC binds to the extracellular domain of a transmembrane protein that contains a coated pit localization signal in its cytoplasmic tail.
Copper binding to N-terminal tandem repeat region of mammalian and avian prion protein. IHornshaw MP; McDermott JR; Candy JM; Lakey JH Biochem Biophys Res Commun 214: 993-9 (1995)Using CD spectroscopy we have investigated the effect of Cu2+ on the secondary structure of synthetic peptides Octa4 and Hexa4 corresponding to tetra-repeats of the octapeptide of mammalian PrP and the hexapeptide of chicken PrP. In addition, fluorescence spectroscopy was used to estimate the dissociation constants (Kd), of Cu2+ binding by both peptides. Both peptides exhibited unusual CD spectra, complicated by the high proportion of aromatic residues, revealing little secondary structure in aqueous solution. Addition of Cu2+ to Hexa4 induced an increase in random coil to resemble Octa4. The fluorescence of both peptides was quenched by Cu2+ and this was used to calculate Kd's of 6.7 microM for Octa4 and 4.5 microM for Hexa4. Other divalent cations showed lesser effects on the fluorescence of the peptides.
Copper binding to the N-terminal tandem repeat regions of mammalian and avian prion protein. IIHornshaw MP; McDermott JR; Candy JM Biochem Biophys Res Commun 207: 621-9 (1995)Mammalian prion protein (PrP) is a normal cellular protein (PrPc) which through post-translational modification produces the infectious prion protein (PrPsc). We have shown, using mass spectrometry, that synthetic peptides containing three or four copies of an octapeptide repeat sequence (PHGGGWGQ), found in a highly conserved N-terminal domain of PrP, preferentially bind copper over other metals. Peptides from the analogous region of chicken PrP, which contains an N-terminal repeat domain of the hexapeptide (NPGYPH), showed similar specificity for copper binding. In addition, gel filtration chromatography demonstrated concentration dependent binding of copper to the mammalian tetra repeat PrP peptide. These results suggest that PrP may be a copper binding protein in vivo.
1. Chicken prion protein gene, complete cds. 969 bp sequenced 29-SEP-1992 Gallus gallus (strain White Leghorn, sub_species domesticus) Eukaryotae; mitochondrial eukaryotes; Metazoa; Chordata; Vertebrata; Archosauria; Aves; Neognathae; Galliformes; Phasianidae; Phasianinae; Gallus. Gabriel,J.-M., Oesch,B., Kretzschmar,H., Scott,M. and Prusiner,S.B. Molecular cloning of a candidate chicken prion protein Proc. Natl. Acad. Sci. U.S.A. 89, 9097-9101 (1992) marllttccllalllaactdvalskkgkgkpsgggwgagshrqp syprqpgyphnpgyphnpgyphnpgyphnpgyphnpgypqnpgyphnpgypgwgqgyn pssggsyhnqkpwkppktnfkhvagaaaagavvgglggyamgrvmsgmnyhfdspdey rwwsensarypnrvyyrdysspvpqdvfvadcfnitvteysigpaakkntseavaaan qtevemenkvvtkviremcvqqyreyrlasgiqlhpadtwlavlllllttlfamh intron 1 <1..34 5'UTR 35..36 exon 35..>969 CDS 37..858 sig_peptide 37..108 mat_peptide 109..855 3'UTR 859..>969 actgccctaacagtgtgtgtccttatgcccgcagccatggctaggctcctcaccacctgc tgcctgctggccctgctgctcgccgcctgcaccgacgtcgccctctccaagaagggcaaa ggcaaacccagtggtgggggttggggcgccgggagccatcgccagcccagctacccccgc cagccgggctaccctcataacccagggtacccccataacccagggtacccccataaccca gggtacccccacaaccctggctatccccataaccccggctacccccagaaccctggctac ccccataacccaggttacccaggctggggtcaaggctacaacccatccagcggaggaagt taccacaaccagaagccatggaaaccccccaaaaccaacttcaagcacgtggcgggggca gcagcggcgggtgctgtggtggggggcttggggggctacgccatggggcgcgttatgtca gggatgaactaccacttcgatagccccgatgagtaccgatggtggagtgagaactcggcg cgttatcccaaccgggtttactaccgggattacagcagccccgtgccacaggacgtcttc gtggccgattgctttaacatcacagtgactgagtacagcattggccctgctgccaagaag aacacctccgaggctgtggcggcagcaaaccaaacggaggtggagatggagaacaaagtg gtgacgaaggtgatccgcgagatgtgcgtgcagcagtaccgcgagtaccgcctggcctcg ggcatccagctgcaccctgctgacacctggctcgccgtcctcctcctcctcctcaccacc ctttttgccatgcactgatgggatgccgtgccccggccctgtggcagtgagatgacatcg tgtccccgtgcccacccatggggtgttccttgtcctcgcttttgtccatctttggtgaag atgtccccc
