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From: TSS ()
Subject: Sensitive Detection of Prion Protein in Human Urine
Date: June 1, 2005 at 11:29 am PST

Sensitive Detection of Prion Protein in Human Urine

HARASH K. NARANG,*2 AYUNA DAGDANOVA, ZHILIANG XIE, QIWEI YANG,

AND SHU G. CHEN,1

*BioTech Global, 22-40 Brentwood Avenue, Newcastle Upon Tyne, NE2 3DH, UK; and Institute of

Pathology, Case Western Reserve University, 2085 Adelbert Road, Cleveland, Ohio 44106

Transmissible spongiform encephalopathies are a group of

infectious diseases typically associated with the accumulation

of a protease-resistant and b-sheet–rich prion protein, PrPSc, in

affected brains. PrPSc is an altered isoform derived from the

host-encoded glycoprotein, PrPC. The expression of PrPC is the

highest in brain tissue, but it can also be detected at low levels

in peripheral tissue. However, it is unclear whether a significant

amount of PrPC is released into body fluid and excreted into

urine. We have developed a simple, rapid method for the reliable

detection of PrPC in urine from normal subjects by Western

blotting. Our method can easily and reliably detect PrPC in

apparently healthy individuals using less than 1 ml of urine in

which the amount of urinary PrPC is estimated to be in the range

of low micrograms/liter. Exp Biol Med 230:343–349, 2005

Key words: urine; prion protein; transmissible spongiform

encephalopathies; bovine spongiform encephalopathy; Creutzfeldt-

Jakob disease

Introduction

Bovine spongiform encephalopathy (BSE) in cattle;

scrapie in sheep and goats; and Creutzfeldt-Jakob

disease (CJD), Gerstmann-Stra¨ussler-Scheinker syndrome,

and kuru in humans are all fatal neurologic disorders

that are collectively known as transmissible spongiform

encephalopathies (TSEs) or prion diseases (1–3). Since the

appearance of BSE in 1985 in the UK (4), a number of newvariant

cases of CJD have been identified and believed to be

caused by the consumption of meat contaminated with BSE

(5, 6). The incubation period between infection and

appearance of clinical symptoms may be several decades.

Following the BSE epidemic in the UK, there have been

small outbreaks of BSE in several other countries. All TSEs

result in the accumulation of a protease-resistant prion

protein (PrPSc) that is derived from its normal counterpart

(PrPC; Refs. 7, 8). The prion protein PrPC is expressed by a

host gene that is predominantly expressed in brain tissue and

detected at low levels in other types of tissue (7, 8). It is

unclear what the normal physiologic function of dominantly

a-helical PrPC may be. It is, however, a membrane-bound,

sialo-glycolipoprotein with a glycophosphatidylinositol

moiety (9), many of which are known to be associated

with transmembrane-signaling functions (10).

The protein sequences of PrPC and PrPSc are identical

(11). However, the two isoforms differ in physicochemical

properties. The normal PrPC isoform exists as a soluble,

dominantly a-helical monomer and is almost completely

degraded by a proteolytic enzyme such as proteinase K

(PK). In contrast, PrPSc has a b-sheet–rich conformation,

and when subjected to PK, a large C-terminal 27- to 30-kDa

segment of PrPSc resists further degradation allowing

detection by Western blotting (12–14). The unique property

of PrPSc in affected brain tissue to PK digestion has been

used in the postmortem diagnosis of TSEs.

Elevated levels of the 14-3-3 protein in the cerebrospinal

fluid of patients with CJD are currently used as

preliminary screen assays for TSEs, but their specificity is

not assured (15–17). The development of a specific,

noninvasive test is critical in assessing the prevalence of

TSEs along with the source of infection and potential

treatment options. Therefore, there is an essential need for a

preclinical diagnostic test for TSEs.

