©1995 by The American Society for Biochemistry and Molecular Biology, Inc.
Truncated Forms of the Human Prion Protein in Normal Brain and in Prion Diseases (*)

(Received for publication, April 19, 1995; and in revised form, June 7, 1995)

Shu G. Chen David B. Teplow (1) Piero Parchi Jan K. Teller Pierluigi Gambetti (§) Lucila Autilio-Gambetti

From theDivision of Neuropathology, Institute of Pathology, Case Western Reserve University, Cleveland, Ohio 44106 and the Department of Neurology (Neuroscience), Harvard Medical School, and Biopolymer Laboratory, Brigham & Women's Hospital, Boston, Massachusetts 02115

ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
FOOTNOTES
ACKNOWLEDGEMENTS
REFERENCES

ABSTRACT

The cellular form of the prion protein (PrP^c) is a glycoprotein anchored to the cell membrane by a glycosylphosphatidylinositol moiety. An aberrant form of PrP^c that is partially resistant to proteases, PrP, is a hallmark of prion diseases, which in humans include Creutzfeldt-Jakob disease (CJD), Gerstmann-Sträussler-Scheinker syndrome, and fatal familial insomnia. We have characterized the major forms of PrP in normal and pathological human brains. A COOH-terminal fragment of PrP^c, designated C1, is abundant in normal and CJD brains as well as in human neuroblastoma cells. Sequence analysis revealed that C1 contains alternative NH(2) termini starting at His-111 or Met-112. Like PrP^c, C1 is glycosylated, anchored to the cell membrane, and is heat-stable. Consistent with the lack of the NH(2)-terminal region of PrP^c, C1 is more acidic than PrP^c and does not bind heparin. An additional fragment longer than C1, designated C2, is present in substantial amounts in CJD brains. Like PrP, C2 is resistant to proteases and is detergent-insoluble. Our data indicate that C1 is a major product of normal PrP^c metabolism, generated by a cleavage that disrupts the neurotoxic and amyloidogenic region of PrP comprising residues 106-126. This region remains intact in C2, suggesting a role for C2 in prion diseases.


INTRODUCTION

The cellular prion protein (PrP^c) (^1)is a glycoprotein anchored to the cell surface by a glycosylphosphatidylinositol (GPI) moiety (Stahl et al., 1987). It is encoded by a single gene on human chromosome 20 and contains 253 amino acids with two potential N-linked glycosylation sites (Kretzschmar et al., 1986; Liao et al., 1986; Puckett et al., 1991). Mature human PrP^c spans residues 23-231 of the polypeptide predicted from the PrP cDNA sequence (Oesch et al., 1985; Kretzschmar et al., 1986; Liao et al., 1986). It results from the removal of the 22-amino acid NH(2)-terminal signal peptide (Hope et al., 1986; Turk et al., 1988) and the cleavage of the 23-amino acid COOH-terminal hydrophobic peptide with the simultaneous addition of the GPI anchor (Stahl et al., 1987, 1990). In mouse neuroblastoma cells, PrP^c turns over with a half-life of 3-6 h (Caughey et al., 1989; Borchelt et al., 1990). A chicken homologue of mammalian PrP^c has been shown to recycle between the plasma membrane and an endosomal compartment with concurrent generation of truncated PrP^c intermediates (Shyng et al., 1993). The role of PrP^c in normal cellular function remains elusive. Expression of PrP^c was found to correlate with neuronal differentiation (Wion et al., 1988) and embryonic development (Mobley et al., 1988; Lazarini et al., 1991; Manson et al., 1992). Transgenic mice devoid of PrP^c lack detectable defects in development and behavior (Büler et al., 1992), but display an impairment of synaptic function in the hippocampus (Collinge et al., 1994).

An aberrant isoform of PrP, PrP, characterized by relative resistance to proteolysis and by insolubility in nondenaturing detergents, is a hallmark of the prion diseases, which include familial, transmitted, and sporadic forms of neurodegenerative disorders such as Creutzfeldt-Jakob disease (CJD), Gerstmann-Sträussler-Scheinker syndrome, kuru, and fatal familial insomnia in humans and scrapie and bovine spongiform encephalopathy in animals (for reviews, see Prusiner(1991) and Prusiner and DeArmond (1994)). Studies using scrapie-infected mouse neuroblastoma cells suggest that PrP is formed from PrP^c, either at the cell surface (Caughey and Raymond, 1991) or along the endocytic pathway (Borchelt et al., 1992). Since there are no detectable differences in primary structure between the two PrP isoforms (Stahl et al., 1993), the formation of PrP is thought to result from the conversion of PrP^c to PrP through a conformational transition. Consistent with this notion is the difference in secondary structure between PrP^c and PrP: PrP^c is rich in alpha-helices, while PrP has a higher content of beta-pleated sheets (Caughey et al., 1991a; Safar et al., 1993; Pan et al., 1993). Several predicted alpha-helical domains of PrP^c form beta-sheet structures and aggregate into amyloid-like fibrils when synthesized as polypeptides (Gasset et al., 1992; Tagliavini et al., 1993). In a recent study, Kocisko et al.(1994) succeeded in converting PrP^c to a protease-resistant form in a cell-free system, suggesting that the conversion process results from a direct interaction between PrP^c and PrP.

