Protein Engineering, Vol. 15, No. 6, 529-537,
June 2002
© 2002 Oxford University Press
High-level expression and characterization of a glycosylated covalently linked dimer of the prion protein
1 Laboratorium für Molekulare Biologie-Genzentrum-Institut für Biochemie der LMU München, Feodor-Lynen Str. 25, D-81377 Munich, Germany and 2 National Institute for Medical Research, Mill Hill, Ridgeway, LondonNW7 1AA, UK
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Abstract |
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There is evidence that prion protein dimers may be involved in the formation of the scrapie prion protein, PrPSc, from its normal (cellular) form, PrPc. Recently, the crystal structure of the human prion protein in a dimeric form was reported. Here we report for the first time the overexpression of a human PrP dimer covalently linked by a FLAG peptide (PrP::FLAG::PrP) in the methylotrophic yeast Pichia pastoris. FLAG-tagged human PrP (aa1-aa253) (huPrP::FLAG) was also expressed in the same system. Treatment with tunicamycin and endoglycosidase H showed that both fusion proteins are expressed as various glycoforms. Both PrP proteins were completely digested by proteinase K (PK), suggesting that the proteins do not have a PrPSc structure and are not infectious. Plasma membrane fractionation revealed that both proteins are transported to the plasma membrane of the cell. The glycosylated proteins might act as powerful tools for crystallization trials, PrPc/PrPSc conversion studies and other applications in the life cycle of prions.
Keywords: covalently linked PrP dimer/glycosylation/Pichia pastoris/prion protein/transmissible spongiform encephalopathies
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Introduction |
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Transmissible spongiform encephalopathies (Weissmann and Aguzzi, 1997
; Prusiner et al., 1998; Lasmézas and Weiss, 2000) are fatal neurodegenerative disorders such as Creutzfeldt–Jakob disease in humans (Creutzfeldt, 1920), bovine spongiform encephalopathy in cattle (Hope et al., 1988) and scrapie in sheep or goat (Dickinson, 1976). They are associated with the accumulation of an abnormal form of the prion protein, PrPSc, derived from the normal cell surface glycoprotein PrPc (Prusiner, 1982). PrPc requires the 37 kDa/67 kDa laminin receptor for internalization (Gauczynski et al., 2001b), a process which is thought to require heparan sulfate proteoglycans (HSPGs) as co-factors mediating the binding of PrPc to its receptor via indirect binding domains (Hundt et al., 2001). Heparan sulfate (HS) binding domains in PrPc have recently been identified (Warner et al., 2002). The conversion of PrPc to PrPSc is thought to take place in compartments of the endocytic pathway such as endosomes, lysosomes or endolysosomes (Gauczynski et al., 2001a). PrPSc and PrPc have very different biochemical properties. PrPc is mainly 
-helical and is readily degradable by proteinase K, whereas PrPSc is characterized by an increase in ß-sheet conformation, a higher tendency to aggregate, insolubility and proteinase K resistance (Prusiner et al., 1984; Meyer et al., 1986; Pan et al., 1993). In cases where the disease is transmitted, prion replication appears to involve the interaction between host PrPc and pathogenic PrPSc from an external source (Prusiner et al., 1984).
There is evidence that prion protein dimers may play a role in the conversion of PrPc to PrPSc. Recently, the crystal structure of the human prion protein in a dimeric form was reported (Knaus et al., 2001). Formation of the dimer involves the three-dimensional swapping of helix 3 and rearrangement of the disulfide bond. The authors suggested that the 3D domain-swapping-dependent oligomerization may be an important step in the PrPc/PrPSc conversion process. Formation of PrP dimers were also observed in N2a cells and in scrapie-infected hamster brains (Priola et al., 1995). They have also been identified as intermediates in the PrP oligo-/multimerization process by fluorescence correlation spectroscopy (Post et al., 1998) and molecular modelling suggested the existence of PrP dimers (Warwicker and Gane, 1996), which could be involved in interspecies transmission (Warwicker, 1997). Recently, covalently linked multimers were observed on western blots of PrPSc purified from hamster brain infected with the 263K strain of scrapie (Callahan et al., 2001). It was suggested that these multimers may be the result of some PrP molecules in the PrPSc aggregates becoming covalently cross-linked in vivo. A monomer–dimer equilibrium was detected under native conditions in at least a fraction of PrPc purified from bovine brains (Meyer et al., 2000). Recently, a dimeric -helical intermediate was observed during the in vitro conversion of recombinant hamster PrP to large insoluble aggregates (Jansen et al., 2001).
