Vo1.184, No. 3,1992
BIOCHEMICAL AND BIOPHYSICAL RESEARCH COMMUNICATIONS
May 15,1992
Pages 1398-1404
A SOLUBLE FORM OF PRION PROTEIN IN HUMAN CEREBROSPINAL FLUID: I m p l i c a t i o n s for p r i o n - r e l a t e d e n c e p h a l o p a t h i e s Fabrizio Tagllavini , Frances Prelli2, Monlca Porro i, Mario Salmona 3, Orso Bugiani I and Blas Frangione2 llstituto Neurologico Carlo Besta, via Celoria 11, 20133 Milano, Italy 2Department of Pathology, New York University, New York, 10016 NY 31stituto di Ricerche Farmacologiche Mario Negri, Mflano, Italy
Received April I, 1992
SUMMARY: The cellular prion protein (PrPc) is a 33-35 kDa sialoglycoprotein anchored to the external surface of neural and n o n - n e u r a l cells by a glycosyl phosphatidylinositol moiety. In addition, a secretory form of PrP c has been found in cell-free translation systems and in cell cultures. On this basis, we investigated h u m a n cerebrospinal fluid for the presence of soluble PrP a n d identified a protein whose molecular weight, antigenic determinants, N-terminal amino acid sequence and sensitivity to protease digestion corresponded to those of PrP C. In prion-related encephalopathies of h u m a n s and animals, the secretory form of PrPc might be converted into the abnormal isoform PrP sc and play a role in the dissemination of the disease process and amyloid formation. © 1992 Academic Press, Inc.
The cellular prion protein (PrP c) is a 33-35 kDa sialoglycoprotein encoded by a gene that in h u m a n s is located on chromosome 20 (1-8). The PrP gene is expressed in neural and n o n - n e u r a l tissues, the highest concentration of mRNA being in n e u r o n s (9-13). The translation product consists of 253 amino acids (14, 15) and undergoes several post-translational modifications. In hamsters, a signal peptide of 22 amino acids is cleaved at the N-terminus, 23 amino acids are removed from the C-terminus on addition of a glycosyl phosphatidylinositol (GPI) anchor,
and
asparagine-linked
oligosaccharides are attached to residues 181 and 197 in a loop formed by a disulfide bond (8,
16-20). In prion-related encephalopathies, such
as
Creutzfeldt-Jakob disease and Gerstmann-Strtiussler-Scheinker disease of h u m a n s , scrapie of sheep and goats, and spongiform encephalopathy of
* To whom reprint requests should be addressed. Abbreviations: CSF: cerebrospinal fluid, GPI: glycosyl phosphatidylinositol, PrP: prion protein, prpC: cellular prion protein, prpSc: scrapie prion protein. 0006-291X/92 $4.00 Copyright © 1992 by Academic Press, Inc. All rights of,reproduction in any form reserved.
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cattle, PrP c is converted into a n altered form designated PrP sc, that is distinguishable from PrP c because only the N-terminal 67 amino acids are removed by proteinase K digestion u n d e r conditions where PrP c is completely degraded (1, 21-25). Several lines of evidence indicate that PrP sc is a key component
of
the
transmissible
agent
responsible
for
prion-related
encephalopathies (26) and that its protease-resistant core is the major structural protein of amyloid fibrils that accumulate intracerebrally in some of these conditions (27-33). PrP c is a m e m b r a n e - b o u n d protein, anchored to the cell surface through the GPI moiety (17-19, 34). Nevertheless, a secretory form of the molecule in addition to the m e m b r a n e form has been found in ce11-free translation
systems
supplemented
with
microsomal
membranes.
Furthermore, spontaneous release of PrP c from cultured cells and from
Xenopus oocytes injected with PrP mRNA has been reported (35-41). On this basis, we analyzed h u m a n cerebrospinal fluid (CSF) for the presence of soluble PrP and identified a protein whose molecular weight, antigenic properties, N-terminal amino acid sequence and sensitivity to protease digestion corresponded to those of PrP c.
