S

Prions

.

and NEIL

#{149} 1

prion

STAHL2

Departments 94143-0518,

proteins

AND STANLEY

of Neurology USA

B. PRUSINER3

and of Biochemistry

and Biophysics,

ABSTRACT Neurodegenerative diseases of animals and humans including scrapie, bovine spongiform encephalopathy, and Creutzfeldt-Jakob disease are caused by unusual infectious pathogens called prions There is no evidence for a nucleic acid in the prion, but diverse experimental results indicate that a host-derived protein called PrPSC is a component of the infectious particle. Experiments with scrapie-infected cultured cells show that PrPSC is derived from a normal cellular protein called PrPC through an unknown posttranslational process. We have analyzed the amino acid sequence and posttranslational modifications of PrP& and its proteolytically truncated core PrP 2 7-30 to identify potential candidate modifications that could distinguish PrP& from PrPC. The amino acid sequence of PrP 2 7-30 corresponds to that predicted from the gene and eDNA. Mass spectrometry of peptides derived from PrP& has revealed numerous modifications including two N-linked carbohydrate moieties, removal of an amino-terminal signal sequence, and alternative COOH termini. Most molecules contain a glycosylinositol phospholipid (GPI) attached at Ser-231 that results in removal of 23 amino acids from the COOH terminus, whereas 15% of the protein molecules are truncated to end at GIy-228. The structure of the GPI from PrPSC has been analyzed and found to be novel, including the presence of sialic acid. Other experiments suggest that the N-linked oligosaccharides are not necessary for PrP formation. Although detailed comparison of PrP& with PrPC is required, there is no obvious way in which any of the modifications might confer upon PFP5c its unusual physical properties and allow it to act as a component of the prion. If no chemical difference is found between PrPC and PrPSC, then the two isoforms of the prion protein may differ only in their conformations or by the presence of bound cellular componentS. Stahl, N.; Prusiner, S B. Prions and prion proteins. FASEBJ. 5: 2799-2807; 1991. Key Words:

scrapie

amyloid

glycosylinositol

pliospholipid

A GROUP OF INFECTIOUS PATHOGEN5 called prions cause transmissible neurodegenerative diseases in both humans and animals (1, 2). In humans, these diseases are kuru, Creutzfeldt-Jakob disease (CJD),4 and GerstmannStr#{228}ussler-Scheinker syndrome (GSS), whereas animal

prion diseases include scrapie, bovine spongiform encephalopathy (BSE), and transmissible mink encephalopathy. Gerstmann-Str#{228}ussler-Scheinker syndrome and familial CJD are unique in that they are inherited and transmissible: these diseases occur in families as an autosomal dominant trait with high penetrance, and extracts of brain tissue from the afflicted individuals can transmit a scrapie-like disease to experimental animals (3-5). Scrapie has been widely recognized

in sheep

for more

than

200 years

recently emerged in the United result of bovine food supplements

whereas

BSE has only

Kingdom, probably as a contaminated with prions

0892-6638/91/0005-2799/$O1 .50. © FASEB

University

of California,

San Francisco,

California

from scrapie-infected sheep offal (6). Although there is a species barrier that prolongs the incubation time observed upon transfer of the disease from one host species to the next (7), the specter of transmission to humans who consume BSEtainted beef has engendered considerable public concern and widened scientific interest in elucidation of the mechanism of

prion infectivity. Prions differ from bacteria,

viruses,

and viroids

by their

unprecedented structure and properties (1, 8). Experiments designed to uncover participation of a nucleic acid in prion structure or infectivity consistently give negative results (9). These approaches include: 1) infectivity measurements after ultraviolet and ionizing radiation (10, 11) or chemical treatments that modify or destroy nucleic acids (12), 2) purification studies aimed at physical detection of prion nucleic acid (13), and 3) a variety of molecular cloning schemes (14, 15).

Prion was defined as an infectious pathogen that requires a protein for infectivity yet is highly resistant to procedures that

modify

or destroy

nucleic

acids

(16).

The unusual nature of the prion has necessitated a tidisciplinary investigation. Several reviews describe our rent understanding of the prion (2), with emphasis molecular genetics (17), pathology (18), prion liposomes glycolipid anchors (19), or human genetics and diagnosis

mulcuron (9), (20,

21). This paper focuses on the protein chemistry of the prion through a comparison of the properties of the normal and abnormal isoforms of prion proteins (PrP).

PrP PURIFICATION Scrapie

infectivity

homogenization,

can

be purified

detergent

from

extraction,

hamster

nuclease

brain

by

digestion,

limited proteolysis with proteinase K, and centrifugation on discontinuous sucrose gradients (22, 23). Analysis of the resulting fractions on silver-stained sodium dodecyl sulfate (SDS) polyacrylamide gels reveals a major band migrating between 27 and 30 kDa; this protein is designated PrP 2 7-30.

