Most Pathogenic Mutations Do Not Alter the Membrane Topology of the Prion Protein
2001; Elsevier BV; Volume: 276; Issue: 3 Linguagem: Inglês
10.1074/jbc.m006763200
ISSN1083-351X
AutoresRichard S. Stewart, David A. Harris,
Tópico(s)Neurological diseases and metabolism
ResumoThe prion protein (PrP), a glycolipid-anchored membrane glycoprotein, contains a conserved hydrophobic sequence that can span the lipid bilayer in either direction, resulting in two transmembrane forms designated NtmPrP andCtmPrP. Previous studies have shown that the proportion ofCtmPrP is increased by mutations in the membrane-spanning segment, and it has been hypothesized that CtmPrP represents a key intermediate in the pathway of prion-induced neurodegeneration. To further test this idea, we have surveyed a number of mutations associated with familial prion diseases to determine whether they alter the proportions of NtmPrP andCtmPrP produced in vitro, in transfected cells, and in transgenic mice. For the in vitro experiments, PrP mRNA was translated in the presence of murine thymoma microsomes which, in contrast to the canine pancreatic microsomes used in previous studies, are capable of efficient glycolipidation. We confirmed that mutations within or near the transmembrane domain enhance the formation of CtmPrP, and we demonstrate for the first time that this species contains a C-terminal glycolipid anchor, thus exhibiting an unusual, dual mode of membrane attachment. However, we find that pathogenic mutations in other regions of the molecule have no effect on the amounts of CtmPrP and NtmPrP, arguing against the proposition that transmembrane PrP plays an obligate role in the pathogenesis of prion diseases. The prion protein (PrP), a glycolipid-anchored membrane glycoprotein, contains a conserved hydrophobic sequence that can span the lipid bilayer in either direction, resulting in two transmembrane forms designated NtmPrP andCtmPrP. Previous studies have shown that the proportion ofCtmPrP is increased by mutations in the membrane-spanning segment, and it has been hypothesized that CtmPrP represents a key intermediate in the pathway of prion-induced neurodegeneration. To further test this idea, we have surveyed a number of mutations associated with familial prion diseases to determine whether they alter the proportions of NtmPrP andCtmPrP produced in vitro, in transfected cells, and in transgenic mice. For the in vitro experiments, PrP mRNA was translated in the presence of murine thymoma microsomes which, in contrast to the canine pancreatic microsomes used in previous studies, are capable of efficient glycolipidation. We confirmed that mutations within or near the transmembrane domain enhance the formation of CtmPrP, and we demonstrate for the first time that this species contains a C-terminal glycolipid anchor, thus exhibiting an unusual, dual mode of membrane attachment. However, we find that pathogenic mutations in other regions of the molecule have no effect on the amounts of CtmPrP and NtmPrP, arguing against the proposition that transmembrane PrP plays an obligate role in the pathogenesis of prion diseases. scrapie isoform of prion protein endoplasmic reticulum glycosyl phosphatidylinositol phosphatidylinositol-specific phospholipase C proteinase K prion protein cellular isoform of prion protein C-terminal transmembrane form of prion protein N-terminal transmembrane form of prion protein secretory form of prion protein phosphate-buffered saline baby hamster kidney Chinese hamster ovary wild-type polyacrylamide gel electrophoresis Prion diseases are neurodegenerative disorders characterized by spongiform destruction of brain tissue and the presence of cerebral amyloid plaques (1Prusiner, S. B. (ed) (1999) Prion Biology and Diseases, Cold