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Prion proteins and ECTO-NOX proteins exhibit similar oscillating redox activities
BBRC Biochemical and Biophysical Research Communications 315 (2004) 1140–1146 www.elsevier.com/locate/ybbrc
Prion proteins and ECTO-NOX proteins exhibit similar oscillating redox activitiesq Chinpal Kim and D. James Morre* Department of Medicinal Chemistry and Molecular Pharmacology, Purdue University, West Lafayette, IN 47907-2064, USA Received 2 February 2004
Abstract Both recombinant full-length mouse prion protein expressed in Escherichia coli and native prion protein (PrPsc ) from mouse brain exhibited NADH oxidase and protein disulfide–thiol interchange activities similar to those formerly thought to be properties exclusive to the growth-related, cell surface ECTO-NOX proteins. The two activities exhibited the complex 2 + 3 pattern of oscillations characteristic of ECTO-NOX proteins where the two activities alternate to generate a period length of 24 min. The oscillations were augmented by copper and diminished by addition of the copper chelator bathocuproene. That the activity might be attributable to a contaminating protein was ruled out by experiments where the purified recombinant prion-containing extracts were resolved by SDS–PAGE and the activity was restricted to a single band corresponding to the predicted Mr of the recombinant prion as verified by Western blot analyses. Ó 2004 Elsevier Inc. All rights reserved. Keywords: Prion protein; ECTO-NOX proteins; Copper; Time keeping properties; NADH oxidation; Protein disulfide–thiol interchange
Our laboratory has described a family of NAD(P)H oxidase (NOX) proteins [1] that exhibit both an oxidative and a protein disulfide isomerase-like activity [1,2]. These proteins are characterized by the property, unprecedented in the biochemical literature, of having two distinct biochemical activities, hydroquinone or (NAD(P)H) oxidation and protein disulfide–thiol interchange, that alternate [1–5]. Present in both plants and animals, they have no flavin, heme nor none-heme iron prosthetic groups and do not require ancillary
q
Abbreviations: BCIP, 5-bromo-4-chloro-3-indoyl phosphate; capsaicin, 8-methyl-N -vanillyl-6-noneamide; CNOX, constitutive and drug-unresponsive cell surface NADH oxidase; DTDP, dithiodipyridine; DTT, dithiothreitol; ECTO-NOX, cell surface and growthrelated NADH oxidases with protein disulfide–thiol interchange activity; Mes, 2-(N -morpholino)ethanesulfonic acid; NBT, nitroblue tetrazolium; PMSF, phenylmethylsulfonyl fluoride; SDS–PAGE, sodium dodecyl sulfate–polyacrylamide gel electrophoresis; tNOX, cancer-associated and drug- (capsaicin-) responsive cell surface NADH oxidase; PrP, prion protein; PrPc, normal prion protein form; PrPsc, pathologic prion protein form. * Corresponding author. Fax: 1-765-494-4007. E-mail address: [email protected] (D.J. Morre). 0006-291X/$ - see front matter Ó 2004 Elsevier Inc. All rights reserved. doi:10.1016/j.bbrc.2004.02.007
proteins for activation [1]. They are referred to as ECTO-NOX proteins because of their cell surface location [6] and to distinguish them from the phox-NOX proteins of host defense [7]. While activities have been most often measured as oxidation of NADH, the physiological substrate for the activity appears to be hydroquinones of the plasma membrane such as reduced coenzyme Q10 [8]. ECTO-NOX proteins have characteristics of prions [9]. They achieve protease (including proteinase K) resistance, impart protease resistance to protease-susceptible proteins, contain a conserved copper binding domain, and form amyloid, all of which are characteristics of prions [8]. Other unusual characteristics that ECTO-NOX proteins have in common with prions include a propensity to form insoluble aggregates, resistance to cyanogen bromide fragmentation, and an ability to form amyloid filaments closely resembling those of transmissible spongiform encephalopathies [10]. The defining prion characteristic of ECTO-NOX proteins is the ability to impart protease resistance to a normally protease-susceptible protein [8] as is characteristic of PrPsc (PrPres ), the presumed infective and proteinase K-resistant form of the scrapie prion.
