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Originally published In Press as doi:10.1074/jbc.M409107200 on August 12, 2004

J. Biol. Chem., Vol. 279, Issue 47, 48817-48820, November 19, 2004
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Immunoglobulins in Urine of Hamsters with Scrapie*

Ana Serban{ddagger}, Giuseppe Legname{ddagger}§, Kirk Hansen¶, Nadia Kovaleva{ddagger}, and Stanley B. Prusiner{ddagger}§||**

From the {ddagger}Institute for Neurodegenerative Diseases and Departments of §Neurology, ||Biochemistry and Biophysics, and Pharmaceutical Chemistry, University of California, San Francisco California 94143

Received for publication, August 9, 2004


   ABSTRACT
TOP
 ABSTRACT
INTRODUCTION
 EXPERIMENTAL PROCEDURES
 RESULTS
 DISCUSSION
 REFERENCES
 
In the prion diseases, a prolonged, asymptomatic incubation period precedes the onset of neurologic dysfunction. At present, a noninvasive test is not available for the presymptomatic diagnosis of prion disease, and thus the report of a test for prions using urine has been of great interest (Shaked, G. M., Shaked, Y., Kariv-Inbal, Z., Halimi, M., Avraham, I., and Gabizon, R. (2001) J. Biol. Chem. 276, 31479–31482). Using Western immunoblots with the anti-prion protein (PrP) 3F4 monoclonal antibody and an anti-mouse IgG secondary antibody, a protease-resistant PrP was reported in the urine of Syrian hamsters and humans with prion disease. Here we have demonstrated that this purportedly "protease-resistant PrP" band in the urine of diseased hamsters is detectable using the anti-mouse IgG secondary antibody in the absence of the 3F4 monoclonal antibody. Mass spectrometric analysis identified an immunoglobulin light chain in the band but found no PrP peptides. No similar band was found in the urine of uninfected hamsters or in brain homogenates from normal or prioninfected hamsters. Moreover, the band in the urine of infected hamsters was not detected using two chimeric human-mouse recombinant anti-PrP antibody fragments followed by an anti-human IgG secondary antibody. Our results indicate that the band detected under previously published conditions is due to the cross-reactivity of the anti-mouse IgG antibody with IgG light chains and possibly heavy chain fragments in urine, but not with PrP.


   INTRODUCTION
TOP
 ABSTRACT
 INTRODUCTION
EXPERIMENTAL PROCEDURES
 RESULTS
 DISCUSSION
 REFERENCES
 
Prions cause neurodegeneration in humans and animals (2). The human prion diseases can manifest as hereditary, sporadic, and infectious disorders. Much evidence argues that prions causing bovine spongiform encephalopathy (BSE)1 have been transferred to humans by ingestion of tainted beef products (3, 4). The resulting disease in humans has been designated variant Creutzfeldt-Jakob disease (vCJD), which occurs primarily in teenagers and young adults. Compared with sporadic CJD that accounts for ~85% of all prion disease in humans, vCJD seems to be caused by a strain of prions that is much more lymphotrophic. Evidence for the lymphotropism of vCJD prions comes from studies of the tonsils and appendix, both of which contain readily measurable levels of the disease-causing isoform (PrPSc) of the prion protein (PrP) (5). The lymphotropism of vCJD prions has raised considerable concern about the safety of the blood supply in Great Britain and led the United States to prohibit blood donations from individuals who have lived in Europe for prolonged periods of time (6). Findings that both scrapie and BSE prions in sheep can transmit disease after blood transfusions in sheep are also of importance (7). Recently, a 69-year-old man who had received a blood transfusion 6.5 years earlier died of vCJD (8). The blood donor died of vCJD three years ago. Because of the advanced age of the transfusion recipient, which contrasts with the young age of all other vCJD cases, it has been suggested that this may be the first case of human prion disease transmitted by blood transfusion.

The foregoing observations argue for the need for a test for prion infection in both humans and animals when they are still asymptomatic. The report of a protease-resistant PrP molecule in the urine of Syrian hamsters, cattle, and humans with prion disease was met with enthusiasm because it seem to herald a means of detecting prions in asymptomatic livestock and humans, perhaps years before they manifest illness (1). It is noteworthy that prion infectivity was not detected in urine. Other approaches to developing antemortem tests include measuring PrPSc in muscle (9–11) and detecting the protease-sensitive form of PrPSc in serum of hamsters (12).

We report here that the Western blot signal of a protease-resistant molecule of ~33 kDa is present as previously described in the urine of prion-infected hamsters and is absent from the urine of uninfected controls (1). Unexpectedly, we found that this ~33-kDa molecule is not PrP but seems to be composed of IgG light chains.


