Doppel is an N-glycosylated, GPI-anchored protein

JBC Papers in Press. Published on June 6, 2000 as Manuscript M003888200

Doppel is an N-glycosylated, GPI-anchored protein: expression in testis and ectopic production in the brains of Prnp0/0 mice predisposed to Purkinje cell loss

Gregory L. Silverman1,2, Kefeng Qin1, Richard C. Moore4, Ying Yang3, Peter Mastrangelo1, Patrick Tremblay4, Stanley B. Prusiner4,5,6, Fred E. Cohen6,7,8,9, and David Westaway1,2*

Centre for Research in Neurodegenerative Diseases, 2Department of Laboratory Medicine and Pathobiology, and 3Mass Spectrometry Laboratory, MMRC, University of Toronto, Toronto, Ontario, Canada M5S 3H2 and 4

Institute for Neurodegenerative Diseases, Departments of 5Neurology, 6Biochemistry and Biophysics, 7Cellular and Molecular Pharmacology, 8Pharmaceutical Chemistry, and 9Medicine, University of California, San Francisco, CA, USA 94143-0518

* To whom correspondence should be addressed at: CRND, Tanz Neuroscience Bldg., 6 Queen's Park Cres. W., Toronto, Ontario, Canada M5S 3H2. Tel: 416-978-1556; Fax: 416-978-1878; E-mail: [email protected]

Running title: "Characterization of the Dpl protein and detection in vivo"

Copyright 2000 by The American Society for Biochemistry and Molecular Biology, Inc.

Downloaded from http://www.jbc.org/ by guest on December 28, 2015

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"Characterization of the Dpl protein and detection in vivo"

SUMMARY

The Prnd gene encodes a homolog of the cellular prion protein (PrPC) called doppel (Dpl). Up-regulation of Prnd mRNA in two distinct lines of PrP gene ablated (Prnp0/0) mice, designated Rcm0 and Ngsk, is associated with death of Purkinje cells. Using recombinant Dpl expressed in E.coli and mouse neuroblastoma cells we demonstrate that wild type (wt) Dpl, like PrPC, adopts a predominantly alpha-helical conformation, forms intramolecular disulfide bonds, has two N-linked oligosaccharides, and is Downloaded from http://www.jbc.org/ by guest on December 28, 2015

presented on the cell-surface via a GPI anchor. Dpl protein was detected in testis of wt mice. Using Triton X-114 phase partitioning to enrich for GPI-anchored proteins, Dpl was detected in brain samples from Rcm0 Prnp0/0 mice but was absent in equivalent samples from wt mice and ZrchI Prnp0/0 mice, indicating that ectopic expression of this protein may cause cerebellar pathology in Rcm0 mice. Biochemical and structural similarities between PrPC and Dpl documented here parallel the observation that ataxic Ngsk Prnp0/0 mice can be rescued by overexpression of wild-type PrP transgenes, and suggest that cell-surface PrPC can antagonize the toxic effect of Dpl expressed in the central nervous system.

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INTRODUCTION

The prion protein gene, Prnp, encodes PrPSc, the major structural component of the etiological agent in disorders such as scrapie and Creutzfeldt-Jakob disease. In uninfected animals Prnp encodes a cellular protein denoted PrPC, which is thought to be converted to PrPSc by a posttranslational mechanism during the course of prion infections. Though Prnp is conserved in mammalian species, the ZrchI and Npu lines of Prnp0/0 gene-ablated mice remain healthy throughout development (1-3): this may

perhaps that other molecules fulfill an overlapping function. An insight into this issue has emerged from large-scale sequencing (4). While Prnp was originally considered to be unique within the mammalian genome it has recently been shown to have a paralog (5). The mouse gene Prnd is located 16 kB downstream of Prnp and encodes the 179 amino acid Dpl protein. Corresponding Dpl coding sequences are located downstream of Prnp in both humans and rats, and most likely in other mammals. Dpl has approximately 25% sequence identity with the C-terminal two-thirds of PrPC and is predicted to contain three alpha helices and two disulphide bonds, but is notably lacking an octapeptide repeat domain like the one present at the N-terminus of PrPC (5). Unlike Prnp, abundant Prnd mRNAs are absent from the brains of wild-type (wt) animals, although species of 1.7 and 2.7 kB are present in high levels in both heart and testis (5). Interestingly, however, inappropriate expression of Prnd mRNA in the CNS is associated with a neurodegenerative syndrome. The Rcm0 and Ngsk lines of Prnp0/0 mice display normal early development but develop a widespread loss of cerebellar

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indicate that PrPC serves a function that is not apparent in a laboratory setting, or


Purkinje cells at around 70 weeks of age, which is accompanied by a progressive ataxia (5,6). These mice differ from asymptomatic Prnp0/0 mice in that they carry chromosomal deletions which extend beyond the Prnp open reading frame (ORF) into intron 2 at the 5’ end, and extending into the 3’ untranslated region of the Prnp mRNA (encoded by exon 3) at the 3’ end. These deletions eliminate the Prnp exon 3 splice acceptor site and result in the generation of high levels of Prnd transcripts in the brain by an unusual mechanism involving exon-skipping and intergenic splicing (5). Although it is possible that long-range perturbations in chromatin structure underlie the ataxic phenotype of the Downloaded from http://www.jbc.org/ by guest on December 28, 2015

Rcm0 and Ngsk lines of Prnp0/0 mice, the parsimonious interpretation of the available data is that Dpl overexpression is toxic to Purkinje cells. Lastly, it is noteworthy that disease in Ngsk Prnp0/0 mice is abrogated by reintroduction of a wt Prnp transgene (7), providing genetic evidence for an interaction between PrPC and the molecules that cause Purkinje cell loss. In this work we sought confirmation of previous predictions regarding the biochemical and structural properties of the Dpl protein. We demonstrate that recombinant Dpl refolded under standard conditions adopts a predominantly α-helical conformation, and that Dpl expressed in mouse neuroblastoma cells possesses two Nlinked oligosaccharides and is attached to the cell surface via a GPI-anchor. These findings show that Dpl and PrPC resemble one another with respect to their posttranslational modifications and sub-cellular localization. We also report that Dpl protein is present in the brains of Rcm0 mice, but is undetectable in the brains of wt FVB/N mice and the ZrchI line of PrP-knockout mice. Our findings are discussed in the context of the transgene rescue experiments indicating interactions between Dpl and PrPC.