It is not found in similar fractions purified from normal brain (22, 23). PrP 27-30 is derived from a larger protein of ‘From the Symposium Prions, Viroids and Infectious Introns presented at the American Society for Biochemistry and Molecular Biology and The American Association of ImmunologistsJoint Meeting, New Orleans, Louisiana, June 7, 1990. 2Current address: Regeneron Pharmaceuticals, 777 Old Saw Mill Road, Tarrytown, NY 10591, USA. 3To whom

ment

all correspondence

Neurology, HSE-78l, cisco, CA 94143-0518, USA. of

4Abbreviations:

GPI,

should

University

glycosylinositol

be addressed,

of California, phospholipid;

at: Depart-

San FranCJD,

C reutzfeldt-Jakob disease; GSS, Gerstmann-Str#{228}ussler-Scheinker syndrome; BSE, bovine spongiform encephalopathy; PrP, prion pro-

teins; SDS-PAGE, trophoresis;

ARIA, Syrian hamster; Tg,

sodium dodecyl sulfate-polyacrylamide acetylcholine transgenic;

gel elec-

receptor-inducing activity; SHa, SAF, scrapie-associated fibrils; AEF,

amyloid-enhancing factor; DLPC, detergent-lipid-protein complexes; HPLC, high-pressure liquid chromatography; PIPLC, phosphatidylinositol

phospholipase

C.

2799

w.fasebj.org by Univ of So Dakota Lommen Hlth Sci Library (192.236.36.29) on September 23, 2018. The FASEB Journal Vol. ${article.issue.getVolume()}, No. ${article.issue.getIssueNum

33-35

kDa called

during

can

limited

PrPS through loss of the NH2 terminus proteolytic digestion (24). Full-length PrPS

be purified

using

procedures

that

avoid

PrP GENE

proteolysis

In hamsters, same

(25-28). No significant difference in the specific infectivity of preparations purified with or without proteinase K digestion has been found (28, 29). Extended incubation with proteinase K revealed a slow disappearance of PrP 27-30 that paralleled the loss of infectivity over several orders of magnitude (30). A great variety of results implicates PrPS or PrP 2 7-30 as an integral component of the infectious scrapie

prion

proteins

are encoded

by a chromosomal

gene,

both PrPC and PrPSC are transcribed

single-copy

gene found

ing frame

is contained

possibility

that

PrPC

within and

from the The open read-

in the host (33).

a single exon,

PrPS

differ

ruling

out the

by alternative

mRNA

splicing. In adult brain, the gene is constitutively expressed as a 2.1-kB mRNA, the appearance and abundance of which is constant throughout the course of scrapie infection (24). The levels of PrP mRNA in the neonatal hamster brain are developmentally regulated and can be increased by injection of nerve growth factor (34). The amount of PrP’- is similar in normal and scrapie-infected animals whereas PrPS is observed only in scrapie-infected brain (24). Low or undetectable levels of PrP mRNA or prion protein are found in other

(1, 2).

Prion

EXPRESSION

not by

a hypothetical nucleic acid within the prion (24). The product of the PrP gene in normal animals is the cellular prion protein, PrPC (24, 26, 31). Adult hamster brain appears to contain -50 sg PrPC/g protein whereas scrapieinfected animals contain -70 eg PrP7g protein, as measured by an ELISA assay (32). PrPC has been purified by immunoaffinity methods (26, 31) but is not available in large

tissues.

PRION

PROTEIN

PRIMARY

STRUCTURE

quantities. A variety of experimental evidence indicates that PrPSC is formed during a posttranslational event from PrPC or a

A detailed analysis of the predicted primary and secondary structures has been published (35). Unusual features include

precursor.

a series

either

The

posttranslational

a chemical

change,

conversion

modification,

or tight binding

nature of the difference PrPS to become a prion ing the difference is one prion research currently that this knowledge will

might

a stable

to other cellular

involve

components.

The

is almost

SHaPSP CHaPrP AHaPrP MOPrP-A M0PVP-B RaPrP ShePrP B0PrF HUPrP 05

1. Predicted

translated Syrian

from hamster

protein

PrP

clones

[SHaPrP

Man-

-1

not homologous

10 LaLFVamWt

syWl

in the middle

conserved

in PrP

from

region,

and

of the protein

that

a variety

of species

that

with

PrP

20 DVOLCKKRPK

.

any

known

may

.

PG

proteins.

A recent

be homologous

-

30 GWNTGGSRY

40 POQOS

.

report

to acetylcholine

50 P

.

P00552

t t S t S C 7 7 7 7 7 7 7 7 7 7 7 7 7 7 7 7 7 7 7 7 7 7 7 7 7 7 7 7 7 7 7 7 vkahlgg I V a g vkahiga I V a S 9c a, V t a 1 gc a, V t a 1

60 . 70 PQ500CWGQPH000WGQPHGGgWGQP800SWGQPH000-WSQGG5tIInQWN

80

.

a a

-

.

90

.

100

a a

a

a g 5 5

Var

S g

-‘ -

5

a g a a

sequences

from (24)],

Chinese hamster [CHaPrP (36)1, Armenian hamster [AHaPrP (36)], NZW mouse [M0PrP-A (37)], I/In mouse (MoPrP-B), rat [RaPrP (38)], human EHuPrP (39)], sheep [ShePrP (40)], bovine [BoPrP (41)], and human variants indicate

perfectly

suggests

SHaPrP CEaPrP ASaPrP MOPrP-A I4oPrP-B RaPrP ShePrP BoPrP H7PrP HuVar

in the NH2-terminal

domain

(Fig. 1). The overall homology between PrPC from rat, mouse, hamster, and human is near 90% whereas the sheep (40) and bovine (41) sequences are somewhat more divergent. The function of PrP is unknown, and its sequence is

between PrPC and PrPSC that allows component is unknown. Identifyof the most important problems in under investigation; it seems likely reveal the mechanism of prion repli-

cation.