Spring Harbor Laboratory Press, Cold Spring Harbor, NYGoogle Scholar, 2Harris D.A. Clin. Microbiol. Rev. 1999; 12: 429-444Crossref PubMed Google Scholar). These disorders include kuru and Creutzfeldt-Jakob disease in humans, "mad cow disease" in cattle, and scrapie in sheep. Prion diseases can have an infectious or genetic origin, or can arise spontaneously. The infectious agent is hypothesized to be PrPSc,1 a conformationally altered isoform of a normal cell-surface glycoprotein of unknown function called PrPC (3Prusiner S.B. Proc. Natl. Acad. Sci. U. S. A. 1998; 95: 13363-13383Crossref PubMed Scopus (5052) Google Scholar). PrPSc from dietary or other infectious sources is thought to act as a catalyst or template to convert endogenous PrPC into more PrPSc, which then accumulates, eventually causing disease. Familial forms of prion disease result from germline mutations in the PrP gene on chromosome 20, which are believed to favor conversion of the protein to the PrPSc form (4Young K. Piccardo P. Dlouhy S. Bugiani O. Tagliavini F. Ghetti B. Harris D.A. Prions: Molecular and Cellular Biology. Horizon Scientific Press, Wymondham, United Kingdom1999: 139-175Google Scholar). Sporadic cases may be due to rare, spontaneous conversion of wild-type PrPC to PrPSc. No covalent modifications that distinguish PrPSc from PrPC have been detected (5Stahl N. Baldwin M.A. Teplow D.B. Hood L. Gibson G.W. Burlingame A.L. Prusiner S.B. Biochemistry. 1993; 32: 1991-2002Crossref PubMed Scopus (533) Google Scholar), but the conformations of the two isoforms are dramatically different, with PrPSc having a much higher content of β-sheet (6Pan K.-M. Baldwin M. Nguyen J. Gasset M. Serban A. Groth D. Mehlhorn I. Huang Z. Fletterick R.J. Cohen F.E. Prusiner S.B. Proc. Natl. Acad. Sci. U. S. A. 1993; 90: 10962-10966Crossref PubMed Scopus (2049) Google Scholar, 7Caughey B.W. Dong A. Bhat K.S. Ernst D. Hayes S.F. Caughey W.S. Biochemistry. 1991; 30: 7672-7680Crossref PubMed Scopus (741) Google Scholar). There are several biochemical properties that distinguish PrPSc from PrPC, the most prominent being protease resistance. After treatment with proteinase K (PK), PrPSc is cleaved near amino acid 90 to yield a protease-resistant core fragment known as PrP 27–30, while under the same conditions PrPC is completely degraded (8Oesch B. Westaway D. Walchli M. McKinley M.P. Kent S.B. Aebersold R. Barry R.A. Tempst P. Teplow D.B. Hood L.E. Prusiner S.B. Weissmann C. Cell. 1985; 40: 735-746Abstract Full Text PDF PubMed Scopus (1238) Google Scholar). PrPSc is found in the brain in most cases of infectious, familial, and sporadic prion disease (3Prusiner S.B. Proc. Natl. Acad. Sci. U. S. A. 1998; 95: 13363-13383Crossref PubMed Scopus (5052) Google Scholar). However, in some inherited prion diseases, for example cases of Gerstmann-Sträussler syndrome due to an A117V mutation in PrP, PrPSc has not been detected and brain material does not appear to be infectious when injected into rodents (9Tateishi J. Kitamoto T. Doh-ura K. Sakaki Y. Steinmetz G. Tranchant C. Warter J.M. Heldt N. Neurology. 1990; 40: 1578-1581Crossref PubMed Google Scholar, 10Tateishi J. Kitamoto T. Brain Pathol. 1995; 5: 53-59Crossref PubMed Scopus (93) Google Scholar, 11Tateishi J. Kitamoto T. Hoque M.Z. Furukawa H. Neurology. 1996; 46: 532-537Crossref PubMed Scopus (86) Google Scholar). These exceptions raise the interesting possibility that PrPSc, although it is the infectious form of the protein, may not be the proximate cause of neurodegeneration in at least some forms of prion disease. Recently, it has been proposed that an alternate form of PrP that is distinct from PrPSc may play an important role in prion pathogenesis. This form, designated CtmPrP, has an unusual transmembrane topology (12Hegde R.S. Mastrianni J.A. Scott M.R. Defea K.A. Tremblay P. Torchia M. DeArmond S.J. Prusiner S.B. Lingappa V.R. Science. 