MATERIALS A N D M E T H O D S Antisera: Two antisera, designated anti-PrPN and anti-PrP 27-30, were used for immunodetection of PrP. Anti-PrPN was raised in rabbits to the synthetic peptide KKRPKPGGWNTGGSRYPGGC, that corresponds to residues 23-40 of the amino acid sequence deduced from the h u m a n PrP cDNA, except for the addition of Gly-Cys at the C-terminus for spacing and coupling. This peptide was synthesized by solid phase techniques using Fmoc-t-butyl-polyamide chemistry, purified by HPLC using a ~t-Bondapak C18 column (Waters), a n d coupled to keyhole limpet hemocyanin with m-maleimidobenzoy1-Nhydroxysuccinimide. Immunization was carried out as described previously (42) and antibody titer was evaluated by ELISA. Anti-PrP 27-30 (kindly provided by Dr. Paul Brown) was generated by immunizing rabbits with the protease-resistant core of PrP sc (i.e, PrP 27-30) purified from scrapie-infected h a m s t e r brains.
F r a c t i o n a t i o n o f CSF proteins: Samples of CSF were collected from three individuals aged 3, 17 and 29 years, who received external s h u n t i n g for tension hydrocephalus due to germinoma of the pineal region, stenosis of the aqueduct a n d gangliocytoma of the cerebellum, respectively. The protein content of the samples was lower t h a n 100 mg per 100 ml, and blood cells were absent. By a m m o n i u m sulphate precipitation four fractions were obtained, that corresponded to the precipitates at salt concentration of 25%, 50% and 70% (designated as P1, P2 and P3, respectively) and to the 70% s u p e m a t a n t (designated as $3). P1, P2 a n d P3 were redissolved in 20 mM Tris-HC1, pH 7.5, a n d dialyzed against distilled water using t u b u l a r m e m b r a n e s with a molecular weight cut off of 12,000-14,000. After centrifugation at 50,000 x g for 30 minutes, pellets (designated as Plp, P2p, a n d P3p, respectively) and s u p e r n a t a n t s (designated as P l s , P2s and P3s, respectively) were lyophflized. $3 was dialyzed against distilled water a n d lyophilized.
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Immunoblot analysis: Aliquots of the fractions were redissolved in 20 mM Tris-HC1, 150 mM NaC1, pH 7.5, and proteins were determined using the Coomassie blue dye-binding assay (Bio-Rad}. Samples containing 50 ~tg protein were subjected to sodium dodecyl sulfate-polyacrylamide gel electrophoresis {10% or 15% monomer) under reducing conditions and eleetrotransferred to ProBlott m e m b r a n e s (Applied Biosystems} using 10 mM 3-cyclohexylamino- 1-propanesulfonic acid buffer, pH 11, 10°/5 methanol. The m e m b r a n e s were either stained with Coomassie blue or immunostained with anti-PrPN antiserum {1: 500), as described previously (32). Proteins specifically detected by the antiserum were identified by using anti-PrPN preabsorbed with 10 mM of the relevant peptide for 60 minutes at 37 ° C. Aliquots of the CSF fraction containing anti-PrPN immunoreactive proteins were treated with proteinase K (20 ~tg/ml) for 60 minutes at 37 ° C. Digestion was terminated by the addition of 20 mM phenylmethylsulfonyl fluoride, and the samples were subjected to immunoblot analysis with anti-PrP 27-30 antiserum (1: 1,000). Protein sequence analysis: Coomassie blue-stained bands corresponding to the bands specifically labeled by anti-PrPN antiserum were excised and analyzed with a 477A microsequencer {Applied Biosystems} for N-terminal amino acid sequence. The resulting phenylthiohydantoin amino acid derivatives were identified using the on-line 120A PTH analyzer and the standard program (Applied Biosystems}.