Figure

of five octa-repeats

a long hydrophobic

conformational

(Hu Var). Uppercase letters conserved residues, lowercase

letters show substitutions, and dashes signify gaps. Human variants (Hu Var): ‘location of 96 or 144 base pair inserts associated with familial CJD (42); 2mutation genetically linked to occurrence of ataxic GSS (43); 3mutation found in patients with dementing GSS (44, 45); 4normal human variant (46); 5mutation found in Libyan Jews (47, 48) and other patients with familial CJD.

SHSPrP CHaPrP Al4aPrP MOPrP-A MOPrP-B RaPrP ShePrP B0PrP HUPrP

110 . KPaKPKTnPKHSAGAAAAGAVVGGLGGYMLGSAMSR

120

130

.

140 Pa,mHFGSDWEDRYY 1 1 I

.

V

n

a 1 S 1

v v

i.2

SHaPrP CHaPrP

RENMnRV

150

j

V

V v

H,aVar

.

V4

160 . PISQVYYRPvDqYn0000PVHDCVNI

170

180

.

1 1 1 11 II

a a

y y

Ii

a

y

. 190 K9HTVCTTTKOENFTE

TI

.

200

AI1aPrP

M0PrP-A M0PrP-8 RMPrP SSePrP BoPrP HuPrP

y y y y S S

a,

a,

a a a

HuVar

S

a

e

a

SHa Pr? CHaPrP ARaPrP MOPrP-A M0PrP-8

r

210 0 1 K I H K H V V E v S V a, V V

RMPrP

V

SSePrP S0PrP HSPEP HuVar

V

a, a, U

a, v

a, a,

a V

a r

Q

. 220 M C t T Q 2 q H K S v v

v

,

K5

QA

. 230 V V S g H r a S -

. 240 a v L F S S P P V

. 250 I L 7. I S F 7. I F 7. a, V 0

C at at

V

V V I I I I

e

r r er er

-q -q

qa ga

vi VI

-q

g

a

-q

g

a,

2800 Vol. 5 October 1991 STAHL AND PRUSNER The FASEB Journal w.fasebj.org by Univ of So Dakota Lommen Hlth Sci Library (192.236.36.29) on September 23, 2018. The FASEB Journal Vol. ${article.issue.getVolume()}, No. ${article.issue.getIssueNum

receptor-inducing activity brain (49). However, this

(ARIA) intriguing

purified from chicken assertion awaits evi-

dence that expression of the isolated clone yields ARIA activity, otherwise ARIA and a putative chicken PrPC homolog may simply copurify. Should the homology prove true, the existence of soluble forms of PrPC (see below) would be consistent with a function as a trophic factor.

Recent experiments have shown that changes in the amino acid sequence of PrPC can have profound consequences. Transgenic (Tg) mice have been constructed that also synthesize Syrian hamster (SHa)PrPC, which differs from mouse PrPC at 16 of 254 residues. Although non-Tg mice are relatively insusceptible to SHa scrapie prions and show extended incubation times before the onset of symptoms, the Tg mice expressing SHaPrPC are highly susceptible to SHa prions (32, 50). In fact, one mouse line bearing -60 copies of the SHa transgene has a shorter incubation time than Syrian hamsters (32). Furthermore, the Tg(SHaPrP) mice infected

with SHa prions synthesize only SHa prions, not mouse (Mo-speciflc prions (32). Transgenic mice that overexpress PrP do not become spontaneously ill in the absence of inoculation with exogenous prions. These results strongly assert that PrP is a component of the infectious agent, and that the sequence of PrP is at least partially responsible for the species barrier. Families with ataxic GSS have a point mutation that changes Pro-102 to Leu in human (Hu)PrPC; occurrence of the disease is tightly linked to this genetic marker (51). Tg(GSSMoPrP)

tion this mice ing

mice

containing

the

corresponding

muta-

in the MoPrP gene have been produced to test whether mutation actually causes the disease. Tg(GSSMoPrP) expressing a MoPrP gene with Leu at the correspond-

position (101 in M0PrP) spontaneously develop a degenerative neurological disease that leads to death whereas non-Tg littermates remain healthy (52). The neuropathology

of Tg(GSSM0PrP) GSS

except

mice resembles

for the absence

that observed

of amyloid

plaques

in human (52).

Brain

tissue from Tg(GSSMoPrP) tion of the protease-resistant

mice show little or no accumulaform of PrPS (52). The occur-

rence

in uninoculated

of neurodegeneration

Tg(GSSMoPrP)

mice argues that 1) a human neurodegenerative disease can be reproduced in animals, and 2) PrP plays a major role in causing

and directing

the course

of the prion

diseases.

Brain

homogenates from the symptomatic Tg(GSSMoPrP) mice have been inoculated in rodents to investigate whether these homogenates can transmit a neurodegenerative disease. If convincing evidence is obtained for the de novo synthesis of infectious prions in Tg(GSSM0PrP) mice, this will argue that prions do not contain foreign nucleic acid and that any nucleic acid required for infectivity must be present in the host. Other forms of GSS and familial CJD are also associated with changes in the PrPC amino acid sequence (42-48), but it remains to be shown whether these mutations

are also causative. Although changes in the PrP amino acid se4uence can lead to disease, much evidence argues that PrP and PrPSC have

identical

amino

acid

sequences.

The

single-copy

PrP

gene shows no rearrangements, no change in PrP mRNA sequence or amount upon infection with scrapie has been detected, and the PrP gene structure gives no opportunity for alternative

sequence

splicing

within

the open

reading

of PrP 2 7-30 has been studied

try and Edman degradation tion with endoproteinase

frame

(33).