In this study, we expressed a covalently linked human PrP dimer (PrP::FLAG::PrP) and full-length human PrP (huPrP::FLAG) in the methylotrophic yeast Pichia pastoris. This powerful expression system makes use of the highly inducible alcohol oxidase promoter to express large amounts of glycosylated protein. The proteins were expressed as fusion proteins to a FLAG peptide and the native prion signal sequence and GPI anchor were included to direct secretion of the protein. Expressions experiments were carried out with tunicamycin, which blocks glycosylation in vivo, to confirm the mixed glycoform expression. Optimization of expression resulted in yields of ~50–100 mg/l. The sensitivity of the expressed FLAG fusion proteins to proteinase K and endoglycosidase H was determined. The fusion proteins were detected in the yeast plasma membrane fraction but not in the media, suggesting trafficking of the proteins to the cell membrane.
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Materials and methods |
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Reagents and antibodies
The monoclonal anti-PrP antibody 3B5 directed against the octapeptide repeat region of human and bovine PrP was a gift from G.Hunsmann, Göttingen, Germany and the 3F4 antibody directed against aa 109–112 of hamster and human PrP was obtained from Chemicon. Anti-FLAG antibody M2, secondary anti-mouse IgG–POD conjugate and tunicamycin were supplied by Sigma. Proteinase K, endoglycosidase H and Pefablock were purchased from Roche Diagnostics.
Plasmid constructions
Construction of pPICZB–huPrP1–227FLAG228–253.
The insertion of a FLAG encoding sequence for the pSFV1–huPrP1–227FLAG228–253 plasmid is described elsewhere (Hundt et al., 2002). The cDNA was amplified by PCR from this plasmid, introducing EcoRI and XbaI restriction sites at the 5' and 3' ends. The amplified fragment was cloned into the P.pastoris expression plasmid pPICZB via EcoRI/XbaI restriction sites, resulting in pPICZB–huPrP1–227FLAG228–253.
Construction of pPICZB–huPrP1–230FLAGhuPrP1–227-FLAG 228–253.
cDNA encoding huPrP1–253 was amplified by PCR and cloned into pSFV1, as described (Krasemann et al., 1996), resulting in pSFV1–huPrP1–253. The cDNA encoding huPrP1–230 was amplified by PCR from this plasmid, introducing EcoRI and HindIII restriction sites at the 5' and 3' ends. A second fragment (FLAGhuPrP23–227FLAG228–253) was amplified by PCR from the pSFV1–huPrP1–227FLAG228–253 plasmid, introducing a HindIII restriction site and a FLAG encoding sequence at the 5' end and an XbaI restriction site at the 3' end. These two fragments were restricted, ligated and cloned into the P.pastoris plasmid pPICZB via EcoRI/ XbaI restriction sites, resulting in pPICZB–huPrP1–230FLAGhuPrP23–227FLAG228–253.
Expression in Pichia pastoris
The P.pastoris expression system uses the promoter from the alcohol oxidase gene, AOX1, to express heterologous proteins. The expression vector pPICZB (EasySelect Pichia Expression Kit, Invitrogen) was digested with EcoRI and XbaI and ligated to the inserts. DH5 cells were transformed with the ligation products and plated on low-salt LB–zeocin medium (0.5% yeast extract, 1% tryptone, 0.5% NaCl and 25 µg/ml zeocin). The transformants were tested by restriction analysis and positive clones were amplified to make larger amounts of DNA.
The nucleotide sequences of the resulting plasmids were confirmed by dideoxy sequencing. Prior to transformation into yeast, the plasmids were digested with SacI. The DNA was transformed into P.pastoris (SMD 1168) according to the manufacturer's instructions and the cells were plated on to YPD–zeocin medium (1% yeast extract, 2% peptone, 2% D-glucose, 0.1 mg/ml zeocin). For secondary selection of multicopy transformants using zeocin, clones were pooled, diluted in sterile water and about 1x104 cells were spread on YPD plates containing increasing concentrations (200, 400, 600 and 1000 µg/ml) of zeocin.