1998; 279: 827-834Crossref PubMed Scopus (613) Google Scholar). Most molecules of PrPC do not span the lipid bilayer and are attached to the cell surface exclusively by a glycosyl phosphatidylinositol (GPI) anchor appended to the C terminus of the polypeptide chain (13Stahl N. Borchelt D.R. Prusiner S.B. Biochemistry. 1990; 29: 5405-5412Crossref PubMed Scopus (223) Google Scholar, 14Lehmann S. Harris D.A. J. Biol. Chem. 1995; 270: 24589-24597Abstract Full Text Full Text PDF PubMed Scopus (158) Google Scholar). In contrast,CtmPrP is thought to span the membrane once, with its C terminus on the exofacial surface and a highly conserved, hydrophobic region in the center of the molecule (amino acids 111–134) serving as a transmembrane anchor. Another form of PrP, NtmPrP, has also been described with the same transmembrane segment, but the reverse orientation (N terminus on the exofacial surface) (12Hegde R.S. Mastrianni J.A. Scott M.R. Defea K.A. Tremblay P. Torchia M. DeArmond S.J. Prusiner S.B. Lingappa V.R. Science. 1998; 279: 827-834Crossref PubMed Scopus (613) Google Scholar). It has been proposed that the relative proportions of these three topological variants is influenced by as yet unidentified accessory proteins that interact with the translocation apparatus in the endoplasmic reticulum (ER) (15Hegde R.S. Voigt S. Lingappa V.R. Mol. Cell. 1998; 2: 85-91Abstract Full Text Full Text PDF PubMed Scopus (86) Google Scholar, 16Hegde R.S. Lingappa V.R. Trends Cell Biol. 1999; 9: 132-137Abstract Full Text Full Text PDF PubMed Scopus (69) Google Scholar). Recent studies have brought the potential biological relevance of transmembrane forms of PrP into sharper focus. These species were originally observed only after translation of PrP mRNA in vitro on rabbit reticulocyte or wheat germ ribosomes in the presence of canine pancreatic microsomes (17Hay B. Prusiner S.B. Lingappa V.R. Biochemistry. 1987; 26: 8110-8115Crossref PubMed Scopus (89) Google Scholar, 18Hay B. Barry R.A. Lieberburg I. Prusiner S.B. Lingappa V.R. Mol. Cell. Biol. 1987; 7: 914-920Crossref PubMed Scopus (100) Google Scholar, 19Lopez C.D. Yost C.S. Prusiner S.B. Myers R.M. Lingappa V.R. Science. 1990; 248: 226-229Crossref PubMed Scopus (112) Google Scholar, 20Yost C.S. Lopez C.D. Prusiner S.B. Myers R.M. Lingappa V.R. Nature. 1990; 343: 669-672Crossref PubMed Scopus (108) Google Scholar, 21De Fea K.A. Nakahara D.H. Calayag M.C. Yost C.S. Mirels L.F. Prusiner S.B. Lingappa V.R. J. Biol. Chem. 1994; 269: 16810-16820Abstract Full Text PDF PubMed Google Scholar). In more recent investigations, however, CtmPrP has been identified in brain membranes from transgenic mice that express PrP molecules carrying mutations within or near the transmembrane domain; in vitro translation experiments had indicated that these mutations increased the relative proportion of CtmPrP (12Hegde R.S. Mastrianni J.A. Scott M.R. Defea K.A. Tremblay P. Torchia M. DeArmond S.J. Prusiner S.B. Lingappa V.R. Science. 1998; 279: 827-834Crossref PubMed Scopus (613) Google Scholar, 22Hegde R.S. Tremblay P. Groth D. DeArmond S.J. Prusiner S.B. Lingappa V.R. Nature. 1999; 402: 822-826Crossref PubMed Scopus (266) Google Scholar). Transgenic mice expressing such CtmPrP-favoring mutations at high levels develop a spontaneous neurodegenerative illness that bears some similarities to scrapie, but without the presence of PrPSc (12Hegde R.S. Mastrianni J.A. Scott M.R. Defea K.A. Tremblay P. Torchia M. DeArmond S.J. Prusiner S.B. Lingappa V.R. Science. 1998; 279: 827-834Crossref PubMed Scopus (613) Google Scholar, 22Hegde R.S. Tremblay P. Groth D. DeArmond S.J. Prusiner S.B. Lingappa V.R. Nature. 