RESULTS AND DISCUSSION Immunoblot analysis of CSF showed that fraction P2p, corresponding to the precipitate at 50% a m m o n i u m sulfate concentration, contained a 33-37 kDa protein band that was specifically labeled by anti-PrPN antiserum (Figure 1). This band was consistently observed in CSF samples of all subjects and was completely degraded by proteinase
K digestion, t h u s
resembling PrP c.
N-terminal sequence analysis of the anti-PrP immunoreactive b a n d
a
b
674330- 0
Figure L Irmnunoblot analysis of CSF fraction P2p showing a broad band with electrophoretic mobility of 33-37 kDa {arrow} that is strongly labeled by the antiserum anti-PrPN {lane a). The immunoreactivity is abolished by absorption of the antiserum with the relevant peptide (lane b}. Protein bands still irnmunoreactive after absorption were regarded as nonspecffic. Molecular weight markers {expressed in kDa} are shown to the left. 15% polyacrylamide minigel; antisera dilutions: 1: 500. 1400
by
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automated E d m a n degradation yielded K X P K (P) G G (P) N T. which aligns at position 24 of the amino acid sequence deduced from h u m a n PrP eDNA, one residue beyond the predicted signal peptide cleavage site (15, 16). This difference might result from nonspecffic proteolysis during collection an d fractionation of CSF or from heterogeneity. In this regard,
N-terminal
heterogeneity of prpSc purified from scrapie-infected h a m s t e r brains with a major sequence starting at position 23 and two minor sequences beginning at residues 24 and 3 I, respectively, h as been reported (8). In the second cycle of the E d m a n degradation, we did not detect the Arg residue predicted from the translated sequence of h u m a n PrP DNA. Absence of Arg signal at this position h a s been observed previously in sequencing studies of h a m s t e r PrP e and PrP sc,
suggesting
that
this
residue
might
undergo
post-translational
modifications which preclude its detection during gas-phase sequencing (8). N-terminal sequences of apolipoproteins E and J, and of the a-chain of complement C4 were also identified in CSF fraction P2p, comigrating with PrP c. These proteins comprised mainly the higher molecular weight sector of the 33-37 kDa protein band, whereas PrP c was found predominantly in the lower portion of the band. The presence of a soluble form of PrP c in h u m a n CSF is in agreement with the following observations. PrP c is secreted from Xenopus oocytes injected with PrP mRNA synthesized in vitro {35). PrP c is spontaneously released from normal and scrapie-infected murine neuroblastoma cells, and from m o u s e C127 cells transfected with the PrP gene cloned from scrapieinfected m o u s e brain (37-39). A secretory form of PrP in addition to a transmembrane
form can be generated in cell-free translation
systems
supplemented with microsomal membranes. The secretory form predominates in the rabbit reticulocyte lysate system, whereas the t r a n s m e m b r a n e form prevails in the wheat germ system, the alternative topology being controlled by a stop-transfer effector domain (26, 35, 36, 40, 41). In situ hybridization s t u d i e s have shown that in the central nervous system PrP mRNA is expressed in a variety of cells, including choroid plexus epithelial cells, ependymal cells and meningeal cells (11). Accordingly, PrP c secreted in h u m a n
CSF may originate from different cell populations.
