Analysis of mammalian scrapie protein by novel monoclonal antibodies recognizing distinct prion...

Brain Research Bulletin 65 (2005) 155–162

Analysis of mammalian scrapie protein by novel monoclonal antibodiesrecognizing distinct prion protein glycoforms: an immunoblot and

immunohistochemical study at the light and electron microscopic levels

Andrea Matuccia, Gianluigi Zanussob, Matteo Gelatib, Alessia Farinazzob, Michele Fiorinib,Sergio Ferrarib, Giancarlo Andrighettoa, Tiziana Cestaria, Maria Caramellic,

Alessandro Negrod, Michela Morbine, Roberto Chiesaf,Salvatore Monacob, ∗, Giuseppe Tridentea

a Section of Immunology, Department of Pathology, University of Verona, Policlinico G.B. Rossi, P. le L.A. Scuro 10, 37134 Verona, Italyb Section of Neurology, Department of Neurological and Visual Sciences, University of Verona, Policlinico G.B. Rossi,

P. le L.A. Scuro 10, 37134 Verona, Italyc CEA, Turin, Italy

d CRIBI Biotechnology Centre, University of Padua, Padua, Italye Istituto Neurologico, C. Besta, Milan, Italy

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Dulbecco Telethon Institute (DTI) and Istituto di Ricerche Farmacologiche Mario Negri, Milan, Italy

Received 27 October 2004; received in revised form 24 December 2004; accepted 30 December 2004

bstract

The availability of specific monoclonal antibodies (mAbs) recognizing the aberrant form (PrPSc) of the cellular prion protein (PrPC) inifferent mammalian species is important for molecular diagnostics, PrPSc typing and future immunotherapy. We obtained a panel of antonoclonal antibodies in PrP0/0 knock-out mice immunized with recombinant human PrP23–231. Two mAbs, recognizing PrP epitopes in-helix 1 (mAb SA65) and�-helix 2 (mAb SA21) regions, immunoreacted with PrPC and PrPSc and its proteolytic product, PrP27–30, frouman, murine, bovine, caprine and ovine brains by Western blot. Remarkably, mAb SA21 recognized unglycosylated and monogrP with the second site occupied by glycan moieties, but not monoglycosylated PrP with the first consensus site occupiedlycosylated species. Immunoblots with mAb SA21 disclosed that PrP glycosylated at the second site accounted for the slowe

orm of the customary monoglycosylated PrP doublet. mAb SA65 immunolabelled all PrP glycoforms by Western blot and was highln detecting tissue PrP by immunohistochemistry in light microscopy and in immunoelectron microscopy. These novel anti-PrP mAools to investigate the subcellular site of PrP deposition in mammalian prion diseases and may also contribute to assess the rolerP glycoforms in human and animal prion diseases.2005 Elsevier Inc. All rights reserved.

eywords: Prion protein; Transmissible spongiform encephalopathies; Sporadic Creutzfeldt–Jakob disease; Bovine spongiform encephalopathrP fragments

. Introduction

Transmissible spongiform encephalopathies (TSEs) areammalian neurodegenerative disorders, which include

∗ Corresponding author. Tel.: +39 045 8074285; fax: +39 045 585933.E-mail address:[email protected] (S. Monaco).

Creutzfeldt–Jakob disease (CJD) in humans, scrapie inand goat and bovine spongiform encephalopathies in c[4,21]. The causative agent of TSEs is thought to consistaberrant isoform (PrPSc) of the cellular prion protein (PrPC),termed prion[20]. PrPSc is generated from the host-encodPrPC by co- and post-translational modifications leadto selection of given PrP glycoforms and conformatio

361-9230/$ – see front matter © 2005 Elsevier Inc. All rights reserved.oi:10.1016/j.brainresbull.2004.12.008

156 A. Matucci et al. / Brain Research Bulletin 65 (2005) 155–162

changes in the PrP backbone. Although PrPC and PrPScsharethe same amino acid sequence, distinctive physicochemicalcharacteristics differentiate the two isoforms[14,20]. As op-posed to PrPC, PrPSc has a high�-sheet content, is insolublein non-denaturing detergents and is partially resistant to pro-tease treatment[14].

