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Abstract

The misfolded form of cellular prion protein (PrP^c) is the main component of the infectious agent of transmissible spongiform encephalopathies and the validated biomarker for these diseases. The expression of PrP^c is highest in the central nervous system and has been found in peripheral tissues. Soluble PrP^c has been detected in cerebrospinal fluid, urine, serum, milk, and seminal plasma. In this study, attempts were made to characterize prion protein in urine samples from normal and scrapie-infected sheep. Urine samples from scrapie-infected sheep and age-matched healthy sheep were collected and analyzed by Western blot following concentration. A protease K-sensitive protein band with a molecular weight of approximately 27–30 kDa was visualized after immunoblotting with anti-PrP monoclonal antibodies to a C-terminal part of PrP^c, but not after immunoblotting with monoclonal antibodies to an N-terminal epitope of PrP^c or with secondary antibodies only. The amount of PrP^c in the urine of 49 animals (control group: n = 16; naturally scrapie-infected group: n = 33) was estimated by comparison with known amounts of ovine recombinant PrP in the immunoblot. Background concentration of PrP^c in urine was found to be 0–0.16 ng/ml (adjusted to the initial nonconcentrated volume of the urine samples). Seven out of 33 naturally scrapie-infected animals had an elevated level (0.3–4.7 ng/ml) of PrP^c in urine. The origin of PrP^c in urine and the reason for the increased level of PrP^c in scrapie-infected sheep urine has yet to be explored.

Keywords

Prion protein scrapie urine

Introduction

Transmissible spongiform encephalopathies (TSEs) are a group of neurodegenerative diseases such as bovine spongiform encephalopathy (BSE) in cattle, scrapie in sheep and goats, chronic wasting disease (CWD) in elk and deer, and Creutzfeldt-Jakob disease (CJD) in humans. TSEs are characterized by amyloid-like deposition of the protease-resistant pathological isoform (PrP^Sc) of host cellular prion protein (PrP^c) primarily in the central nervous system and lymphoid organs. 21,27 The expression of PrP^c is highest in neural tissues and also has been found in non-neural tissues. 4,5,7,11,17 PrP^c is located on the cellular surface, but soluble PrP^c has also been detected in cerebrospinal fluid, 20,28 blood plasma, 29 milk, 8,15 ovine seminal plasma, 6 and human urine. 18 It is unknown whether TSE-associated PrP (PrP^Sc) circulates in body fluids and is eventually eliminated from the body. Blood has been repeatedly stated to be infectious, 3,12,16,25 and recently, results on CWD transmission by saliva have been reported. 16 Urine also has been found to be infectious in cases of coincident scrapie infection and chronic kidney inflammation in autoimmune mice 22 and in scrapie-infected hamsters, 13 but not in CWD-infected deer. 16

A protease-resistant isoform of the prion protein was reported once in the urine of scrapie-infected hamsters, BSE-infected cattle, and humans with CJD, 24 but later these results were declared as a consequence of cross-reaction of secondary antibodies with either contaminating bacterial proteins 9 or immunoglobulin fragments excreted in urine. 10,14,23 TSE-infectivity in urine of scrapie-infected hamsters 13 and mice with coincident TSE and kidney inflammation 22 reopened an inquiry into the presence of TSE-associated proteins in urine. In this study, attempts were made to characterize prion protein in urine samples from normal and scrapie-infected sheep, to determine the conditions for its optimal detection, and to quantify the amount of PrP in urine of healthy and scrapie-infected animals.

Materials and methods

Animals

Scrapie-infected sheep were sourced from a farm quarantined for scrapie and were identified by a PrP^Sc immunohistochemistry (IHC) assay of the “third eyelid” or the nictitating membrane (NM)-associated lymphoid tissue. 19 NM-test positive animals used in this study were kept in a designated animal facility at Ottawa Laboratory Fallowfield (Ottawa, ON, Canada). Age-matched control animals were housed in a different “clean” building. All experimental procedures involving animals were carried out according to Canadian Council on Animal Care guidelines.

Sheep urine samples collection and processing

Urine samples (450–600 ml) from scrapie-positive and age-matched control animals were collected in the morning after the intramuscular administration of 100 mg of furosemide. a Urine was cleared of debris by centrifugation for 20 min at 4,000 × g and kept frozen at −20°C until use. Urine samples supplemented with 1 mM phenylmethylsul-phonylfluoride c (PMSF) were concentrated 250-fold in Amicon Ultra Centrifugal Filter 5000 MWCO Devices, d dialyzed against phosphate buffered saline, and processed for immunoblotting with anti-PrP mAbs. Total protein concentration in the concentrated urine samples was determined using a Coomassie Dry Protein Assay Kit b according to the manufacturer's protocol.