2. Gallus gallus prion-like protein mRNA, complete cds 2188 bp sequenced 19-SEP-1991 Harris,D.A., Falls,D.L., Johnson,F.A. and Fischbach,G.D. A prion-like protein from chicken brain co-purifies with an acetylcholine receptor-inducing activity Proc. Natl. Acad. Sci. U.S.A. 88, 7664-7668 (1991) marllttccllalllaactdvalskkgkgkpsgggwgagshrqp syprqpgyphnpgyphnpgyphnpgyphnpgypqnpgyphnpgypgwgqgynpssggs yhnqkpwkppktnfkhvagaaaagavvgglggyamgrvmsgmnyhfdrpdeyrwwsen sarypnrvyyrdysspvpqdvfvadcfnitvteysigpaakkntseavaaanqtevem enkvvtkviremcvqqyreyrlasgiqlhpadtwlavlllllttlfamh signal peptide 172..414 CDS 172..975 gaattccctcggcagccagctcctccctctcgctatttattcctttctcccccccctacg ctggatctggatcatctcaagccgagcggtgacggcttcttggatcgctcatacataaat atctgtgagtcagaggaagcaaccaccgaccccaagacctcaccccgagccatggctagg ctcctcaccacctgctgcctgctggccctgctgctcgccgcctgcaccgacgtcgccctc tccaagaagggcaaaggcaaacccagtggtgggggttggggcgccgggagccatcgccag cccagctacccccgccagccgggctaccctcataacccagggtacccccataacccaggg tacccccacaaccctggctatccccataaccccggctacccccagaaccctggctacccc cataacccaggttacccaggctggggtcaaggctacaacccatccagcggaggaagttac cacaaccagaagccatggaaaccccccaaaaccaacttcaagcacgtggcgggggcagca gcggcgggtgctgtggtggggggcttggggggctacgccatggggcgcgttatgtcaggg atgaactaccacttcgatagacccgatgagtaccgatggtggagtgagaactcggcgcgt tatcccaaccgggtttactaccgggattacagcagccccgtgccacaggacgtcttcgtg gccgattgctttaacatcacagtgactgagtacagcattggccctgctgccaagaagaac acctccgaggctgtggcggcagcaaaccaaacggaggtggagatggagaacaaagtggtg acgaaggtgatccgcgagatgtgcgtgcagcagtaccgcgagtaccgcctggcctcgggc atccagctgcaccctgctgacacctggctcgccgtcctcctcctcctcctcaccaccctt tttgccatgcactgatgggatgccgtgccccggccctgtggcagtgagatgacatcgtgt ccccgtgcccacccatggggtgttccttgtcctcgcttttgtccatctttggtgaagatg tccccccgctgcctccccgcaggctctgatttgggcaaatgggaggggattttgtcctgt cctggtcgtggcaggacggctgctggtggtggagtgggatgcccaaaaaatggccttcac cacttcctcctcctcttcctttctggggcggagatatgggctcgtccagcccttattgtc cctgcaagagcgtatctgaaaatcctctttgctaacaagcagggttttacctaatctgct tagccccagtgacagcagagcgcctttccccagggcacaccaaccccaagctgaggtgct tggcagccacacgtcccatggaggctgatgggttttggggcgtcccaagcaacaccctgg gctactgaggtgcaattgtagctctttaatctgccaatcccaaccctaccgtgtagatag gaactgcctgctctgcattttgcatgctgcaaacacctcctgccgcagcgcccccaaaat agagtgatttgggaatagtgaggctgaagccacagcagcttgggattgggctcatcatat caatccatgatgctttgcttccagctgagcctcactgcccttttatagcctgcccagagg aagggagcgctgctaaatgcccaaaaaggtaacactgagcaaaagcttatttcaatgtat gatagagaacgagtgcatctcgcacagatcagccatgggagcatcgtttgccatcagccc caaaacccaaaggatgctaaaatgcagccaaaggggaatcaagcacgcagggaaggactt gaatcagctcaactggattgaaatggcaaaaggcatgagtagaacgaacggcaaggggat gctggagatccacctcctgtgagcaaattgttcgatgcagccaatggaactattgcttct tgtgcttcagttgctgctgatgtgtacataggctgtagcatatgtaaagttacacgtgtc aagctgctcgcaccgcgtagagctaatatgtatcatgtatgtgggcactgaatgccaccg ttggccatacccaaccgtcctaaacgattttcacgtcgctgtaacttaagtggagataca ctttcagtatattcagcaaaaggaattc 3. SWISSPROT:PRIO_CHICK P27177; Ref 1 Gabriel j.m., Oesch b., Kretzschmar h., Scott m.,Prusiner s.b.; PNAS 89:9097-9101(1992). Ref 2 HARRIS D.A., FALLS D.L., JOHNSON F.A., FISCHBACH G.D.; PNAS 88:7664-7668(1991). Ref 3 FALLS D.L., HARRIS D.A., JOHNSON F.A., MORGAN M.M., CORFAS G., FISCHBACH G.D.; COLD SPRING HARB. SYMP. QUANT. BIOL. 55:397-406(1990). signal 1 24 chain 25 273 major prion protein homolog. domain 42 89 8 x 6 aa tandem repeats of [hr]-[nq]-p-g-y-p. repeat 42 47 1. repeat 48 53 2. repeat 54 59 3. repeat 60 65 4. repeat 66 71 5. repeat 72 77 6. repeat 78 83 7. repeat 84 89 8. carbohyd 194 194 potential. carbohyd 209 209 potential. carbohyd 218 218 potential. disulfid 192 237 by similarity. conflict 78 83 missing (in ref. 2). conflict 156 156 s -> r (in ref. 2). SQ SEQUENCE 273 AA; 29909 MW; 6F7255C6 CRC32; >PRIO_CHICK marllttccllalllaactdvalskkgkgkpsgggwgagshrqpsyprqpgyphnpgyph npgyphnpgyphnpgyphnpgypqnpgyphnpgypgwgqgynpssggsyhnqkpwkppkt nfkhvagaaaagavvgglggyamgrvmsgmnyhfdspdeyrwwsensarypnrvyyrdys spvpqdvfvadcfnitvteysigpaakkntseavaaanqtevemenkvvtkviremcvqq yreyrlasgiqlhpadtwlavlllllttlfamh