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In this report, the question addressed is do prions have properties considered to be unique to ECTO-NOX proteins? The findings show that both purified recombinant mouse prion and native PrPsc purified from mouse brain exhibit the copper-dependent oscillations in oxidative and protein disulfide–thiol interchange enzymatic activities that define ECTO-NOX proteins. These commonalities extend further the functional links between ECTO-NOX proteins and prions of our previous studies [9]. Materials and methods Materials. Polyclonal rabbit anti-prion antisera were generated by Immunodynamics (San Diego, CA) by using a synthetic peptide corresponding to amino acids 88–105 of the N-terminus of the human prion or purchased from Research Diagnostics (amino acids 79–97 of the N-terminus of the human prion protease-resistant protein, catalog number: RDI-PrionabG, Flanders, NJ). Alkaline phosphatase-conjugated S-protein was from Novagen (Madison, WI). Alkaline phosphatase-conjugated anti-polyhistidine monoclonal antibody was from Sigma (St. Louis, MO). The sample of PrPsc purified from brains of scrapie-infected mice [11] was graciously provided by Dr. Gabriella Saborio, Department of Cellular Biochemistry, Serrano Pharmaceutical Research Institute, Geneva, Switzerland. Isolation of total RNA from mouse brain. A freshly excised mouse brain was snap-frozen by dipping into liquid nitrogen and ground using a mortar and pestle. Total RNA (11 lg) was isolated by using Qiaamp RNA blood mini kit (Quagen, Valencia, CA). RT-PCR of mouse prion mRNA. Open reading frame of mouse prion mRNA was reverse-transcribed by using reverse primer derived from mouse prion mRNA or oligo(dT) and SuperScript II RNase H reverse transcriptase (Invitrogen, Carlsbad, CA). One microgram of total RNA was used for the synthesis of first-stand cDNA in 20 ll reaction volume. The open reading frame of mouse prion cDNA was amplified by PCR using forward and reverse primers and Taq DNA polymerase. Primers used for the reaction were: 50 -AGAGTCCGGGAGCTC TAGCGAACCTTGGCTACTGGCTG-30 (forward primer) and 50 -ATGACTGGATCCTCAT CCCACGATCAG GAAGAT-30 (reverse primer). A SacI restriction site was incorporated into the forward primer and a BamHI restriction site was incorporated into the reverse primer. Conditions for PCR amplification were 45 s at 94 °C followed by 30 cycles of 45 s at 94 °C, 45 s at 60 °C, and 1 min 10 s at 72 °C, one cycle of 45 s at 94 °C, 45 s at 60°, and 7 min at 72 °C. Construction of mouse prion expression vector. The open reading frame of mouse prion cDNA amplified by PCR and plasmid pET43.1a(+) (Novagen, Madison, WI) were digested with SacI and BamHI, purified by agarose gel electrophoresis, and ligated together to construct the mouse prion expression vector (pET43-prion). Plasmid pET-43.1(+) was utilized as a means to express the prion protein as a soluble fusion protein with NusA (495 aa). After construction of pET43-prion, BL21(DE3) cells were transformed with the plasmid and plated on LB plates containing 100 lg/ml ampicillin. Colonies carrying plasmid pET43-prion were screened by PCR using forward and reverse primers. Colonies carrying the pET43-prion were further screened for the expression of the mouse prion by Western blot using anti-prion antibody or alkaline phosphatase-conjugated S-protein. Briefly, colonies were inoculated in 5 ml LB media containing ampicillin (100 lg/ ml) and grown for 16 h at 37 °C. Aliquots of 50 ll from each sample were used for SDS–PAGE and Western blot analysis. From a colony expressing recombinant mouse prion, plasmid pET43-prion was purified with Plasmid Midi Kit (Qiagen, Valencia, CA) and the DNA insert of the plasmid was sequenced (Genomics Core Facility, Purdue University, West Lafayette, IN).
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Expression of recombinant mouse prion protein. BL21(DE3) cells carrying pET43-prion were grown in 300 ml LB medium containing 100 lg/ml ampicillin at 37 °C for 16 h and harvested. Cells were resuspended in 60 ml of 20 mM Tris–HCl, pH 8.0, containing 1 mM PMSF, 1 mM benzamidine–HCl, and 10 mM of 6-amino caproic acid. Proteins were extracted by French press disruption (3 passages at 20,000 psi). The extracts were centrifuged for 20 min at 10,000g. Supernatants were collected and used for purification of mouse prion. Purification of recombinant mouse prion by hydroxyapatite chromatography. The prion protein was precipitated with ammonium sulfate (40% saturation) and resuspended in 10 ml of 10 mM Na2 HPO4 , pH 7.8, and centrifuged (20 min, 10,000g). Supernatants were collected and dialyzed against 10 mM Na2 HPO4 . Proteins were applied to a hydroxyapatite column (EconoPac HTP, 5 ml, Bio-Rad, Hercules, CA). The column was washed with 10 mM Na2 HPO4 . Proteins were eluted with a linear gradient of 50 ml of 10 mM Na2 HPO4 to 50 ml of 400 mM Na2 HPO4 with a flow rate of 1 ml/min. Protein elution was monitored at 280 nm and peaks were collected. Enzyme assays. NADH oxidation was measured spectrophotometrically from the disappearance of NADH measured at 340 nm [3]. Protein disulfide–thiol interchange was determined either by restoration of activity to scrambled and inactive RNase prepared from native RNase A as described with cCMP as the RNase substrate [12] or spectrophotometrically from the cleavage of dithiodipyridine (DTDP) [13]. Proportionality of enzymatic activity to protein concentration with the purified prion or with the bacterial extracts was verified for all three assays. Protein concentrations were estimated by the bicinchoninic acid (BCA) procedure [14] with bovine serum albumin as standard.
Results Identification of colonies expressing mouse prion Positive colonies expressing fusion prion protein were screened by Western blot analysis using alkaline phosphatase-conjugated S-protein. Colonies expressed prion fusion protein either as a truncated form or as a fulllength protein (arrows in Fig. 1A). Colonies 15 and 17 were further analyzed by Western blot using rabbit polyclonal anti-prion antisera. The antisera confirmed the expression of prion protein as a full-length protein or as a truncated form (arrows in Fig. 1B). Colony 17 was used to isolate a plasmid for DNA sequencing and to express the prion protein. DNA sequencing revealed no errors. Purification of recombinant mouse protein Recombinant mouse prion protein was purified by 40% ammonium sulfate precipitation followed by hydroxyapatite chromatography. Proteins were eluted with increasing concentration of sodium phosphate from 10 to 400 mM and fractions were collected. Protein composition of eluates was analyzed by silver staining of SDS–polyacrylamide gel and Western blot by using alkaline-phosphatase conjugated anti-polyhistidine monoclonal antibody. Fractions 2–4 contained recombinant mouse prion (arrows in Figs. 2A and B). Fraction 2 was used to investigate enzymatic activities.
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Fig. 1. Screening of colonies expressing recombinant mouse prion protein by Western blot analysis. Colonies expressed either full length or truncated forms of prion proteins (arrows in (A,B)). Numbers at the bottom of the figures indicate individual colonies carrying pET43prion except number zero which indicates a colony grown without carrying the pET43-prion. For (A), the nitrocellulose membrane was incubated with alkaline phosphatase-conjugated S-protein followed by BCIP/NBT color detection. For (B), the nitrocellulose membrane was stained with Ponceau 5 (a) or incubated with rabbit anti-prion polyclonal antibody (Immunodynamics) followed by AP-conjugated antirabbit IgG antibody (b) or with goat anti-prion polyclonal antibody (RDI Research Diagnotics, Flanders, NJ) followed by AP-conjugated anti-goat IgG antibody (c). Detection was with BCIP/NBT.