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EXPERIMENTAL PROCEDURES Expression, purification, and refolding of mouse Dpl 27-154. The DNA fragment coding for mouse Dpl (MoDpl) 27-154 was amplified by the polymerase chain reaction from cosmid I/LnJ-4 using the oligonucleotide primers: (1) N-terminal primer: 5’GGGGCATATGTCTAGGGGCATAAAGCACAGG-3’; and (2) C-terminal primer: 5’GGGGGATCCTATCACCTTTCCAGCCAGAAATCGCA-3’. The amplified gene was cloned into the plasmid pRBI-PDI-T7 via the Nde I and BamH I restriction enzyme sites.

gene is under the control of the T7 promoter/lac operator sequence (8,9). According to the N-end rule in bacteria (10), we introduced a Ser at the amino-terminus of MoDpl 27154 to minimize proteolytic degradation in the cytoplasm. The recombinant MoDpl 27154 consists of 130 amino acid residues with the correct gene sequence verified by DNA sequencing. Cells of E.coli BL21 (DE3) including pMoDpl27-154 were grown at 37 ºC in 1 L LB media containing ampicillin (100 µg/ml). At OD600nm = 0.8-1.0, IPTG was added to a final concentration of 1 mM and the culture was incubated at 37 ºC for another 16 h. Cells were harvested by centrifugation at 3,000 g for 10 min and suspended in 20 ml of suspension buffer (50 mM Tris-HCl, pH 8.0, 1 mM MgCl2, 0.4 mg/ml DNase I, 0.4 mg/ml RNase A, 1 mg/ml lysozyme, 1 mM PMSF), then shaken at 37 ºC for 2 h and at room temperature for 1 h. The lysate was centrifuged at 4 ºC at 39,000 g for 1 h. The insoluble inclusion bodies were washed twice with 10 ml wash buffer (20 mM Tris-HCl, pH 8.0, 23% sucrose (w/v), 0.5% Triton X-100 (v/v), 1 mM EDTA, 1 mini-complete protease inhibitor cocktail tablet, EDTA-free (Boehringer

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This expression plasmid was named pMoDpl27-154. In this plasmid, the mouse Dpl


Mannheim)) and solubilized in 10 ml of dissolve buffer (10 mM Tris-HCl, pH 8.0, 50 mM DTT, 1 mM EDTA, 8 M urea, 1 mini-complete proteases inhibitor cocktail tablet, EDTAfree). After centrifugation at 39,000 g at 22 ºC for 1 h, 3 ml of supernatant was applied to a SP-Sepharose column (20 ml: Pharmacia) equilibrated with 10 mM MOPS-NaOH, pH 7.0, 5 mM DTT, 1 mM EDTA, 8 M urea, using a BioLogic HR chromatography system (Bio-Rad). The isocratic rate was 1 ml/min and fractions were collected in 1.5 ml/tube. MoDpl 27-154 was eluted with a linear NaCl gradient (0-0.6 M). Fractions containing MoDpl 27-154 were pooled and diluted with 50 mM Tris-HCl, pH 8.7, 8 M

concentration of 1 µM, and the solution stirred for 2 h at room temperature. The oxidation was quenched by addition of 1 mM EDTA. The solution was dialyzed against ddH2O and concentrated by Centriplus 3 kDa molecular weight cut-off concentrators (Amicon) to 1 mg/ml, and stored at -20 ºC. Recombinant mouse PrP (MoPrP23-231) was prepared as described previously (11).

Electrophoretic analyses and N-terminal amino acid sequencing: Proteins were boiled in the gel loading buffer for 5 min, electrophoresed on 10-20% Tricine gels and stained with Coomassie Blue. For N-terminal amino acid sequencing, the protein was electrophoresed on a 10-20% Tricine gels, transferred to a PVDF membrane, and stained by Coomassie Blue. The membrane was sliced and individual bands sequenced using a Porton Gas-phase Microsequencer Model 2090 and on-line PTH analysis.

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urea to a protein concentration of 0.05 mg/ml. CuSO4 was added to a final


Amino acid analysis and determination of protein concentration: The amino acid analysis was performed on a Waters PICO-TAG System calibrated in triplicate using a collection of derivatized amino acid standards. Dried MoDpl 27-154 was hydrolyzed using 6 M HCl with 1% phenol at 110 ºC for 24 h. After hydrolysis, excess HCl was removed from the hydrolysis tube under vacuum, the sample was derivatized, dissolved in sample diluent (pH 7.4) and an aliquot injected into a Waters PICO-TAG column running on a Waters PICO-TAG gradient, with a column temperature of 33 ºC. For tabulation of individual amino acids, yields are expressed as percentage weight or Downloaded from http://www.jbc.org/ by guest on December 28, 2015

percentage content with a molecular weight correction for loss of one water molecule per residue.

S-carbamidomethylation of cysteines: MoDpl 27-154 (1.2 mg/ml) in 50 µl Scarbamidomethylation buffer (0.1 M Tris-HCl, pH 8.0, 1 mM EDTA, 6 M guanidine hydrochloride) was incubated with or without 10 mM dithiothreitol (DTT) at room temperature for 1 h. The reduced or non-reduced MoDpl 27-154 was incubated with 50 mM iodoacetamide (IAM) at room temperature in the dark for 30 min. Iodoacetamide reacts with free SH group of cysteine to yield a carbamidomethyl cysteine, with a concomitant increase in molecular weight of 57 Da. 1 µl of reduced or non-reduced MoDpl 27-154 treated with IAM was then used for matrix-assisted laser desorption/ionization mass spectrometric (MALDI-MS) analysis without further purification.

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Trypsin digestion of MoDpl 27-154: Trypsin digestion was carried out with MoDpl 27154 (1 mg/ml) in 20 µl of 100 mM ammonium bicarbonate buffer, pH 8.0, 1 mM CaCl2. 1 µl of modified trypsin (sequencing grade, Promega) solution (2 mg/ml in 50 mM acetic acid) was added to yield a final enzyme protein ratio of 1:10. After incubation at 37 ºC for 2 h, 2 µl of 1 N HCl was added to bring the mixture to pH 3.0 and inactivate the trypsin. One half of the sample (10 µl) was moved to another tube and mixed with 2 µl of 0.1 M Tris-(2-carboxyethyl)phosphin (TCEP, Pierce) in 0.1 M citrate buffer, pH 3.0.

containing tryptic peptides. 1µl aliquots of reduced or non-reduced tryptic peptide mixture were used for MALDI-MS analysis without further purification.