A definite molecular diagnosis of TSE is obtained fol-lowing the demonstration of PrPSc in affected tissues byimmunoblotting and conformation-dependent immunoassay,whereas the diagnostic use of antibodies immunocapturingexclusively PrPSc but not PrPC is under scrutiny[12,15,17].In different TSEs, PrPSc shows distinct conformations, de-duced by the size of the unglycosylated PrP27-30 fragment,and strain-specific glycosylation patterns[6,18,22,27]. PrPSc

typing represents a useful mean of assessing strain signaturein prion diseases, and is currently used to screen small rumi-nants for the presence of bovine spongiform encephalopathy(BSE)[13]. In the recent past, this biochemical approach hasbeen of crucial importance in assessing a link between BSEand variant CJD[6]. However, the search for molecular simi-larities among human, bovine, caprine and ovine prion strainsrequires the use of appropriate antibodies which have beenobtained only in a few studies[13,24,29].

In the present study, we generated anti-PrP monoclonalantibodies (mAbs) by immunizing PrP knock-out mice withr P,t t andc PrPb dis-c conds

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hypoxantine/aminopterin/thymidine-containing media. Hy-bridoma cell supernatant was screened by ELISA. Ninety-six-well plates (Maxisorp, Nunc, Denmark) were coatedovernight with 100�l of a PrP23–231 solution (1�g/ml di-luted in carbonate buffer pH 9.6). Wells were blockedwith 3% bovine serum albumin (Sigma) for 2 h at 37◦C,washed in phosphate buffered saline (PBS) and incubatedwith 100�l of undiluted conditioned supernatant at 37◦Cfor 30 min. After washing, a horseradish peroxidase (HRP)-conjugated goat anti-mouse antibody (1:5000, Sigma) wasadded for 30 min. The reaction was visualized by 3,3′,5,5′-tetramethylbenzidine ELISA-substrate (Sigma) and blockedwith 1 M sulfidric acid. ELISA plates were read at 450 nmon Titertek Multiscan Spectrophotometer (ICN Biomedicals,Milan, Italy). Seventy positive clones were identified at thefirst screening, whereas at follow-up only 35 clones retainedtheir specificity. Two of these clones were chosen for fur-ther studies and their isotype was determined by IsoStripIsotyping kit (Roche). All procedures involving the animalswere performed under authorization of the Italian Ministry ofHealth, in accordance with NIH Guide for the USE and Careof Laboratory Animals, and with the European CommunitiesCouncil Directive (86/609/EEC).

2.3. Double immunofluorescent staining andfl

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ecombinant human PrP23–231. In addition to human Prhese new mAbs cross-reacted with PrP from sheep, goaows and appeared useful for the immunodetection ofy post-embedding immunoelectron microscopy and inriminating monoglycosylated PrP species with the seite occupied.

. Materials and methods

.1. Brain tissues

Pathologically and biochemically definite sporadic CsCJD) cases for the present study have been previescribed[28]. Frozen and formalin fixed brain tissu

rom cattle affected by bovine amyloidotic spongifoncephalopathy (BASE) and BSE[4] and from sheep anoats affected with scrapie[26] were provided by thurveillance Center for Animal TSEs (CEA, Turin, Italy)

.2. Generation of mAbs

Twelve-week-old male C57BL/6J× CBA/JPrn-p0/0mice3,5] were subcutaneously and intraperitoneally immunith 100�g of recombinant human PrP23–231emulsified inreund’s adjuvant (Sigma, Poole, Dorset, UK). Four defore somatic fusion, each immunized mice was boo

ntravenously with 90�g of protein in 100�l PBS. Miceere sacrificed and single cell spleen suspensions

used with murine X63-Ag8.653 myeloma and cultivate

uorescence-activated cell sorting (FACS) analysis

A human leukocyte suspension containing 1,000ells was incubated with fluorescein isothiocyanate-labffinity-purified mAbs and phycoerythrin-conjugated mclonal anti-mouse surface leukocyte markers on ice5 min. Cells were washed and fixed in 1% paraformayde and analyzed in a Coulter EPICS XL cytometer (C

er, Fullerton, CA). Each sample contained at least 5000nd experiments were repeated three times.

.4. Epitope mapping

The epitopes recognized by different antibodies wereermined by using nine synthetic 20-residue overlapeptides, corresponding to amino acids 90–110, 105–20–140, 135–155, 150–170, 165–185, 180–200, 195nd 210–231 of human PrP (Primm, Milan, Italy). Peptere dissolved in carbonate buffer, pH 9.6, and used as

ng peptides in the direct ELISA test described above.