Mouse PrPsc exhibits both NADH oxidase and protein disulfide–thiol interchange activities As an extension of our previous work with ECTONOX proteins where these proteins share characteristics in common with prions, native PrPsc from mouse brain was examined for its ability to oxidize NADH. Not only did the native PrPsc from mouse brain oxidize NADH (Fig. 3), the NADH oxidative activity oscillated as with ECTO-NOX oxidation of NADH [15]. The pattern of oscillations consisted of two maxima separated by an interval of 6 min. This two-peak pattern of NADH oxidation then repeated at intervals of 24 min. A second enzymatic activity associated with ECTONOX proteins is that of protein disulfide–thiol interchange [15]. As measured by cleavage of synthetic dipyridyl–dithio substrates, recombinant prion protein also showed protein disulfide–thiol interchange and, as with ECTO-NOX proteins, the protein disulfide–thiol interchange activity alternated with that of NADH oxidation (Fig. 3B). The protein disulfide–thiol interchange activity of the mouse PrPsc also exhibited a characteristic
Fig. 2. Purification of recombinant prion protein. Prion protein was purified by 40% ammonium sulfate precipitation and hydroxyapatite chromatography. Numbers at the bottom are fraction numbers. (A) Silver staining of 10% SDS–polyacrylamide gel. (B) Western blot (incubation with alkaline phosphatase-conjugated monoclonal anti-polyhistidine antibody and color detection with BCIP/NBT) corresponding to the region of the gel shown by the arrow in (A).
oscillatory activity. However, for protein disulfide–thiol interchange three activity maxima spaced at intervals of 4.5 min were observed. As with NADH oxidation, the activity maxima repeated with an average period length of 24 min. The determinations of Fig. 3 used two spectrophotometers operated in parallel. Equal amounts of PrPsc were used with NADH as substrate in machine A and dithiodipyridine as substrate in machine B. Since the two determinations were in parallel the experiment shows unequivocally that the two activities alternate. Oscillatory oxidation of NADH also exhibited by recombinant mouse prion Full length recombinant mouse prion expressed in bacteria also exhibited oscillatory NADH oxidase activity (Fig. 4A) although the specific activity was lower than that of the PrPsc from mouse brain possibly due to the propensity of the recombinant prion to form inactive protein aggregates. The period length was 24 min for the PrPsc of Fig. 3A. With both the native (not shown) and the recombinant prion, activity was inhibited by bathocuproene (Fig. 4B) suggestive of the involvement of a metal. To demonstrate that the measured oscillatory activity was associated with the recombinant prion and not with low levels of some contaminating bacterial protein, the recombinant protein was resolved by SDS–PAGE and proteins were eluted from 0.5 cm gel segments. NADH oxidase activity from the gel slices was restored as
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Fig. 3. Mouse prion protein (PrPsc ) exhibits a pattern of oscillating alternation of NADH oxidation (A) and protein disulfide–thiol interchange (B) with a period length of 24 min that is indistinguishable from that of a mammalian constitutive NOX (CNOX) protein, i.e., the mouse prion has enzymatic activities equivalent to those of an ECTO-NOX protein. The two determinations used simultaneous measurements from two spectrophotometers operated in parallel. Equal amounts of PrPsc were added to curvettes in both instruments with NADH as substrate in machine A and dithiodipyridine as substrate in machine B, the latter for determination of protein disulfide–thiol interchange.
previously described [16] by first incubating for 10 min with 100 lM GSH in the presence of 125 lM NADH to reduce disulfide bonds and to permit refolding. This was followed by addition of 0.03% H2 O2 to reform disulfide bridges in the refolded protein. Activity overall was greatest in fraction 4 corresponding to the molecular weight of 88.7 kDa for the recombinant protein (Fig. 5). When analyzed separately for oscillatory activity only fraction 4 corresponding to the molecular weight of the recombinant protein contained the oscillatory activity. A longer period of analysis through four full cycles (Fig. 6) revealed the ca. 5-peak pattern of Figs. 3 and 4 within each cycle. The recombinant mouse prion protein
also exhibited protein disulfide–thiol interchange activity as determined by cleavage of dithiodipyridine or by restoration of activity to denatured and inactive (scrambled) RNase but lacked the ability to oxidize hydroquinones. Oscillations in NADH oxidation by recombinant mouse prion appear metal dependent The periodic nature of oxidation of NADH by the recombinant mouse prion was inhibited by addition of 1 lM bathocuproene (Fig. 4), a copper chelator, and restored by addition of 10 lM copper chloride. When
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Fig. 6. Analyses over 96 min of 1.5 and 2.0 cm gel slices from fraction 3 (A) and fraction 4 (B). Only in the 2.0 cm slice (fraction 4) was the oscillating pattern of NADH oxidation observed. The results shown for the 1.5 cm gel fraction from fraction 3 (A) are representative of results from fractions 1, 2, and 5 to 10.
Fig. 4. NADH oxidation by recombinant mouse prion (A) and inhibition by 1 lM bathocuproene (B). The two assays were conducted using two spectrophotometers operated in parallel and yielding simultaneous measurements as described for Fig. 3 except that NADH was the substrate for both. Bathocuproene was added to the curvette of machine B after 48 min. Arrows indicate maxima separated by intervals of 24 min.
tion of oscillations (not shown). The oscillations could be restored by the addition of 0.1 lM CuCl2 . The addition of copper initially stimulated the entire 5 peak pattern but, after the first cycle, one peak was disproportionately enhanced relative to the others.