The molecular mechanism that leads to this conversion and to subsequent prion replication remains unclear. An essential step toward the complete clarification of the conversion of PrP^c to PrP is the detailed knowledge of PrP^c metabolism. In this study, we have characterized the PrP forms present in normal and pathological human brains and in neuroblastoma cells. Using epitope mapping and radiosequencing, we have demonstrated the presence of truncated forms of PrP^c under normal conditions, in addition to full-length PrP^c. Moreover, an additional NH(2)-terminally truncated fragment that is insoluble in detergents and resistant to protease is present in the brains of subjects with prion diseases.


MATERIALS AND METHODS

Antibodies

The following antibodies were used: anti-N, a rabbit antiserum to a synthetic peptide corresponding to human PrP residues 23-40 (B. Ghetti, Indiana University); alpha90-104, a rabbit antiserum to a synthetic peptide corresponding to hamster PrP residues 90-104 (M. Shinagawa, Obihiro University of Agriculture and Veterinary Medicine, Obihiro, Japan); 3F4, a monoclonal antibody that recognizes an epitope of human PrP residues 109-112 (R. Kascsak, New York State Institute for Basic Research in Developmental Disabilities); and anti-C, a rabbit antiserum to synthetic human PrP residues 220-231 (Monari et al., 1994).

Brain Tissues

Tissues were obtained at autopsy from seven histologically normal brains from subjects free of neurological diseases (mean age of 66 years, range of 44-84 years; post-mortem interval of 4-14 h) and from four brains from subjects with sporadic CJD confirmed by histological examination and the presence of PrP (mean age of 65 years, range of 52-75 years; post-mortem interval of 9-17 h). Tissues were also obtained from two histologically normal human brain biopsies performed for diagnostic purposes. All tissues were frozen as soon as collected and stored at -80 °C. All tissue preparations were carried out at 4 °C in the presence of protease inhibitors (1 mM phenylmethylsulfonyl fluoride, 10 µg/ml leupeptin, 3 mM EDTA), unless otherwise indicated.

Total Brain Homogenate

Brain homogenate was prepared as described previously (Monari et al., 1994). Tissues from the cerebral cortex were extracted in 9 volumes of lysis buffer (100 mM NaCl, 10 mM EDTA, 0.5% Nonidet P-40, 0.5% sodium deoxycholate, 10 mM Tris, pH 7.5). Prior to immunoblot analysis, the total brain homogenates were mixed with an equal volume of 2 electrophoresis sample buffer (6% SDS, 5% 2-mercaptoethanol, 4 mM EDTA, 20% glycerol, 125 mM Tris, pH 6.8) and boiled for 10 min.

Subcellular Fractionation

A 10% (w/v) homogenate was prepared in 0.32 M sucrose, 5 mM EDTA, 20 mM Tris, pH 7.5. After centrifugation at 1000 g for 10 min, the postnuclear supernatant (S1) was centrifuged at 100,000 g for 1 h to obtain a cytosolic fraction (S2) and a membrane fraction (P2).

To release GPI-anchored membrane proteins, the P2 fraction was resuspended in a hypotonic buffer (20 mM Tris, 2 mM EDTA, pH 7.5). The sample was then adjusted to 20 mM Tris, 150 mM NaCl, 2 mM EDTA, pH 7.5 (TNE buffer), and incubated for 2 h at 37 °C with 1 unit/ml recombinant Bacillus thuringiensis phosphatidylinositol-specific phospholipase C (PIPLC) purified according to Deeg et al.(1992) or obtained from Oxford Glycosystems. The mixture was then diluted with 10 volumes of TNE buffer and centrifuged at 100,000 g for 1 h to separate PIPLC-released proteins (supernatant) from the rest of the membrane components (pellet).

To examine the detergent solubility and protease resistance of PrP in normal and CJD brains, the membrane fraction (P2) was resuspended in lysis buffer (without protease inhibitors) and centrifuged at 100,000 g to obtain detergent-soluble (supernatant) and detergent-insoluble (pellet) fractions. Protease treatment was carried out by incubation of samples in lysis buffer with 10 µg/ml proteinase K at 37 °C for 1 h.

Cultured Cells

The human neuroblastoma cell line M17 BE(2)-C (kindly provided by B. Spengler and J. Biedler, Memorial Sloan-Kettering Cancer Center, New York) was maintained at 37 °C in Opti-MEM (Life Technologies, Inc.) supplemented with 5% calf serum and antibiotics. The medium was changed twice a week. Cells were differentiated by addition of 5 µM retinoic acid to the medium for 7-10 days until long neurites were formed. To release PrP from the cell surface, cells on 150-mm (148-cm^2) dishes with 80% confluence were incubated in 10 ml of serum-free Opti-MEM containing 0.2 unit/ml PIPLC for 8-12 h. After addition of protease inhibitors, the medium was collected, clarified by centrifugation at 1000 g for 5 min, and stored at -20 °C. In general, 0.3 ml of the medium (10 µg of protein) was used for a single lane of SDS-PAGE unless stated otherwise.