Ten clones with high zeocin resistance were selected for a test expression. Single colonies were used to inoculate 10 ml of BMGY (1% yeast extract, 2% peptone, 1.34% yeast nitrogen base without amino acids, 0.00004% biotin, 1% glycerol, 50 µg/ml kanamycin, 0.1 M potassium phosphate buffer, pH 6.0). The cultures were grown overnight at 28°C to an A600 of 2–6 and then harvested (2000 g, 5 min, room temperature). The cultures were resuspended in medium that contained 0.5% methanol instead of glycerol in order to induce the yeast cells to express the heterologous protein. Aliquots of 1ml of culture were removed every 24 h and centrifuged at 6000 r.p.m. for 2 min in a microcentrifuge. A 60 µl volume of the supernatant was added to 30 µl of 3x SDS-loading buffer. The pellet was resuspended in 0.5 ml of distilled water and 60 µl were added to 30 µl of 3x SDS-loading buffer. Expression of the recombinant protein was monitored by SDS–PAGE followed by western blotting and detection with anti-PrP specific antibodies (3F4 or 3B5) or the anti-FLAG M2 antibody.
Larger scale expression and optimization
The highest expressing clones of the covalently linked dimer and monomer, as determined by western blot analysis, were used to inoculate 25 ml cultures of BMGY. The cultures were grown at 28°C (230 r.p.m.) to an A600 = 2–6. After centrifugation the cultures were resuspended in 100 ml of BMMY containing 0.5, 1.0 or 2% methanol (to an A600 of 1) in 1 l baffled flasks and shaken at 28°C (200 r.p.m.) for 72 h. Aliquots of 1 ml were removed every 24 h for determination of protein expression.
Expression in the presence of tunicamycin
Tunicamycin was used to block in vivo glycosylation. It was added to 10 ml cultures of the covalently linked dimer and monomer (from a stock solution of 1 mg/ml in 0.1 M NaOH) to a final concentration of 15 µg tunicamycin/ml culture. Small-scale expression was carried out essentially as described above, with tunicamycin being included in the BMGY and BMMY culture media. Aliquots of 1 ml were removed 24 h after induction and expression of the covalently linked dimer and monomer in the cell lysate was analysed by SDS–PAGE and western blotting. The monoclonal antibody 3B5, which recognizes the octarepeat region of human and bovine PrP, was used for protein detection.
Cell lysis and sensitivity to proteinase K
Cell pellets containing over-expressed FLAG-tagged covalently linked dimer and monomer, isolated from 2 ml of each culture, were resuspended in 1 ml of lysis buffer (10 mM Tris–HCl buffer, pH 7.5, containing 10 mM EDTA, 100 mM NaCl, 0.5% Triton X-100 and 0.5% deoxycholate). An equal volume of glass beads (500 µm) was added to each suspension and the cells were broken by vortexing for a total of 4 min in bursts of 30 s alternating with cooling on ice for 30 s. The glass beads were separated by centrifugation (4000 r.p.m. for 10 min, 4°C).
The resistance of the covalently linked PrP dimer and monomer to proteinase K was assessed. Aliquots of 100 ml of the supernatants were incubated with proteinase K (0–4 mg/ml) at 37°C for 1 h. Digestion was stopped by the addition of Pefablock to a final concentration of 1 mM and samples were analysed by immunoblotting (with the 3B5 and 3F4 antibody) after SDS–PAGE.
Sensitivity to endoglycosidase H
Cell pellets containing overexpressed FLAG-tagged covalently linked PrP dimer and monomer were lysed as above, but in the lysis buffer 40 mM sodium citrate, pH 5.5, 0.05% SDS, 0.5 mM PMSF. Aliquots of 50 ml of the supernatants were incubated with or without 0.5 units/ml endoglycosidase H at 37°C for 3 h. The reaction was stopped by addition of 3x SDS-loading buffer and heating at 95°C for 5 min. Deglycosylation was monitored by SDS–PAGE followed by western blotting and detection with the 3B5 antibody.