Discussion
Fig. 5. Analysis of 10% SDS–PAGE gel slices for oscillatory oxidation of NADH with a period length of 24 min. The activity was located predominantly in fraction 4 which corresponded to a molecular weight of ca. 89 kDa. Slices were homogenized in 50 mM Tris–Mes, pH 7, and incubated overnight at 4 °C to elute the protein. After removal of gel fragments by centrifugation, full enzymatic activity was restored using an established protocol of incubation in the presence of 150 lM NADH with 100 lM reduced glutathione followed by 10 min of incubation with 0.03% hydrogen peroxide [16].
dialyzed to remove copper, the specific activity of the recombinant prion for oxidation of NADH was 20– 30 nmol/min/mg protein with nearly complete elimina-
We have described a family of cell surface NADH oxidases (hydroquinone oxidases) that exhibit unusual characteristics including resistance to proteases, resistance to cyanogen bromide digestion, and an ability to form amyloid filaments closely resembling those of spongiform encephalopathies and all of which are characteristic of PrPsc (PrPres ), the presumed infective and proteinase K-resistant particle of the scrapie prion [17,18]. A drug-responsive ECTO-NOX form, tNOX, from the HeLa cell surface co-purified with authentic glyceraldehyde-3-phosphate dehydrogenase (muscle form) (GAPDH) [16]. Surprisingly, the tNOX-associated muscle GAPDH also was proteinase K resistant [9]. In our studies, we showed that combination of authentic rabbit muscle GAPDH with tNOX rendered the GAPDH resistant to proteinase K digestion. This property, that of converting the normal form of a protein into a likeness of itself, is one of the defining characteristics of the group of proteins designated as prions [17,18]. In this report, the functional similarities of tNOX and prion proteins are expanded by the observation that recombinant mouse prion exhibited both of the enzymatic activities normally associated with NOX proteins,
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i.e., oxidation of NADH and protein disulfide–thiol interchange. In addition to exhibiting the enzymatic activities, the activity of the recombinant proteins oscillated with the same 2 + 3 pattern of alternating fast and slow rates exhibited by tNOX [2,19]. The oscillatory activity was enhanced by copper. tNOX has a copper binding site [2] conserved with that of superoxide dismutase [20]. However, the activity and oscillations, unlike those of tNOX, are insensitive to inhibition by the classic tNOX inhibitors such as capsaicin. Taken together with the period length of 24 min, the ECTONOX-like enzymatic activity of the recombinant mouse prion more closely resembles those of the constitutive CNOX forms rather than the tumor-associated tNOX form. It is unlikely that either the oscillations or the copper response of the recombinant protein was influenced by the fusion with nus A as the characteristics of PrPc from mouse brain lacking the tag were similar to those of the recombinant protein. The ECTO-NOX pattern of oscillations associated with the oxidation of NADH within the 24 min period consists of 1 or 2 maxima separated by 6 min. These maxima alternate with 3 additional maxima separated by intervals of 4.5 min associated with the protein disulfide–thiol interchange activity of the protein. As shown by data of Fig. 3, this pattern of activity is fully duplicated by the recombinant mouse prion supporting our earlier contention [9] that the ECTO-NOX proteins and prions were structurally and functionally similar. Like ECTO-NOX proteins, the prion proteins (PrP) are Cu2þ binding cell surface proteins [21,22]. The copper is bound to the histidines of the prion octarepeats [repeats of the sequence P(Q/H)GG(G/S)WGQ]. A second copper site around histidines 96 and 111 has also been indicated from fluorescence and NMR data [23]. In birds the highly conserved mammalian octarepeat is replaced by a hexarepeat which also binds copper but with different coordination geometry than that of the mammalian octarepeat [19]. Several lines of evidence have suggested that copper ions play a role in the biology of both PrPc and PrPsc , the normal and pathologic forms, respectively, of the prion protein. Copper rapidly and reversibly induced PrPc to become protease-resistant and detergent insoluble both properties commonly associated with PrPsc . However, based on a conformation-dependent immunoassay, the copper-treated PrPc appears to be structurally distinct from PrPsc [22]. One suggestion has been that PrPc serves as a receptor for cellular uptake of copper [24,25]. An enzymatic function for PrPc has also been claimed based on the observation that copper binding confers superoxide dismutase activity on the protein [24]. Based on an examination of the phenotype of prion knockout mice, a number of biochemical changes have been observed which, when taken together, have suggested that a lack of expression of the
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prion protein results in a neuronal phenotype sensitive to oxidative stress [26]. Other responses to loss of prion protein expression in mice include altered circadian activity rhythms and sleep patterns [27]. The prion loss is the first null mutation that has been shown to affect sleep regulation. Moreover, one of the inherited prion diseases, fatal familial insomnia, involves a profound alteration in sleep and daily rhythms as well. The invariably fatal neuro-degenerative diseases, including both genetic or sporadic diseases and transmissible spongiform encephalopathies caused by prions, require the development of both PrPc and PrPsc [28]. PrPsc differs from PrPc in having a tertiary structure rich in b-structure. In fact, PrPsc has been suggested to be a totally dehydrated protein with an anhydrous environment, probably a thin carbon dioxide gas gap, to explain why it is highly resistant not only to proteases but to heat and radiation as well and to chemical disinfectants in the water phase except sodium hydroxide and sodium hypochlorite under certain conditions [29]. The unusual properties of prions also extend to ECTO-NOX proteins which are heat and protease resistant [2,16] and extremely resistant to any conditions of denaturation [30]. Such properties may be extremely important to ECTO-NOX proteins which carry out functions in the enlargement phase of cell growth [31] and in biological time keeping [32] from a location at the external cell surface [6,33]. How these normal cellular functions associated with a cell surface protein having an oscillatory activity relate to normal and aberrant prion protein functions in neural tissue remains unexplained. However, the observation of oscillatory oxidative and protein disulfide–thiol interchange activities in both types of proteins may indicate functional roles for prions that may parallel in some way those of the ECTO-NOX proteins.