Heparin Binding

The medium (0.5 ml) of PIPLC-treated cells was incubated with 30 µl of heparin-agarose beads (Sigma, type I-A) at room temperature for 1 h, followed by centrifugation at 1000 g for 3 min. Proteins bound to heparin were eluted by boiling the beads in 1% SDS for 10 min.

Heat Resistance

The culture medium containing PIPLC-released proteins was adjusted to 2 mM dithiothreitol, 100 mM Tris, pH 7.5, containing protease inhibitors. The mixture was boiled for 15 min, followed by cooling on ice for 1 h. After centrifugation at 16,000 g for 20 min, the heat-stable (supernatant) and heat-unstable (pellet) fractions were obtained.

Enzymatic Deglycosylation

Proteins were precipitated from the medium of PIPLC-treated cells with 4 volumes of methanol for 2 h at -20 °C. Following centrifugation at 16,000 g for 10 min, the pellet was denatured by boiling for 5 min in 0.5% SDS, 1% beta-mercaptoethanol, 25 mM sodium phosphate, pH 7.5. After addition of Nonidet P-40 to 1%, Asn-linked oligosaccharides were removed by incubation with recombinant PNGase F at 37 °C for 2 h as specified by the supplier (New England Biolabs Inc.).

Immunoblots

Protein samples were separated by 14% SDS-PAGE (Laemmli, 1970) or by two-dimensional gel electrophoresis (NEPHGE gel) (O'Farrell et al., 1977). Proteins were then transferred to Immobilon P (Millipore Corp.) for 2 h at 60 V using a mini-transblot cell (Bio-Rad). After blocking with 10% nonfat milk in Tris-buffered saline, pH 7.5, blots were probed with the appropriate antibodies. The immunoreactivity was visualized on Kodak X-Omat film by enhanced chemiluminescence as specified by the manufacturer (Amersham Corp.). Quantitation of the immunoblots was performed using a computer-assisted laser densitometric scanner (Pharmacia Biotech Inc.).

Radioiodination

Twenty ml of the medium from PIPLC-treated cells were concentrated to 1 ml with a Centriprep 10 device (Amicon, Inc.). The heat-stable fraction prepared as described above was injected into a polymer-based reverse-phase HPLC column (type PLRP-S, 300 Å, 2.5 mm (inner diameter) 250 mm, Polymer Laboratories). The column was equilibrated in buffer A (1% acetonitrile, 20 mM Tris, 10 mM betaine HCl, pH 8.2) and eluted with a gradient of 0-95% buffer B (70% acetonitrile, 20 mM Tris, 10 mM betaine HCl, pH 8.2) at a flow rate of 1 ml/min. The fractions reacting with anti-C on immunoblots were pooled and vacuum-concentrated using a SpeedVac (Savant Instruments, Inc.). After resuspending in 0.5% SDS, the sample was iodinated with 0.5 mCi of carrier-free NaI (DuPont NEN) in a 12 75-mm glass tube coated with 20 µg of IODO-GEN (Pierce) for 30 min at room temperature. The reaction was terminated by transferring the solution to a microcentrifuge tube containing 2 mM dithiothreitol, followed by freezing at -80 °C.

Immunoprecipitation

Immunoprecipitation was performed essentially as described (Stahl et al., 1987). In brief, I-labeled proteins were precipitated with 4 volumes of methanol for 2 h at -20 °C. Following centrifugation at 16,000 g for 15 min at 4 °C, the pellet was resuspended in 1 ml of 10 mM Tris, 150 mM NaCl, 1% Sarkosyl, 0.5 mM phenylmethylsulfonyl fluoride, pH 7.5 (TNS buffer). After addition of 50 µl of 5% bovine serum albumin and 5 µl of anti-C, the mixture was incubated at 4 °C for 12-15 h, followed by incubation with 30 µl (packed volume) of protein A-agarose (Zymed Laboratories, Inc.) for 1 h at room temperature. The protein A beads were washed five times with TNS buffer (pH adjusted to 8.2) and then once with Tris-buffered saline, pH 7.5.

Radiosequencing

Immunoprecipitated radioactive samples were deglycosylated with PNGase F and loaded onto 14% SDS-PAGE mini-gels (Bio-Rad) with 0.1 mM sodium thioglycolate in the cathode chamber. Proteins were transferred to Problott membrane (Applied Biosystems Inc.) for 2 h at 60 V at 4 °C. The membrane was then rinsed with distilled H(2)O and air-dried. After visualization of I-labeled proteins on Kodak X-Omat film, the bands of interest were excised and stored at -70 °C until sequencing. Radiosequence analysis was performed by cutting the sample strips into pieces of 1 mm^2 in area and then loading them on top of Polybrene-coated trifluoroacetic acid-treated glass-fiber filters (Applied Biosystems Inc.). Automated Edman chemistry was carried out on an Applied Biosystems Model 475A instrument essentially as described by the manufacturer, except that anilinothiazolinone-derivatives were collected without being converted into phenythiohydantoins. Anilinothiazolinone-derivative samples were counted in a Packard Cobra series -counter.