1999; 402: 822-826Crossref PubMed Scopus (266) Google Scholar). There is also indirect evidence thatCtmPrP accumulates in mice expressing wild-type PrP during the course of scrapie infection (22Hegde R.S. Tremblay P. Groth D. DeArmond S.J. Prusiner S.B. Lingappa V.R. Nature. 1999; 402: 822-826Crossref PubMed Scopus (266) Google Scholar). Based on these results, it has been hypothesized that CtmPrP represents a common intermediate in the pathogenesis of both infectious and genetic prion diseases (22Hegde R.S. Tremblay P. Groth D. DeArmond S.J. Prusiner S.B. Lingappa V.R. Nature. 1999; 402: 822-826Crossref PubMed Scopus (266) Google Scholar). In this view, CtmPrP is the ultimate cause of neurodegeneration, and PrPSc acts indirectly by increasing the amount of CtmPrP. We have previously carried out extensive studies of the properties of mutant PrP molecules expressed in cultured cells and transgenic mice (23Lehmann S. Harris D.A. J. Biol. Chem. 1996; 271: 1633-1637Abstract Full Text Full Text PDF PubMed Scopus (133) Google Scholar, 24Lehmann S. Harris D.A. Proc. Natl. Acad. Sci. U. S. A. 1996; 93: 5610-5614Crossref PubMed Scopus (97) Google Scholar, 25Daude N. Lehmann S. Harris D.A. J. Biol. Chem. 1997; 272: 11604-11612Abstract Full Text Full Text PDF PubMed Scopus (84) Google Scholar, 26Chiesa R. Piccardo P. Ghetti B. Harris D.A. Neuron. 1998; 21: 1339-1351Abstract Full Text Full Text PDF PubMed Scopus (278) Google Scholar, 27Chiesa R. Drisaldi B. Quaglio E. Migheli A. Piccardo P. Ghetti B. Harris D.A. Proc. Natl. Acad. Sci. U. S. A. 2000; 97: 5574-5579Crossref PubMed Scopus (137) Google Scholar). The mutations analyzed in those investigations lie outside of the conserved hydrophobic region that serves as a transmembrane anchor in CtmPrP and NtmPrP. To test the hypothesis that transmembrane PrP is part of a general pathway for prion-related neurodegeneration, we undertook here to determine whether these mutations induce the formation of NtmPrP andCtmPrP in vitro, in cultured cells, and in transgenic mice. We confirm that mutations in the central, hydrophobic region enhance formation of transmembrane PrP, and we demonstrate for the first time that CtmPrP contains a C-terminal GPI anchor as well as a transmembrane segment, thus exhibiting two modes of membrane attachment. However, we find that pathogenic mutations in other regions of the molecule have no effect on the amounts ofCtmPrP and NtmPrP, arguing against the possibility that transmembrane PrP plays an obligate role in the pathogenesis of prion diseases. P45–66 antibody, raised against a synthetic peptide encompassing amino acids 45–66 of mouse PrP, has been described previously (14Lehmann S. Harris D.A. J. Biol. Chem. 1995; 270: 24589-24597Abstract Full Text Full Text PDF PubMed Scopus (158) Google Scholar). Monoclonal antibody 3F4, which recognizes an epitope from hamster and human PrP encompassing residues 109–112 (28Bolton D.C. Seligman S.J. Bablanian G. Windsor D. Scala L.J. Kim K.S. Chen C.M. Kascsak R.J. Bendheim P.E. J. Virol. 1991; 65: 3667-3675Crossref PubMed Google Scholar), was a gift of Richard Kascsak (Institute for Basic Research, New York). R20 antibody, raised against a synthetic peptide comprising residues 218–232 of mouse PrP (29Caughey B. Raymond G.J. Ernst D. Race R.E. J. Virol. 