Preparation of brain tissue homogenates

Sheep brain tissue was obtained from normal or scrapie-infected animals with clinical signs confirmed by histopathology and PrP^Sc IHC. A 20% (w/v) homogenate was prepared in cold homogenization buffer (10 mM Tris-HCl. pH 7.2, 300 mM sucrose) using a FastPrep Cell Disrupter e at a speed setting of 6.5 for 45 sec. The homogenate was cleared of cell debris by centrifugation at 1,500 × g for 20 min.

Proteinase K digestion

Concentrated and dialyzed urine samples and brain tissue homogenates of scrapie or control healthy sheep were incubated with 1–50 μg/ml proteinase K c for 60 min at 37°C. The reaction was stopped by the addition of PMSF to a final concentration of 5 mM.

Peptide: N-Glycosidase F treatment of urine and brain PrP

Deglycosylation was performed using a Peptide: N-Glycosidase F (PNGase F) kit f according to the manufacturer's protocol. A volume of 50 μL of 250-fold concentrated and dialyzed sheep urine or 50 μL of 10% (w/v) ovine brain tissue homogenate were denatured by heating for 10 min at 100°C in the presence of 0.5% sodium dodecyl sulfate (SDS) and 1% β-mercaptoethanol, and incubated for 60 min at 37°C in buffer containing 100 mM sodium phosphate, 10 mM Tris-HCl, 1% NP-40, and 1–4 units of PNGase F. Treatment was stopped by the addition of 2X SDS loading sample buffer followed by incubation at 100°C for 5 min.

Sodium dodecyl sulfate polyacrylamide gel electrophoresis and Western blot detection of PrP

For Western blot analysis, samples were subjected to sodium dodecyl sulfate Polyacrylamide gel electrophoresis (SDS-PAGE) using reducing conditions and were subsequently transferred to a polyvinylidene difluoride (PVDF) membrane g by semidry blotting in 10 mM 3-(cyclohexylamino)-1-propanesulfonic acid c (CAPS) buffer plus 10% methanol, pH 11.0. Membranes were blocked with 1% casein in 10 mM Tris-HCl, 150 mM NaCl, 0.1% Triton X-100, pH 7.5 (Tris buffered saline Tween [TBST]) overnight at 4°C, and then incubated for 60 min at ambient temperature with 1 μg/ml of one of the following anti-PrP monoclonal antibodies: F99/97.6.1, h M2188, M2197, M2932. 1 Hybridization with primary antibodies was omitted in control experiments on verification of the specificity of the PrP binding. The membrane was washed 3 times with 20 ml of TBST for 20 min and incubated with peroxidase-conjugated AffmiPure donkey anti-mouse IgG (H+L) (DAM-HRP) i with minimal cross-reaction to bovine, chicken, goat, guinea pig, Syrian hamster, horse, human, rabbit, and sheep serum proteins at a 1/5,000 dilution for 60 min at ambient temperature. All dilutions of detection antibodies were in TBST with 1% casein. After a final washing of the membranes with TBST, prion protein bands were visualized by enhanced chemiluminescence j using the ChemiDoc System g or Bioflex MSI film. k

Mass spectrometry analysis of urine proteins

A urine sample from a scrapie-infected animal was processed as described above and loaded into 2 lanes on an SDS-PAGE gel. One line was analyzed by Western blotting with an anti-PrP monoclonal antibody M2188. A segment in the unprocessed parallel lane was excised according to the position of the immunoreactive band on the Western blot and analyzed by liquid chromatography-tandem mass spectroscopy (LC-MS/MS) at the Eastern Quebec Proteomics Core Facility (Quebec City, Canada). The peptide masses and sequences obtained were matched to proteins using the Mascot Search program (http://www.matrixscience.com).

Quantitative analysis of PrP in urine samples

The amount of PrP in urine samples was estimated by comparison with a known amount of sheep recombinant PrP_25–233 ARQ (alanine-136 arginine-154 glutamine-171). 1 Urine concentrates (equivalent to approximately 2.5 ml of nonconcentrated urine) were tested in parallel with 0.03–4.0 ng of sheep recombinant PrP_25–233. Western blotting was performed as described above. The amount of PrP was quantified by densitometry according to the intensity of PrP bands with known protein concentration using a ChemiDoc System g and a Quantity One 1-D Analysis Software, version 4.4. g Skewness of data was assessed using the Kolmogorov-Smirnov test. The nonparametric Mann-Whitney U-test was used to calculate the statistical difference in the PrP^c concentration between scrapie infected and control animals (MedCalc 4.20 software m ).