RESULTS

Presence of Truncated Forms of PrP^cin Normal Human Brain

PrP^c and the regions recognized by the antibodies used in this study are shown diagrammatically in Fig.1A. Immunoblots of normal brain tissues showed a major PrP^c band of 35 kDa that reacted with antibodies specific for epitopes located in the NH(2)-terminal (anti-N), COOH-terminal (anti-C), and middle (3F4) regions of PrP^c (Fig.1B). Some of the faster migrating bands of 26-30 kDa reacted only with antibodies anti-C and 3F4, but not with anti-N (Fig.1B, lanes 1-3). Tissue homogenates were treated with PNGase F to reduce the heterogeneity of the PrP^c bands due to the presence of Asn-linked oligosaccharides. Enzymatic deglycosylation resulted in a PrP^c band of 27 kDa (Fig.1B, lanes 4-6), which is consistent with the molecular mass of GPI-containing, deglycosylated full-length PrP^c (Haraguchi et al., 1989). An additional prominent band of 18 kDa, which we have named C1, was recognized by anti-C (Fig.1B, lane6), while 3F4 immunoreacted with a faint band of 21 kDa (lane5). Neither of these PrP^c fragments was recognized by anti-N (Fig.1B, lane4), indicating that both are NH(2)-terminal truncated forms of PrP^c. These findings were confirmed with fresh brain tissues obtained by biopsy in the presence of various protease inhibitors such as tosylphenylalanyl chloromethyl ketone, phenylmethylsulfonyl fluoride, and leupeptin (data not shown). Absorption of anti-N and anti-C was carried out with respective synthetic peptide antigens. No bands were detected in immunoblots with either anti-N (Fig.1C, first and secondlanes) or anti-C (ninth and tenth lanes) following the absorption. The specificity of 3F4 immunoreactivity has been established (Kascsak et al., 1987; Bolton et al., 1991). Since C1 is recognized by anti-C but not by 3F4, the NH(2)-terminal PrP^c region up to residue 109 must be absent in this fragment. C1 must be glycosylated since it is sensitive to PNGase F. Quantitation by densitometric analysis shows that the amount of C1 in the cerebral cortex of normal autopsy brains was 30-50% that of full-length PrP^c, while the amount of the 21-kDa band recognized by 3F4 (lane5 in Fig.1B and the sixthlane in Fig. 1C) was <5%. Thus, C1 is present in normal human brain in substantial amounts.


Figure 1: Characterization of truncated forms of PrP in normal human brain. A, shown is a schematic representation of human PrP, with the underlined regions recognized by the three main antibodies used in this study. The amyloidogenic and neurotoxic domain (residues 106-126) (Tagliavini et al., 1993; Forloni et al., 1993) of PrP is shaded. Post-translational modifications of PrP include two N-linked glycosylation sites (indicated by Y-shaped symbols) at residues 181 and 197, respectively, and a GPI linkage at the COOH-terminal residue 231 (Prusiner, 1991). Numbering corresponds to human PrP amino acid sequence (Kretzschmar et al., 1986). B, shown is the presence of truncated forms of PrP^c in the normal human brain. Brain homogenates were either left untreated(-) or treated (+) with PNGase F to remove N-linked oligosaccharides. Samples were analyzed on immunoblots with the anti-N (a-N), 3F4, and anti-C (a-C) antibodies. A prominent PrP fragment, designated C1, reacted with anti-C, but not with 3F4 and anti-N. C, PrP immunoreactivity was eliminated following absorption of anti-N and anti-C with their respective peptide antigens.



Subcellular Localization of C1 in Human Brain

Since it is recognized by the anti-C antibody, C1 is likely to contain an intact COOH-terminal region and, like PrP^c, to be anchored to the cell membrane. To investigate this issue, we obtained cytosolic and membrane fractions and probed them with the 3F4 and anti-C antibodies. Most of C1 and PrP^c were recovered in the membrane fraction (Fig.2A, P2), and both were released from the membrane fraction after treatment with PIPLC (Fig.2B). Therefore, most of C1, like PrP^c, is linked to the cell membrane by a GPI anchor.


Figure 2: Subcellular localization of C1 in human brain. A, a postnuclear brain fraction (S1) was used to prepare cytosol (S2) and membrane (P2) fractions. The fractions were deglycosylated prior to immunoblotting with anti-C and 3F4. C1 was recovered mainly in the membrane fraction, while a small amount was detectable in the cytosol. B, the membrane fraction (P2) obtained in A was treated with PIPLC and then centrifuged at 100,000 g for 1 h. The resulting fractions were analyzed in immunoblots with anti-C to detect PrP forms released by PIPLC (supernatant) and those remaining associated with the membrane (pellet). Like PrP^c, C1 was released by PIPLC and recovered in the supernatant.



Small amounts of C1 and PrP^c were also present in the cytosolic fraction (Fig.2A, S2). Both of these forms may represent PrP^c molecules that have slowly lost their GPI linkage to the membrane as observed previously in primary cultures of neonatal hamster brain (Borchelt et al., 1993).