Mass spectrometric molecular weight determination and peptide mapping - MALDI timeof-flight (TOF) MS analyses were carried out using a Perseptive Biosystem Voyager-DE STR mass spectrometer (Perseptive Biosystems Inc.), equipped with a pulsed UV nitrogen laser (337 nm, 3 ns pulse) and a dual microchannel plate detector. For molecular weight determination of full-length MoDpl 27-154 protein, spectra were acquired at linear-DE mode, acceleration voltage set to 25 kV, grid voltage at 95% of the acceleration voltage, guide wire voltage at 0.150%, delay time at 320 ns, and low mass gate was set at 1000 Da. The mass to charge ratio was calibrated with the molecular weight of a mixture of proteins (MW 5734.58 Da to 16952.56 Da). For analysis of tryptic peptides, the spectra was acquired at reflectron-DE mode with acceleration voltage set to 20 kV, grid voltage at 72% of the acceleration voltage, guide wire voltage at 0.050%, delay time at 200 ns, and low mass gate at 250 Da. The mass

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The mixture was incubated at 37 ºC for 30 min for reduction of the disulfide bond-


to charge ratio was calibrated with the mass of a mixture of standard peptides (MW 904.46 Da to 5734.58 Da). Saturated α-cyano-4-hydroxycinnamic acid in 70% acetonitrile containing 0.1% trifluoroacetic acid (TFA) was used as the matrix for analysis of tryptic peptides, and saturated sinapinic acid in 50% acetonitrile containing 0.1% TFA was used as the matrix for protein analysis. 1 µl of a solution of MoDpl 27154, reduced or non-reduced S-carbamidomethyl MoDpl 27-154, or reduced or nonreduced tryptic peptide mixture was applied on the MALDI plate followed by 1 µl of saturated matrix solution. Spectra were recorded after evaporation of the solvent and

were calculated by the Peptide Mass program on the ExPASy Home Page (http://expasy.cbr.nrc.ca).

Circular Dichroism: Far-UV circular dichroism (CD) measurements were performed by a JASCO J-715 spectropolarimeter. Using a cell of path length 1 mm, scans were conducted between 190 and 250 nm at a scan speed of 20 nm/min with a sensitivity of 50 mdeg. All CD spectrum measurements were performed at room temperature, in 20 mM phosphate buffer (PB), pH 7.0. Spectra were subject to curve-smoothing and where noted converted into [θ], mean residue ellipticities, in degree cm2/dmol based on the protein concentration determined by amino acid analysis using the PICO-TAG system.

Antibodies - The anti-Dpl rabbit polyclonal Ab, D7177, was raised against the synthetic Dpl-2 peptide as described previously (5).

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processed using GRAMS software for data collection and analysis. Predicted masses


Cell lines - The mouse neuroblastoma cell line, N2a, was obtained commercially (ATCC) and used to establish all cell lines. The cDNA for mouse Dpl was cloned into the mammalian expression vector pcDNA3.0 (Invitrogen) and used to establish the stably transfected line of Dpl expressing cells, called Dpl-3, as described previously (5). The Dpl-7 cell line, which expresses no Dpl mRNA or protein when assayed by northern and western blotting respectively (5), and untransfected N2a cells were used as negative controls.

DMEM supplemented with 10% FBS. Media for Dpl-3 and Dpl-7 cultures was additionally supplemented with 0.3 mg/ml G418 (Life Technologies). Cells were washed 3X in HBSS, scraped into HBSS, pelleted at 1000 g at 4 °C and then resuspended in cell lysis buffer (0.5% Triton X-100, 0.5% Na-deoxycholate, 150 mM NaCl, 20 mM TrisHCl, pH 7.4) containing one mini-complete protease inhibitor tablet. Cells were disrupted by agitating for 10 min at 4 °C and then centrifuging at 20,000 g for 10 min at 4 °C to remove insoluble debris. Cleared supernatant was transferred to a new tube and stored at -80 °C until needed.

Preparation of tissue homogenates - Ten percent (w/v) homogenates of tissues were prepared in PBS (pH 7.4) containing a mini-complete protease inhibitor tablet on ice using a Dounce homogenizer. Homogenates were spun at 3000 g for 10 min at 4 °C and the cleared supernatant was aliquoted and stored at -80 °C until needed. The

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Preparation of cell lysates - Cells were grown to 80-90% confluency in 10 cm2 dishes in


concentration of total protein in each sample was determined using a Bradford assay as per the manufacturer's instructions (Bio-Rad).

PNGaseF digestion - Aliquots of cell lysate or tissue homogenates were thawed, 0.1 vol of 10X denaturing buffer (5% SDS, 10% β-mercaptoethanol) was added to each sample, and tubes were boiled for 10 min. To each sample the following was added: 0.1 vol 10% NP-40, 0.1 vol 0.5 M Na-phosphate buffer (pH 7.5). 0.2 vol of PNGaseF (1 unit/µl in 50 µM Na-phosphate buffer, pH 7.5; New England Biolabs) was added to all

phosphate buffer (pH 7.5) alone was added. Samples were incubated for 0, 15, 30, 45, 60 or 120 min at 37 °C with gentle shaking. 0.25 vol of 4X Laemmli buffer was added to each sample following incubation, and samples were analyzed by western blotting.

PIPLC incubation - Dpl-3 and Dpl-7 (negative control) cell lines were grown to 80-90% confluency on 10 cm2 dish as noted above. Cells were washed 3X in HBSS, and then incubated in serum-free OptiMEM (Life Technologies) with or without 0.2 units/ml PIPLC (Molecular Probes) for 1 hr at 37 °C. Cell media was recovered and transferred to a tube containing 0.1 vol of 10X TNE (500 mM Tris-HCl, pH 7.5, 1.5 M NaCl, 50 mM EDTA) . Recovered media was centrifuged at 2500 g for 5 min at 4 °C to remove any cellular debris. The cleared media was concentrated using a 10 kDa molecular weight cut-off centricon tube (Amicon), and then methanol precipitated. Precipitated proteins were resuspended in 1X Laemmli buffer and stored at -80 °C for later analysis. After removal of the media, cells were washed 3X in HBSS, scraped into 5 ml HBSS, and

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samples except those to be used as negative controls to which 0.2 vol of 50 mM Na-


pelleted at 1000 g for 5 min at 4 °C. The supernatant was removed and the cell pellet was resuspended in cell lysis buffer and transferred to a clean microfuge tube. Cells were disrupted by agitating for 10 min at 4 °C and then centrifuging at 20,000 g for 10 min at 4 °C to remove insoluble debris. Cleared supernatant was transferred to a new tube, 0.25 vol of 4X Laemmli buffer was added, and samples were analyzed by western blotting.