Although PrPC is predominantly expressed in brain

tissue, it is unclear whether a significant amount of PrPC is

circulated in body fluids and eventually eliminated from the

body. Shaked et al. (18) used an ultracentrifugation and

dialysis technique to show that PrP can be detected by the

mouse monoclonal antibody (mAb) 3F4 in 10 ml to 50 ml

This work was supported, in part, by grants from the U.S. National Institutes of

Health, the Department of Agriculture, and the Department of Defense, and by a gift

from Mr. Ken Bell. H.K.N. and A.D. contributed equally to this work.

1 To whom correspondence should be addressed at Institute of Pathology, Case

Western Reserve University, 2085 Adelbert Road, Cleveland, OH 44106. E-mail:

shu.chen@case.edu

2 Current address: West Acres, West Road, Hexham NE46 3DD, UK.

Received May 12, 2004.

Accepted February 21, 2005.

343

1535-3702/05/2305-0343$15.00

Copyright  2005 by the Society for Experimental Biology and Medicine

of urine from normal and diseased subjects. However, the

validity and reproducibility of this finding has been

challenged by two recent reports (19, 20). The evidence of

a cross-reactivity of the anti-mouse IgG with either

contaminating bacterial proteins (19) or urinary IgG fragments

(20) was used to argue that Shaked et al. (18)

mistakenly identified nonspecific urinary proteins as PrP.

Therefore, the presence of either PrPC or PrPSc in urine was

never reliably demonstrated in these studies (18–20).

Nevertheless, it is widely hoped that the ability to

demonstrate the presence of PrP in urine, as well as in

blood, will provide a useful marker of preclinical TSEs.

In the present study, we describe a simple, reliable ioncapture

method that can be used to concentrate PrP from

small or large volumes (i.e., 1 ml to 1 liter) of urine samples.

Following solid-phase extraction, normal PrP in less than 1

ml of urine collected from healthy individuals was sufficient

for detection by Western blotting. We demonstrated the

successful detection of normal PrP in all urine specimens

with the anti-C antibody (21, 22) against the C-terminal

region of PrP, but not with 3F4 mAb (23) recognizing an

epitope in the N-terminal region of PrP that was also used

unsuccessfully in the two recent studies (19, 20). Our

findings highlight the importance of understanding the

unique structural properties of urinary PrP in devising an

appropriate analytic strategy.

Materials and Methods

Urine Collection. In this study, we examined urine

samples from 50 apparently healthy individuals, both male

and female, aged between 25 years and 60 years. The first

morning urine specimens were collected from these normal

individuals who were not affected by any disease conditions

at the time of urine collection. From six of these individuals

we collected three urine samples daily (i.e., first-morning

pass, midday, evening) for 5 days. For the assessment of

kidney function, creatinine levels in the urine samples were

determined at the core laboratory at the University Hospitals

of Cleveland using a Dimension clinical chemistry system

(Dade Behring Inc., Newark, DE).