The

by mass spectrome-

of peptides generated Lys-C (53). Through

by digesthis proce-

dure we have identified every peptide expected from translated gene sequence and found that these molecules actly match this sequence (N. Stahl, M. A. Baldwin,

the exD.

Teplow, and S. B. Prusiner, unpublished results). Sequencing of purified PrPC (26) and PrP C (25, 26, 28) revealed identical NH2 termini and removal of the signal sequence. As there is no reason to believe that the sequence of PrPC does not correspond to that predicted from the gene, it is hghly

probable

that the primary

structures

of PrPS

and PrPL are

identical. Unless a small fraction of PrPSC has an altered amino acid sequence and is responsible for causing scrapie, it is likely that some other difference distinguishes PrPC and PrPS.

PROTEASE

RESISTANCE

The enhanced

AGGREGATION

to protease digestion is a it from PrPC (30, 54, 55). Whereas PrPC is completely degraded upon incubation with proteinase K, PrPSC loses an NH2-terminal domain containing the octarepeats to give PrP 27-30 (24). Furthermore, PrPC is soluble in the presence of various detergents cardinal

resistance

AND

feature

that

of PrPS

distinguishes

whereas PrPS forms insoluble aggregates (56). PrP 2 7-30 polymerizes into rod-shaped structures in the presence of detergent. The prion rods behave as amyloid; they show green birefringence under polarized light upon staining with Congo red (57). It has been claimed that scrapie-associated fibrils (SAF) are synonymous with prion

rods and are composed of PrP although SAF was repeatedly distinguished from amyloids (8, 58-65). Amyloid is characterized by three criteria: the existence of unbranched fibrils, binding of Congo red to the fibrils in a fashion green birefringence under polarized light, and the of cross /3-pleated sheets in the fibrils (66). Amyloid exclusively observed extracellularly, although some thought to be synthesized in macrophages (67).

preparations stead show

of PrPSC do not contain

amyloid

that gives existence is almost forms are Purified

fibrils, but in-

an amorphous web of material by electron microscopy (29). Formation of amyloid rods from these preparations of crude microsomal fractions can be achieved through action of proteinase K or trypsin in the presence of fairly strong detergents such as Sarkosyl and SDS (29). The

exact morphology of the amyloid rods varies somewhat depending on the choice of protease. Incubation of the membranes with proteinase K, followed by the protease inhibitor phenylmethylsulfonylfluoride before the addition of detergent, cleaves PrPS to PrP 27-30 but does not result in amyloid rod formation (29). It is not clear whether collapse of the amorphous material into the rod results from removal of the NH2 terminus of PrPSC or digestion of another copurifying molecule that is bound to PrPS and preventing rod formation. Although PrP 2 7-30 appears to be the

predominant rods,

we

molecule have

also

purifying

in preparations

observed

copurification

of prion of

other

molecules, one of which contains alkali-sensitive fatty acid (68). Whether molecules other than PrP 27-30 participate in scrapie infectivity and prion rod formation is still unknown. The behaviors of PrPSC and PrP 27-30 are similar to other amyloidogenic proteins which require partial proteolysis before amyloid formation is observed (69-71). Scrapie prion infectivity is unchanged by limited proteolysis and amyloid rod

formation (29). Gajdusek (72) has proposed

that PrPSC serves as a nuclea-

tion nidus for the formation of amyloid by recruiting molecules of PrPC, and draws comparison to amyloidenhancing factor (AEF) which can dramatically accelerate the formation of AA type amyloid in mouse models (73). In fact, the proposition that AEF is a nucleation seed (74) is disputed, and a variety of evidence suggests that AEF is a pro-

PRIONSAND PRION PROTEINS 2801 w.fasebj.org by Univ of So Dakota Lommen Hlth Sci Library (192.236.36.29) on September 23, 2018. The FASEB Journal Vol. ${article.issue.getVolume()}, No. ${article.issue.getIssueNum

tease that can cleave the amyloidogenic precursor SAA (75-77). Under certain conditions, this protease can aggreate (75). Although binding and aggregation of PrPC to PrP is a viable mechanism, evidence argues that formation of amyloid is probably irrelevant to the course of scrapie in animals. Several rodent models of scrapie show no accumulation of amyloid in the brain (78, 79). Cultured cells that synthesize scrapie infectivity also show no evidence of amyloid fibril accumulation (54). PrPS, not PrP 27-30, accumulates in scrapie-infected brain tissue (24, 25, 28). Although PrPSC aggregates, it does not form fibrils (29). Proteolysis of PrPSC in the presence of detergent is required before PrP 27-30 polymerizes into amyloid rods that show Congo red birefringence (29). Prion rods can be functionally solubilized by bath sonication in a buffer containing detergents and phospholipids (80). Functional solubilization in this instance signifies that 50-90% of the PrP 2 7-30 remains in the supernatant fraction upon centrifugation at 100,000 g for 1 h. Extraction of the phospholipid and detergent with organic solvents results in reformation of the rods (80). PrPSC (or PrP 2 7-30) and prion infectivity copartition by all of these procedures, strengthening the assertion that PrP is a component of the prion (9). Furthermore, the infectivity of the preparation frequently increases by 10- to 100-fold after this functional solubilization. The actual structure and aggregation state of PrPS or PrP 27-30 in the detergent-lipid-protein complexes (DLPC) is uncertain. PrPSC remains protease resistant in the DLPC or cellular membranes (29), and is specifically retained by immunoaffinity chromatography with an anti-PrP monoclonal antibody (27). PrP 27-30 is more resistant to chemical crosslinking when solubilized in DLPC than when aggregated as prion rods, but some residual cross-linking of DLPC is still observed (R. Gabizon, unpublished results). Brown, Gajdusek, and colleagues (81) have recently reported that scrapie infectivity is unaffected by boiling in SDS-polyacrylamide gel electrophoresis (PAGE) sample buffer and it can be recovered from polyacrylamide gels in a fraction that coelutes with PrP 27-30 at an Mr near 30,000. Although the PrP is protease-sensitive after this procedure, they did not report whether the extracted infectivity was also protease-sensitive. Similar results were obtained by Safar et a!. (82) upon purification of SDS-boiled PrPSC by size-exclusion high-pressure liquid chromatography (HPLC). Purification of scrapie infectivity by gel elecTABLE