Acknowledgments This work was supported in part by R01 CA75461, P50 AT00477, and NASA NAG-2-1344.
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Prion proteins and ECTO-NOX proteins exhibit similar oscillating redox activitiesq Chinpal Kim and D. James Morre* Department of Medicinal Chemistry and Molecular Pharmacology, Purdue University, West Lafayette, IN 47907-2064, USA Received 2 February 2004
Abstract Both recombinant full-length mouse prion protein expressed in Escherichia coli and native prion protein (PrPsc ) from mouse brain exhibited NADH oxidase and protein disulfide–thiol interchange activities similar to those formerly thought to be properties exclusive to the growth-related, cell surface ECTO-NOX proteins. The two activities exhibited the complex 2 + 3 pattern of oscillations characteristic of ECTO-NOX proteins where the two activities alternate to generate a period length of 24 min. The oscillations were augmented by copper and diminished by addition of the copper chelator bathocuproene. That the activity might be attributable to a contaminating protein was ruled out by experiments where the purified recombinant prion-containing extracts were resolved by SDS–PAGE and the activity was restricted to a single band corresponding to the predicted Mr of the recombinant prion as verified by Western blot analyses. Ó 2004 Elsevier Inc. All rights reserved. Keywords: Prion protein; ECTO-NOX proteins; Copper; Time keeping properties; NADH oxidation; Protein disulfide–thiol interchange
Our laboratory has described a family of NAD(P)H oxidase (NOX) proteins [1] that exhibit both an oxidative and a protein disulfide isomerase-like activity [1,2]. These proteins are characterized by the property, unprecedented in the biochemical literature, of having two distinct biochemical activities, hydroquinone or (NAD(P)H) oxidation and protein disulfide–thiol interchange, that alternate [1–5]. Present in both plants and animals, they have no flavin, heme nor none-heme iron prosthetic groups and do not require ancillary
q
Abbreviations: BCIP, 5-bromo-4-chloro-3-indoyl phosphate; capsaicin, 8-methyl-N -vanillyl-6-noneamide; CNOX, constitutive and drug-unresponsive cell surface NADH oxidase; DTDP, dithiodipyridine; DTT, dithiothreitol; ECTO-NOX, cell surface and growthrelated NADH oxidases with protein disulfide–thiol interchange activity; Mes, 2-(N -morpholino)ethanesulfonic acid; NBT, nitroblue tetrazolium; PMSF, phenylmethylsulfonyl fluoride; SDS–PAGE, sodium dodecyl sulfate–polyacrylamide gel electrophoresis; tNOX, cancer-associated and drug- (capsaicin-) responsive cell surface NADH oxidase; PrP, prion protein; PrPc, normal prion protein form; PrPsc, pathologic prion protein form. * Corresponding author. Fax: 1-765-494-4007. E-mail address: [email protected] (D.J. Morre). 0006-291X/$ - see front matter Ó 2004 Elsevier Inc. All rights reserved. doi:10.1016/j.bbrc.2004.02.007
proteins for activation [1]. They are referred to as ECTO-NOX proteins because of their cell surface location [6] and to distinguish them from the phox-NOX proteins of host defense [7]. While activities have been most often measured as oxidation of NADH, the physiological substrate for the activity appears to be hydroquinones of the plasma membrane such as reduced coenzyme Q10 [8]. ECTO-NOX proteins have characteristics of prions [9]. They achieve protease (including proteinase K) resistance, impart protease resistance to protease-susceptible proteins, contain a conserved copper binding domain, and form amyloid, all of which are characteristics of prions [8]. Other unusual characteristics that ECTO-NOX proteins have in common with prions include a propensity to form insoluble aggregates, resistance to cyanogen bromide fragmentation, and an ability to form amyloid filaments closely resembling those of transmissible spongiform encephalopathies [10]. The defining prion characteristic of ECTO-NOX proteins is the ability to impart protease resistance to a normally protease-susceptible protein [8] as is characteristic of PrPsc (PrPres ), the presumed infective and proteinase K-resistant form of the scrapie prion.
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In this report, the question addressed is do prions have properties considered to be unique to ECTO-NOX proteins? The findings show that both purified recombinant mouse prion and native PrPsc purified from mouse brain exhibit the copper-dependent oscillations in oxidative and protein disulfide–thiol interchange enzymatic activities that define ECTO-NOX proteins. These commonalities extend further the functional links between ECTO-NOX proteins and prions of our previous studies [9]. Materials and methods Materials. Polyclonal rabbit anti-prion antisera were generated by Immunodynamics (San Diego, CA) by using a synthetic peptide corresponding to amino acids 88–105 of the N-terminus of the human prion or purchased from Research Diagnostics (amino acids 79–97 of the N-terminus of the human prion protease-resistant protein, catalog number: RDI-PrionabG, Flanders, NJ). Alkaline phosphatase-conjugated S-protein was from Novagen (Madison, WI). Alkaline phosphatase-conjugated anti-polyhistidine monoclonal antibody was from Sigma (St. Louis, MO). The sample of PrPsc purified from brains of scrapie-infected mice [11] was graciously provided by Dr. Gabriella Saborio, Department of Cellular Biochemistry, Serrano Pharmaceutical Research Institute, Geneva, Switzerland. Isolation of total RNA from mouse brain. A freshly excised mouse brain was snap-frozen by dipping into liquid nitrogen and ground using a mortar and pestle. Total RNA (11 lg) was isolated by using Qiaamp RNA blood mini kit (Quagen, Valencia, CA). RT-PCR of mouse prion mRNA. Open reading frame of mouse prion mRNA was reverse-transcribed by using reverse primer derived from mouse prion mRNA or oligo(dT) and SuperScript II RNase H reverse transcriptase (Invitrogen, Carlsbad, CA). One microgram of total RNA was used for the synthesis of