1991; 65: 6597-6603Crossref PubMed Google Scholar), was a gift of Byron Caughey (Rocky Mountain Laboratories, Hamilton, MT). Anti-calnexin antibody was fromStressgen (Vancouver, British Columbia, Canada). All mouse PrP cDNAs were cloned into the vector pcDNA3 (Invitrogen), and carried an epitope tag for monoclonal antibody 3F4 created by changing residues 108 and 111 to methionine. The following mutations were introduced into the wild-type PrP cDNA using polymerase chain reaction as described previously (14Lehmann S. Harris D.A. J. Biol. Chem. 1995; 270: 24589-24597Abstract Full Text Full Text PDF PubMed Scopus (158) Google Scholar): PG11 (six-octapeptide insertion), PG14 (eight-octapeptide insertion), K109I/H110I, 3AV (Ala → Val at 112, 114, and 117), A116V, D177N, V179I, F197S, E199K, and V209I. Plasmids were linearized with XbaI and gel-purified. In vitro transcriptions were performed with the mMessage mMachine T7 kit (Ambion, Austin, TX). Messenger RNAs were translated with [35S]methionine in a final volume of 25 μl containing 50% nuclease-treated, rabbit reticulocyte lysate (Promgea, Madison, WI) according to the manufacturer's instructions. Canine pancreatic microsomal membranes were purchased from Promega, or were prepared by the method of Walter and Blobel (30Walter P. Blobel G. Methods Enzymol. 1983; 96: 84-93Crossref PubMed Scopus (470) Google Scholar). Microsomes from BW5147.3 mouse thymoma cells were prepared as described (31Vidugiriene J. Menon A.K. EMBO J. 1995; 14: 4686-4694Crossref PubMed Scopus (25) Google Scholar), except that cells were lysed by Dounce homogenization and passage through a 27-gauge needle. Pancreatic and thymoma microsomes were used at 1.5 and 5 μl per translation reaction, respectively, to equalize the amounts of ER marker proteins added. In some reactions, an oligosaccharide acceptor peptide (Bz-N-G-T-Ac; Bachem, Torrance, CA) was used at a final concentration of 0.5 mm to inhibit N-linked glycosylation. Translation reactions were incubated at 30 °C for 60 min. To detect protease-protected products, 5 μl aliquots of translation reactions were incubated in a final volume of 50 μl with 100 μg/ml PK (Roche Molecular Biochemicals) in 50 mm Tris-HCl (pH 7.5) and 1 mm CaCl2 for 60 min at 4 °C, followed by addition of 5 mm phenylmethylsulfonyl fluoride to terminate digestion. Some digestion reactions also contained 0.5% Triton X-100 to solubilize membranes. Samples were either analyzed directly by SDS-PAGE and autoradiography, or they were first subjected to immunoprecipitation or treatment with phosphatidylinositol-specific phospholipase C (PIPLC) as described below. Radioactive bands on gels were quantitated using a PhosphorImager (Molecular Dynamics, Sunnyvale, CA). Aliquots of the translation reactions were boiled in the presence of 1% SDS for 5 min to denature the proteins, and were then diluted with 10 volumes of radioimmune precipitation assay buffer (50 mm Tris-HCl (pH 7.5), 150 mm NaCl, 0.1% Triton X-100, 0.5% sodium deoxycholate, 0.1% SDS) plus protease inhibitors (pepstatin A and leupeptin, 1 μg/ml; phenylmethylsulfonyl fluoride, 5 mm). One μl of the appropriate anti-PrP antibody was added, and samples incubated on ice for 60 min. Protein A-Sepharose beads were added, and samples were rotated at 4 °C for 30 min. Beads were collected by low speed centrifugation and washed three times with radioimmune precipitation assay buffer, after which proteins were eluted with 1% SDS in 50 mm Tris-HCl (pH 7.5) and subjected to deglycosylation with peptide:N-glycosidase F (New England Biolabs, Beverly, MA) according to the manufacturer's directions. Following methanol precipitation, proteins were analyzed by SDS-PAGE and autoradiography. Five μl aliquots of translation reactions were diluted to 200 μl with 1% Triton X-114 (precondensed as described (Ref. 32Bordier C. J. Biol. Chem. 1981; 256: 1604-1607Abstract Full Text PDF PubMed Google Scholar)) in phosphate-buffered saline (PBS). After incubation at 4 °C for 20 min, samples were subjected to phase partitioning by incubation at 37 °C for 10 min, followed by centrifugation at 16,000 × g for 10 min to separate the phases. The aqueous phase was removed, and the detergent