1. Summary of properties and posttranslational Property

NH2-terminal removed

unpublished results). There is no obvious reason crepancy as similar isolates of SHa scrapie prions

(263K and Sc237) were used, and Brown et al. (81) reported no unusual lengthening of incubation interval for a given titer. Other denaturation protocols, such as incubation with guanidine hydrochloride, reproducibly result in complete loss of scrapie infectivity.

Resolution

of

this

discrepancy

POSTTRANSLATIONAL

Inspired by the possibility that a chemical modification constituted the difference between PrPC and PrP, we have tried to create a complete catalog describing the types and structures of all posttranslational modifications of both PrP isoforms. Thus far, no chemical difference between the two proteins has been reported. At least eight posttranslational modifications have been described for both PrPC and PrP& (Table 1). The proteins are synthesized with an NH2-

PrP

Comments

Lys-23

NH2 terminus

+

+

Structures

known

+

+

+

+

GPI attached

+

+

Structures

+

-

Native

+

+

Denatured

Arg-25

+

+

Arg-37

?

+

?

Glysss

carbohydrate

Unknown

modification

COOH-terminal

removed

be-

MODIFICATIONS

+

Disulfide bond COOH-terminal peptide GPI anchor present PIPLC releasable from cellular membranes GPI is PIPLC-sensitive

is important

cause convincing data demonstrating that infectivity is associated with monomeric PrP 27-30 would have profound implications for prion structure and replication. The cause of the protease resistance of PrPS and PrP 27-30 is unknown. One possibility is aggregation, for which there are many precedents (85). A second possible explanation is tight binding of PrPS to another molecule. For example, binding of proteins or peptides to proteoglycans often increases their resistance to proteases (86). A third possibility is a posttranslational chemical modification that restricts access of the protease to the protein. Whereas glycosylation of mucins renders them resistant to proteolysis (87), this mechanism seems unlikely with respect to PrPS (see next section). Fourth, it is conceivable that PrPSC folds into an alternative conformation in a manner that masks sites on the protein which must be hydrolyzed initially by a protease to induce further unfolding and proteolysis.

+

N-linked

for the dis-

mod/lcations for PrPC and PrP& PrPC

signal sequence

trophoresis was reported 13 years ago (83), but proved not to be reproducible (84). Those early studies reported that scrapie infectivity in purified fractions was DNase-sensitive but resistant to proteinase K digestion (83). These findings were also not reproducible (84). Experiments from our laboratory generally reveal infectivity losses of 10- to 1000-fold upon boiling prions in SDS, and low levels of scrapie infectivity are uniformly distributed over the entire molecular weight range upon either SDS-PAGE purification (84) or size-exclusion HPLC (D. Groth and S. B. Prusiner,

for

in SHaPrP

PrPSC

to Ser-231 in SHaPrP for SHaPrP&

known

PrP

PrP

of

truncation

-

15%

of PrPS

of soluble PrP sequence corresponds to that predicted from gene

?

truncated; COOH terminus forms of PrPc unknown

+

STAHL AND PRUSINER The FASEB Journal 2802 Vol. 5 October 1991 w.fasebj.org by Univ of So Dakota Lommen Hlth Sci Library (192.236.36.29) on September 23, 2018. The FASEB Journal Vol. ${article.issue.getVolume()}, No. ${article.issue.getIssueNum

terminal signal sequence that targets the proteins to the endoplasmic reticulum where the peptide is removed, leaving Lys-23 as the amino-terminal residue for both PrPC (26) and PrPSC (25, 26, 28). There are two consensus acceptor sites for the addition of N-linked sugars, both of which are glycosylated in the majority of protein molecules (88, 89). Both isoforms of SHaPrP have two cysteines, which form a disulfide bridge creating a loop that contains both sites of Nlinked carbohydrate (26). A glycosylinositol phospholipid (GPI) anchor is attached to the COOH terminus of both isoforms (68). In SHaPrP, 23 amino acids are removed from the COOH terminus upon attachment of the GPI anchor at Ser-231 (90). The exact GPI attachment point for PrPC is not yet known, but is likely to occur at the same site. There are unidentified modifications at Arg-25 and Arg-37 in PrPS based on absent or novel signals at the corresponding Edman cycles 6, 91); an absent signal in Edman cycle at Arg-25 of PrP was also observed (26). This unidentified Arg modification is either variable or labile as unmodified Arg is frequently observed as well (25, 26, 28, 82; N. Stahl