first-stand cDNA in 20 ll reaction volume. The open reading frame of mouse prion cDNA was amplified by PCR using forward and reverse primers and Taq DNA polymerase. Primers used for the reaction were: 50 -AGAGTCCGGGAGCTC TAGCGAACCTTGGCTACTGGCTG-30 (forward primer) and 50 -ATGACTGGATCCTCAT CCCACGATCAG GAAGAT-30 (reverse primer). A SacI restriction site was incorporated into the forward primer and a BamHI restriction site was incorporated into the reverse primer. Conditions for PCR amplification were 45 s at 94 °C followed by 30 cycles of 45 s at 94 °C, 45 s at 60 °C, and 1 min 10 s at 72 °C, one cycle of 45 s at 94 °C, 45 s at 60°, and 7 min at 72 °C. Construction of mouse prion expression vector. The open reading frame of mouse prion cDNA amplified by PCR and plasmid pET43.1a(+) (Novagen, Madison, WI) were digested with SacI and BamHI, purified by agarose gel electrophoresis, and ligated together to construct the mouse prion expression vector (pET43-prion). Plasmid pET-43.1(+) was utilized as a means to express the prion protein as a soluble fusion protein with NusA (495 aa). After construction of pET43-prion, BL21(DE3) cells were transformed with the plasmid and plated on LB plates containing 100 lg/ml ampicillin. Colonies carrying plasmid pET43-prion were screened by PCR using forward and reverse primers. Colonies carrying the pET43-prion were further screened for the expression of the mouse prion by Western blot using anti-prion antibody or alkaline phosphatase-conjugated S-protein. Briefly, colonies were inoculated in 5 ml LB media containing ampicillin (100 lg/ ml) and grown for 16 h at 37 °C. Aliquots of 50 ll from each sample were used for SDS–PAGE and Western blot analysis. From a colony expressing recombinant mouse prion, plasmid pET43-prion was purified with Plasmid Midi Kit (Qiagen, Valencia, CA) and the DNA insert of the plasmid was sequenced (Genomics Core Facility, Purdue University, West Lafayette, IN).
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Expression of recombinant mouse prion protein. BL21(DE3) cells carrying pET43-prion were grown in 300 ml LB medium containing 100 lg/ml ampicillin at 37 °C for 16 h and harvested. Cells were resuspended in 60 ml of 20 mM Tris–HCl, pH 8.0, containing 1 mM PMSF, 1 mM benzamidine–HCl, and 10 mM of 6-amino caproic acid. Proteins were extracted by French press disruption (3 passages at 20,000 psi). The extracts were centrifuged for 20 min at 10,000g. Supernatants were collected and used for purification of mouse prion. Purification of recombinant mouse prion by hydroxyapatite chromatography. The prion protein was precipitated with ammonium sulfate (40% saturation) and resuspended in 10 ml of 10 mM Na2 HPO4 , pH 7.8, and centrifuged (20 min, 10,000g). Supernatants were collected and dialyzed against 10 mM Na2 HPO4 . Proteins were applied to a hydroxyapatite column (EconoPac HTP, 5 ml, Bio-Rad, Hercules, CA). The column was washed with 10 mM Na2 HPO4 . Proteins were eluted with a linear gradient of 50 ml of 10 mM Na2 HPO4 to 50 ml of 400 mM Na2 HPO4 with a flow rate of 1 ml/min. Protein elution was monitored at 280 nm and peaks were collected. Enzyme assays. NADH oxidation was measured spectrophotometrically from the disappearance of NADH measured at 340 nm [3]. Protein disulfide–thiol interchange was determined either by restoration of activity to scrambled and inactive RNase prepared from native RNase A as described with cCMP as the RNase substrate [12] or spectrophotometrically from the cleavage of dithiodipyridine (DTDP) [13]. Proportionality of enzymatic activity to protein concentration with the purified prion or with the bacterial extracts was verified for all three assays. Protein concentrations were estimated by the bicinchoninic acid (BCA) procedure [14] with bovine serum albumin as standard.
Results Identification of colonies expressing mouse prion Positive colonies expressing fusion prion protein were screened by Western blot analysis using alkaline phosphatase-conjugated S-protein. Colonies expressed prion fusion protein either as a truncated form or as a fulllength protein (arrows in Fig. 1A). Colonies 15 and 17 were further analyzed by Western blot using rabbit polyclonal anti-prion antisera. The antisera confirmed the expression of prion protein as a full-length protein or as a truncated form (arrows in Fig. 1B). Colony 17 was used to isolate a plasmid for DNA sequencing and to express the prion protein. DNA sequencing revealed no errors. Purification of recombinant mouse protein Recombinant mouse prion protein was purified by 40% ammonium sulfate precipitation followed by hydroxyapatite chromatography. Proteins were eluted with increasing concentration of sodium phosphate from 10 to 400 mM and fractions were collected. Protein composition of eluates was analyzed by silver staining of SDS–polyacrylamide gel and Western blot by using alkaline-phosphatase conjugated anti-polyhistidine monoclonal antibody. Fractions 2–4 contained recombinant mouse prion (arrows in Figs. 2A and B). Fraction 2 was used to investigate enzymatic activities.
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Fig. 1. Screening of colonies expressing recombinant mouse prion protein by Western blot analysis. Colonies expressed either full length or truncated forms of prion proteins (arrows in (A,B)). Numbers at the bottom of the figures indicate individual colonies carrying pET43prion except number zero which indicates a colony grown without carrying the pET43-prion. For (A), the nitrocellulose membrane was incubated with alkaline phosphatase-conjugated S-protein followed by BCIP/NBT color detection. For (B), the nitrocellulose membrane was stained with Ponceau 5 (a) or incubated with rabbit anti-prion polyclonal antibody (Immunodynamics) followed by AP-conjugated antirabbit IgG antibody (b) or with goat anti-prion polyclonal antibody (RDI Research Diagnotics, Flanders, NJ) followed by AP-conjugated anti-goat IgG antibody (c). Detection was with BCIP/NBT.