Characterization of C1 in Human Neuroblastoma Cell Lines

To determine whether C1 is a product of the normal cellular processing of PrP^c and not an artifact generated during tissue preparation, we examined cell-surface expression of C1 in the human neuroblastoma cell line M17 BE(2)-C. Cells were grown in the absence or presence of retinoic acid (a differentiation agent), followed by treatment with PIPLC for 2 h to release GPI-anchored proteins. As shown in Fig.3, immunoblot analysis revealed sets of heterogeneous bands of 28-35 kDa that were reactive with anti-C (lanes1 and 2) and 3F4 (lanes5 and 6). Deglycosylation with PNGase F (lanes3, 4, 7, and 8) resulted in the identification of 27-kDa PrP^c recognized by both anti-C and 3F4 and of 18-kDa C1 recognized by anti-C (lanes3 and 4), but not by 3F4 (lanes7 and 8). No detectable C1 and PrP^c were found in the medium if PIPLC treatment was omitted (data not shown). These data demonstrate that, consistent with our earlier findings in brains, C1 in cultured cells contains Asn-linked sugars and is anchored to the cell surface by a GPI linkage. Moreover, both C1 and PrP^c increased in amount following retinoic acid-induced differentiation (lanes2, 4, 6, and 8). This agrees with the reported increase in PrP^c gene expression during neuronal differentiation (Wion et al., 1988) and development (Mobley et al., 1988; Lazarini et al., 1991; Manson et al., 1992).


Figure 3: Presence of C1 on the cell surface of human neuroblastoma cells and increased expression with differentiation. M17 BE(2)-C cells were cultured in the absence(-) or presence (+) of retinoic acid for 7-10 days. Cells were then incubated in fresh medium containing PIPLC for 2 h at 37 °C to release PrP molecules from the cell surface. The samples were analyzed on immunoblots with anti-C (lanes 1-4) or 3F4 (lanes 5-8) before (-PNGase) or after (+PNGase) deglycosylation.



Since human PrP^c is encoded by a single exon of a single copy gene on chromosome 20 (Liao et al., 1986; Puckett et al., 1991), C1 is likely to be a product of PrP^c processing. The lack of C1 immunoreactivity with 3F4, whose epitope includes PrP residues 109-112, suggests that C1 is generated by a cleavage beyond residue 109. This cleavage may constitute an important event in normal PrP^c metabolism. To characterize the exact cleavage site(s) in PrP^c that generated C1, we partially purified C1 from the medium of PIPLC-treated cells by reverse-phase HPLC, radioiodinated Tyr residues of C1 with I and IODO-GEN, and performed NH(2)-terminal radiosequencing of C1. After radioiodination and SDS-PAGE, C1 appeared as a doublet of 22 kDa (Fig.4A, lane2). This shift in mobility is not surprising since incorporation of I into Tyr residues results in an increased mass of C1 and could produce additional mobility shifts due to perturbation in SDS binding. To characterize the doublet bands of C1 (designated as C1-upper and C1-lower, respectively), they were each subjected to NH(2)-terminal radiosequencing. The results show that both bands have the same NH(2) termini. Both C1-upper and C1-lower produced major I peaks at cycles 17 and 18, consistent with C1 starting at either His-111 or Met-112 (Fig.4B). Therefore, C1 is generated by an alternative cleavage around His-111 of PrP^c. Since no apparent doublet is seen in unlabeled C1 (Fig.4A, lane1), the observed I-C1 doublet may represent the same C1 molecules with anomalous behavior on SDS-PAGE due to radioiodination.


Figure 4: Radiosequencing of C1. C1 from PIPLC-conditioned culture medium was separated from other proteins by its resistance to heat and by chromatography on a reverse-phase HPLC column. A, C1 was either left untreated (lane1) or radioiodinated with NaI by the IODO-GEN method (lane2). Unlabeled C1 was detected on immunoblots with anti-C (lane1), while labeled C1 was immunoprecipitated with anti-C and visualized by autoradiography (lane2). After iodination, C1 appeared as doublet bands at 22 kDa designated as C1-upper and C1-lower, respectively. B, Problott membranes containing the C1-upper and C1-lower bands shown in A were excised and radiosequenced. The sequences of C1 molecules beginning at His-111 and Met-112 are superimposed to illustrate the concordance of radioactivity with positions at which tyrosine residues should occur.



To compare the biochemical properties of C1 to those of full-length PrP^c present in cultured cells, we first examined the capacity of these two molecules to bind heparin. PrP^c has been shown to interact with heparin and other glycosaminoglycans, which prevent the formation of PrP (Gabizon et al., 1993; Caughey and Raymond, 1993; Caughey et al., 1994). Fig.5shows that at variance with PrP^c (lane6), C1 did not bind heparin (lane4). The lack of the PrP^c NH(2)-terminal region in C1 suggests that this region is important for heparin binding. Taking advantage of the different heparin binding characteristics of C1 and PrP^c, we have attempted to clarify the changes in electrophoretic mobility of these two proteins following deglycosylation. It is clear that the 18-kDa deglycosylated C1 isoform stems from its 28-kDa glycoform (lanes3 and 4), while 27-kDa PrP^c is derived from a glycosylated 35-kDa form (lanes5 and 6). The 8-10-kDa reduction in molecular mass for both C1 and PrP^c following deglycosylation is consistent with the presence of two Asn-linked glycans at residues 181 and 197 (Endo et al., 1989; Haraguchi et al., 1989).