Whether
from
it
derives
membrane-bound
molecules
by
endogenous
GPI-releasing activities, or it corresponds to a C-terminal truncated PrP c derivative h a s to be established. Two observations argue for the latter possibility. First, sequencing studies of C-terminal peptides derived from enzymatic digestion of PrP sc purified from scrapie-infected h a m s t e r brains have shown that approximately 15% of the molecules do not contain the GPI a n c h o r and are truncated at the C-terminus, ending at Gly228 rather t h a n at Ser231 (18). Second, PrP c secreted from cultured cells into the m e d i u m does
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not reacts with a n antiserum that specifically binds a n epitope generated by phosphatidylinositol phospholipase C cleavage of m e m b r a n e - b o u n d PrP c (26). The identification of a soluble form of PrP c in CSF of normal individuals raises the question as to whether a soluble form of PrP sc is generated in prion-related encephalopathies. If this were the case, PrP sc secreted from cells might play a crucial role in the dissemination of the disease process. In this regard, it is noteworthy that the inoculation of CSF from patients with Creutzfeldt-Jakob disease and animals with n a t u r a l or experimental scrapie induces a spongfform encephalopathy in the recipient animal (43). The putative secretory form of PrP sc might correspond to the non-GPI-linked, C-terminal-truncated molecules that have been isolated from scrapie-infected hamster brains (18). Conversely, it seems unlikely that it could derive from GPI-linked PrPsc, since this accumulates within cytoplasmic vesicles of cultured cells instead of being exported to the plasma m e m b r a n e (44). The secretory form of PrP sc could serve as a substrate for amyloid protein formation. If so, amyloidogenesis in prion-related encephalopathies would resemble the pathologic process that occurs in most systemic and some cerebral amyloidoses, in which amyloid proteins are derived by proteolysis of soluble precursors (45).
Acknowledgments" This study was supported by the Italian Ministry of Health, Department of Social Services, and by U.S. National Institutes of Health (grants NS30455 and AR02591 to B.F.).
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Brown H.R., Goner N.L., Rudelli R.D., Merz G.S., WoKe G.C,, Wisniewski H.M., Robakis N.K. (1990) Acta Neuropathol. 80, 1-6. C a s h m a n N.R., Loertscher R., Nalbantoglu J., Shaw I., K a s c s a k tLJ., Bolton D.C., Bendheim P.E. (1990) Cell 61, 185-192. Bendheim P.E., Brown H.R., Rudelli R.D., Scala L.J., Goller N.L., Wen G.Y., Kascsak R.J., C a s h m a n N.R., BoRon S.C. (1992) Neurology 42, 149-156. Kretzschmar H.A., Stowring L.E., Westaway D., Stubblebine W.H., Prusiner S.B., DeArmond S.J. (1986) DNA 5, 315-324. Puckett C., Concarmon P., Casey C., Hood L. (1991) Am. J. Hum. Genet. 49,320-329. Hope J., Morton L.J.D., F a r q u h a r C.F., Multhaup G., Beyreuther K., Kimberlin R.H. (1986) EMBOJ. 5, 2591-2597. Stahl N., Borchelt D.R., Hsiao K., Prusiner S.B. (1987) Cell 51, 229-240. Stahl N., Baldwin M.A., Burlingame A.L., Prusiner S.B. (1990) Biochemistry 29, 8879-8884. Stahl N., Borchelt D.R., Prusiner S.B. (1990) Biochemistry 29, 5405-5412. S a f a r J . , Wang W., Padgett M.P., Ceroni M., Piccardo P., Zopf D., G a j d u s e k D.C., Gibbs C.J.Jr. (1990) Proc. Natl. Acad. ScL USA 87, 6373-6377. BoRon D.C., McKinley M,P., Prusiner S.B. (1982) Science 218, 1309-1311. McKinley M.P., BoRon D.C., Prusiner S.B. (1983) Cell 35, 57-62. Bolton D.C., McKinley M.P., Prusiner S.B. (1984) Biochemistry 23, 5898-5905. Prusiner S.B., Groth D.F., Bolton D.C., Kent S.B., Hood L.E. (1984) Cell 38, 127-134. Bolton D.C., Bendheim P.E., Marmorstein A.D., Potempska A. (1987) Arch. BiocherrL Biophys. 