and M. Baldwin, unpublished results). Approximately 15% of purified PrPSC and PrP 2 7-30 molecules are truncated at the COOH terminus, resulting in loss of three amino acids and the GPI anchor (90). This catalog of posttranslational modifications is probably complete for most protein molecules every peptide expected to arise from PrP 27-30 has been identified and mapped by mass spectrometry as mentioned previously (53). However, it is possible that a minority of PrP 27-30 molecules contain an unidentified modification. Comprehensive mapping of PrPC by similar procedures must await methods for purification of PrPC in substantial quantities. The structures of the N-linked sugars attached to PrP 27-30 have been examined. The structures are complex and quite heterogenous; many carbohydrate chains contain sialic acid and fucose (92). PrPC shows heterogeneity by twodimensional gel electrophoresis with a pattern of acidic species that disappears after enzymatic removal of the N-linked carbohydrate (88). Ablation of either or both N-linked sugar loci by site-directed mutagenesis prevents PrPC from reaching the cell surface in cultured cells; thus the carbohydrate may play a role in proper targeting of the protein (89). Several experiments suggest that the N-linked carbohydrate on PrP does not play a pivotal role in formation of PrP&. Cultured mouse neuroblastoma (Nsa) cells that are chronically infected synthesize scrapie prions and PrPSC (54). Pulse-chase radiolabeling experiments in the presence of the glycosylation inhibitor tunicamycin reveal that nascent PrP containing no N-linked sugars can still become protease resistant during a chase period (93). Furthermore, N2a cells that synthesize a PrP molecule whose N-linked sugar consensus sequences have been ablated by site-directed mutagenesis also make protease-resistant PrPS (93). Because the presence of protease-resistant PrPS has never been uncoupled from the presence of scrapie infectivity, we anticipate that the mutant protein will be associated with infectivity. Experiments are in progress to assess whether SHa prions can be synthesized in Tg mice expressing unglycosylated SHaPrPC that possesses no N-linked sugar acceptor sequences. Although both PrPC and PrPS contain GPI anchors, the possibility remains that their exact structures differ. A partial structure for the carbohydrate core from the SHaPrP 27-30 anchor has been determined (94). The intact GPI glycan was cleaved from the COOH-terminal peptide by incubation in 50% aqueous HF, then permethylated and analyzed by tandem mass spectrometry (94). This procedure revealed both the carbohydrate composition of the various glycan forms as

well as the branching pattern. Our first results indicate three main forms of the GPI glycan as shown in Fig. 2. The smallest form is a linear chain containing four hexoses, a free hexosamine, and inositol whereas the larger forms also contain N-acetylhexosamine with and without another terminal hexose. The smallest two forms correspond to structures observed for rat brain Thy-i, whereas the largest species is novel. Recently we also used mass spectrometry and other techniques to identify the presence of two other GPI glycan species that both contain neuraminidase-sensitive sialic acid (N. Stahl, M. A. Baldwin, R. Hecker, and S. B. Prusiner, unpublished results). Carbohydrate compositional analysis confirms the presence of sialic acid as well as galactose. Galactose has not been reported as a component of mammalian GPI anchors, and sialic acid has not previously been observed in GPI anchors from any source. The structure of the SHaPrPC GPI is unknown. Elucidation of the PrPC GPI structure is required before determining whether the presence of the novel GPI structures in PrP 27-30 may feature in scrapie. PrPC and PrPS differ in their ability to be released from cellular membranes by phosphatidylinositol phospholipase C

(PIPLC)

(55, 95). Using

membranes

isolated

from normal

and scrapie-infected hamster brains, PrPC is released large! by PIPLC whereas PrPS is resistant to release. Both PrP and PrPSC were accessible to added reagents in these cell membrane preparations (95). In contrast, purified denatured PrPSC and PrP 27-30 are sensitive to digestion with PIPLC (95). Although the most likely explanation for this difference is that the conformation or aggregation of PrP bound to membranes prevents access of PIPLC to the COOH terminus, other interpretations must also be considered (95). Unfortunately, the GPI anchor on PrP does not give any clear clues to its function. Although several GPI-anchored proteins serve as adhesion factors or cell-surface enzymes, there is no unifying function displayed by this class of proteins (96, 97). There is no evidence that PrPC binds to itself, nor is there evidence for binding of PrPS or PrP 27-30 to PrPC (98). At least two other PrP binding proteins have been described, one of which is a 45-kDa protein that was shown to be glial fibrillary acidic protein (98). The significance of this interaction is unknown. Another posttranslational modification of PrPS and PrP 27-30 is the existence of protein molecules that are truncated at the carboxyl terminus (90). Analysis of COOH-terminal peptides generated by endoproteinase Lys-C digestion revealed that most were GPI linked through ethanolamine attached at serine 231, but - 15% ended at Gly-228. This truncation was not due to purification of PrP 2 7-30 with

Hex- Hex- Hex Hex-HexNH2-

mo

Hex- Hex- Hex\ Hex-HexNH 2 mo -

HexNAc

Hex- Hex- Hex\

,Hex-HexNH2- mo Hex

-

HexNAc

Figure

2.