Triton X-114 phase partitioning - Prior to conducting Triton X-114 phase partitioning,

was thoroughly mixed at 4 °C to solubilize the detergent and then incubated overnight at 37 °C to induce phase transition. The upper aqueous phase was discarded and the lower detergent phase was mixed with 0.1 vol of 10X TNE to create a Triton X-114 stock. The hydrophobicity of cellular proteins was determined by harvesting cells as described above and resuspending the cell pellet in 1X TNE containing 0.25 vol of Triton X-114 stock. Recovered media was first cleared, concentrated and methanol precipitated as described above; proteins were then resuspended in 1X TNE containing 0.25 vol of Triton X-114 stock. All samples were cooled to 4 °C and then centrifuged at 3000 g for 10 min at 4 °C to remove any insoluble aggregates. Supernatant was transferred to a clean microfuge tube, heated at 37 °C for 10 min to induce phase transition, then centrifuged at 3000 g for 10 min to pellet detergent-protein micelles. The upper aqueous phase was transferred to a new tube, again mixed with 0.25 vol of Triton X-114 stock at 4 °C, and then heated to 37 °C to induce phase transition. This

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stock solutions were prepared as follows. A 3% (v/v) aqueous solution of Triton X-114


repartitioning of the aqueous phase was repeated two more times resulting in the generation of four separate detergent phases and a single aqueous phase. Detergent phases one, two and three were pooled in a single tube. All samples were then precipitated by adding 5-10 vol of methanol, incubating overnight at -20 °C, and then centrifuging at 3000 g for 30 min at 4 °C. Samples were resuspended in 1X Laemmli buffer and analyzed by western blotting. Triton X-114 phase partitioning of mouse tissue homogenates was conducted as follows. Volumes of ten percent (w/v) tissue homogenate corresponding to 50 µg of total

Samples were mixed at 4 °C and then partitioned as described above.

Western blotting - Prior to fractionation by SDS-PAGE, samples were boiled for 5 min and the centrifuged at 20,000 g for 5 min. Samples were loaded onto 10-20% Tricine gradient gels (Novex) and electrophoresed at 125V for approximately 90 min. Samples were transferred to 0.2 µm nitrocellulose (Schleicher & Schuell) for 60 min at 100V in 20 mM Tris buffer (pH 8.5) containing 150 mM glycine, 20% (w/v) methanol, 0.01% SDS. Blots were blocked with 5% (w/v) milk powder in TBS (pH 7.4) for 3 hr at room temperature and then probed with D7177 Ab at a concentration of 1:1,250 in 0.5% (w/v) milk powder in TBS (pH 7.4). Where D7177 Ab was preincubated with the immunogenic Dpl-2 peptide, 20 µg peptide was added to the antibody and incubated with gentle shaking at room temperature for 3 hr prior to use. The secondary Ab used was goat anti-rabbit HRP conjugated antisera (Bio-Rad). Blots were visualized using ECL or ECL-

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protein were thawed and mixed 1:4 with 1X TNE containing 0.25 vol Triton X-114 stock.


Plus detection reagents (Amersham Pharmacia) as per the manufacturer's instructions and exposed on X-OMAT AR film (Kodak).


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RESULTS Expression and authentication of recombinant MoDpl 27-154: Dpl is predicted to contain N- and C-terminal signal peptides, directing expression into the secretory pathway and addition of a glycolipid anchor, respectively ((5), this paper). Accordingly, an expression cassette was constructed omitting these signal peptide regions (MoDpl 27-154: Figure 1) by amplifying DNA from the I/LnJ-4 cosmid clone (12). The N-terminus of the reading frame was modified by the addition of a methionine. Since the wild-type N-terminal Arg is predicted to be a destabilizing residue in E.coli according to the N-end rule, (10), a

demonstrated a single species at 14.9 kDa (data not shown) and Edman degradation yielded the anticipated N-terminal sequence SRGIKHRF. MALDI-MS molecular weight analysis of MoDpl 27-154 showed a single charged protein signal ([M+H]+) at m/z14917.0 (±1.5) Da (Table 1, Figure 2A), in excellent agreement with the calculated molecular weight 14916.9 Da ([M+H]+). The only other strong signal present in this analysis (Figure 2A) is a double charged protein signal at m/z 7458.9 Da, underscoring the purity of the protein sample. Lastly, as detailed below, the masses of tryptic peptides derived from the recombinant protein are also in very close accord with those predicted from the nucleotide sequence of Prnd (Table 1).

Disulfide bonds in MoDpl 27-154: Our preparation scheme for MoDpl 27-154 included exposure to 1 µM Cu(II), to favour formation of an intramolecular of disulfide bond (or bonds). Whereas PrPC has a single disulfide bond, according to computer modeling studies four Cys residues in the C-terminus of Dpl (residues 95, 109, 143 and 148) are

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Ser residue was also introduced. Electrophoretic analysis of purified MoDpl 27-154


predicted to participate in two disulfide bonds (5). We therefore employed the Scarbamidomethylation method, previously used to analyze PrP23-231 (11), to titrate the number of free cysteines in MoDpl 27-154 prepared by this standard oxidation-refolding regimen. After incubation with 6 M guanidine hydrochloride in the presence or absence of DTT at pH 8.0, MoDpl 27-154 was incubated with iodoacetamide (IAM) for Scarbamidomethylation of cysteines and analysed by MALDI-TOF-MS. “Native” MoDpl 27-154 (i.e., non-reduced) treated with IAM did not exhibit a significant change in mass:

14918.4 Da, well within the error of 0.05% (~7.5 Da) for this type of analysis (Figure 2A and 2B). This indicates an absence of free cysteines. After reduction with DTT, carbamidomethylated MoDpl 27-154 increased in mass by 231 units to 15149.0 Da (Figure 2C), consistent with the addition of four adducts (predicted mass 15144.9 Da). These data are compatible with 2 disulfide bridges per MoDpl 27-154 molecule.

Secondary structure and disulfide bonds in MoDpl 27-154: A solution of MoDpl 27-154 in 20 mM PB, pH 7.0 was examined by far-UV circular dichroism (CD), using mouse PrP23-231 produced by a similar refolding regimen as a control (9,11). The Dpl27-154 sample exhibited a CD spectrum indicative of a high α-helical content, as did MoPrP23231, with minima at 208 and 222 nm and a maximum at 192 nm (Figure 3). Standard deconvolution algorithms indicated α-helical content for Dpl27-154 between 28 and 35%, with β-sheet estimated at ~15%. These data are compatible with the prediction

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the starting material had a mass of 14916.9 and IAM-treated material had a mass of


that Dpl has three alpha helices analogous to those present in mouse PrPC and that it could have a short β-sheet region like PrPC (5,13-15). Tryptic peptides of Dpl (see Table 1, Figures 4 and 5) were analyzed by mass spectroscopy to ascertain the connectivity of disulfide linkages. Mass spectra of tryptic digests of native and reduced MoDpl 27-154 samples are presented in Figure 4A and 4B. As predicted from the analysis with IAM, four signals present in the reduced sample were either absent or present at considerably lower levels than in the native sample (see also Figure 5B: Table 1). These are tryptic peptides 12 (T12) at m/z 2953.9 Da,