.5. Western blot analysis

Brain tissues from humans with sCJD, sheep and gith scrapie, cattle with BSE and BASE, and normal c

rols, including golden hamster and cat, were homogenn 9 volumes of lysis buffer (100 mM sodium chloride, 10 mDTA, 0.5% Nonidet P-40, 0.5% sodium deoxychol0 mM Tris, pH 7.4). Aliquots of brain homogenates wigested with proteinase K (PK) (100�g/ml) and incubatet 37◦C for 1 h. PK digestion was terminated by the

A. Matucci et al. / Brain Research Bulletin 65 (2005) 155–162 157

dition of phenylmethylsulfonyl fluoride to 3 mM. Samples,equivalent to 300�g of wet tissue, were resolved on 12%sodium dodecyl sulfate-polyacrylamide gel electrophoresis(SDS-PAGE) gels, and then transferred onto polyvinylidenedifluoride membrane (Immobilon P, Millipore) for 2 h at 60 V.Membranes were blocked with 1% non-fat dry milk in TBST(10 mM Tris, 150 mM sodium chloride, 0.1% Tween-20, pH7.5) for 1 h at 37◦C and incubated overnight at 4◦C withdifferent mAbs. After washing in TBST, membranes wereincubated with peroxidase-labeled anti-mouse immunoglob-ulins (1:3000, Amersham, Uppsala, Sweden). Blots were de-veloped with an enhanced chemiluminescence system (ECL,Amersham) and PrP visualized on an autoradiography film(Hyperfilm, Amersham).

2.6. Immunohistochemistry

Brain sections from human and animal controls and TSE-affected subjects were processed for immunostaining afterhydrolytic autoclaving. The sections were deparaffinized, re-hydrated and immersed in 98% formic acid for 1 h at roomtemperature. Endogenous peroxidase was blocked by im-mersion in 8% hydrogen peroxide in methanol for 10 min.Sections were immersed in 1.5 mM HCl and autoclaved at121◦C for 10 min. After rinsing, slides were incubated withd ncu-b 1 ha olu-t gt opedw withh

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onsw ickelg min.A acid

for 10 min. Residual aldehyde groups were quenched with0.05 M glycine (Sigma) in PBS, pH 4, for 5 min. After rins-ing in PBS, sections were incubated with 10% normal goatserum in PBS for 20 min followed by mAb SA65 (1:20) inPBS overnight at 4◦C. After washing in PBS, grids wereincubated with 18-nm gold-conjugated goat anti-mouse sec-ondary antibody (Jakson ImmunoResearch, West Grove, PA)diluted 1:50 for 3 h. Grids were post-fixed in 1% glutaralde-hyde and in 1% OsO4, counterstained with uranile acetate andlead citrate and viewed under a EM109 electron microscope(Zeiss).

3. Results

3.1. Immunofluorescent staining of PrPC in humanleukocytes

mAb SA65 stained about 90% of the total peripheral bloodmononuclear cell (PBMC) population. We used two-colorimmunofluorescent staining and flow cytometry to furtherdefine the expression of PrPC in different cell types. mAbSA65 detected PrPC in 33% of CD3+ T cells, 33% of CD4+

T cells, 20% of CD8+ T cells, 12% of CD19+, 82% of CD45+

and 66% of CD14+ cells. On the contrary, mAb SA21 stainedabout 5% of the total PBMC population, including 4% ofC

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ifferent mAbs overnight. Sections were subsequently iated with a biotinylated goat anti-mouse antibody fort room temperature, and then with the avidin–biotin s

ion (ABC-Elite Kit, Vector lab, Burlingame, CA) accordino the manufacturer’s instruction. Samples were develith 3,3′-diaminobenzidine (Sigma) and counterstainedaematoxylin.

.7. Immunohistochemistry on semithin sections andmmunoelectron microscopy

Brain tissues from controls and TSE-affected subjexed in formaldehyde, were washed in 0.1 M phospuffer, pH 7.4, and post-fixed with 2.5% glutaraldehypecimens were dehydrated in acetone and embeddpurr resin. Semithin sections (2�m-thick) were collecte

n Vectabond (Vector Laboratories, Burlingame, CA)-coalides and dried at 60◦C. Slides were immersed in Potassiethoxide/18-Crown-6, aqueous dimethyl sulfoxide s

ion for 2 h, washed in methanol and rehydrated in ethaections were treated with 98% formic acid for 10 m

insed and incubated with anti-PrP SA65 mAb (1:100). Sequently, slides were incubated with a biotinylated aouse IgG secondary antibody (Vector Laboratories) fot room temperature and with avidin–biotin-complex (Alite, Vector Laboratories). The reaction was visualized u,3′-diaminobenzidine as a chromogen.