Enrichment of PrP From Urine. Proteins in urine

were concentrated by ion-capture–based, solid-phase extraction

using the urine concentration kit (GB98/00374 and

9601054; BioTec Global, Newcastle Upon Tyne, UK). All

buffers and ion-capture resin mentioned here were supplied

with the kit. The procedure was performed at room

temperature. Urine samples were centrifuged at 1000 g for

10 mins to sediment the occasional debris. The supernatant

was transferred to fresh tubes, and 1 ml of the concentrate

buffer (250 mM sodium phosphate, pH 7.5; 68 mM

potassium chloride; and 3 M sodium chloride) was added

to each 50-ml urine sample. After a gentle mix, the samples

were subdivided into 1-, 3-, 5-, and 10-ml aliquots. To each

tube that contained 1- to 5-ml urine samples, we added 100

ll of the ion-capture resin (i.e., calcium phosphate, which

was supplied with the kit), and 200 ll was added to the 10-

ml urine sample. The samples were gently mixed by hand

flicking and were then left on a shaking platform (Red

Rotor; Hoefer Pharmacia Biotech, San Francisco, CA) at the

speed setting of 3 for 60 mins and agitated by hand every 10

mins to ensure that the resin was well-dispersed in

suspension. After the protein adsorption, the tubes were

centrifuged at 500 g for 5 mins and the supernatant was

discarded. The resulting pellet was resuspended in 0.75-ml

of wash buffer (10 mM sodium phosphate, pH 7.5; 3 mM

potassium chloride; and 137 mM sodium chloride), transferred

to 1-ml microfuge tubes, and centrifuged at 16,000 g

for 10 secs. The supernatant was discarded and replaced

with 30 ll of the sodium dodecyl sulfate (SDS) sample

buffer (63 mM Tris-hydrochloride, pH 6.8; 2 mM EDTA,

3% SDS, 10% glycerol, and 1% b-mercaptoethanol).

Samples were then boiled for 10 mins and centrifuged at

16,000 g for 30 secs. The supernatant containing eluted

proteins was used for Western blotting.

Western Blotting. Samples were applied to a 12% or

16% Tris-glycine SDS polyacrylamide gel electrophoresis

(SDS-PAGE; precast gels; Invitrogen, Carlsbad, CA) in a

mini-cell apparatus (Bio-Rad, Hercules, CA) and subsequently

transferred to polyvinylidene fluoride membranes

(Millipore, Bedford, MA) at 70 V for 2 hrs at 48C. The

membranes were then blocked using blocking buffer

containing 3% fat-free milk and 1% bovine serum albumin

(BSA) in Tris-buffered saline supplemented with 0.1%

Tween 20 (TBS-T; pH 7.6) for 1 hr. Then, the membranes

were incubated with one of the following primary antibodies:

(i) the rabbit anti-C antiserum (21, 22) against

human PrP residues 220–231 at a dilution of 1:3000, and (ii)

mouse 3F4 mAb (23) recognizing an epitope of human PrP

residues 109–112 at a dilution of 1:50,000 in antibody

dilution buffer (1% [v/v] normal goat serum, 0.05% [w/v]

BSA, 0.1% [w/v] thimerosal in TBS) for 2 hrs at room

temperature or overnight at 4oC. The membranes were then

rinsed four times in TBS-T for 15 mins each, followed by

incubation with an appropriate secondary antibody (donkey

anti-rabbit IgG F(ab9)2 fragment (catalog number NA9310)

and sheep anti-mouse IgG F(ab9)2 fragment (catalog number

NA9340) conjugated with horseradish peroxidase (Amersham

Biosciences, Piscataway, NJ) for 1 hr at room

temperature. After being rinsed another four times in

TBS-T, PrP was visualized on Kodak X-Omat films by

enhanced chemiluminescence (ECL Plus kit; Amersham

Biosciences).

To evaluate the amount of PrP in urine sample, protein

concentrates were prepared from 0.5- to 10-ml urine

samples as previously described. Protein concentrates were

run in parallel with 1 ng to 8 ng of the recombinant human

PrP 23–231 (Abcam, Cambridge, MA). Western blotting

was performed as previously described. The amount of PrP

was quantified by densitometry according to the intensity of

PrP bands using the UN-SCAN-IT software (Silk Scientific,

Orem, UT).

344 NARANG ET AL

Peptide: N-Glycosidase F (PNGase F) Treatment.

Deglycosylation was performed using PNGase F

and other reagents provided by the supplier (New England

BioLabs, Beverly, MA). Proteins were concentrated from 10

ml of urine as previously described. The final pellet was

suspended in 30 ll of denaturing buffer (0.5% SDS, 1% bmercaptoethanol)

and boiled for 10 mins. The supernatant

containing eluted and denatured proteins was supplemented

with G7 buffer (50 mM sodium phosphate, pH 7.5) and 1%

NP-40 and digested with 3 ll of PNGase F (500,000 U/ml)

for 60 mins at 37oC. Digestion was stopped by the addition

of SDS sample buffer followed by boiling for 10 mins.

Samples were applied to a 10%- to 20%-gradient Tristricine

SDS-PAGE using precast gels (Invitrogen) and were,

subsequently, subjected to Western blotting as previously

described.