(Parenthetically, the T12 peptide has a propensity to form Na+ adducts - secondary and tertiary peaks immediately to the right of the signal at 2953.9 - under the conditions of this analysis, although the significance of this phenomenon is currently unclear). Conversely, additional signals at high mass-to-charge ratios and with large peak heights were present in the un-reduced “native” sample. The peak with the largest amplitude in this analysis was detected at m/z 2920.0 Da and is assignable as T13-S-S-T19 (Figure 4A: predicted mass 2920.3 Da). This indicates a disulfide bond between residues 109 and 143, exactly analogous to the PrPC disulfide bond between residues 178 and 213 linking helices B and C. A strong signal was also detected at m/z 4058.6 Da, assignable as T12-S-S-T20 and indicating a Cys95-Cys148 connectivity. However, we also observed signals with small amplitudes indicative of two other disulfide bond connectivities not predicted by computer modeling (Table 1, Figure 4A and “dashed-line” tryptic peptide maps in Figure 5B). First, a signal at m/z 1754.4 Da, possibly corresponding to a Cys143-Cys148 bond (predicted mass of the T19-S-S-T20 di-peptide 1755.0 Da), though

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T13 at m/z 2271.6 Da, T19 at m/z 650.5 Da, and T20 at m/z 1106.0 Da.


a peptide indicative of a reciprocal connectivity of T12-T13 was not detected at the predicted mass of 5223.7 Da (not shown). Second, a smaller signal at m/z 3375.6 Da, assignable as T13-S-S-T20 (predicted mass 3375.7 Da), though again, a signal indicating a reciprocal connectivity of T12-S-S-T19 (predicted mass 3603.0 Da) was not detected. We conclude that while most MoDpl 27-154 molecules prepared by a standard oxidative refolding scheme contain the Cys109-Cys143 and Cys95-Cys148 linkages predicted by computer modeling (5), lower abundance species with other connectivities or free sulphydryl groups can be detected under these particular

Dpl has two N-linked glycosylation sites - Previous experiments involving immunoblots of Dpl-3 cell lysates showed a heterodisperse signal between ~32-41 kDa when blotted with the D7177 Ab. This signal is reduced to a single band at ~17 kDa when cell lysates were incubated with PNGaseF prior to blotting, indicating that Dpl is heavily glycosylated (5). When Dpl cell lysates were incubated with PNGaseF for different periods of time it was possible to identify a single intermediate form of Dpl at ~27 kDa, presumably representing the monoglycosylated protein (Figure 6). Prolonged incubations with PNGaseF did not reduce Dpl beyond ~17 kDa. These results indicate the presence of two N-linked glycosylation sites within Dpl as had been predicted previously (5).

Dpl is a GPI-anchored cell surface protein - Analysis of the translated protein sequence of Prnd failed to reveal a classical GPI recognition sequence (5), although a

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conditions of proteolysis and mass spectrometric analysis.


hydrophobic C-terminal extension was broadly compatible with this notion. Resolution of this issue was deemed of importance, as it bears upon the biological site of action of Dpl. Since the D7177 Ab exhibits cross-reactivity with protein species that do not correspond to Dpl ((5): this paper) it was considered unsuitable for immunohistochemical studies of cell cultures using the GPI-anchor cleaving enzyme PIPLC (16). Instead, we sought direct biochemical evidence for a GPI anchor. Figure 7A shows an immunoblot of cell lysates and media from the Dpl-expressing neuroblastoma cell line, Dpl-3. When a monolayer of intact Dpl-3 cells was incubated with the enzyme

from the cell lysate. These results contrast with those obtained when non-PIPLC treated Dpl-expressing cells were western blotted. In this case, Dpl was detected in the cell lysate but was not present in the recovered media. Dpl was not observed in the pellet or supernatant fractions of PIPLC and non-PIPLC treated Dpl-7 cells used as negative controls (data not shown). Triton X-114 phase-partitioning of PIPLC treated and non-treated Dpl-3 cells and media was used as another means of confirming the presence of Dpl's GPI anchor. When cell lysates were subjected to Triton X-114 phase partitioning prior to immunoblotting, Dpl was detected in pooled detergent phases 1-3, but was not present in either detergent phase 4 (included as an internal negative control), or the aqueous phase (Figure 7B). When recovered media from PIPLC-treated Dpl-3 cells was subjected to Triton X-114 phase partitioning Dpl was found to have shifted from the detergent phase to the aqueous phase. Together these results indicate that the Dpl protein is present on the cell surface and is attached via a GPI anchor.

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PIPLC prior to collection and lysis, Dpl was present in the recovered media but absent


Detection of Dpl in mouse tissue samples - Homogenates of testis and brain from wt FVB/N mice were incubated with or without PNGaseF for 3 hours prior to western blotting. When these samples were electrophoresed and probed with D7177 Ab, Dpl could be observed in the testis samples, but was not detected within brain samples (Figure 8A). Untreated testis contained a heterodisperse signal from ~29-37 kDa, while PNGaseF treated testis displayed a single band at ~17 kDa which corresponded to unglycosylated Dpl protein. These signals were not observed in blots probed with

pre-immune sera (data not shown). As Prnd mRNA levels are low in the CNS of wt FVB/N and C57BL6 mice (5) (i.e., undetectable by standard northern blotting procedures), Triton X-114 phase partitioning was used to enrich for Dpl, and thereby enhance the possibility of detecting the Dpl protein in brain samples. As Dpl with an intact GPI anchor partitioned within the detergent phase (Figure 7B), tissue homogenates were subjected to repetitive partitioning and detergent fractions 1 to 3 were pooled and used for western blot analysis. When partitioned brain extracts from Rcm0, ZrchI and wt FVB/N mice (using asymptomatic animals in the case of the Rcm0 knock-out line) were probed with D7177, a ~30-33 kDa signal corresponding to Dpl could be detected in brains from Rcm0 mice, as well as in detergent phases 1-3 from testis of wt animals. A comparable signal was absent from similarly processed brain samples of ZrchI and wt FVB/N mice, as well as the aqueous phase of testis homogenates included as a negative control (Figure 8B, left-hand panel). A duplicate blot probed with D7177 Ab preincubated with a synthetic

20


D7177 that had been preincubated with Dpl-2 peptide, or when blots were probed with


Dpl peptide competitor confirmed that the signals attributed to Dpl within wt testis and Rcm0 brain were not due to cross-reactivity of the antibody with a non-specific antigen (Figure 8B, right-hand panel). These results indicate that while the Dpl protein is present in the testis of wt FVB/N mice, it is only present in the brains of Rcm0 mice, as suggested by prior northern blot and RT-PCR analyses (5).