258, 579-590. Prusiner S.B. (1991) Science 252, 1515-1522. Bendheim P.E., Barry tLA., DeArmond S.J., Stites D.P., Prusiner S.B. (1984) Nature310, 418-421. DeAn~ond S.J., McKinley M.P., Barry R.A., Braunfeld M.B., McColloch J.R., Prusiner S.B. (1985) CeU41, 221-235. Kitamoto T., Tateishi J., Tashima T., Takeshita I., Barry R.A., DeArmond S.J., Prusiner S.B. (1986) Ann. Neurol. 20, 204-208. Roberts G.W., Lofthouse R., Brown R., Crow T.J., Barry R.A., Prusiner S.B.(1986) N. Engl. J. Med. 315, 1231-1233. Ghetti B., Tagliavini F., Masters C.L., Beyreuther K., Giaccone G., Verga L., Farlow M.R., Cormeally P.M., Dlouhy S.R. Azzarelli B., Bugiani O. (1989) Neurology 39, 1453-1461. Tagliavini F., Prelli F., Ghiso J., Bugiani O., Serban D., Prusiner S.B., Farlow M.R., Ghetti B., Frangione B. (1991) EMBO. J. 10, 513-519. Kitamoto T., Yamaguchi K., Doh-ura K., Tateishi J. (1991) Neurology 41,306-310. Safar J., Ceroni M., Gajdusek D.C., Gibbs C.J.Jr. (1991) J. Infect. Dis. 163, 488-494. Hay B., Prusiner S.B., Lingappa V.R. {1987) Biochemistry 26, 8110-8115. Hay B., Barry R.A., Lieberburg I., Prusiner S.B., Lingappa V.R. (1987) Mol. Cell. Biol. 7,914-920. Caughey B., Race R.E., Vogel M., Buchmeier M.J., Chesebro B. (1988) Proc. Natl. Acad. Sci, USA 85, 4657-4661. Caughey B., Race R.E., Ernst D., Buchmeier M.J., Chesebro B. (1989) J. Virol. 63, 175-181. Borctlelt D.R., Scott M., Taraboulos A., Stahl N., Prusiner S.B. (1990) J. Cell. Biol. 110, 743-752. Lopez C.D., Yost C.S., Prusiner S.B., Myers R.M., Lingappa V.R. (1990) Science 248, 226-229. 1403
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Yost C.S., Lopez C.D., Prusiner S.B., Myers R.M., Lingappa V.R. (1990) Nature 343,669-672. G h i s o J . , TagliaviniF., TimmersW.F., FrangioneB. (1989) Biocherrr Biophys. Res. Commun. 163, 430-437. Brown P. (1980) EpidemioI. Rev. 2, 113-135. Taraboulos A., Serban D., Prusiner S.B. (i990) J. Cell. Biol. 110, 2117-2132. Castafio E.M., Wisniewski T., Frangione B. (1991) Current Opinion in Neurobiology 1,448-454.
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Pages 1398-1404
A SOLUBLE FORM OF PRION PROTEIN IN HUMAN CEREBROSPINAL FLUID: I m p l i c a t i o n s for p r i o n - r e l a t e d e n c e p h a l o p a t h i e s Fabrizio Tagllavini , Frances Prelli2, Monlca Porro i, Mario Salmona 3, Orso Bugiani I and Blas Frangione2 llstituto Neurologico Carlo Besta, via Celoria 11, 20133 Milano, Italy 2Department of Pathology, New York University, New York, 10016 NY 31stituto di Ricerche Farmacologiche Mario Negri, Mflano, Italy
Received April I, 1992
SUMMARY: The cellular prion protein (PrPc) is a 33-35 kDa sialoglycoprotein anchored to the external surface of neural and n o n - n e u r a l cells by a glycosyl phosphatidylinositol moiety. In addition, a secretory form of PrP c has been found in cell-free translation systems and in cell cultures. On this basis, we investigated h u m a n cerebrospinal fluid for the presence of soluble PrP a n d identified a protein whose molecular weight, antigenic determinants, N-terminal amino acid sequence and sensitivity to protease digestion corresponded to those of PrP C. In prion-related encephalopathies of h u m a n s and animals, the secretory form of PrPc might be converted into the abnormal isoform PrP sc and play a role in the dissemination of the disease process and amyloid formation. © 1992 Academic Press, Inc.