   EXPERIMENTAL PROCEDURES
TOP
 ABSTRACT
 INTRODUCTION
 EXPERIMENTAL PROCEDURES
RESULTS
 DISCUSSION
 REFERENCES
 
Animals and Inocula—Syrian hamsters (LVG:Lak) were inoculated intracerebrally with Sc237 prions. They were sacrificed after 60–65 days, and their brains were collected and immediately frozen in liquid N2.

Urine Collection—Urine from normal and hamsters inoculated with prions 60–65 days earlier was collected overnight in metabolic cages (Rodent Metabolic Cage; Nalgene, Naperville, IL). In the morning, this urine was centrifuged at 500 x g for 5 min and then dialyzed against 0.85% NaCl with three changes as described (1). The dialysis bags were 6000–8000 molecular weight cutoff, either CelluSep T2 (Membrane Filtration Products, Seguin, TX) or Spectra/Por (Spectrum Chemical). After this stage, the samples were aliquoted and kept at –80 °C.

Sample Preparation—Each urine sample consisted of 2 ml of dialyzed urine from normal or ill Syrian hamsters. The urine sample was concentrated by methanol precipitation at a ratio of 1:10 (v/v), for 2 h at –20 °C and centrifuged at 2500 x g for 30 min. The pellet was resuspended in 500 µl of STE buffer (10 mM Tris-HCl, pH 7.5, 10 mM NaCl, 1 mM EDTA, 2% Sarkosyl) (1). Each sample was divided into two equal parts. One half was digested with proteinase K (PK) (Invitrogen) at a final concentration of 40 µg/ml for 60 min in a water bath at 37 °C. The reaction was stopped with 2 mM phenylmethylsulfonyl fluoride. The other sample was kept as an undigested control.

Ten percent (w/v) brain homogenates were prepared from hamsters killed in the terminal stages of disease. The brains were homogenized in phosphate-buffered saline without calcium and magnesium chloride (Invitrogen) with 2% Sarkosyl by three 15-s strokes of a PowerGen homogenizer (Fisher Scientific International, Tustin, CA). The homogenate was centrifuged at 500 x g for 5 min, and the supernatant was used for the preparation of the sample.

SDS-PAGE/Western Blotting—The urine was analyzed on a 12% SDS-PAGE. We added an equal volume (250 µl) of 2x sodium dodecyl sulfate (SDS) sample buffer with 1.5 M urea to each sample. The samples were boiled for 7 min, cleared by centrifugation for 1 min in the tabletop microfuge, and 60 µl was applied to the gel. SDS-PAGE was performed in a 1.5-mm thick, 12% polyacrylamide gel and then electrotransferred to a cellulose nitrate membrane (Hoefer/Pharmacia Biotech, San Francisco, CA). The membrane was blocked for 30 min with 5% nonfat milk in Tris-buffered saline with 0.05% Tween 20 (TBST) at room temperature. As suggested to reduce the background due to non-specific IgG in the urine (1), we performed a second blocking with a mixture of goat anti-rabbit IgG heavy and light chain (H&L) and rabbit anti-mouse IgG (Pierce), both diluted 1:3000 in TBST. We developed the blot with the primary monoclonal antibody (mAb) 3F4 (13), diluted 1:5000, followed by the secondary antibody goat anti-mouse IgG (H&L) conjugated to alkaline phosphatase (AP) (Promega, Madison, WI) diluted 1:7500 in TBST. The substrate was the Lumi-Phos WB chemiluminescent substrate (Pierce). The blots were exposed to CL-Xposure film (Pierce).

Electrospray Ionization Mass Spectrometry—In-gel digestion was carried out by excising bands from the unstained half of the gel. After reduction and alkylation, gel slices were resuspended in ~25 µl of 5 ng/µl trypsin (Promega) and digested for 12 h at 37 °C. Peptides were extracted from gel slices using 50% acetonitrile, 2% formic acid; sample volumes were concentrated and resuspended in 0.1% formic acid. The extracted peptides were injected onto a reverse-phase column using a nanoflow (~350 nl/min) HPLC system (Eldex, Napa, CA). The column effluent was coupled directly via a fused silica capillary transfer line to a LTQ linear ion trap mass spectrometer (Thermo Finnegan, San Jose, CA) equipped with a nanospray ion source. Liquid chromatography runs of 50-min duration were monitored by sequentially recording the precursor scan (MS), a zoom scan to allow for correct charge state assignment (narrow window MS) followed by collision-induced dissociation acquisitions (MS/MS). Automatic gain control was used to define acquisition times, with the average for all three scan types taking ~1.5 s. Singly charged ions were excluded from collision-induced dissociation selection. Normalized collision energies were employed using nitrogen as the collision gas.