phase diluted to 200 μl with PBS containing protease inhibitors and 100 μg/ml bovine serum albumin. One unit of PIPLC from Bacillus thuringiensis (prepared as described in Ref. 33Shyng S.L. Moulder K.L. Lesko A. Harris D.A. J. Biol. Chem. 1995; 270: 14793-14800Abstract Full Text Full Text PDF PubMed Scopus (148) Google Scholar) was added to one half of the diluted detergent phase, and both halves were incubated at 4 °C for 2 h. The phase separation was repeated, and proteins in the second set of aqueous and detergent phases either were precipitated with methanol and analyzed by SDS-PAGE or were subjected to immunoprecipitation with 3F4 antibody. BHK cells were maintained in α-minimal essential medium supplemented with 10% fetal calf serum, nonessential amino acids, and penicillin/streptomycin. CHO cells were grown in α-minimal essential medium supplemented with 7.5% fetal calf serum and penicillin/streptomycin. Transfections were performed with LipofectAMINE (Life Technologies, Inc.) according to the manufacturer's instructions. Cells were harvested 24 h after transfection by brief trypsinization or by mechanical detachment, rinsed twice with PBS, and resuspended in 250 μl of 0.25m sucrose, 10 mm HEPES (pH 7.4), 1 μg/ml pepstatin A, and 1 μg/ml leupeptin. After 5 min on ice, cells were lysed by 10 passages through silastic tubing (0.3 mm, inner diameter) connecting two syringes with 27-gauge needles (34Lehmann S. Chiesa R. Harris D.A. J. Biol. Chem. 1997; 272: 12047-12051Abstract Full Text Full Text PDF PubMed Scopus (134) Google Scholar). A post-nuclear supernatant was prepared by centrifugation at 5,000 ×g for 2 min. PK protection assays were performed by incubating post-nuclear supernatants in 50 mm Tris-HCl (pH 7.5), 250 μg/ml PK, and in some cases 0.5% Triton X-100. After 60 min at 22 °C, digestion was terminated by addition of 5 mm phenylmethylsulfonyl fluoride. Samples were deglycosylated with peptide:N-glycosidase F prior to analysis by Western blotting. Fresh brain tissue (∼0.5 g wet weight) was homogenized using a Dounce apparatus in 5 ml of Buffer B (0.25m sucrose, 10 mm HEPES (pH 7.5), 100 mm KOAc, 1 mm Mg(OAc)2, 1 μg/ml pepstatin, 1 μg/ml leupeptin), followed by passage (five times each) through 16-, 18-, 21-, 23-, and 27-gauge needles. The homogenate was centrifuged first at 12,000 × g for 10 min in a microcentrifuge, and then at 541,000 × g for 20 min in a Beckman TLA100.3 rotor, and the microsomal pellet was resuspended in 250 μl of Buffer B. PK protection assays were carried out as described above for transfected cells, except that PK was used at 50 μg/ml. PrP is attached to the cell surface via a C-terminal GPI anchor (13Stahl N. Borchelt D.R. Prusiner S.B. Biochemistry. 1990; 29: 5405-5412Crossref PubMed Scopus (223) Google Scholar, 14Lehmann S. Harris D.A. J. Biol. Chem. 1995; 270: 24589-24597Abstract Full Text Full Text PDF PubMed Scopus (158) Google Scholar). In carrying out studies of the membrane topology of PrP after in vitro translation, it would thus be desirable to utilize microsomal membranes that are capable of attaching the GPI anchor to newly synthesized polypeptides. Previous investigations have employed microsomes derived from canine pancreas, which do not effectively carry out GPI anchor addition (35Fasel N. Rousseaux M. Schaerer E. Medoff M.E. Tykocinski M.L. Bron C. Proc. Natl. Acad. Sci. U. S. A. 1989; 86: 6858-6862Crossref PubMed Scopus (15) Google Scholar). We therefore tested microsomes derived from BW5147.3 murine thymoma cells, which have been reported to be much more efficient in GPI anchoring (31Vidugiriene J. Menon A.K. EMBO J. 1995; 14: 4686-4694Crossref PubMed Scopus (25) Google Scholar, 36Vidugiriene J. Menon A.K. J. Cell Biol. 1993; 121: 987-996Crossref PubMed