Protease Digestion. Proteins were concentrated

from 10-ml aliquots of urine samples as previously

described. After the pellet was resuspended in 200 ll of

wash buffer, 20 ll of trypsin (2 mg/ml) or PK (2 mg/ml)

was added for on-resin digestion. Control was made in

which no enzyme was added. All tubes were incubated for

60 mins at 378C. Following enzyme digestion, the samples

were centrifuged for 30 secs at 16,000 g in microfuge tubes,

and the supernatant was removed. The pellet was washed

with 0.75 ml of wash buffer and resuspended in 30 ll of

SDS sample buffer followed by boiling for 10 mins.

Samples were run on 10%- to 20%-gradient Tris-tricine

SDS-PAGE gels and were subjected to Western blotting as

previously described.

Spiking of Brain Homogenate Into Urine. To

model the possibility of our method being used for the PrPSc

detection in urine, brain homogenate of both normal and

scrapie-adapted (i.e., 263K prion) hamsters was spiked into

the urine samples. The total brain homogenate (10% [w/v])

of normal and 263K scrapie hamsters was made in

phosphate-buffered saline (pH 7.5) followed by brief

centrifugation. Urine (1 ml) was mixed with 20 ll of the

concentrate buffer and 100 ll of the ion-capture resin. The

clarified brain homogenate (10 ll) was spiked into these

urine samples. The samples were left for incubation on a

shaking platform (Red Rotor; Hoefer Pharmacia Biotech) at

the speed setting of 3 for 60 mins at room temperature. After

adsorption of proteins onto the resin, samples were

centrifuged at 16,000 g for 10 secs and the supernatant

was discarded. The resulting pellet was resuspended in 0.75

ml of wash buffer. After centrifugation again at 16,000 g for

10 secs, the supernatant was discarded and 0.3 ml of fresh

wash buffer was added. Each sample was then divided into

two groups: 0.1 ml for the control sample without the

addition of PK and 0.2 ml for PK digestion at final enzyme

concentration of 50 lg/ml. All samples were incubated for

60 mins at 378C. Reaction was stopped by adding 1 ll of

100 mM Pefa block (Roche Molecular Biochemicals,

Indianapolis, IN). Samples were centrifuged for 20 secs at

16,000 g, and the supernatant was removed. The pellet was

resuspended in 30 ll of SDS sample buffer and processed as

previously described for Western blotting.

Results

The apparently healthy individuals that we tested had

creatinine levels ranging from 60 mg/dl to 300 mg/dl in

morning urine samples, with no medical conditions

indicative of kidney malfunction. We intended to use these

samples in developing our techniques for the detection of

PrP in human urine.

On Western blots probed by the rabbit anti-C antiserum

(21, 22) recognizing human PrP residues 220–231,

detectable amounts of PrPC migrating at 28 kDa to 30

kDa were observed in urine samples of all healthy

individuals (N = 50). When urine samples were subdivided

into 1-, 3-, and 5-ml aliquots and all proteins concentrated

from the samples were applied, a proportional increase in

the amount of PrPC was demonstrated (Fig. 1, Lanes 1–3).

The total protein loading in these urine samples displayed a

similar trend of increase as judged by silver staining (Fig. 1,

Lanes 4–6), which demonstrates a quantitative recovery of

urinary proteins by the solid-phase extraction. However, no

bands were observed after immunoblotting with 3F4 mAb

(Fig. 1, Lanes 7–9). To test if the binding of the secondary

IgG to nonspecific proteins such as bacterial outer

membrane proteins (19) or urinary human IgG fragments

(20) accounted for the false positive detection of PrP as

reported previously (18), control experiments were performed

with the use of secondary antibodies (i.e., donkey

anti-rabbit IgG, sheep anti-mouse IgG, F(ab9)2 fragment) in

the absence of the respective primary antibodies (i.e., rabbit

anti-C antibody, mouse 3F4 mAb). No immunoreactive

bands were observed for either secondary anti-rabbit IgG

(Fig. 1, Lanes 10–12) or secondary anti-mouse IgG (Fig. 1,

Lanes 13–15) under our experimental conditions, confirming

that our method reliably detected PrPC in human urine,

not any other nonspecific proteins. The detection of PrPC

could be achieved in as little as 1 ml of urine (Fig. 1) or less

(Fig. 2) from normal individuals (N = 50).