The cellular prion protein (PrP c) is a 33-35 kDa sialoglycoprotein encoded by a gene that in h u m a n s is located on chromosome 20 (1-8). The PrP gene is expressed in neural and n o n - n e u r a l tissues, the highest concentration of mRNA being in n e u r o n s (9-13). The translation product consists of 253 amino acids (14, 15) and undergoes several post-translational modifications. In hamsters, a signal peptide of 22 amino acids is cleaved at the N-terminus, 23 amino acids are removed from the C-terminus on addition of a glycosyl phosphatidylinositol (GPI) anchor,
and
asparagine-linked
oligosaccharides are attached to residues 181 and 197 in a loop formed by a disulfide bond (8,
16-20). In prion-related encephalopathies, such
as
Creutzfeldt-Jakob disease and Gerstmann-Strtiussler-Scheinker disease of h u m a n s , scrapie of sheep and goats, and spongiform encephalopathy of
* To whom reprint requests should be addressed. Abbreviations: CSF: cerebrospinal fluid, GPI: glycosyl phosphatidylinositol, PrP: prion protein, prpC: cellular prion protein, prpSc: scrapie prion protein. 0006-291X/92 $4.00 Copyright © 1992 by Academic Press, Inc. All rights of,reproduction in any form reserved.
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cattle, PrP c is converted into a n altered form designated PrP sc, that is distinguishable from PrP c because only the N-terminal 67 amino acids are removed by proteinase K digestion u n d e r conditions where PrP c is completely degraded (1, 21-25). Several lines of evidence indicate that PrP sc is a key component
of
the
transmissible
agent
responsible
for
prion-related
encephalopathies (26) and that its protease-resistant core is the major structural protein of amyloid fibrils that accumulate intracerebrally in some of these conditions (27-33). PrP c is a m e m b r a n e - b o u n d protein, anchored to the cell surface through the GPI moiety (17-19, 34). Nevertheless, a secretory form of the molecule in addition to the m e m b r a n e form has been found in ce11-free translation
systems
supplemented
with
microsomal
membranes.
Furthermore, spontaneous release of PrP c from cultured cells and from
Xenopus oocytes injected with PrP mRNA has been reported (35-41). On this basis, we analyzed h u m a n cerebrospinal fluid (CSF) for the presence of soluble PrP and identified a protein whose molecular weight, antigenic properties, N-terminal amino acid sequence and sensitivity to protease digestion corresponded to those of PrP c.
MATERIALS A N D M E T H O D S Antisera: Two antisera, designated anti-PrPN and anti-PrP 27-30, were used for immunodetection of PrP. Anti-PrPN was raised in rabbits to the synthetic peptide KKRPKPGGWNTGGSRYPGGC, that corresponds to residues 23-40 of the amino acid sequence deduced from the h u m a n PrP cDNA, except for the addition of Gly-Cys at the C-terminus for spacing and coupling. This peptide was synthesized by solid phase techniques using Fmoc-t-butyl-polyamide chemistry, purified by HPLC using a ~t-Bondapak C18 column (Waters), a n d coupled to keyhole limpet hemocyanin with m-maleimidobenzoy1-Nhydroxysuccinimide. Immunization was carried out as described previously (42) and antibody titer was evaluated by ELISA. Anti-PrP 27-30 (kindly provided by Dr. Paul Brown) was generated by immunizing rabbits with the protease-resistant core of PrP sc (i.e, PrP 27-30) purified from scrapie-infected h a m s t e r brains.