DISCUSSION Features of MoDpl 27-154 expressed in E. coli - Though a number of difficulties were

feasibility of expressing Dpl in this system. Dpl produced by a standard folding paradigm adopts an α-helix rich conformation, a feature it shares in common with full-length PrPC, and demonstrated here in side-by-side CD spectral analyses (Figure 3). However, since Dpl likely contains two, rather than one intramolecular disulfide bond, novel re-folding protocols may need to be developed to obtain stoichiometric formation of two disulfide bridges without accruing molecules with extraneous connectivities. Though tryptic dipeptides containing the Cys95-Cys148 and Cys109-Cys143 disulfide bonds predicted from computer modeling were the species with the highest peak-heights in the mass spectrometric analyses of re-folded MoDpl 27-154 (Figure 4A), tryptic peptides indicative of alternative connectivities were present of low levels. In particular, Cys 148 located in tryptic peptide 20 has a tendency to form linkages to Cys 143 and Cys 109. Whether the alternative disulfide linkages represented by T13-S-S-T20 and T19-S-ST20 di-peptides and the low levels of “free” T13 and T20 peptides represent interchange occurring during refolding, or under the conditions of proteolysis and mass

21


encountered expressing PrPC in E.coli (17,18) our experiments demonstrate the


spectrometric analysis of the “non-reduced” samples remains to be determined. Nonetheless, there are two implications of our current findings for future studies. First, that our figure for the α-helical content of Dpl27-154 may be an underestimate of the true value: this would be consistent with Dpl lacking the unstructured N-terminal region present in PrP. Second, that experiments to verify the Cys95-Cys148 and Cys109-Cys143 disulfide bonds in Dpl purified from mouse tissue samples would appear prudent before embarking upon high resolution structural studies of Dpl polypeptides refolded in vitro.

the form N-X-T occurring at residues 181 and 196. While only the first site is conserved in mouse Dpl (predicted to occur at residue 111 in the helix B region), a second nonconserved N-V-T Asn-linked glycosylation site was predicted at residue 99. Our data is compatible with the prediction that mature Dpl is glycosylated at two sites (Figure 6), though further studies will be required to demonstrate that these involve carbohydrate adducts at residues 99 and 111. The Dpl oligosaccharide side-chains are resistant to digestion by endoglycosidase H (G.L. Silverman, unpublished data) but are sensitive to PNGaseF, indicating that Dpl is processed in the ER and Golgi, as expected. PrPC is attached to the cell surface via a GPI-anchor (16) and our data now confirms that this is also the case for Dpl, even though it lacks a GPI-addition signal peptide closely related to that of PrP. The shift of Dpl protein from the cell lysate to the recovered media after incubation of live cells with PIPLC provides biochemical evidence that most Dpl in N2a cells is exposed on the cell surface and modified by the addition of a GPI-anchor. Triton X-114 phase partitioning was used as an additional means of

22


Glycobiology of Dpl - Mouse PrP has two consensus Asn-linked glycosylation sites of


verifying this conclusion. Proteins with an intact GPI-anchor normally partition in the detergent phase, but treatment with PIPLC removes the hydrophobic diacylglycerol moiety of the anchor and results in the recovery of treated proteins in the aqueous phase (19,20). Using this experimental paradigm, Dpl was found to shift from the detergent phase to the aqueous phase after PIPLC treatment. While the attachment sites for GPI anchors often correspond to Asp or Ser residues (21), being Ser 231 in the case for PrPC, addition at other amino acid residues is not without precedent. Indeed, GPI-anchor signal peptides are somewhat degenerate and addition can occur at a

or serine (22,23). In the case of Dpl, prior computer modeling predicts addition to Gly 155 (5). It is also noteworthy that even in the case of PrP, conservation of Ser 231 is not ubiquitous: thus rabbit PrPC lacks serine at position 231 and GPI-attachment is thought to occur at the adjacent glycine (24).

Dpl physiology. Dpl does not appear to be present in the brains of adult wt animals and is associated with neurodegeneration when expressed ectopically in this location (see below). It is therefore reasonable to anticipate that while Dpl may play a role in the initial development of the brain, the principal physiological function likely lies within other tissues. Northern and western blot data indicates that Dpl is expressed at high levels in the testes of adult wt mice (Figure 8), and is also present in the heart (5). We anticipate that experiments to determine the temporal and spatial expression patterns of Prnd and to ablate the Prnd coding region will be required to obtain definitive insights into the normal function of this novel PrP-like protein. Nonetheless, some experiments reported

23


variety of different small amino acids, including alanine, aspartate, asparagine, glycine


here can be interpreted to indicate that Dpl biogenesis is modulated, perhaps in a tissue-specific manner. For example, the Asn-linked side-chains associated with (ectopic) expression in the brain appear different to those observed in testis and N2a cells, as indicated by the ~30-33 and ~27-39 kDa species detected by western blotting. Secondly, although Prnd mRNA can be detected in testis of wt mice and in the brains of (asymptomatic) Rcm0 mice by northern analysis (5), Dpl protein is more abundant in the former location than the latter (Figure 8B). This suggests that Dpl protein expressed in the CNS is unstable, or that translation of Dpl from chimeric mRNA in the brains of

untranslated region encoded by Prnd exons 1A and 1B (5).

Dpl, PrPC and pathways to neuronal death. Prior analyses of Rcm0 and Ngsk Prnp0/0 mice have attributed the cerebellar ataxia present in these mice, but not in other Prnp0/0 lines such as Zrch I and Npu, to an organization of the residual Prnp gene favouring overexpression of Prnd mRNA. This correlation now extends to a fifth Prnp0/0 line designated ZrchII (25). Though experiments to express Dpl under the control of a heterologous promoter will be required to cement this relationship, results presented here lend additional support this hypothesis, and begin to suggest the type of events that may initiate neuronal death. Thus, Dpl protein can be detected in the CNS of Rcm0 mice, but not in the brains of wt or ZrchI mice. Further, we demonstrate that Dpl possesses two N-linked oligosaccharides and is attached to the cell surface via a GPI anchor. Thus Dpl not only resembles PrPC with regard to its amino acid sequence, but also with respect to synthesis in the secretory pathway, certain post-translational

24


Rcm0 mice may be less efficient than translation of Prnd mRNA containing a 5’


modifications, and sub-cellular localization to the external face of the plasma membrane. It is likely that Dpl’s GPI anchor directs it to caveolae-like membrane domains (26,27), though this remains to be established experimentally. The biochemical similarities between Dpl and PrPC are germane to the observation that Purkinje cell death in Ngsk mice can be abrogated by overexpression of wt PrPC encoded by the Tg4053 transgene array (7). Notably, these similarities suggest that PrPC, by virtue of a C-terminal domain structurally similar to that of Dpl, can antagonize a deleterious activity associated with ectopic expression of Dpl in CNS neurons.

