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From: TSS ()
Subject: Detection of Type 1 Prion Protein in Variant Creutzfeldt-Jakob Disease
Date: January 9, 2006 at 8:15 am PST

Detection of Type 1 Prion Protein in Variant

Creutzfeldt-Jakob Disease

Helen M. Yull,* Diane L. Ritchie,*

Jan P.M. Langeveld,† Fred G. van Zijderveld,†

Moira E. Bruce,‡ James W. Ironside,* and

Mark W. Head*

From the National CJD Surveillance Unit,* School of Molecular

and Clinical Medicine, University of Edinburgh, Edinburgh,

United Kingdom; Central Institute for Animal Disease Control

(CIDC)-Lelystad, † Lelystad, The Netherlands; Institute for Animal

Health, Neuropathogenesis Unit, ‡ Edinburgh, United Kingdom

Molecular typing of the abnormal form of the prion

protein (PrPSc) has come to be regarded as a powerful

tool in the investigation of the prion diseases. All evidence

thus far presented indicates a single PrPSc molecular

type in variant Creutzfeldt-Jakob disease (termed

type 2B), presumably resulting from infection with a

single strain of the agent (bovine spongiform encephalopathy).

Here we show for the first time that the PrPSc

that accumulates in the brain in variant Creutzfeldt-

Jakob disease also contains a minority type 1 component.

This minority type 1 PrPSc was found in all 21

cases of variant Creutzfeldt-Jakob disease tested, irrespective

of brain region examined, and was also

present in the variant Creutzfeldt-Jakob disease tonsil.

The quantitative balance between PrPSc types was maintained

when variant Creutzfeldt-Jakob disease was

transmitted to wild-type mice and was also found in

bovine spongiform encephalopathy cattle brain, indicating

that the agent rather than the host specifies their

relative representation. These results indicate that PrPSc

molecular typing is based on quantitative rather than

qualitative phenomena and point to a complex relationship

between prion protein biochemistry, disease phenotype

and agent strain. (Am J Pathol 2006, 168:151–157;

DOI: 10.2353/ajpath.2006.050766)



In the apparent absence of a foreign nucleic acid genome

associated with the agents responsible for transmissible

spongiform encephalopathies or prion diseases,

efforts to provide a molecular definition of agent strain

have focused on biochemical differences in the abnormal,

disease-associated form of the prion protein, termed

PrPSc. Differences in PrPSc conformation and glycosylation

have been proposed to underlie disease phenotype

and form the biochemical basis of agent strain. This

proposal has found support in the observation that the

major phenotypic subtypes of sCJD appear to correlate

with the presence of either type 1 or type 2 PrPSc in

combination with the presence of either methionine or

valine at codon 129 of the prion protein gene.2 Similarly,

the PrPSc type associated with vCJD correlates with the

presence of type 2 PrPSc and is distinct from that found in

sCJD because of a characteristically high occupancy of

both N-linked glycosylation sites (type 2B).6,11 The

means by which such conformational difference is detected

is somewhat indirect; relying on the action of proteases,

primarily proteinase K, to degrade the normal

Figure 6. Type 1 PrPSc is a stable minority component of PrPSc from the vCJD

brain. Western blot analysis of PrP in a sample of cerebral cortex from a case

of vCJD during digestion with proteinase K is shown. Time points assayed

are indicated in minutes (T0, 5, 10, 30, 60, 120, 180). Duplicate blots were

probed with 3F4, which detects both type 1 and type 2 PrPSc, and with 12B2,

which detects type 1. The insert shows a shorter exposure of the same time

course study from a separate experiment also probed with 3F4. Both blots

included samples of cerebral cortex from a case of sporadic CJD MM1 (Type

1) and molecular weight markers (Markers) indicate weights in kd.

Figure 7. A minority type 1-like PrPSc is found in vCJD tonsil, vCJD transmitted

to mice and in BSE. Western blot analysis of PrPSc in a concentrated

sample of tonsil from a case of vCJD (Tonsil), in a concentrated brain sample

of a wild-type mouse (C57BL) infected with vCJD and in a sample of cattle

BSE brain (BSE) is shown. Tissue extracts were digested with proteinase K.