Structures

identified

for

the

Syrian

hamster PrP 27-30 GPI glycan (94). Hex: hexose, HexNAc: N-acetylhexosamine, samine,

GIcNH2: glucoinositol.

mo:

PRIONSAND PRION PROTEINS 2803 w.fasebj.org by Univ of So Dakota Lommen Hlth Sci Library (192.236.36.29) on September 23, 2018. The FASEB Journal Vol. ${article.issue.getVolume()}, No. ${article.issue.getIssueNum

proteinase K since a similar fraction of PrPS, which is purified in the absence of proteases, was also truncated (90). Although the mechanism of formation of this truncated COOH terminus is unknown, one possibility is that processing occurs at the sequence Gly-Arg-Arg, which is an established site for proteolysis and maturation of bioactive peptides (99). Sequential proteolysis by a dibasic-specific protease and a carboxypeptidase B-like protease would leave the observed product. Several viral fusion proteins containing the Gly-Arg-Arg sequence are processed by this pathway to give a product identical to that observed for PrP (100). A similar activity has been proposed for the release of cellsurface proteoglycans, one of which is also GPI-anchored (101, 102). It seems likely that PrPC also can be truncated near the COOH terminus, although this has not been definitively shown (19). Soluble forms of PrPC are released from cultured cells (19, 103). Examination of this soluble form suggests that most and perhaps all of the GPI anchor is missing, consistent with cleavage of the protein near Gly-228 (D. R. Borchelt, N. Stahl, M. Rogers, and S. B. Prusiner, unpublished results). BIOSYNTHESIS

OF PRION

AND

CELLULAR

LOCALIZATION

PROTEINS

PrPC and PrPS differ with respect to their synthesis, degradation, and cellular localization. Pulse-chase radiolabeling experiments in cultured cells revealed that PrPC was synthesized rapidly and could reach the cell surface in 20 mm, then disappeared with a half-time of -6 h (104). In contrast, protease-resistant PrPSC was synthesized slowly with a halftime of more than 3 h in N2a cells and no degradation could be detected (104). The fact that protease-resistant PrPS appeared only during the nonradioactive chase period demonstrates that it is synthesized as a protease-sensitive precursor, and provides direct evidence confirming previous suppositions that PrPSC acquires its unique properties as the result of a posttranslational event (104). Recent experiments in cultured cells have indicated that a substantial fraction of PrPS accumulates intracellularly (105). Although the majority of PrPC is targeted to the cell surface, immunofluorescent confocal microscopy showed that PrPS is localized to an internal vesicular compartment. Immunoelectron microscopy suggests that this compartment may be largely secondary lysosomes (106). The ultrastructural localization of PrPS in scrapie-infected brain tissue is unknown. It is likely that only a small fraction of PrPSC forms amyloid plaques in the extracellular space (29). The aberrant localization of PrPS could obviously play a significant role in the development of scrapie.

tional event (104). Protein chemical studies have confirmed that the amino acid sequence of the majority of PrP 27-30 molecules matches that predicted from the gene and eDNA sequences, and thus probably corresponds to that of PrPC (53). Posttranslational chemical modifications of PrP 2 7-30 molecules have been catalogued through mass spectrometric analysis of every peptide. Although several modifications have been identified and structurally characterized (90, 92, 94) and a final comparison to those of PrPC has not yet been completed, there is no obvious means by which the chemical modifications of PrP 2 7-30 could result in its distinct physical properties. We are left with the possibilities that either a minority of PrPSC molecules, as yet undetected, contain an unusual chemical modification that distinguishes it from PrPC, or that the two isoforms differ through conformation or tight binding of other molecules. One possible explanation for the difference between PrPS and PrPC is that they differ only in their tertiary structure. The altered conformation of PrPS might act as a template that binds PrPC and allows it to be converted to PrP. Denaturation of PrPS or PrP 27-30 with guanidine hydrochloride disrupts the tertiary structure and results in sensitivity to proteases and disaggregation (90), along with the loss of scrapie infectivity. Attempts to regain protease resistance and scrapie infectivity as well as reform aggregates by various protocols for removal of the denaturant have been

AND

SUMMARY

Although our understanding of PrPC and PrPS has progressed significantly in the last few years, the mechanism of prion replication remains elusive. Recent results with Tg mice argue that PrP participates in the transmission and susceptibility to scrapie (32, 50). Notable is the ability of the Leu-102 point mutation in PrP to cause a neurodegenerative disease in a Tg mouse (52), and experiments are in progress to determine whether de novo synthesis of prions occurs in these animals. If this occurs, it will indicate that foreign nucleic acid is not required for prion infectivity but will not eliminate the possible involvement of a host-encoded molecule. There is excellent evidence that the protease resistance of PrPSC is acquired as a result of a posttransla-

2804

Vol. 5

October

1991

The

and

S. B. Prusiner,

unpublished

results). Thus PrP 2 7-30 in prion rods differs from some amyloidogenic proteins where spontaneous reformation of amyloid fibrils is observed upon removal of the denaturant (107, 108). Whether this means that specific environmental conditions, binding of another component, or an unknown variable is required for amyloid formation remains to be established. It has also been observed that the PrP containing Leu-102 in human GSS also accumulates in amyloid plaques (109), but does not appear to aggregate spontaneously into amyloid when expressed in vitro or in cell culture (D. R. Borchelt and S. B. Prusiner, unpublished results). An Indiana kindred with a form of GSS accumulates an 11-kDa proteolytic fragment of PrP that begins at Gly-58 within amyloid

plaques

(110).

Another possibility is that other cellular components are tightly bound to PrP and contribute to its novel properties. Although purified prion rods contain primarily PrP 27-30, we have identified other molecules that also copurify (68; N. Stahl and M. Baldwin, unpublished results). It is unclear whether these molecules play any role in the structure or function of prions. All varieties of amyloid plaques examined seem to contain glycosaminoglycans (111) and many of them also contain serum component P (112). More highly purified preparations of prions are needed to evaluate the role of other

OVERVIEW

(D. Groth

unsuccessful

molecules.