Purification of yeast plasma membrane fraction
The plasma membrane fractions of yeast overexpressing the FLAG fusion proteins were purified using standard procedures (Panaretou and Piper, 1996). P.pastoris culture pellets (from 50 ml cultures) were resuspended in 10 ml of cold lysis buffer (25 mM imidazole, pH 7.0, 2 mM EDTA, 0.4 M sucrose). The cells were re-pelleted by centrifugation and the supernatants discarded. Amounts of 2 ml of glass beads and 2 ml of lysis buffer were added and cells were broken by vortexing as described above. A 9 ml volume of cold lysis buffer was added and the cell debris and glass beads were pelleted by centrifugation (530 g, 20 min, 4°C). The supernatant was removed and centrifuged (22 000 g, 30 min, 4°C) to pellet the plasma membrane and mitochondria fractions. The supernant (cytosolic fraction) was removed and the pellet taken up in TBS containing 5% Triton X-100. This was further diluted to 20 ml with TBS containing 0.1% sarcosine, 0.1% NP-40 and 100 mM dithiothreitol.
Immunoprecipitation
The FLAG fusion proteins were immunoprecipitated with 200 µl of a 50% slurry of protein A-Sepharose (Pharmacia) and 10 µl of 3B5 antibody as described previously (Caughey et al., 1999).
Removal of GPI anchor by cleavage with enterokinase
Enterokinase cleaves the final lysine of the FLAG peptide and was used here to remove the GPI anchor of huPrP::FLAG. The expressed dimer was also treated with enterokinase even though it has two potential cleavage sites. Yeast cells were lysed in TBS, 0.1% Triton X-100 and 100 µl of each supernatant were incubated with CaCl2 (final concentration 10 mM) and enterokinase (50 µl added, 1 unit/µl) at 37°C for 20 h. The reaction was terminated with EDTA (20 mM).
SDS–PAGE and immunoblotting
Protein samples were separated on 12% Mighty Small gels according to the manufacturer's protocol (Hoefer, Pharmacia Biotech, San Francisco, CA) and transferred electrophoretically on to pre-wetted poly(vinyl difluoride) membranes. The blots were incubated with an anti-PrP antibody (3F4, 3B5, 1:5000 dilution) or with an anti-FLAG M2 antibody (1:600 dilution). The incubation steps were preformed as described previously (Weiss et al., 1995, 1996) and the bound antibody was visualized with 3,3'-diaminobenzidine tetrahydrochloride.
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Results |
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Expression of covalently linked human PrP dimer and huPrP::FLAG proteins in Pichia pastoris
A covalently linked dimer of the human PrP (PrP::FLAG::PrP), with the FLAG octapeptide (DYKDDDDK) as a linker and at its C-terminus (Figure 1A) was expressed in P.pastoris. The FLAG peptide is used as an epitope tag for the detection and purification of recombinant proteins and was chosen here because of its highly charged, polar sequence. For comparison, we also expressed a C-terminally FLAG-tagged human PrP molecule (Figure 1B, huPrP::FLAG).
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Plasmids pPICZB–huPrP1–227FLAG228–253 and pPICZB– huPrP1–230FLAGhuPrP1–227FLAG228–253, transformed into the protease deficient P.pastoris strain SMD 1168, exhibited high levels of intracellular production of the FLAG-tagged proteins (Figure 2
). Antibody 3B5 (and also 3F4 and anti-FLAG M2; results not shown) recognized three bands with apparent molecular masses ranging from ~25 to ~33 kDa for huPrP::FLAG (lanes 1 and 2) and approximately five bands for PrP::FLAG::PrP (lanes 3 and 4), indicating that the fusion proteins were glycosylated. Higher molecular mass bands were also detected for huPrP::FLAG at approximately the same molecular mass as the dimer bands, which suggests that the expressed PrP::FLAG forms covalently linked dimers. Priola et al. also observed a PrP dimer of 60 kDa derived from hamster PrP expressed in murine neuroblastoma cells (Priola et al., 1995). This 60 kDa PrP was not dissociated under several harsh denaturing conditions.