Results

Urine samples from scrapie-infected and control sheep were collected, concentrated 250-fold, and analyzed by Western blot with anti-PrP monoclonal antibodies (mAbs). A diffuse protein band (in some preparations a double band) with MW 27–30 kDa was visualized in some urine samples after immunoblotting with anti-PrP mAb M2188 (binds with epitope SRPLIHFG; Fig. 1A). Sheep PrP peptides YYRENMYRY (amino acid positions 129–138) and VVEQMCITQYQRE (amino acid positions 189–202) were identified by LC-MS/MS in an anti-PrP antibody immunoreactive segment of the SDS-PAGE gel. To examine if nonspecific binding of the secondary antibodies to urinary immunoglobulin fragments or contaminating bacterial proteins occurred during the immunoblotting procedure as previously reported, 9,10,23 control immunoblotting experiments were performed using secondary antibodies only (omitting the hybridization step with primary antibodies). No immunoreactive bands were detected with secondary anti-mouse antibodies (Fig. IB) or with non-PrP antibodies (anti-Salmonella mAb M105, data not shown), confirming that PrP^c was detected in sheep urine. Concentrated urine samples were also probed with various anti-PrP monoclonal antibodies (Fig. 2A). Thus, PrP^c in urine was detected by the monoclonal antibody M2197 (binds with epitope HVAGAAAAGAVVG; Fig. 2B), F99/97.6.1 (binds with epitope QYQRES; Fig. 2B), but not after immunoblotting with mAb M2932 (binds with epitope YPPQGGGGW; Fig. 2B). Digestion of concentrated urine samples with proteinase K eliminated the immunostaining of PrP^c on Western blots (Fig. 2A), indicating that PrP^c excreted in urine was in a proteinase K-sensitive form.

Figure 1.

Western blot analysis of concentrated urine specimens from sheep naturally infected with scrapie. A, the immunoblot was probed for PrP^c with an anti-PrP monoclonal antibody M2188 and DAM-HRP as a secondary antibody. B, the immunoblot was probed for nonspecific binding proteins with DAM-HRP only. Line MWM indicates molecular weight markers. The numbers on the left are the molecular mass markers in kilodaltons (kDa). Identification numbers of animals are indicated on the top of the image.

Treatment of concentrated urine samples with PNGase F (an enzyme that removes oligosaccharides from N-linked glycoproteins) resulted in the shift of the molecular weight from 27–30 kDa to 17–18 kDa (Fig. 2C). In contrast, the deglycosylated PrP^c from brain tissue had a molecular weight of approximately 25 kDa (Fig. 2C). After partial cleavage with PNGase F, 3 isoforms of urine PrP^c were identified: 28–30 kDa, 23–24 kDa, and 17–18 kDa (data not shown). These observations suggested that the urinary PrP^c was excreted in a diglycosylated-truncated form. A PK titration (1–50 μg/ml) was performed to compare the protease sensitivity of PrP^c from urine and brain tissue homogenate samples. PrP^c in urine from a scrapie-infected sheep was more PK sensitive than PrP^c in brain tissue homogenate from a scrapie negative sheep (Fig. 3).

It was essential to analyze whether the excretion of PrP^c in urine was associated with the scrapie status of the sheep. The amount of PrP^c in the urine of 49 animals (control group: n = 16; naturally scrapie-infected group: n = 33) was estimated by comparison with known amounts of sheep recombinant PrP in the immunoblot (Fig. 4A, 4B). The limit of detection of recombinant sheep PrP by Western blot was approximately 30 pg. Concentration of PrP^c in control sheep urine samples was found to be 0–0.16 ng/ml (median 0.04 ng/ml, 95% CI for the median 0.00–0.10), after adjusting to the initial nonconcentrated volume of the urine samples. This was lower than the reported average concentration of cellular PrP in sheep milk (∼1 ng/ml) 8 or PrP^c concentration in urine of healthy human individuals (∼7 ng/ml). 18 Concentration of PrP^c in urine samples from the majority (n = 26) of naturally scrapie-infected sheep was in the same range as a control group (median 0.04 ng/ml; 95% CI for the median 0.02–0.10). Seven of 33 animals from the naturally scrapie-infected group had an elevated level of PrP^c in urine (0.6–4.7 ng/ml; Fig. 4C). There was no correlation between the increased PrP^c concentration and the total protein concentration (2.5–38.8 μg/ml; mean 15.4; SD 9.9) in urine samples. No statistically significant difference was found in the PrP^c concentration in the urine of scrapie-infected and control sheep (Mann-Whitney U-test; P = 0.1813). Several animals have been monitored during the course of the disease, and no clear correlation was found between the urinary PrP^c concentration and progress of scrapie-associated clinical symptoms (data not shown).