Figure 5: Binding of C1 and PrP^c to heparin. Medium containing PIPLC-released proteins from cells was incubated with heparin-agarose for 1 h at room temperature, followed by centrifugation at 500 g. An aliquot of the untreated sample served as control (ORIGINAL). Samples were analyzed on immunoblots with anti-C before(-) and after (+) deglycosylation with PNGase F. C1 did not bind heparin and was recovered in the supernatant (UNBOUND), while full-length PrP was bound to heparin immobilized on agarose beads (BOUND).



We also studied the heat resistance of PrP^c isolated from cultured cells. PIPLC-released proteins were boiled in the presence of protease inhibitors, followed by cooling and centrifugation to pellet the heat-sensitive protein aggregates. Both C1 and full-length PrP^c were recovered in the supernatant as heat-stable proteins (Fig.6, lanes3 and 4). In contrast, most of the other proteins were heat-sensitive and were recovered in the pellet.


Figure 6: Thermal stability of PrP^c and C1. Cells were treated with fresh Opti-MEM containing PIPLC for 2 h at 37 °C. The medium was collected and adjusted to 1 mM phenylmethylsulfonyl fluoride, 10 µg/ml leupeptin, 2 mM EDTA, 1 mM dithiothreitol, 100 mM Tris, pH 7.5. After boiling for 15 min followed by cooling for 1 h at 4 °C, the sample was centrifuged at 16,000 g for 15 min at 4 °C. The resulting supernatant (heat-stable) and pellet (heat-sensitive) were analyzed on immunoblots with anti-C before(-) and after (+) deglycosylation with PNGase F.



Two-dimensional gel electrophoresis of the PIPLC-released and deglycosylated proteins from neuroblastoma cells revealed that C1 and full-length PrP^c migrated to regions of the NEPHGE gel of pH 6.0 and 7.8, respectively (Fig.7). Thus, C1 is more acidic than PrP^c. Since the NH(2)-terminal region of PrP^c contains several basic amino acid residues, an acidic shift in the pI of C1 is consistent with the loss of this region. Some charge microheterogeneity was evident for both C1 and full-length PrP^c. This may be due to the presence of sialic acid in some of the GPI anchors of PrP^c (Stahl et al., 1992).


Figure 7: Two-dimensional gel electrophoresis of C1 and PrP^c. PIPLC-released proteins from M17 BE(2)-C cells were concentrated and deglycosylated prior to NEPHGE for 4 h at 400 V, followed by SDS-PAGE (14% separating gel). An immunoblot was prepared using anti-C. C1 (arrowhead) and PrP^c migrated to NEPHGE regions of pH 6.0 and 7.8, respectively.



PrP Fragments in CJD Brains

Brain homogenates were prepared from CJD subjects to investigate whether PrP^c is processed differently in prion diseases, relative to its processing in normal brain. Like normal brains, CJD brains contained C1, recognized by anti-C but not by 3F4, and full-length PrP^c (Fig.8). However, all CJD brains had, in addition, a substantial amount of a fragment that after deglycosylation migrated at 20 kDa and that we have termed C2. Unlike C1, C2 reacted not only with anti-C, but also with 3F4 (Fig.8) and with alpha90-104 (data not shown), recognizing hamster PrP residues homologous to human PrP residues 90-104 (Shinagawa et al., 1986). However, like C1, C2 did not react with anti-N antibody (data not shown). Thus, C2 is also truncated at the NH(2) terminus and is likely to extend from approximately residue 90 to the COOH terminus.


Figure 8: Presence of the C2 fragment in CJD brains. Brain homogenates from one control (CO) and three CJD subjects were deglycosylated and analyzed on immunoblots with anti-C and 3F4. In the CJD brains, there were significant amounts of the C2 fragment, which reacted with both anti-C and 3F4.



PrP is known to be insoluble in nondenaturing detergents, a property that allows it to be separated from normal PrP^c by differential centrifugation (Caughey et al., 1991b; McKinley et al., 1991). To investigate the possibility that C2 shares these characteristics with PrP, detergent-soluble and detergent-insoluble membrane fractions from a CJD brain as well as from a normal brain were obtained by extraction with lysis buffer and centrifugation. As shown by immunoblots (Fig.9) with anti-C (upperpanel) and 3F4 (lowerpanel), C2 was found only in the detergent-insoluble fraction of the CJD brain (lanes2), along with PrP. Deglycosylated 27-kDa PrP was seen as a doublet (lanes2). The two bands of the doublet reacted with both anti-C and 3F4 (Fig.9) as well as with anti-N (data not shown), suggesting that they may be the same in length, but differ in an unidentified post-translational modification. Immunoblots of the detergent-insoluble fraction, following proteinase K digestion (lanes6), revealed a single band that had the same electrophoretic mobility and immunostaining characteristics as C2. Both C1 and full-length PrP^c were recovered in the detergent-soluble fraction (lanes1 and 3) and were sensitive to the protease treatment (lanes5 and 7). Therefore, C2 is likely to correspond to the PrP fragment generated in vitro by proteinase K digestion, which includes residues 90-231 (Bolton et al., 1987). The amount of C2 appeared to correlate with the amount of abnormal PrP fragment generated in vitro by proteinase K treatment (data not shown). Our findings indicate that the NH(2)-terminally truncated PrP form, likely to be the same as that generated in vitro from brains of patients with prion diseases following proteinase K digestion, is already present in CJD brains in vivo. This conclusion is consistent with the observation that a PrP isoform truncated at residue 90 is present in scrapie-infected mouse neuroblastoma cells (Caughey et al., 1991b).