Mouse PrPsc exhibits both NADH oxidase and protein disulfide–thiol interchange activities As an extension of our previous work with ECTONOX proteins where these proteins share characteristics in common with prions, native PrPsc from mouse brain was examined for its ability to oxidize NADH. Not only did the native PrPsc from mouse brain oxidize NADH (Fig. 3), the NADH oxidative activity oscillated as with ECTO-NOX oxidation of NADH [15]. The pattern of oscillations consisted of two maxima separated by an interval of 6 min. This two-peak pattern of NADH oxidation then repeated at intervals of 24 min. A second enzymatic activity associated with ECTONOX proteins is that of protein disulfide–thiol interchange [15]. As measured by cleavage of synthetic dipyridyl–dithio substrates, recombinant prion protein also showed protein disulfide–thiol interchange and, as with ECTO-NOX proteins, the protein disulfide–thiol interchange activity alternated with that of NADH oxidation (Fig. 3B). The protein disulfide–thiol interchange activity of the mouse PrPsc also exhibited a characteristic
Fig. 2. Purification of recombinant prion protein. Prion protein was purified by 40% ammonium sulfate precipitation and hydroxyapatite chromatography. Numbers at the bottom are fraction numbers. (A) Silver staining of 10% SDS–polyacrylamide gel. (B) Western blot (incubation with alkaline phosphatase-conjugated monoclonal anti-polyhistidine antibody and color detection with BCIP/NBT) corresponding to the region of the gel shown by the arrow in (A).
oscillatory activity. However, for protein disulfide–thiol interchange three activity maxima spaced at intervals of 4.5 min were observed. As with NADH oxidation, the activity maxima repeated with an average period length of 24 min. The determinations of Fig. 3 used two spectrophotometers operated in parallel. Equal amounts of PrPsc were used with NADH as substrate in machine A and dithiodipyridine as substrate in machine B. Since the two determinations were in parallel the experiment shows unequivocally that the two activities alternate. Oscillatory oxidation of NADH also exhibited by recombinant mouse prion Full length recombinant mouse prion expressed in bacteria also exhibited oscillatory NADH oxidase activity (Fig. 4A) although the specific activity was lower than that of the PrPsc from mouse brain possibly due to the propensity of the recombinant prion to form inactive protein aggregates. The period length was 24 min for the PrPsc of Fig. 3A. With both the native (not shown) and the recombinant prion, activity was inhibited by bathocuproene (Fig. 4B) suggestive of the involvement of a metal. To demonstrate that the measured oscillatory activity was associated with the recombinant prion and not with low levels of some contaminating bacterial protein, the recombinant protein was resolved by SDS–PAGE and proteins were eluted from 0.5 cm gel segments. NADH oxidase activity from the gel slices was restored as
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Fig. 3. Mouse prion protein (PrPsc ) exhibits a pattern of oscillating alternation of NADH oxidation (A) and protein disulfide–thiol interchange (B) with a period length of 24 min that is indistinguishable from that of a mammalian constitutive NOX (CNOX) protein, i.e., the mouse prion has enzymatic activities equivalent to those of an ECTO-NOX protein. The two determinations used simultaneous measurements from two spectrophotometers operated in parallel. Equal amounts of PrPsc were added to curvettes in both instruments with NADH as substrate in machine A and dithiodipyridine as substrate in machine B, the latter for determination of protein disulfide–thiol interchange.
previously described [16] by first incubating for 10 min with 100 lM GSH in the presence of 125 lM NADH to reduce disulfide bonds and to permit refolding. This was followed by addition of 0.03% H2 O2 to reform disulfide bridges in the refolded protein. Activity overall was greatest in fraction 4 corresponding to the molecular weight of 88.7 kDa for the recombinant protein (Fig. 5). When analyzed separately for oscillatory activity only fraction 4 corresponding to the molecular weight of the recombinant protein contained the oscillatory activity. A longer period of analysis through four full cycles (Fig. 6) revealed the ca. 5-peak pattern of Figs. 3 and 4 within each cycle. The recombinant mouse prion protein
also exhibited protein disulfide–thiol interchange activity as determined by cleavage of dithiodipyridine or by restoration of activity to denatured and inactive (scrambled) RNase but lacked the ability to oxidize hydroquinones. Oscillations in NADH oxidation by recombinant mouse prion appear metal dependent The periodic nature of oxidation of NADH by the recombinant mouse prion was inhibited by addition of 1 lM bathocuproene (Fig. 4), a copper chelator, and restored by addition of 10 lM copper chloride. When
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Fig. 6. Analyses over 96 min of 1.5 and 2.0 cm gel slices from fraction 3 (A) and fraction 4 (B). Only in the 2.0 cm slice (fraction 4) was the oscillating pattern of NADH oxidation observed. The results shown for the 1.5 cm gel fraction from fraction 3 (A) are representative of results from fractions 1, 2, and 5 to 10.
Fig. 4. NADH oxidation by recombinant mouse prion (A) and inhibition by 1 lM bathocuproene (B). The two assays were conducted using two spectrophotometers operated in parallel and yielding simultaneous measurements as described for Fig. 3 except that NADH was the substrate for both. Bathocuproene was added to the curvette of machine B after 48 min. Arrows indicate maxima separated by intervals of 24 min.
tion of oscillations (not shown). The oscillations could be restored by the addition of 0.1 lM CuCl2 . The addition of copper initially stimulated the entire 5 peak pattern but, after the first cycle, one peak was disproportionately enhanced relative to the others.