Discussion

The sheep urine samples were concentrated approximately 250-fold, providing an opportunity to estimate a concentration of PrP in the urine of scrapie-infected and control animals by Western blot. Proteinase K-sensitive PrP (i.e., PrP^c) was identified by a range of anti-PrP monoclonal antibodies that bind to distinct epitopes located at the C-terminus of PrP. Sheep PrP peptides were identified by LC-MS/MS in an anti-PrP antibody immunoreactive segment of the SDS-PAGE gel. No proteins were detected by Western blotting when monoclonal antibodies with other than prion protein specificity were used or when secondary antibodies were applied alone. Therefore, the detection of PrP^c in sheep urine was not a result of the cross-reaction with bacterial proteins or immunoglobulins excreted in urine as was previously reported for the false-positive detection of protease K-resistant PrP in urine samples. 9,10,23,24 Interestingly, the urinary prion protein did not react with anti-PrP mAb M2932, which binds to an N-terminal epitope of PrP. After deglycosylation with PNGase F, urinary PrP^c had a molecular weight of 17–18 kDa, which was lower than the molecular weight of deglycosylated PrP^c from brain tissue (approx. 25 kDa). This combined data suggested that PrP^c was excreted in urine in an N-terminally truncated form. The exact origin of the urinary PrP^c is yet to be determined. The blood-borne PrP^c may filter though the kidney as the molecular weight threshold for protein excretion is ∼40 kDa. In addition, detectable expression of PrP^c in kidneys has been reported 17 ; it is feasible that PrP^c can be shed from the cell surface and contribute to the overall PrP concentration in urine. The N-terminally truncated PrP^c was previously detected in the urine of healthy human individuals 18 and in the ram reproductive fluid along with the C-terminally truncated and full-length PrP^c forms 6 ; only full-length and C-terminally truncated forms of PrP^c were reported in ovine milk, 15 suggesting tissue-specific variations in proteolytic cleavage/shedding of cellular PrP.

Figure 2.

Detection of PrP in urine and brain tissue homogenate samples by anti-PrP monoclonal antibodies (mAbs). A, urine and brain tissue specimens from sheep #539 were treated with proteinase K (50 μg/ml) and subjected to immunoblotting with various anti-PrP mAbs as described in the text. Note that there was no signal in the PK-treated brain tissue homogenate sample and urine samples when immunoblotting was performed with mAb M2932, which binds to an N-terminal part of PrP. B, amino acid sequence of sheep prion protein. Antigenic epitopes of mAbs used in detection of PrP in urine samples are designated in bold; corresponding mAbs are indicated on the top of the sequence. C, urine and brain tissue specimens from sheep #539 were treated with PNGase F and subjected to immunoblotting with mAbs M2188 as described in the text. The minus (—) symbol designates undigested samples; the plus (+) symbol designates samples after enzymatic digestion. The molecular weight markers based on migration of protein standards are indicated on the left in kilodaltons (kDa).

Figure 3.

Proteinase K titration and digestion of urine and brain tissue-derived PrP^c. Total protein concentration in samples was adjusted to 3–4 mg/ml. PrP^c was detected by Western blot using monoclonal antibody M2197 as a primary antibody. Fraction of the proteolysed PrP^c is presented as a function of PK concentrations. Results are expressed as the means ± SD.

Figure 4.