Scopus (120) Google Scholar). We translated mouse PrP mRNA in vitro using rabbit reticulocyte lysate in the presence of pancreatic or thymoma microsomes, and then incubated Triton X-114 lysates of the membranes with or without PIPLC, a bacterial phospholipase that cleaves the GPI anchor. By partitioning the lysate into detergent and aqueous phases, we could then score the amount of PrP that had been rendered hydrophilic by removal of the anchor. As shown in Fig.1, all translation reactions produced two groups of products of 32 and 25 kDa, representing, respectively, core-glycosylated and unglycosylated forms of PrP. The latter correspond to molecules that had not been translocated into the lumen of the microsomes, since they are susceptible to digestion with PK (see below). Less than 10% of the PrP chains translated in the presence of canine pancreatic microsomes shifted into the aqueous phase after PIPLC treatment, indicating that very few molecules carried a GPI anchor (lanes 1–4). In contrast, about 50–60% of the glycosylated protein produced in the presence of thymoma microsomes shifted into the aqueous phase, indicating relatively efficient GPI anchoring (lanes 5–8). Untranslocated PrP chains, although they lack a GPI anchor, are retained in the detergent phase, presumably because they contain both N- and C-terminal signal peptides that are hydrophobic. Of note, PrP molecules shifted into the aqueous phase migrated with a slightly lower mobility than those that remained in the detergent phase (compare lanes 7and 8), a phenomenon that is typical for polypeptides that have lost their GPI anchor (37Rosenberry T.L. Cell Biol. Int. Rep. 1991; 15: 1133-1150Crossref PubMed Scopus (19) Google Scholar). We believe that the actual efficiency of anchor addition by thymoma microsomes may be even higher than 60%, and that the persistence of some glycosylated PrP in the detergent phase after PIPLC treatment is likely to reflect inefficient PIPLC digestion and phase partitioning. In support of this idea, when samples were denatured in SDS prior to PIPLC treatment, to improve accessibility of proteins to the phospholipase, essentially all of the glycosylated PrP was converted to the more slowly migrating band (not shown). In contrast, PIPLC treatment after SDS denaturation did not alter the gel mobility of PrP synthesized with pancreatic microsomes. To detect transmembrane forms of PrP, translation products were subjected to digestion by PK, which cleaves off portions of the polypeptide chain residing on the external side of the microsomal membrane and leaves transmembrane and lumenal domains intact. Samples were then immunoprecipitated with anti-PrP monoclonal antibody 3F4 to eliminate globin contamination contributed by the reticulocyte lysate, which obscures visualization of low molecular weight PrP fragments. Proteins were also subjected to enzymatic deglycosylation to reveal differences in the sizes of PrP species independent of glycosylation state. After PK digestion of translation reactions containing either canine pancreatic or thymoma microsomes, wild-type PrP was resolved into three forms of 25, 19, and 15 kDa (Fig.2, A and B,lane 2). The 25-kDa form is the same size as PrP before protease treatment (lane 1) and therefore represents molecules that are completely protected from digestion, and which therefore have been fully translocated into the microsome lumen; for consistency with an earlier report (12Hegde R.S. Mastrianni J.A. Scott M.R. Defea K.A. Tremblay P. Torchia M. DeArmond S.J. Prusiner S.B. Lingappa V.R. Science. 