For database searches, the Mascot Distiller program (Matrix Science, Boston, MA) was used to create centroided peak lists from the raw spectra. These peak lists were submitted for database searching using an in-house Mascot server (Version 2.0; Matrix Science). In these searches, we allowed mass tolerances of ± 2.0 Da for MS peaks and ± 0.5 Da for MS/MS fragment ions. Samples were searched against all species entries in the UniProt (www.pir.uniprot.org) and nrNCBI (www.ncbi.nlm.nih.gov) databases. The modifications allowed in the searches included oxidation of methionine residues, N-acetylation of proteins, pyroglutamic acid formation at the peptide N termini, and up to two trypsin missed cleavages. Homology searches were carried out using the program MS-Homology (proteinprospector.ucsf.edu, in-house version 4.7).


   RESULTS
TOP
 ABSTRACT
 INTRODUCTION
 EXPERIMENTAL PROCEDURES
 RESULTS
DISCUSSION
 REFERENCES
 
Protease-resistant Proteins in Urine of Hamsters—Urine samples were collected from normal and symptomatic Syrian hamsters. For comparison, we prepared brain homogenates from Syrian hamsters dying of prion disease. We evaluated these samples in parallel on a Western blot, which was developed with the 3F4 mAb (13), followed by immunostaining with a goat anti-mouse IgG-AP secondary antibody (Fig. 1A). A control gel was probed with only the secondary antibody (Fig. 1B).



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  		FIG. 1.
Western immunoblot of PrP in urine of normal and prion-diseased Syrian hamsters compared with brain homogenates of ill hamsters. A, Western blot probed with mAb 3F4 followed by the goat anti-mouse IgG-AP secondary antibody. B, Western blot probed only with the goat anti-mouse IgG-AP secondary antibody used as a control. C, Western blot probed with 3F4 mAb followed by the goat anti-mouse IgG-horseradish peroxidase secondary antibody. The minus (–) symbol designates undigested samples; the plus (+) symbol represents samples after limited PK digestion. Paired lanes are lanes 1, 4, urine from normal hamsters; lanes 2, 5, urine from ill hamsters; lanes 3, 6, 10% brain homogenates from hamsters with prion disease. Molecular mass markers based on migration of protein standards are shown in kDa.

 
At 29 kDa, a protease-sensitive band was present in the normal urine (Fig. 1A, lane 1), whereas urine from ill hamsters had two strong bands, at 29 and 33 kDa (Fig. 1A, lane 2). Although the 29-kDa band in the urine of ill hamsters was protease-sensitive like that in normal urine, it is unclear if the 33-kDa band is resistant to digestion. The 33-kDa band seen after digestion (Fig. 1A, lane 2) is narrower than the band before digestion, indicating that it is less heterogeneous. Possibly, both the 33-kDa band seen after digestion and the faint 34-kDa band above it are generated from larger proteins during limited proteolysis. Brain homogenates from ill hamsters presented the typical pattern with the characteristic shift in molecular mass after limited proteolysis (Fig. 1A, lane 3).

Surprisingly, the same signals were detected on the control blot that stained with only the secondary antibody (Fig. 1B). The protease-sensitive, 29-kDa band and the protease-resistant 33-kDa band were found in the urine of both normal and diseased hamsters with the secondary antibody alone. In contrast, we detected neither full-length PrPSc nor N-terminally truncated PrPSc (PrP 27–30) in diseased brain homogenates subjected to limited PK digestion using the secondary antibody alone (Fig. 1B, lane 6).

When we developed the Western blot with the goat anti-mouse IgG conjugated to horseradish peroxidase, the same pattern was observed, with a weaker signal corresponding to the 33-kDa band (Fig. 1C). This was likely due to the low concentration of antigen in the urine sample and the lower sensitivity of the horseradish peroxidase system.

PrP Signals Were Not Detected Using Recombinant Fabs That Bind PrP—To characterize the 33-kDa band detected in the urine of ill hamsters, a Western blot was developed with two antibody fragments (Fab) known to react avidly with Syrian hamster PrP. These mouse Fabs were isolated by phage display (14) and then engineered into an expression vector that created human-mouse (HuM) chimeras denoted HuM-D18 and HuM-P (Refs. 15 and 16, respectively). HuM-P binds to an epitope comprised of PrP residues 96–105 (16), which is adjacent to the 3F4 mAb epitope at residues 104–113 (17, 18). HuM-D18 binds to an epitope in the region of PrP between residues 133–157 (15). Both Fabs were detected with an anti-human IgG secondary antibody.