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Optimum expression was obtained with a 0.5–1.0% methanol concentration and an induction time of 24 h (Figure 2
). After longer induction times, degradation of the fusion proteins occurred. Our data represent the first high-level expression of PrP in P. pastoris, with an approximate expression yield of 50–100 mg fusion protein/l. Effect of tunicamycin and endoglycosidase H sensitivity
HuPrP::FLAG has two potential glycosylation sites (N-X-S/T), whereas the covalently linked dimer has four sites. To investigate whether the higher molecular mass bands were due to glycosylated protein, we expressed the fusion proteins in media containing tunicamycin, which blocks glycosylation in vivo, and analysed the cell lysates by SDS–PAGE and western blotting (Figure 3). In the presence of tunicamycin there was no detectable glycosylated human PrP::FLAG (Figure 3A). With the covalently linked dimer, the bands corresponding to the tri- and tetraglycosylated forms were strongly reduced (Figure 3B).
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Endoglycosidase H cleaves high-mannose sugars and was used to confirm the expression of various glycoforms of the fusion proteins. Cell lysate supernatants containing overexpressed huPrP::FLAG or PrP::FLAG::PrP were incubated with endoglycosidase H (0.5 units/ml) for 3 h at 37°C. Separation of proteins by SDS–PAGE and immunodetection with the 3B5 antibody (Figure 4
) showed no detectable higher molecular mass bands of huPrP::FLAG, corresponding to the mono- and diglycosylated forms. In contrast to the PrP::FLAG::PrP, there is some residual glycosylation which may be consistent with the covalent prion dimer having some tertiary structure.
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Proteinase K sensitivity
In order to analyse the resistance of the covalently linked dimer to proteinase K (PK) and to compare it with huPrP::FLAG expressed in the same system, the cell lysate supernatants were incubated with 0, 2 and 4 µg/ml PK for 1 h at 37°C. Analysis by SDS–PAGE and western blotting employing the 3B5 antibody (Figure 5) and the 3F4 antibody (data not shown) showed that the fusion proteins have similar PK sensitivity, both being completely digested by 4 µg/ml PK. Evidently PK is able to degrade the prion monomer and covalent dimer equivalently.
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Secretion of the fusion proteins to the plasma membrane
The plasma membrane fractions of P.pastoris overexpressing huPrP::FLAG and PrP::FLAG::PrP were isolated and analysed by western blotting (Figure 6B and C). Both fusion proteins were detected in the plasma membrane fraction and in the cytosolic fraction of the cells. Coomassie Brilliant Blue staining confirmed that the covalently linked dimer is overexpressed and transported to the cell membrane (Figure 6A, lane 1). This finding is in harmony with the fact that both of our proteins are glycosylated.
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Immunoprecipitation
The FLAG fusion proteins were immunoprecipitated with the anti-PrP antibody 3B5 directed against the octapeptide repeat region of human and bovine PrP and protein A-Sepharose. The beads were washed and analysed by SDS–PAGE and immunoblotting (Figure 7), demonstrating that the various glycosylation forms of both the dimer and monomer are specifically recognized by PrP antibodies in solution, under non-denaturing conditions.
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Enterokinase cleavage
Enterokinase is a highly specific serine protease which cleaves after the C-terminal lysine of the recognition sequence Asp–Asp–Asp–Asp–Lys. These are the last five amino acids of the FLAG-tag. Enterokinase was used to remove the final lysine of the FLAG peptide and the GPI anchor of huPrP::FLAG. The expressed dimer was also treated with enterokinase even though it has two potential cleavage sites.
Comparison of the digested HuPrP::FLAG (Figure 8, lane 2) with the undigested protein (Figure 8, lane 1) shows a slight reduction in molecular mass. However, no difference in apparant molecular masses was observed in the case of the dimer (Figure 8, lanes 3 and 4). Since the dimer contains two FLAG-tags, one as the linker peptide and one close to the C-terminus, we would expect a reduction in the amount of dimer and the appearance of monomer bands after cleavage with enterokinase. The results obtained indicate that the dimer may have some tertiary structure, which might protect the internal cleavage site from the protease.
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Discussion |
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We used the methylotrophic yeast P.pastoris to express high levels of non-, mono- and diglycosylated full-length human PrP and various glycoforms of a covalently linked human PrP dimer. Over the last few years, interest in the P.pastoris expression system has grown since it has the potential for high-level expression. It has been reported that in some cases up to several grams of the target recombinant protein per litre of culture have been obtained (Romanos, 1995
); however, it is normally necessary to carry out fermentation to achieve this level of protein expression. In mammalian cells high-mannose sugars are added to PrPc in the endoplasmic reticulum and are subsequently modified in the Golgi, becoming endoglycosidase H resistant. In yeast, no modification of the high-mannose sugars occurs and the glycosyl groups remain endoglycosidase H sensitive.