Quantitation of PrPC present in the urine samples was

accomplished through comparison with known amounts of

human recombinant PrP run in parallel on SDS-PAGE gels.

Variations, ranging from 2 ng/ml to 25 ng/ml of the firstmorning

urine specimens (with an average of 7.2 6 6.8 ng/

ml) were observed among normal individuals who were not

affected by any medical conditions (mean 6 SD; N = 50).

As shown in Figure 2, the amount of urinary PrPC was

approximately 10 ng/ml in the urine of one healthy

individual. To clarify whether PrPC is excreted into urine

throughout the day, urine samples from six individuals were

collected in the morning, midday, and evening for 5 days.

Regardless of the time of specimen collection, PrPC was

detectable by Western blotting with the anti-C antibody in

all urine specimens (data not shown).

Because PrPC is a glycoprotein that contains two

DETECTION OF PRION PROTEIN IN URINE 345

consensus sites for asparagine-linked glycosylation,

PNGase F digestion was performed to remove glycans

and reveal the protein backbone of urinary PrPC.

Following deglycosylation (Fig. 3), the heterogeneous 28

kDa to 30 kDa urinary PrPC bands (Lane 1) shifted mainly

to a lower molecular weight band of 18 kDa (Lane 2). This

is consistent with the similar, approximately 10-kDa shift

expected from the removal of two asparagine-linked

complex glycans from PrP, as shown in cultured cells

and brain tissue (21). Therefore, our detection of urinary

PrP is highly specific, without artifacts associated with the

unrelated proteins (18–20). As expected, PrPC in the urine

of normal individuals was sensitive to digestion by both

trypsin and PK, as exogenous protease digestion almost

completely degraded PrP into small peptides of less than

7 kDa (Fig. 3).

It is essential to test whether our method is capable of

capturing PrPSc as effectively as PrPC because PrPSc

associated with TSEs has an altered protein conformation

that is different from normal PrPC. This possibility was

tested in experiments in which small quantities of scrapie

brain homogenate containing PrPSc was spiked into urine.

As shown on Western blots probed by the anti-C antibody,

PrP bands were detected in all PK-untreated samples (i.e.,

control urine, urine spiked with normal or scrapie brain

homogenate). Following PK digestion, PK-resistant PrP

derived from PrPSc was only found in the urine sample

spiked with scrapie hamster brain homogenate (Fig. 4, upper

panel). When probed by 3F4 mAb (Fig. 4, lower panel), no

immunoreactive PrP bands were found in nonspiked control

urine, a finding that is consistent with the results from the

previous experiments (Fig. 1). However, strong PrP bands

were detected by 3F4 mAb in urine spiked with either

normal or scrapie brain homogenate before PK digestion.

This result, as expected, suggests that 3F4 mAb readily

recognizes PrP from the brain, (23, 24) but not from urine.

As with anti-C antibody, PK-resistant PrPSc was detected on

Western blots probed with 3F4 in the urine sample spiked

with scrapie brain homogenate (Fig. 4, lower panel). Taken

together, we have shown that the present method was able to

recover and detect both PrPC and PrPSc from urine samples

spiked with normal and scrapie brain homogenate.

Figure 1. Detection of prion protein (PrP) in human urine. A urine sample from a normal individual was subdivided into 1-, 3-, and 5-ml aliquots.