Discussion
Fig. 5. Analysis of 10% SDS–PAGE gel slices for oscillatory oxidation of NADH with a period length of 24 min. The activity was located predominantly in fraction 4 which corresponded to a molecular weight of ca. 89 kDa. Slices were homogenized in 50 mM Tris–Mes, pH 7, and incubated overnight at 4 °C to elute the protein. After removal of gel fragments by centrifugation, full enzymatic activity was restored using an established protocol of incubation in the presence of 150 lM NADH with 100 lM reduced glutathione followed by 10 min of incubation with 0.03% hydrogen peroxide [16].
dialyzed to remove copper, the specific activity of the recombinant prion for oxidation of NADH was 20– 30 nmol/min/mg protein with nearly complete elimina-
We have described a family of cell surface NADH oxidases (hydroquinone oxidases) that exhibit unusual characteristics including resistance to proteases, resistance to cyanogen bromide digestion, and an ability to form amyloid filaments closely resembling those of spongiform encephalopathies and all of which are characteristic of PrPsc (PrPres ), the presumed infective and proteinase K-resistant particle of the scrapie prion [17,18]. A drug-responsive ECTO-NOX form, tNOX, from the HeLa cell surface co-purified with authentic glyceraldehyde-3-phosphate dehydrogenase (muscle form) (GAPDH) [16]. Surprisingly, the tNOX-associated muscle GAPDH also was proteinase K resistant [9]. In our studies, we showed that combination of authentic rabbit muscle GAPDH with tNOX rendered the GAPDH resistant to proteinase K digestion. This property, that of converting the normal form of a protein into a likeness of itself, is one of the defining characteristics of the group of proteins designated as prions [17,18]. In this report, the functional similarities of tNOX and prion proteins are expanded by the observation that recombinant mouse prion exhibited both of the enzymatic activities normally associated with NOX proteins,
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i.e., oxidation of NADH and protein disulfide–thiol interchange. In addition to exhibiting the enzymatic activities, the activity of the recombinant proteins oscillated with the same 2 + 3 pattern of alternating fast and slow rates exhibited by tNOX [2,19]. The oscillatory activity was enhanced by copper. tNOX has a copper binding site [2] conserved with that of superoxide dismutase [20]. However, the activity and oscillations, unlike those of tNOX, are insensitive to inhibition by the classic tNOX inhibitors such as capsaicin. Taken together with the period length of 24 min, the ECTONOX-like enzymatic activity of the recombinant mouse prion more closely resembles those of the constitutive CNOX forms rather than the tumor-associated tNOX form. It is unlikely that either the oscillations or the copper response of the recombinant protein was influenced by the fusion with nus A as the characteristics of PrPc from mouse brain lacking the tag were similar to those of the recombinant protein. The ECTO-NOX pattern of oscillations associated with the oxidation of NADH within the 24 min period consists of 1 or 2 maxima separated by 6 min. These maxima alternate with 3 additional maxima separated by intervals of 4.5 min associated with the protein disulfide–thiol interchange activity of the protein. As shown by data of Fig. 3, this pattern of activity is fully duplicated by the recombinant mouse prion supporting our earlier contention [9] that the ECTO-NOX proteins and prions were structurally and functionally similar. Like ECTO-NOX proteins, the prion proteins (PrP) are Cu2þ binding cell surface proteins [21,22]. The copper is bound to the histidines of the prion octarepeats [repeats of the sequence P(Q/H)GG(G/S)WGQ]. A second copper site around histidines 96 and 111 has also been indicated from fluorescence and NMR data [23]. In birds the highly conserved mammalian octarepeat is replaced by a hexarepeat which also binds copper but with different coordination geometry than that of the mammalian octarepeat [19]. Several lines of evidence have suggested that copper ions play a role in the biology of both PrPc and PrPsc , the normal and pathologic forms, respectively, of the prion protein. Copper rapidly and reversibly induced PrPc to become protease-resistant and detergent insoluble both properties commonly associated with PrPsc . However, based on a conformation-dependent immunoassay, the copper-treated PrPc appears to be structurally distinct from PrPsc [22]. One suggestion has been that PrPc serves as a receptor for cellular uptake of copper [24,25]. An enzymatic function for PrPc has also been claimed based on the observation that copper binding confers superoxide dismutase activity on the protein [24]. Based on an examination of the phenotype of prion knockout mice, a number of biochemical changes have been observed which, when taken together, have suggested that a lack of expression of the
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prion protein results in a neuronal phenotype sensitive to oxidative stress [26]. Other responses to loss of prion protein expression in mice include altered circadian activity rhythms and sleep patterns [27]. The prion loss is the first null mutation that has been shown to affect sleep regulation. Moreover, one of the inherited prion diseases, fatal familial insomnia, involves a profound alteration in sleep and daily rhythms as well. The invariably fatal neuro-degenerative diseases, including both genetic or sporadic diseases and transmissible spongiform encephalopathies caused by prions, require the development of both PrPc and PrPsc [28]. PrPsc differs from PrPc in having a tertiary structure rich in b-structure. In fact, PrPsc has been suggested to be a totally dehydrated protein with an anhydrous environment, probably a thin carbon dioxide gas gap, to explain why it is highly resistant not only to proteases but to heat and radiation as well and to chemical disinfectants in the water phase except sodium hydroxide and sodium hypochlorite under certain conditions [29]. The unusual properties of prions also extend to ECTO-NOX proteins which are heat and protease resistant [2,16] and extremely resistant to any conditions of denaturation [30]. Such properties may be extremely important to ECTO-NOX proteins which carry out functions in the enlargement phase of cell growth [31] and in biological time keeping [32] from a location at the external cell surface [6,33]. How these normal cellular functions associated with a cell surface protein having an oscillatory activity relate to normal and aberrant prion protein functions in neural tissue remains unexplained. However, the observation of oscillatory oxidative and protein disulfide–thiol interchange activities in both types of proteins may indicate functional roles for prions that may parallel in some way those of the ECTO-NOX proteins.
Acknowledgments This work was supported in part by R01 CA75461, P50 AT00477, and NASA NAG-2-1344.
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