F r a c t i o n a t i o n o f CSF proteins: Samples of CSF were collected from three individuals aged 3, 17 and 29 years, who received external s h u n t i n g for tension hydrocephalus due to germinoma of the pineal region, stenosis of the aqueduct a n d gangliocytoma of the cerebellum, respectively. The protein content of the samples was lower t h a n 100 mg per 100 ml, and blood cells were absent. By a m m o n i u m sulphate precipitation four fractions were obtained, that corresponded to the precipitates at salt concentration of 25%, 50% and 70% (designated as P1, P2 and P3, respectively) and to the 70% s u p e m a t a n t (designated as $3). P1, P2 a n d P3 were redissolved in 20 mM Tris-HC1, pH 7.5, a n d dialyzed against distilled water using t u b u l a r m e m b r a n e s with a molecular weight cut off of 12,000-14,000. After centrifugation at 50,000 x g for 30 minutes, pellets (designated as Plp, P2p, a n d P3p, respectively) and s u p e r n a t a n t s (designated as P l s , P2s and P3s, respectively) were lyophflized. $3 was dialyzed against distilled water a n d lyophilized.
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Immunoblot analysis: Aliquots of the fractions were redissolved in 20 mM Tris-HC1, 150 mM NaC1, pH 7.5, and proteins were determined using the Coomassie blue dye-binding assay (Bio-Rad}. Samples containing 50 ~tg protein were subjected to sodium dodecyl sulfate-polyacrylamide gel electrophoresis {10% or 15% monomer) under reducing conditions and eleetrotransferred to ProBlott m e m b r a n e s (Applied Biosystems} using 10 mM 3-cyclohexylamino- 1-propanesulfonic acid buffer, pH 11, 10°/5 methanol. The m e m b r a n e s were either stained with Coomassie blue or immunostained with anti-PrPN antiserum {1: 500), as described previously (32). Proteins specifically detected by the antiserum were identified by using anti-PrPN preabsorbed with 10 mM of the relevant peptide for 60 minutes at 37 ° C. Aliquots of the CSF fraction containing anti-PrPN immunoreactive proteins were treated with proteinase K (20 ~tg/ml) for 60 minutes at 37 ° C. Digestion was terminated by the addition of 20 mM phenylmethylsulfonyl fluoride, and the samples were subjected to immunoblot analysis with anti-PrP 27-30 antiserum (1: 1,000). Protein sequence analysis: Coomassie blue-stained bands corresponding to the bands specifically labeled by anti-PrPN antiserum were excised and analyzed with a 477A microsequencer {Applied Biosystems} for N-terminal amino acid sequence. The resulting phenylthiohydantoin amino acid derivatives were identified using the on-line 120A PTH analyzer and the standard program (Applied Biosystems}.
RESULTS AND DISCUSSION Immunoblot analysis of CSF showed that fraction P2p, corresponding to the precipitate at 50% a m m o n i u m sulfate concentration, contained a 33-37 kDa protein band that was specifically labeled by anti-PrPN antiserum (Figure 1). This band was consistently observed in CSF samples of all subjects and was completely degraded by proteinase
K digestion, t h u s
resembling PrP c.