Figure 9: Insolubility in detergent and resistance to protease of the C2 fragment. Membrane fractions of a CJD brain and of a normal brain (Control) were prepared as described under ``Materials and Methods.'' The P2 fraction was resuspended in detergent-containing lysis buffer (DET) and centrifuged at 100,000 g for 1 h at 4 °C. The resulting supernatant and pellet were referred to as detergent-soluble (S) and detergent-insoluble (I) fractions, respectively. They were treated in the absence(-) or presence (+) of 10 µg/ml proteinase K (PK) in the above buffer for 1 h at 37 °C. All samples were then deglycosylated prior to immunoblotting with anti-C (upperpanel) and 3F4 (lowerpanel). C2 and PrP were insoluble in detergents, while C1 and PrP^c were soluble.



C2 was found to be more basic than C1 on two-dimensional gel electrophoresis (Fig.10). This finding is consistent with the known presence of additional basic amino acids between residues 90 and 110. C2 thus contains residues 106-126, the region that has been shown in vitro to be highly amyloidogenic and neurotoxic (Tagliavini et al., 1993; Forloni et al., 1993).


Figure 10: Two-dimensional gel electrophoresis of the C2 fragment. Brain homogenate from a CJD brain was deglycosylated prior to NEPHGE for 4 h at 400 V, followed by SDS-PAGE (14% separating gel). Immunoblots were prepared using anti-C and 3F4. C2 (open arrowheads) migrated to a more basic NEPHGE region than C1 (solidarrowhead), while full-length PrP migrated to an even more basic region at 27 kDa.




DISCUSSION

This study indicates that a COOH-terminal fragment of PrP^c, which we have termed C1, is present in significant amount in human brain (Fig.1). This fragment is unlikely to be the product of post-mortem autolysis since it is also present in brain tissue obtained by biopsy and in a human neuroblastoma cell line. C1 is NH(2)-terminally truncated and is the main product of normal PrP^c processing. It shares many features with the COOH-terminal half of PrP^c, including glycosylation, membrane attachment by a GPI anchor, and heat stability in aqueous solutions. The thermal stability of C1 and PrP^c in solution, which to our knowledge has never been reported, provides a simple way to purify PIPLC-released PrP^c forms because of the relatively small number of heat-stable proteins.

Biochemical differences between C1 and full-length PrP^c are related to the lack of the NH(2)-terminal region of PrP^c in C1. C1 polypeptide exhibited a relatively acidic pI when compared with PrP^c. This shift in electrophoretic mobility is to be expected as a result of the loss of the NH(2)-terminal region of PrP^c, which contains 10 basic residues up to position 110. Basic residues have also been shown to be crucial for heparin binding (Margalit et al., 1993), providing a possible mechanism for the lack of heparin binding of C1. On the basis of antibody competition, PrP^c-heparin binding had been proposed to occur between residues 142 and 174 (Gabizon et al., 1993). How the polyclonal antibody recognizing PrP residues 142-174 affected heparin binding in that study is not clear. Our present finding argues that the first 110 NH(2)-terminal residues of PrP^c are necessary for heparin binding since C1 lacks affinity for heparin even though it contains residues 142-174. The difference in heparin binding affinity allows for the separation of C1 from full-length PrP^c.

The cellular compartment in which human PrP^c is cleaved to generate C1 in vivo is unknown. PrP fragments comparable to C1 have been noted previously. In mouse neuroblastoma cells transfected with chicken PrP^c constructs, PrP^c has been shown to recycle between the cell surface and the endosomal compartment to produce a COOH-terminal fragment (Shyng et al., 1993) by a cleavage between chicken PrP^c residues 116 and 137 (Harris et al., 1993), corresponding to human PrP^c residues 109-130 (Gabriel et al., 1992). A similar fragment in hamster brain has been suggested to result from a cleavage between hamster PrP^c residues 111 and 138, corresponding to residues 112-134 of the human PrP sequence (Pan et al., 1992). We have performed NH(2)-terminal radiosequencing and found that human PrP^c is cleaved to produce C1 that has the NH(2) terminus starting at either His-111 or Met-112. The sequence information obtained from the present study will help to reconstruct the individual steps of PrP^c metabolism and C1 formation.

There is mounting evidence that the region between residues 100 and 130, containing the C1 cleavage site, plays a critical role in the conformational changes underlying the conversion of PrP^c into PrP, the abnormal isoform present in all prion diseases. Gasset et al.(1992) have identified the PrP^c sequence comprising residues 109-122 as one of the putative alpha-helical regions that, as synthetic peptide, acquires a beta-sheet structure and forms amyloid-like fibrils, a property shared by PrP purified from scrapie-infected brains (Caughey et al. 1991a; Gasset et al., 1992; Safar et al., 1993). Tagliavini et al.(1993) have also shown that a synthetic peptide encompassing human PrP^c residues 106-126 forms fibrils similar to those present in the PrP amyloid plaques of prion diseases such as CJD and Gerstmann-Sträussler-Scheinker syndrome. Moreover, this peptide is toxic to neurons and causes astrocytic proliferation in culture (Forloni et al., 1993).