HuM-P recognized PrPSc in hamster brain homogenates but did not detect PrP in any of the urine samples (Fig. 2A, left panel). Similarly, the Fab HuM-D18 did not detect any PrP in the urine samples but clearly recognized PrPSc in brain homogenates (Fig. 2B, left panel). In contrast to the results presented in Fig. 1B, the goat anti-human IgG secondary antibody alone did not detect any macromolecules in the urine of hamsters (Fig. 2, A and B, right panels).



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  		FIG. 2.
Western immunoblot of PrP from urine of normal and prion-diseased Syrian hamsters compared with brain homogenates of ill hamsters. A, Western blot probed with recombinant Fab HuM-P followed by the goat anti-human IgG (Fab')2-AP secondary antibody. The right panel was blotted with the secondary antibody alone. B, Western blot probed with recombinant Fab HuM-D18 followed by the goat anti-human IgG (Fab')2-AP secondary antibody. The right panel was developed with the secondary antibody alone. The minus (–) symbol designates undigested samples; the plus (+) symbol represents samples after limited PK digestion. Paired lanes are lanes 1, 4, urine from normal hamsters; lanes 2, 5, urine from ill hamsters; lanes 3, 6, 10% brain homogenates from hamsters with prion disease. Molecular mass markers based on migration of protein standards are shown in kDa.

 
Mass Spectrometric Identification of {kappa} Light Chain Peptides—Urine from ill hamsters was digested with PK (40 µg/ml) for 60 min at 37 °C. The digested sample was loaded into two lanes on an SDS-PAGE gel. One lane was analyzed by Western blotting to establish approximate molecular mass. At ~33 kDa in an unstained parallel lane, a 20-mm segment was excised. From this segment, 10 equal bands were cut and numbered from 1 to 10 from lowest to highest molecular mass (bottom to top of the gel segment). These 10 samples were placed in individual tubes and subjected to in-gel reduction, alkylation, and digestion with trypsin. The 10 samples were sequentially analyzed by tandem mass spectrometry, resulting in ~5000 collision-induced dissociation sequence spectra that were submitted for protein database searching. At this time, the databases used for these searches contain no hamster immunoglobulin sequences; therefore, any matches are from completely homologous peptide sequences of other species. Four peptides extracted from band 4 were identified as belonging to immunoglobulin light chain from various species. Two of the spectra that gave unambiguous identifications for the peptides LLIYWASTR and DSTYSLSSTLTLTK are shown in Fig. 3.



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  		FIG. 3.
Mass spectra of {kappa} light chain peptides. A, product ion spectra (MS/MS) used to identify the sequence LLIYWASTR, which matches the Uniprot entry for human Ig {kappa} chain. The probability that the matched sequence is a random event is 4.5e–06. B, spectra used to identify the sequence DSTYSLSSTLTLTK matching the nrNCBI database entry for mouse Ig {kappa} chain. The peptide DSTYSMSSTLTLTK appears to be the most common homologue of this peptide, with 281 entries in the nrNCBI database almost exclusively part of the light chain sequences. The probability that the matched sequence is a random event is 3.5e–05. Only selected ions are labeled for simplicity. The superscript o on b ions represents the loss of H2O.

 
To confirm that the peptide sequence is unique to the immunoglobulin {kappa} chain, these peptides were submitted to a protein homology database search. With no species specified and allowing for isoleucine/leucine substitutions, 501 (Ile -> Leu) and 1 (Leu -> Ile) peptides from the nrNCBI database were found to match the two sequences. All peptides appeared to be derived from immunoglobulin light chains from various species. None of the database searches or manual data analysis resulted in the identification of any PrP peptides even when using very low statistical significance threshold criteria (p < 0.5).


   DISCUSSION
TOP
 ABSTRACT
 INTRODUCTION
 EXPERIMENTAL PROCEDURES
 RESULTS
 DISCUSSION
REFERENCES
 
The development of antemortem tests for prion diseases is of utmost importance. Such tests would have immediate application in the routine testing of human blood and the testing of livestock, such as cattle and sheep, as well as free-ranging animals, including deer and elk. Thus, the report of a protease-resistant form of PrP in the urine of Syrian hamsters, cattle, and humans with prion disease was met with considerable enthusiasm (1). Interestingly, no prion infectivity could be recovered from the urine of Syrian hamsters infected with Sc237 prions (1).