The physical state of the recombinant prion protein, monomer or covalent dimer, is unclear at present. The endoglycosidase H studies suggest that the covalently linked PrP dimer has some three-dimensional structure, stable enough to interfere with the deglycosylation by the enzyme. Equally the effects of tunicamycin in abolishing glycosylation are less complete with the covalent dimer. The proteinase K sensitivity status of the FLAG tagged prion protein and the covalently linked PrP dimer, however, proved to be similar. This suggests that neither recombinant protein has the PrPSc structure, which is PK resistant (Taraboulos et al., 1990). The similarity in their cleavage properties is, however, not inconsistent with the covalent dimer retaining some tertiary structure. We suppose that the structure of the covalently linked PrP dimer reported here might be different from the structure of the crystallized PrP dimer where the N- and C-termini of the two chains appear to be very far apart (Knaus et al., 2001). The organization of our covalently linked dimer might also be different from other PrP dimers observed.
The availablility of large amounts of recombinant PrP expressed in Escherichia coli has allowed the solution structure of mouse, hamster, human and bovine PrP to be determined by NMR spectroscopy (Donne et al., 1997; Riek et al., 1997; Lopez Garcia et al., 2000; Zahn et al., 2000). However, these recombinant proteins lack two glycosyl groups and a glycosylphosphatidylinositol (GPI) membrane anchor. Very little is known about the effect of these two post-translational modifications on the structure and function of PrP.
We proved that our recombinant FLAG tagged prion protein expressed in P.pastoris is highly glycosylated and differs in this respect from other non-glycosylated bacterially expressed prion proteins (Riek et al., 1996, 1997). Further structural studies with our glycosylated prion protein will prove whether glycosylations will influence the secondary/tertiary structure of the prion protein.
The generation of a covalently linked enzymatically active dimer has been described for the protease of human immunodeficiency virus (HIV) type one, composed of two copies of the protease sequence linked by a structurally flexible hinge region (Krausslich, 1991). The expressed dimer was stable and active against HIV polyprotein substrates. It was reported recently that human PrP crystallizes in a dimeric form (Knaus et al., 2001). Formation of the dimer involves 3D swapping of the C-terminal helix and rearrangement of the disulfide bond. The authors suggested that this oligomerization may be an important step in the PrPc/PrPSc conversion process. We hypothesize that the covalently linked PrP dimer might be a useful tool in cell-free conversion assays (Horiuchi et al., 2000). It could be used as a template in the assay or added to investigate whether the rate conversion of PrPc to PrPSc is altered. In addition, the covalently linked PrP dimer might be a suitable tool in cell culture studies of non-infected or scrapie-infected neuroblastoma cells, investigating again its role in the PrPc and PrPSc propagation process.
Recently, the 37 kDa/67 kDa laminin receptor has been identified as the cell surface receptor for cellular PrP (Gauczynski et al., 2001b). This process involves cell surface HSPGs identified as co-factors for the 37 kDa/67 kDa laminin receptor-dependent PrP binding and internalization process (Hundt et al., 2001). Further studies will prove whether the covalently linked PrP dimer will be bound or become internalized differently from monomeric PrP or might interfere with the PrPc/PrPSc internalization process on neuronal cells.
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3 To whom correspondence should be addressed. E-mail: weiss{at}lmb.uni-muenchen.de
M.L.Riley and C.Leucht contributed equally to this work
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Acknowledgments |
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We are grateful to G.Hunsmann for the 3B5 antibody and to S.Krasemann for plasmid pSFV1-huPrP1. We thank A.Pahlich, K.Krüger and S.Janetzky for excellent technical assistance. S.Weiss thanks R.Grosschedl and E.-L. Winnacker for continuous support and valuable advice. This work was funded by grant FAIR-CT-98-7020 from the European Union.
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Received November 14, 2001; revised February 26, 2002; accepted March 8, 2002.
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