A fascinating aspect of scrapie prions is the existence of distinct isolates or strains that display different properties, such as the incubation time between inoculation and the onset of symptoms (113). For example, Syrian hamsters infected with the Sc237 isolate have an incubation time of approximately 75 days, whereas animals inoculated with the 139H isolate take approximately 160 days before showing symptoms (114). Although more than 20 strains of the scrapie agent have been reported (113), some but not all of these results are probably due to differences in the PrP amino acid sequence between mice with long and short incubation periods (79). Although strains of viroids and viruses arise from variations in the sequences of their nucleic acid genomes, it is more difficult to imagine how prions, if they are devoid of nucleic acid, might store and propagate informa-

FASEB Journal

STAHL AND

PRUSINER

w.fasebj.org by Univ of So Dakota Lommen Hlth Sci Library (192.236.36.29) on September 23, 2018. The FASEB Journal Vol. ${article.issue.getVolume()}, No. ${article.issue.getIssueNum

tion. Some investigators scrapie strains require

insist that the results of studies with the presence of a scrapie-specific nucleic acid within the prion particle, but it is conceivable that prion-encoded information is stored in distinct heritable conformations or in non-nucleic acid second components. This

work

was

supported

by research

grants

from

the

National

Institutes of Health (AG02132, NS14069, AG08967, and NS22786) and American Health Assistance Foundation as well as by gifts from Sherman Fairchild Foundation and National Medical Enterprises. REFERENCES 1. Prusiner,

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37. Westaway,

D., Goodman,

P. A.,

Mirenda,

C. A.,

McKinley,

M. P., Carlson, G. A., and Prusiner, S. B. (1987) Distinct prion proteins in short and long scrapie incubation period mice. Cell 51, 651-662 38. Liao, Y. -C., Tokes, Z., Lim, E., Lackey, A., Woo, C. H., Button, J. D., and Clawson, G. A. (1987) Cloning of rat “prionrelated protein” cDNA. Lab. Invest. 57, 370-374 39. Kretzschmar, H. A., Stowring, L. E., Westaway, D., Stubblebine, W. H., Prusiner, S. B., and DeArmond, S. J. (1986) Molecular cloning of a human prion protein cDNA. DNA 5, 315-324

40. Goldmann, W., Hunter, N., Foster,J. D., Salbaum,J. M., Beyreuther, K., and Hope, J. (1990) Two alleles of a neural protein gene linked to scrapie in sheep. Proc. Natl. Acad. Sci. USA 87, 2476-2480

41. Goldmann, W., Hunter, N., Martin, T., Dawson, M., and Hope, J. (1991) Different forms of the bovine PrP gene have five or six copies of a short, G-C-rich element within the proteincoding exon. j Gen. ViroL 72, 201-204 42. Owen, F, Poulter, M., Lofthouse, R., Collinge, J., Crow, T. J., Risby, D., Baker, H. F, Ridley, R. M., Hsiao, K., and Prusiner, S. B. (1989) Insertion in prion protein gene in familial Creutzfeldt-Jakob disease. Lancet 1, 51-52 43. Goldgaber, D., Goldfarb, L. G., Brown, P., Asher, D. M., Brown, W. T., Lin, S., Teener,J. W., Feinstone, S. M., Rubenstein, R., Kascsak, R. J., Boellaard, J. W., and Gajdusek, D. C. (1989) Mutations in familial Creutzfeldt-Jakob disease and Gerstmann-Str#{227}ussler-Scheinker’s syndrome. Exp. NeuroL

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45. Nochlin,

C. M., mentia

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Gerstmann-Str#{228}ussler

deof

46. Owen, F, Poulter, H., Collinge, J., and Crow, T (1990) Codon 129 changes in the prion protein gene in Caucasians. Am. j Hum. Genet. 46, 1215-1216 47. Goldfarb, L., Korczyn, A., Brown, P., Chapman, J., and Gajdusek, D. C. (1990) Mutation in codon 200 of scrapie amyloid precursor

gene

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and non-Libyan

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in

Lancet 2,

637-638 48. Gabizon, R., Meiner, Z., Cass, C., Kahana, E., Kahana, I., Abrahami, D., Abramsky, 0., Scarlatto, G., Prusiner, S. B., and Hsiao, K. K. (1991) Prion protein gene mutation in Libyan Jews with Creutzfeldt-Jakob disease. Neurology 41, 160 (abstr.) 49. Harris, D. A., Falls, D. L., Walsh, G. D., and Fishbach, G. D. (1989) Molecular cloning of an acetylcholine receptor-inducing protein. Soc. Neurosci. 15, 70.7 (abstr.) 50. Scott, M., Foster, D., Mirenda, C., Serban, D., Coufal, F, W#{228}lchli, M., Torchia, M., Groth, D., Carlson, G., DeArmond, S. J., Westaway, D., and Prusiner, S. B. (1989) Transgenic mice

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PRIONS AND PRION PROTEINS 2807 w.fasebj.org by Univ of So Dakota Lommen Hlth Sci Library (192.236.36.29) on September 23, 2018. The FASEB Journal Vol. ${article.issue.getVolume()}, No. ${article.issue.getIssueNum

Prions and prion proteins.

Neurodegenerative diseases of animals and humans including scrapie, bovine spongiform encephalopathy, and Creutzfeldt-Jakob disease are caused by unus...
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