N-terminal sequence analysis of the anti-PrP immunoreactive b a n d
a
b
674330- 0
Figure L Irmnunoblot analysis of CSF fraction P2p showing a broad band with electrophoretic mobility of 33-37 kDa {arrow} that is strongly labeled by the antiserum anti-PrPN {lane a). The immunoreactivity is abolished by absorption of the antiserum with the relevant peptide (lane b}. Protein bands still irnmunoreactive after absorption were regarded as nonspecffic. Molecular weight markers {expressed in kDa} are shown to the left. 15% polyacrylamide minigel; antisera dilutions: 1: 500. 1400
by
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automated E d m a n degradation yielded K X P K (P) G G (P) N T. which aligns at position 24 of the amino acid sequence deduced from h u m a n PrP eDNA, one residue beyond the predicted signal peptide cleavage site (15, 16). This difference might result from nonspecffic proteolysis during collection an d fractionation of CSF or from heterogeneity. In this regard,
N-terminal
heterogeneity of prpSc purified from scrapie-infected h a m s t e r brains with a major sequence starting at position 23 and two minor sequences beginning at residues 24 and 3 I, respectively, h as been reported (8). In the second cycle of the E d m a n degradation, we did not detect the Arg residue predicted from the translated sequence of h u m a n PrP DNA. Absence of Arg signal at this position h a s been observed previously in sequencing studies of h a m s t e r PrP e and PrP sc,
suggesting
that
this
residue
might
undergo
post-translational
modifications which preclude its detection during gas-phase sequencing (8). N-terminal sequences of apolipoproteins E and J, and of the a-chain of complement C4 were also identified in CSF fraction P2p, comigrating with PrP c. These proteins comprised mainly the higher molecular weight sector of the 33-37 kDa protein band, whereas PrP c was found predominantly in the lower portion of the band. The presence of a soluble form of PrP c in h u m a n CSF is in agreement with the following observations. PrP c is secreted from Xenopus oocytes injected with PrP mRNA synthesized in vitro {35). PrP c is spontaneously released from normal and scrapie-infected murine neuroblastoma cells, and from m o u s e C127 cells transfected with the PrP gene cloned from scrapieinfected m o u s e brain (37-39). A secretory form of PrP in addition to a transmembrane
form can be generated in cell-free translation
systems
supplemented with microsomal membranes. The secretory form predominates in the rabbit reticulocyte lysate system, whereas the t r a n s m e m b r a n e form prevails in the wheat germ system, the alternative topology being controlled by a stop-transfer effector domain (26, 35, 36, 40, 41). In situ hybridization s t u d i e s have shown that in the central nervous system PrP mRNA is expressed in a variety of cells, including choroid plexus epithelial cells, ependymal cells and meningeal cells (11). Accordingly, PrP c secreted in h u m a n
CSF may originate from different cell populations.
Whether
from
it
derives
membrane-bound
molecules
by
endogenous
GPI-releasing activities, or it corresponds to a C-terminal truncated PrP c derivative h a s to be established. Two observations argue for the latter possibility. First, sequencing studies of C-terminal peptides derived from enzymatic digestion of PrP sc purified from scrapie-infected h a m s t e r brains have shown that approximately 15% of the molecules do not contain the GPI a n c h o r and are truncated at the C-terminus, ending at Gly228 rather t h a n at Ser231 (18). Second, PrP c secreted from cultured cells into the m e d i u m does
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not reacts with a n antiserum that specifically binds a n epitope generated by phosphatidylinositol phospholipase C cleavage of m e m b r a n e - b o u n d PrP c (26). The identification of a soluble form of PrP c in CSF of normal individuals raises the question as to whether a soluble form of PrP sc is generated in prion-related encephalopathies. If this were the case, PrP sc secreted from cells might play a crucial role in the dissemination of the disease process. In this regard, it is noteworthy that the inoculation of CSF from patients with Creutzfeldt-Jakob disease and animals with n a t u r a l or experimental scrapie induces a spongfform encephalopathy in the recipient animal (43). The putative secretory form of PrP sc might correspond to the non-GPI-linked, C-terminal-truncated molecules that have been isolated from scrapie-infected hamster brains (18). Conversely, it seems unlikely that it could derive from GPI-linked PrPsc, since this accumulates within cytoplasmic vesicles of cultured cells instead of being exported to the plasma m e m b r a n e (44). The secretory form of PrP sc could serve as a substrate for amyloid protein formation. If so, amyloidogenesis in prion-related encephalopathies would resemble the pathologic process that occurs in most systemic and some cerebral amyloidoses, in which amyloid proteins are derived by proteolysis of soluble precursors (45).
Acknowledgments" This study was supported by the Italian Ministry of Health, Department of Social Services, and by U.S. National Institutes of Health (grants NS30455 and AR02591 to B.F.).
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