Duplicate blots were probed with either 3F4 or 6H4, both of which detect

type 1 and type 2 PrPSc, and with 12B2, which detects type 1. The blots

included samples of cerebral cortex from a case of sporadic CJD MM1 (Type

1) and molecular weight markers (Markers) indicate weights in kd.

Type 1 PrPSc in Variant Creutzfeldt-Jakob Disease 155

AJP January 2006, Vol. 168, No. 1

cellular form of PrP and produce a protease-resistant

core fragment of PrPSc that differs in the extent of its

N-terminal truncation according to the original


A complication has recently arisen with the finding that

both type 1 and type 2 can co-exist in the brains of

patients with sCJD.2,5-8 More recently this same phenomenon

has been demonstrated in patients with iatrogenically

acquired and familial forms of human prion disease.

9,10 The existence of this phenomenon is now

beyond doubt but its prevalence and its biological significance

remain a matter of debate.

Conventional Western blot analysis using antibodies

that detect type 1 and type 2 PrPSc has severe quantitative

limitations for the co-detection of type 1 and type 2

PrPSc in individual samples, suggesting that the prevalence

of co-occurrence of the two types might be underestimated.

We have sought to circumvent this problem by

using an antibody that is type 1-specific and applied this

to the sole remaining human prion disease where the

phenomenon of mixed PrPSc types has not yet been

shown, namely vCJD.

These results show that even in vCJD where susceptible

individuals have been infected supposedly by a

single strain of agent, both PrPSc types co-exist: a situation

reminiscent of that seen when similarly discriminant

antibodies were used to analyze experimental BSE in

sheep.14,17 In sporadic and familial CJD, individual

brains can show a wide range of relative amounts of the

two types in samples from different regions, but where

brains have been thoroughly investigated a predominant

type is usually evident.2,6,10 This differs from this report

on vCJD, where type 1 is present in all samples investigated

but always as a minor component that never

reaches a level at which it is detectable without a type

1-specific antibody. It would appear that the relative balance

between type 1 and type 2 is controlled within

certain limits in the vCJD brain. A minority type-1-like

band is also detected by 12B2 in vCJD tonsil, in BSE

brain and in the brains of mice experimentally infected

with vCJD, suggesting that this balance of types is agent,

rather than host or tissue, specific. Interestingly the “glycoform

signature” of the type 2 PrPSc found in vCJD (type

2B) is also seen in the type 1 PrPSc components, suggesting

that it could legitimately be termed type 1B.

PrPSc isotype analysis has proven to be extremely

useful in the differential diagnosis of CJD and is likely to

continue to have a major role in the investigation of human

prion diseases. However, it is clear, on the basis of

these findings, that molecular typing has quantitative limitations

and that any mechanistic explanation of prion

replication and the molecular basis of agent strain variation

must accommodate the co-existence of multiple

prion protein conformers. Whether or not the different

conformers we describe here correlate in a simple and

direct way with agent strain remains to be determined. In

principle two interpretations present themselves: either

the two conformers can be produced by a single strain of

agent or vCJD (and, therefore, presumably BSE) results

from a mixture of strains, one of which generally predominates.

Evidence for the isolation in mice of more than one

strain from individual isolates of BSE has been presented


One practical consequence of our findings is that the

correct interpretation of transmission studies will depend

on a full examination of the balance of molecular types

present in the inoculum used to transmit disease, in addition

to a thorough analysis of the molecular types that

arise in the recipients. Another consequence relates to

the diagnostic certainty of relying on PrPSc molecular

type alone when considering the possibility of BSE infection

or secondary transmission in humans who have a

genotype other than methionine at codon 129 of the

PRNP gene. In this context it is interesting to note that this

minority type 1B component resembles the type 5 PrPSc

described previously to characterize vCJD transmission

into certain humanized PRNP129VV transgenic mouse

models.12,20 This apparently abrupt change in molecular

phenotype might represent a selection process imposed

by this particular transgenic mouse model. Irrespective of

whether this proves to be the case, the results shown

here point to further complexities in the relationship between

the physico-chemical properties of the prion protein,

human disease phenotype, and prion agent strain.



Type 1 PrPSc in Variant Creutzfeldt-Jakob Disease 157

AJP January 2006, Vol. 168, No. 1 ...TSS

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