Proteins were concentrated by solid-phase extraction as described in the Materials and Methods section. Samples equivalent to 1 ml (first lanes

in each panel), 3 ml (middle lanes in each panel), and 5 ml (last lanes in each panel) of urine were analyzed on Western blots. Rabbit anti-C

antibody was used to detect PrP (Lanes 1–3; note the gradient increase of PrP from 1- to 5-ml urine samples). Total proteins captured by solidphase

extraction from 1 ml to 5 ml of urine showed a similar increase as judged by silver staining (Lanes 4–6). No detectable bands were

recognized by using 3F4 monoclonal antibody (mAb) (Lanes 7–9). Nonspecific immunoreactivity was not found in the control experiments in

which the secondary antibodies (i.e., anti-rabbit IgG, Lanes 10–12; anti-mouse IgG, Lanes 13–15) were used without the respective primary

antibodies. The position (in kDa) of molecular-weight markers run in parallel on each blot is indicated on the left.

Figure 2. Estimation of amounts of urinary prion protein (PrP).

Samples equivalent to 0.5 ml, 1 ml, 2 ml, 5 ml, and 10 ml of urine

(Lanes 1–5) were loaded along with known quantity (i.e., 1 ng, 2 ng, 4

ng, and 8 ng) of recombinant human PrP23-231 (Lanes 6–9).

Western blotting was performed using the anti-C antibody as

described in the Materials and Methods section. The position (in

kDa) of molecular-weight markers is indicated on the left.

346 NARANG ET AL

Discussion

The simple ion-capture method presented in the present

study effectively extracted proteins from urine onto small

quantities of resin, thus providing a convenient way for the

enrichment of excreted protein from large volumes of urine.

We have shown that normal PrP was detectable in less than

1 ml of urine in all healthy individuals examined. The PrP

that was concentrated from the urine was detected using the

anti-C antibody, an antibody that is immunoreactive to the

C-terminus of human PrP (21, 22). No detectable urinary

PrP was recognized by 3F4 mAb, which is contrary to an

earlier report by Shaked et al. (18) whose findings were

challenged in more recent studies (19, 20). Attempts to

detect PrPC in either human or hamster urine using the 3F4

mAb by two other groups have failed (19, 20). Instead, a

nonspecific cross-reactivity to other contaminating proteins

was reported (19, 20). In the present study, no bands were

detected when a secondary antibody was used alone.

Therefore, the detection of PrPC using the anti-C antibody

is not due to contamination with bacterial outer-membrane

proteins (19) or endogenous IgG (20) as observed by other

groups when using 3F4 mAb. Because 3F4 mAb is well

known for its ability to recognize brain PrP (23, 24), its

failure to detect urinary PrPC suggests that the 3F4 epitope

located in the N-terminal region is absent in urinary PrPC.

This conclusion is consistent with the fact that the anti-C

antibody against the C-terminus of PrP readily detects

urinary PrPC. Our findings not only highlight the unique

structural properties of urinary PrP that are apparently

different from brain PrP, but also help clarify the recent

controversy involving the attempts to detect urinary PrP

using 3F4 (18–20).

Semiquantitative studies in which recombinant PrP was

applied along with urinary protein concentrates from 0.5- to

10-ml samples revealed that the lower detection limit on

Western blots is better than 1 ng of PrPC in urine. On

average, each healthy individual excreted approximately 7

ng of PrPC per ml of urine. If a healthy person produces 1

liter of urine a day, the daily excretion of a microgram

amount of normal PrPC into the urine is expected. Variations

in the amounts of PrPC per ml of urine were observed

among normal individuals. The reason for the observed

variations is unclear, but they are not unexpected because

protein excretion per ml of urine is likely to fluctuate among

different individuals in untimed-morning urine used in the

present study. A more accurate estimation of the rate of

PrPC excretion and its normalization among healthy

individuals may require a correlation analysis of baseline

parameters such as total urine protein levels, creatinine

clearance, and the use of accurately timed–urine collection

(e.g., 24-hr urine specimens). Moreover, the exact origin of

urinary PrPC is yet to be determined. Nevertheless,

consistent detection of PrP in the urine of normal, healthy

individuals raises the question of whether the protein

Figure 3. Digestion of urinary prion protein (PrP) by PNGase F and

proteases. A urine sample from a normal individual was subdivided

into 10-ml aliquots. Proteins were concentrated and treated with

either PNGase F or proteases (i.e., trypsin, proteinase K [PK]),

followed by Western blotting with the anti-C antibody as described in

the Materials and Methods section, except that 10% to 20% Tristricine

SDS-PAGE gels were used to reveal the presence of small

peptides. Following PNGase F digestion, the diffused bands of

approximately 28-kDa PrP in urine (Lane 1) was deglycosylated and

shifted to a sharper band at approximately 18-kDa PrP (Lane 2).