Our data demonstrate that in normal human brain and in neuroblastoma cells, PrP^c undergoes a proteolytic cleavage that breaks the potentially pathogenic region. Thus, the cleavage that generates C1 from PrP^c may be the crucial step in the constitutive pathway of PrP^c metabolism that precludes the accumulation of a pathogenic fragment (Hope and Chong, 1994). It is thus critical to know whether perturbations in this metabolic pathway play a role in prion diseases. In scrapie, PrP^c and PrP have been shown to have identical amino acid sequence and are thought to differ only in conformation. Hence, prion replication would occur by conversion of PrP^c into PrP through dimerization (Cohen et al., 1994) or nucleation-dependent polymerization (Jarrett and Lansbury, 1993). In brains from subjects with sporadic CJD, a PrP COOH-terminal fragment that we named C2 was markedly increased compared with control brains (Fig.8). C2 is insoluble in nondenaturing detergents (Fig.9), a property shared by PrP (Bolton et al., 1987). C2 is recognized by alpha90-104, 3F4, and anti-C and has the same electrophoretic mobility as the protease-resistant core fragment of PrP that spans residues 90-231 (Bolton et al., 1987; Stahl et al., 1993). Therefore, C2 is the ``in vivo homologue'' of the PrP fragment generated in vitro by proteinase K digestion and is likely to derive from limited NH(2)-terminal trimming of PrP in CJD brains. The concurrent presence of both full-length PrP and C2 is consistent with this notion. The finding that NH(2)-terminal truncation of PrP occurs in vivo in scrapie-infected mouse brain (Hope et al., 1988) and scrapie-infected neuroblastoma cells (Caughey et al., 1991b) further supports this conclusion. Alternatively, C2 may derive from PrP^c through activation of an otherwise minor metabolic pathway that differs from the constitutive pathway generating C1 and that preserves the pathogenic PrP^c region comprising residues 106-126. In scrapie-infected cells, it has been shown that a PrP^c COOH-terminal fragment, comparable to C2, can be directly converted into a protease-resistant form (Rogers et al., 1993). Therefore, the possibility that in CJD and in other prion diseases the production of significant amounts of C2 through an abnormal metabolic pathway is an important pathogenetic event, and C2 is directly converted to the insoluble form, cannot be ruled out. Regardless of which of these two possibilities is correct, the present findings suggest that normal processing of PrP^c in human brain occurs at a cleavage site within the amyloidogenic region of PrP^c, generating C1. In prion disease, either PrP^c or PrP is also cleaved at a different site, resulting in a longer fragment, C2, which contains the entire amyloidogenic region.

The pathogenic region of PrP^c bears resemblance to the amyloid beta-protein (Come et al., 1993), a pathogenic peptide of 39-42 amino acids derived from a large precursor protein (amyloid beta-precursor protein) (Selkoe, 1994). Amyloid beta-precursor protein is constitutively cleaved at residue 16 of amyloid beta-protein, a cleavage that prevents the production of intact amyloid beta-protein (Selkoe, 1994).

Amyloid beta-protein, upon conformational change, may become toxic and resistant to proteases and form amyloid fibrils (Nordstedt et al., 1994; Maury, 1995). Although small amounts of amyloid beta-protein are secreted by cells in vitro, intact amyloid beta-protein is not detectable in normal human brains. However, it is abundantly present in the brains of subjects with Alzheimer's disease (Tabaton et al., 1994). The presence of significant amounts of a pathogenic peptide, perhaps as a result of altered metabolic processing, might be a critical event in both Alzheimer's and prion diseases.


FOOTNOTES

*
This work was supported by Grants AG08155 and AG08992 from the National Institutes of Health. The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore by hereby marked ``advertisement'' in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

§
To whom correspondence should be addressed: Div. of Neuropathology, Inst. of Pathology, Case Western Reserve University, 2085 Adelbert Rd., Cleveland, OH 44106. Tel.: 216-844-1808; Fax: 216-844-1810.

^1
The abbreviations used are: PrP^c, normal cellular isoform of the prion protein; PrP, protease-resistant abnormal isoform of the prion protein; PrP, prion protein; GPI, glycosylphosphatidylinositol; CJD, Creutzfeldt-Jakob disease; PIPLC, phosphatidylinositol-specific phospholipase C; PAGE, polyacrylamide gel electrophoresis; PNGase F, peptide:N-glycosidase F; NEPHGE, nonequilibrium pH gradient gel electrophoresis; HPLC, high performance liquid chromatography.


ACKNOWLEDGEMENTS

We thank Drs. Bernardino Ghetti, Richard Kascsak, and Morikazu Shinagawa for gifts of various antibodies. We also thank Drs. Terrone Rosenberry and Robert B. Petersen for providing purified recombinant PIPLC, Dr. Lucia Monari for participation in the initial phase of this study, Wen Wang for skillful technical assistance, and Anu Arora for maintaining cell cultures.


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