initiate a signal leading to cell death, whereas PrPC, when overexpressed, competes for this binding site without initiating a signal transduction event. This type of model was proposed by Schmerling et al. to account for the pathogenic properties of internallydeleted PrP molecules and was first discussed in the context of a putative PrP-ligand denoted PrPL. These workers noted that while ZrchI Prnp0/0 mice exhibit normal development, the introduction of Prnp transgenes encoding PrP molecule where amino acids 32-121 (PrP∆32-121) or 32-134 (PrP∆32-134) had been deleted caused these mice to develop ataxia and cerebellar granule cell degeneration (28). Our data do not speak to the hypothetical PrP ligand PrPL, but as Dpl resembles PrPC minus the conserved N-terminal Cu(II) binding domain, they do suggest that expression of the ∆32-121 and ∆32-134 “Dpl-like” versions of PrPC in ZrchI mice may activate a Dpl dependent pathway of neurotoxicity, with pathogenic consequences somewhat akin to those observed in Rcm0 and Ngsk mice (25). Here, partially analogous to the case with Ngsk mice, pathology could be abrogated in ZrchI mice expressing N-terminally deleted

25


One mechanism would be that Dpl binds to another cell-surface molecule to


PrP by reintroduction of a single copy of Prnp encoding full-length wt PrPC (28), suggestive of competitive inhibition. Identification of a receptor for Dpl with some affinity for PrPC therefore comprises one line of future research, with the potential to yield insights into the physiological and pathogenic attributes of both proteins.


26


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Moore, R. C., Lee, I. Y., Silverman, G. L., Harrison, P. M., Strome, R., Heinrich, C., Karunaratne, A., Pasternak, S. H., Chishti, M. A., Liang, Y., Mastrangelo, P., Wang, K., Smit, A. F., Katamine, S., Carlson, G. A., Cohen, F. E., Prusiner, S. B., Melton, D. W., Tremblay, P., Hood, L. E., and Westaway, D. (1999) J Mol Biol 292(4), 797-817

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28


13.


FOOTNOTES

This work was supported by grants from the National Institutes of Health, the American Health Assistance Foundation, the Sherman Fairchild Foundation (SBP and FEC), and the Medical Research Council of Canada, Health Canada, and the Alzheimer Society of Ontario (DW). GLS was supported by an Ontario Student Opportunity Trust Fund award, and RCM by a Human Frontier Science Program long term fellowship award.

Chris Stenland for useful discussions and Rudi Glockshuber for the pRBI-PDI-T7 plasmid.

1

Abbreviations used are: PrPC, cellular isoform of prion protein; PrPSc, scrapie isoform of

prion protein; Dpl, doppel (downstream, prion protein-like); MoDpl, mouse doppel protein; Prnp, mouse prion protein gene; Prnd, mouse Dpl gene; Prn, mouse prion gene complex comprised of Prnp and Prnd; PNGaseF, N-glycosidase F; PIPLC, phosphatidylinositol-specific phospholipase C; GPI, glycophosphatidylinositol; ECL, enhanced chemiluminescence; Ab, antibody; HBSS, Hank's balanced saline solution; DMEM, Dulbecco's modified Eagle medium; FBS, fetal bovine serum; TBS, Trisbuffered saline; wt, wild-type; CNS, central nervous system; RT-PCR, polymerase chain reaction analysis of reverse transcriptase synthesized cDNA.

29


Acknowledgments: The authors thank Paul Mathews, Doug Lee, Robert Strome and


FIGURE LEGENDS

Table 1. MALDI-MS peptide mapping analysis of tryptic peptides, disulfide-linked peptides, and full-length protein of MoDpl 27-154.

Figure 1. Predicted structural features of Dpl. Diagram of the mouse Dpl protein with its predicted post-translational modifications. Shaded residues 1-27 represent the cleaved N-terminal signal sequence, while shaded residues 155-179 represent the C-

155. Two N-linked glycosylation sites and two disulphide bonds predicted from computer modeling are indicated. The coordinates of the synthetic Dpl-2 peptide used to raise the D7177 antibody are also indicated.

Figure 2. MALDI mass spectra of MoDpl 27-154 and determination of two disulfide bonds. A. MALDI mass spectrum of purified recombinant MoDpl 27-154. B. Scarbamidomethylation of cysteine in MoDpl 27-154. Mass spectrum of non-reduced MoDpl 27-154 incubated with IAM. MoDpl 27-154 (1.2 mg/ml) in 0.1 M Tris-HCl, pH 8.0, 1 mM EDTA, 6 M guanidine hydrochloride was incubated without DTT at room temperature for 1 h. The non-reduced MoDpl 27-154 was incubated with 50 mM iodoacetamide (IAM) at room temperature in the dark for 30 min. C. Mass spectrum of reduced MoDpl 27-154 incubated with IAM. MoDpl 27-154 (1.2 mg/ml) in 0.1 M TrisHCl, pH 8.0, 1 µM EDTA, 6 M guanidine hydrochloride was incubated with 10 µM DTT

30


terminal domain thought to be removed upon attachment of a GPI anchor to residue


at room temperature for 1 h. The reduced MoDpl 27-154 was incubated with 50 mM IAM at room temperature in the dark for 30 min.

Figure 3. CD spectrum of MoDpl 27-154. CD spectrum of 10 µM MoDpl27-154 measured in 20 mM PB, pH 7.0 (solid line) at room temperature. The dashed line represents a spectrum from mouse PrP23-231 prepared and purified by a similar method.

A. MALDI mass spectra of peptides after tryptic digestion without reduction. T20: 1106.4 Da; T8: 1202.4 Da; T7: 1315.4 Da; T10+T11: 1335.7 Da; T8+T9: 1444.0 Da; T19-S-ST20: 1754.4 Da; T13: 2272.0 Da; T13-S-S-T19: 2920.0 Da; T13-S-S-T20: 3375.6 Da; T12-S-S-T20: 4058.6 Da. B. MALDI mass spectra of peptides after tryptic digestion with reduction by TCEP. T19: 650.5 Da; T20: 1106.0 Da; T8: 1202.0 Da; T7: 1315.1 Da; T10+T11: 1335.7 Da; T8+T9: 1444.0 Da; T13: 2271.6 Da; T12: 2953.9 Da. Note secondary and tertiary peaks adjacent to T12 has the mass predicted for Na+ adducts.