Urinary PrP was sensitive to degradation by trypsin (Lane 3) and PK

(Lane 4). The position (in kDa) of molecular-weight markers is

indicated on the left.

Figure 4. Detection of hamster brain prion protein, PrPSc, spiked into

human urine. Aliquots of urine (1 ml each) from a normal individual

were taken as controls and as samples to which brain homogenate

from normal and scrapie-affected hamsters was spiked. Following

the solid-phase extraction of proteins, samples were either untreated

with proteinase K (PK–) or treated with PK (PKþ) at a final

concentration of 50 lg/ml for 1 h at 37oC as described in the

Materials and Methods section. The detection of PrP on Western

blots was achieved by using either the anti-C antibody (Upper panel)

or 3F4 monoclonal antibody (Lower panel).

DETECTION OF PRION PROTEIN IN URINE 347

generated in the body is efficiently reutilized. It is possible

that most proteins are, indeed, reutilized. However, if even a

small proportion of PrPC is not reutilized, it could filter

through the kidney.

Deglycosylation by PNGase F is often used to reduce

the heterogeneity of PrP molecules. Treatment with PNGase

F revealed that PrPC in urine was glycosylated, but the

protein was truncated. Following deglycosylation, the size

of urinary PrPC shifted from 28–30 kDa to 18 kDa. As the

full-length PrPC following deglycosylation has an electrophoretic

mobility of about 27 kDa on SDS-PAGE gels (20),

our data suggest that PrPC in human urine is mostly

truncated with a size much smaller than that expected from

the full-length protein (25). Such a truncation is unlikely an

artifact generated during our experimental procedures

because the same results were obtained from fresh urine

samples containing the added protease inhibitors following

concentration at a lower temperature (10oC) or when

proteins were precipitated in cold methanol at 20oC.

Therefore, the truncated PrPC in human urine is likely the

result of proteolytic processing that occurred in vivo before

the excretion, a normal metabolic event previously shown in

human neuroblastoma cells and the brain (21), possibly by a

calpain-dependent proteolytic process (26).

It would be desirable to study a large number of

individuals from whom 24-hr urine samples were obtained.

Furthermore, it would be essential to investigate whether

there is any difference in the amounts of PrPC excreted into

the urine between normal individuals and those with TSEs.

A previous report (18) on the detection of the urinary PrPSc

in TSEs using 3F4 mAb has recently been challenged as an

artifact (19, 20), possibly due to lack of the 3F4 epitope in

urinary PrP as demonstrated in the present study. Therefore,

it is unclear whether TSEs lead to the excretion of urinary

PrP with the PrPSc-like conformation. Assuming this is the

case, the ability to detect PrPC may lay the foundation for a

future technique to be used in PrPSc detection. Furthermore,

the positive data obtained in our experiments in which brain

PrPSc was spiked into urine suggest the potential of our

method for identifying urinary PrPSc in TSEs which may

have, so far, evaded detection due its extremely low

concentration. Equally important is a further analysis of

other biochemical properties of urinary PrP between normal

individuals and those affected by CJD. In the present study,

we present a simple, convenient, and efficient means to

concentrate and detect trace amounts of PrP in urine. Our

assay may be further developed to determine whether any

unique characteristics of urinary PrP, or its interaction with

other molecules, might be associated with a preclinical state

of TSEs. Finally, it is our hope that the technique presented

here will evoke further interests and improvements in

diagnostic strategies for TSEs.

We thank Fang Zhang for technical assistance and Dr. Robert

Petersen for helpful discussion.

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