Multiple attempts to identify the protease-resistant form of PrP reported to be present in urine of animals and humans with prion disease (1) were initially judged to be reproducible in our studies of urine collected from prion-infected Syrian hamsters. Western blots developed with the anti-PrP 3F4 mAb showed the presence of a PK-resistant macromolecule in the urine of ill hamsters. However, when we analyzed the Western blot with the anti-mouse IgG secondary antibody alone, we detected the same signal. This indicates that the band detected on Western blot was not PrP as initially thought but is a protein or another macromolecule that shares epitopes with mouse IgG. That the band is likely to be mouse IgG is supported by additional studies in which an anti-human IgG secondary antibody was used and no signal was detected. We also found no immunoreactivity with the anti-human IgG secondary antibody, which we used for the detection of two different anti-PrP recombinant HuM chimeric antibodies.

Among all the antibodies used in our studies, only the anti-mouse IgG secondary antibody detected a macromolecule in the urine of prion-infected hamsters. This finding argues for cross-reactivity with a non-PrP hamster antigen. Furthermore, we found by means of electrospray ionization mass spectrometry that this band contains immunoglobulin light chain fragments. We suggest that this band might be Bence Jones protein. The Bence Jones protein is a mixture of aggregated {kappa} and {lambda} light chains from immunoglobulins (19). The molecular mass of the Bence Jones protein varies from low molecular mass fragments to 22-kDa monomers or 44-kDa dimers (20). It has been documented that the Bence Jones proteins or light chain immunoglobulins form amyloid structures, which can be stained with Congo red (21). Polymerization of such proteins into amyloid might explain the apparent protease-resistance of these urinary proteins.

It is unclear why Bence Jones proteins should accumulate in the urine of humans, cattle, and rodents infected with prions. Whether prions cause a nephrotic syndrome or central nervous system damage leading to inactivity that, in turn, results in a nephrotic syndrome remains uncertain.

The report of a protease-resistant PrP in urine prompted speculation that urine may carry infectious prions and that horizontal transmission of scrapie in flocks or chronic wasting disease in herds of deer may have occurred through prion contamination of pastures via urine (22, 23). To date, no one has convincingly identified prions in urine, but a much more likely source of prions is fecal matter.

Miyazawa et al. (24) reported finding protease-resistant PrP in urine of seven of nine CJD patients examined. Possibly, these investigators did not perform control studies in which they omitted the primary anti-PrP antibody. Furukawa et al. (25) reported their experience with patients suffering from CJD, Alzheimer's disease, and non-dementing disorders as well as healthy controls. Twenty-nine of 38 CJD patients had protease-resistant IgG bands in urine, similar to results reported here for urine of Syrian hamsters inoculated with prions; 2 of 19 non-demented patients were also positive. None of the Alzheimer's disease patients (n = 20) and healthy controls (n = 19) exhibited protease-resistant IgG bands in urine. Those findings and these reported here provide a compelling argument that if urine contains any protease-resistant PrP, then the levels must be quite low. Moreover, it seems doubtful that measuring protease-resistant IgG bands in urine will form the basis for a useful diagnostic test for prion disease. Such measurements will be fraught with all the difficulties encountered by medical investigators who have attempted to use elevated levels of the stress protein 14–3-3 in cerebrospinal fluid as a diagnostic marker for prion disease (5, 26, 27).


   FOOTNOTES
 
* This work was supported by National Institutes of Health Grants AG02132 and AG010770 as well as by a gift from the G. Harold and Leila Y. Mathers Charitable Foundation. A. Serban, G. Legname, and S. B. Prusiner have financial interest in InPro Biotechnology. The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact. Back

** To whom correspondence should be addressed: Institute for Neurodegenerative Diseases, Box 0518, University of California, San Francisco, CA 94143-0518. Tel.: 415-476-4482; Fax: 415-476-8386; E-mail: stanley{at}ind.ucsf.edu.

1 The abbreviations used are: BSE, bovine spongiform encephalopathy; vCJD, variant Creutzfeldt-Jakob disease; PrP, prion protein; PrPSc, disease-causing isoform of PrP; mAb, monoclonal antibody; AP, alkaline phosphatase; PK, proteinase K; Fab, antibody fragment; HuM, human-mouse; MS, mass spectrometry. Back


   ACKNOWLEDGMENTS
 
We thank the staff at the Hunters Point Animal Facility and Dr. Ruth Gabizon for many helpful discussions.



   REFERENCES
TOP
 ABSTRACT
 INTRODUCTION
 EXPERIMENTAL PROCEDURES
 RESULTS
 DISCUSSION
 REFERENCES
 

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