Figure 5. Tryptic peptides from MoDpl27-154. A. Amino acid sequence and tryptic peptide mapping of MoDpl 27-154. Trypsin cleavage sites (T) and Cys (C) residues are shown. B. Connectivity of disulfide linkages. The top panel indicates disulfide linkages between tryptic fragments as deduced from prior computer modeling of Dpl structure. The connection between Cys109 and Cys 143 is analogous to that found in PrPC. The lower two panels with tryptic peptides denoted by dashed lines indicate other

31


Figure 4. MALDI mass spectrometric disulfide bond mapping of MoDpl 27-154.


connectivities detected at low levels by mass spectrometric analysis (see also Figure 4, Table 1).

Figure 6. Time course PNGaseF digestion of Dpl. PNGaseF digestion of Dpl. Aliquots of Dpl cell lysate were digested with PNGaseF for 0, 15, 30, 45, 60 or 120 min as indicated by the figure legend. D = diglycosylated Dpl; M = monoglycosylated Dpl; and U = unglycosylated Dpl.

hydrophobicity by Triton X-114 phase partitioning. A. Dpl-3 cells were incubated with or without PIPLC as indicated by the figure legend. When cells were untreated, Dpl was detectable in the cell lysate but not in the media. In PIPLC-treated samples Dpl was detectable in the recovered media but not in the cell lysate. B. Dpl-3 cells were incubated with or without PIPLC. Cell lysates and recovered media were then subjected to repeated Triton X-114 phase partitioning. Detergent phases 1-3 (Dt 1-3) were combined, while detergent phase 4 (Dt 4) was kept separate as an internal control for pipeting error. The detergent phases and the aqueous phase (Aq) were then analyzed by SDS-PAGE. Lanes 1-3, Triton X-114 fractions from untreated Dpl-3 cells. Lanes 4-6, Triton X-114 fractions from PIPLC-treated Dpl-3 cells. Lanes 7-9, Triton X-114 fractions of recovered media from PIPLC-treated cell. Dpl is only present in lanes 1 and 9.

Figure 8. Western blot of wt FVB/N testes and brain homogenates. A. Testes and brain homogenates from were prepared from adult wt FVB/N mice. Lanes 1 and 3

32


Figure 7. Detection of Dpl after PIPLC treatment and determination of relative


represent untreated tissue homogenates, while lanes 2 and 4 were incubated with PNGaseF prior to SDS-PAGE analysis. Diglycosylated Dpl is present in lane 1, while the unglycosylated Dpl can be seen in lane 2. Dpl is not detectable in lanes 3 or 4. B. Brain homogenates from wt FVB/N, ZrchI and Rcm0 mice were subjected to Triton X114 phase partitioning to enrich samples for Dpl protein. Samples were split and loaded on identical SDS-PAGE gels. The blot in the left-hand panel was probed with D7177 Ab alone, while the blot in the right-hand panel was probed with D7177 Ab that had been preincubated with Dpl-2 peptide. Left-hand panel: lane 1, detergent phases 1-3 of wt

detergent phases 1-3 of ZrchI brain; lane 4, detergent phases 1-3 of Rcm0 brain; lane 5, aqueous phase of wt FVB/N testes (negative control). Dpl is only detectable in lane 1 and lane 4 (as indicated by the arrowhead). Right-hand panel: Lane 6, detergent phases 1-3 of wt FVB/N testes; lane 7, detergent phases 1-3 of wt FVB/N brain; lane 8, detergent phases 1-3 of ZrchI brain; lane 9, detergent phases 1-3 of Rcm0 brain; lane 10, aqueous phase of wt FVB/N testes. The signals corresponding to Dpl protein in the left-hand panel are not present here.

33


FVB/N testes (positive control); lane 2, detergent phases 1-3 of wt FVB/N brain; lane 3,

27

Figure 1

1

143 148

S-S S-S

155

GPI


111

109 99

CHO

88 95

D7177 Ab 68

CHO MoDpl 27-154

179


T8+T9*

T20

T19

T13

T8 T12

T7

MoDpl 27-154

Protein or peptide

65-77

52-65

147-154

141-146

103-123

52-62 78-102

39-51

27-154

partial sequence

4058.4

1335.5

1443.7

1106.2

650.7

2271.5

1202.4 2954.2

1315.5

14916.9

2920.0

4058.6

1335.7

1444.0

1106.4

650.5

2271.6

1202.2 2954.0

1315.4

14916.9

Table 1

T10+T11*

(78-102)-S-S-(147-154)

2920.3

1754.4

(observed)

T12-S-S-T20

(103-123)-S-S-(141-146)

1755.0

3375.6

[M+H] +

T13-S-S-T19

(141-146)-S-S-(147-154)

3375.7

(calculated)

T19-S-S-T20

(103-123)-S-S-(147-154)

[M+H] +

T13-S-S-T20

* Partial digestion products.

Figure 2 MoDpl 2+

7458.9

[M+2H]

[M+H]+ MoDpl 27-154 14916.9

A

10000

MoDpl + IAM

12000 14000

6000

12000 14000

8000

16000

18000

MoDpl 27-154 (4 carbamidomethyl cysteines) 15149.0

MoDpl + DTT + IAM

6000

18000

14918.4 10000

7575.6

C

8000

16000

MoDpl 27-154 (no free cysteines)

7459.5

B

8000

10000

12000 14000

Mass (m/z)

16000

18000


6000

20

10

-10

-20 190 200 210 220 230 240

Wavelength (nm)

250


0

[θ] x 103 (deg cm2 / dmol)

Figure 3


Figure 5 A 27

35

45

55

65

75

SRGIKHRFKW NRKVLPSSGG QITEARVAEN RPGAFIKQGR KLDIDFGAEG T1 T2 T3 T4

T5 T6

T7

85

95

T8 T9 T10

105

109

115

125

T11

T12

135

143 145

T13 T14

154

QDSKLHQRVL WRLIKEICSA KHCDFWLER T15

T16

T17 T18

T19

T20

B 95

S -S

T12

T13 109

148

T19

S -S 143

T19 S -S

109

T13

Mass [M+H]+ Calculated

Observed

4058.4

4057.8

T20

143

S -S

Tryptic fragments

T12 - T20

148

T13 - T17

2920.3

2919.8

T19 - T20

1755.0

1754.4

T13 - T20

3375.7

3374.6

T20 148

T20


NRYYAANYWQ FPDGIYYEGC SEANVTKEML VTSCVNATQA ANQAEFSREK




Protein Structure and Folding: Doppel is an N-glycosylated GPI-anchored protein: expression in testis and ectopic production in the brains of Prnp0/0 mice predisposed to Purkinje cell loss Gregory L.Silverman, Kefeng Qin, Richard C. Moore, Ying Yang, Peter Mastrangelo, Patrick Tremblay, Stanley B. Prusiner, Fred E. Cohen and David Westaway J. Biol. Chem. published online June 6, 2000

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