Assay of PrP^c concentration in sheep urine. A, Western blot analysis of concentrated sheep urine samples with anti-PrP monoclonal antibodies M2188 (see text). Identification numbers of animals are indicated on the top of the image. Calibration standards are serial 2-fold dilutions of recombinant sheep PrP (shrecPrP) ranging from 30 to 1,000 pg. Line MWM indicates molecular weight markers. The numbers on the left are the molecular mass markers in kilodaltons (kDa). B, calibration curve for the quantitative Western blot assay of PrP. Densitometric readings of the immunoblot with calibration standards prepared as serial 2-fold dilutions of shrecPrP ranging from 30 to 1,000 pg were performed using a ChemiDoc System and a Quantity One 1-D Analysis Software, version 4.4. C, concentration of PrP^c in sheep urine. Individual results for each urine sample were plotted in a diagram. Group 1: control animals, n = 16; group 2: naturally infected scrapie NMT-positive animals, n = 33. The P value compares the PrP^c concentration in the urine of scrapie-infected and control sheep.

Considerable variation in the amount of excreted urinary N-terminally truncated PrP^c was observed among sheep from a naturally scrapie-infected flock. Thus, the majority of NM-test positive sheep had urinary PrP^c concentration in the same range as control animals: the median value for both groups was 0.04 ng/ml. Seven of 33 NM-test positive animals had an elevated level of PrP in urine: 0.6–4.7 ng/ml. The reason and consequences of the observed variation in urinary PrP excretion in scrapie-infected animals is unclear. No correlation was found between the urinary PrP^c concentration, the total protein concentration in urine samples, and severity of scrapie-associated clinical signs. The authors were unable to detect proteinase K-resistant PrP in scrapie-infected sheep urine samples, possibly as a result of levels being below the limit of detection of this method or the lack of proteinase K-resistant PrP excreted in urine. Attempts of other researchers to identify protease K-resistant PrP in urine samples of TSE-affected animals failed, 9,10,23,24 but urine has been reported to be infective in the case of coincident scrapie infection and chronic kidney inflammation in autoimmune mice 22 and scrapie-infected hamsters. 13 A recent study 26 reported immunohistochemical detection of the disease-associated N-terminally truncated form of PrP in kidneys of 48% of experimentally scrapie-infected sheep cases and of 25% of the natural scrapie cases. BSE infectivity has been detected by mouse bioassay in the kidney of ARQ/ARQ Romney sheep orally infected with BSE. 2 Based on this data, the possibility of TSE infectivity in urine of scrapie-infected sheep has to be carefully investigated. The present study provides experimental evidence of the elevated level of N-terminally truncated PrP^c in urine of 21% of naturally scrapie-infected sheep. Although these results do not provide the basis of a useful diagnostic test for scrapie, it remains to be established by bioassay whether the elevated level of urinary PrP^c is accompanied by scrapie infectivity in urine and potentially contributes to the horizontal transmission of scrapie.

Acknowledgements

The authors are grateful to Brian Cathcart, Pierre Gauthier, and Dean McIntyre for help with sheep urine samples collection; Hongsheng Huang for help with brain tissue homogenates preparation and prion protein quantification; and TSE unit staff for processing biopsies and immunostaining. This work was supported through a collaborative research agreement between Diachemix LLC. Milwaukee, Wisconsin, and the Canadian Food Inspection Agency (RPS#NBR 0421). Copyright by the Crown in right of Canada.

Footnotes

a.

Furosemide Injection Sabex Standard 5% solution, Sabex. Inc., Quebec, Canada.

b.

Millipore, Billerica, MA.

c.

Sigma-Aldrich Co., Oakville, Ontario, Canada.

d.

Pierce, Rockford, IL.

e.

Qbiogene, Carlsbad, CA.

f.

New England BioLabs, Inc., Pickering, Ontario, Canada.

g.

Bio-Rad, Mississauga, Ontario, Canada.

h.

VMRD, Inc., Pullman, WA.

i.

Jackson Immunoresearch Laboratories, Inc., West Grove, PA.

j.

Amersham, Piscataway, NJ.

k.

Clonex Co., Markham, Ontario, Canada.

l.

Alicon AG, Schlieren, Switzerland.

m.

MedCalc Software, Mariakerke, Belgium.

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Prion protein in sheep urine

Abstract

Keywords

Introduction

Materials and methods

Animals

Sheep urine samples collection and processing

Preparation of brain tissue homogenates

Proteinase K digestion

Peptide: N-Glycosidase F treatment of urine and brain PrP

Sodium dodecyl sulfate polyacrylamide gel electrophoresis and Western blot detection of PrP

Mass spectrometry analysis of urine proteins

Quantitative analysis of PrP in urine samples

Results

Discussion

Acknowledgements

Footnotes

a.

b.

c.

d.

e.

f.

g.

h.

i.

j.

k.

l.

m.

References