1998; 279: 827-834Crossref PubMed Scopus (613) Google Scholar), we refer to this form asSecPrP. The two smaller fragments are derived from transmembrane species whose cytoplasmic domains have been digested by the protease, and whose lumenal and transmembrane domains have been protected by the microsome. No PrP is detected after PK treatment in the presence of a detergent that disrupts the microsomal membrane (lane 3), confirming that the 19- and 15-kDa fragments do not represent intrinsically protease-resistant portions of the molecule. As will be shown below, the 19-kDa fragment derives fromCtmPrP, a species whose C terminus resides in the ER lumen, and the 15-kDa fragment from NtmPrP, a species whose N terminus is lumenal. Consistent with previous studies (12Hegde R.S. Mastrianni J.A. Scott M.R. Defea K.A. Tremblay P. Torchia M. DeArmond S.J. Prusiner S.B. Lingappa V.R. Science. 1998; 279: 827-834Crossref PubMed Scopus (613) Google Scholar, 22Hegde R.S. Tremblay P. Groth D. DeArmond S.J. Prusiner S.B. Lingappa V.R. Nature. 1999; 402: 822-826Crossref PubMed Scopus (266) Google Scholar), we find that two mutations in the central hydrophobic region of the PrP molecule increase the relative amount of CtmPrP with little change in the amount of NtmPrP (Fig. 2, A and B,lanes 4–9; TableI). A116V is the mouse homologue of a mutation in human PrP (A117V) that is associated with Gerstmann-Sträussler syndrome (38Tranchant C. Doh-ura K. Warter J.M. Steinmetz G. Chevalier Y. Hanauer A. Kitamoto T. Tateishi J. J. Neurol. Neurosurg. Psychiatry. 1992; 55: 185-187Crossref PubMed Scopus (49) Google Scholar, 39Mastrianni J.A. Curtis M.T. Oberholtzer J.C. Da Costa M.M. DeArmond S. Prusiner S.B. Garbern J.Y. Neurology. 1995; 45: 2042-2050Crossref PubMed Scopus (69) Google Scholar); 3AV is a triple alanine → valine mutation at residues 112, 114, and 117 that is not found in human PrP but that produces neurological disease in transgenic mice (12Hegde R.S. Mastrianni J.A. Scott M.R. Defea K.A. Tremblay P. Torchia M. DeArmond S.J. Prusiner S.B. Lingappa V.R. Science. 1998; 279: 827-834Crossref PubMed Scopus (613) Google Scholar). The effect of the A116V mutation was weaker than that of the 3AV mutation.Table IProportions of three topological forms of PrP produced by in vitro translationPancreasSecCtmNtmWT42.313.744.0(n = 7)(11.2)(4.8)(13.0)3AV17.735.047.3(n = 7)(9.9)(5.9)(12.9)A116V22.625.052.4(n = 4)(1.3)(3.1)(4.2)ThymomaSecCtmNtmWT79.68.811.6(n = 6)(12.3)(6.3)(6.9)3AV61.725.412.6(n = 6)(8.6)(5.4)(6.7)A116V72.511.715.9(n = 2)(6.9)(4.2)(2.7)Messenger RNAs encoding wild-type (WT), 3AV, or A116V PrP were translated in the presence of microsomes from canine pancreas or murine thymoma cells. Samples were subjected to PK digestion, and PrP was immunoprecipitated, deglycosylated, and analyzed by SDS-PAGE as in Fig.2. The percentage of each topological form was determined by phosphorImager analysis of SDS-PAGE gels. Each entry represents the mean of n experiments, with the S.D. given in parentheses below each entry. Values were corrected for the differences in methionine content among the three protease-protected products. Open table in a new tab Messenger RNAs encoding wild-type (WT), 3AV, or A116V PrP were translated in the presence of microsomes from canine pancreas or murine thymoma cells. Samples were subjected to PK digestion, and PrP was immunoprecipitated, deglycosylated, and analyzed by SDS-PAGE as in Fig.2. The percentage of each topological form was determined by phosphorImager analysis of SDS-PAGE gels. Each entry represents the mean of n experiments, with the S.D. given in parentheses below each entry. Values were corrected for the differences in methionine content among the three protease-protected products. We found that, for wild-type PrP, as well as for the two mutant PrPs, both NtmPrP and CtmPrP were produced in considerably smaller amounts in the presence of thymoma m
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