For immunoelectron microscopy, 80 nm-thick sectiere placed on 200 mesh formvar-carbon coated nrids (EMS) and etched in 1% sodium periodate for 60ntigen expression was enhanced with 40% formic

D45+ and 2% of CD14+ cells (Fig. 1).

.2. Immunoblot analysis

We characterized mAb reactivity by Western blot anis of normal and TSE brain tissues from humans andals. mAb SA65 immunoreacted with different glycoforf the full length PrPC from human, cattle, sheep, goat aouse brains, migrating in a molecular mass range bet7 and 35 kDa (Fig. 2A), as well as from golden hamsnd cat (data not shown). In addition, mAb SA65 starP27–30 molecules from different sCJD subtypes, cSE and BASE, and sheep and goat scrapie (Fig. 2B). Note-orthy, in subjects with classical sCJD, mAb SA65 decorrecently identified 17-kDa truncated PrP fragment[27], thusroviding an additional marker for PrPSc typing in sCJD. Inifferent human and animal TSEs, distinct migration pattnd glycosylation state of each disease-associated PrPSc typeere revealed by SA65, as well as molecular similarities

ween sCJD cases with type 2 PrPSc and cattle BASE.The second antibody, mAb SA21, recognized unglyc

ated PrP and the upper band of the monoglycosylatedoublet, but not the highly glycosylated forms immunodrated by mAb SA65, either in normal brains or in differuman and animal prion diseases (Fig. 2C–E). In keepingith recent observations[16], the slower electrophoretic mration of the monoglycosylated PrP species recognizeAb SA21 is consistent with this form being glycosylat the second consensus site, which contains tri- andntennary glycans in higher amounts than the first conseite[23].


Fig. 1. Detection of PrPC on the cell surface of human PBMCs as analyzed by FACScan. PrP expression was observed in cell lines exhibiting positivity toCD3, CD4, CD8, CD14, CD19 and CD45 by double direct immunolabeling.


Fig. 2. Western immunoblot detection of specificity of PrPC and PrP27–30in different species. (A) Homogenates from human, cattle, sheep, goat andmouse normal brain immunoblotted with mAb SA65 (diluted 1:5000) showthe presence of the PrPC triplet in all examined species, (B) proteinase K-treated brain samples from classical (type 1 PrPSc) and ataxic (type 2 PrPSc)sCJD cases, cattle BSE and BASE, and from scrapied sheep and goats, showthe presence of different PrPSc types at immunoblot with mAb SA65 diluted1:5000, (C) the same brain samples as in (B) immunoblotted with mAbSA21 (diluted 1:5000) resulted in immunolabeling of the unglycosylatedand slower migrating monoglycosylated PrP27-30 bands, but not of highlyglycosylated species, (D and E) comparison of the banding patterns obtainedin sCJD with type 1 and type 2 PrPSc with mAbs SA65 and SA21.

3.3. PrP epitopes recognized by SA65 and SA21 mAbs

Based on the pattern of mAb SA65 and SA21 immunore-activity at immunoblot analysis of PrPC and PK-resistant PrPforms, we used a gridded array of 20 amino acid peptides,corresponding to human PrP90–231, to assess the sequencerecognized by each antibody. By direct ELISA, mAb SA65immunoreacted with the sequence 135–155, but not 120–140or 150–170, suggesting that the PrP epitope recognized bythis antibody is located within the highly conserved�-helix1 region, between amino acids 141 and 149. On the contrary,mAb SA21, which failed to recognize highly glycosylatedPrP forms and faster migrating monoglycosylated species,was reactive with the PrP sequence 165–185, indicating thatthe epitope recognized is glycan-dependent and included thefirst Asn glycosylation site within the�-helix 2 region.

3.4. Immunohistochemical pattern of mAb SA65

Only mAb SA65 immunostained the disease-associatedform of PrP in tissue sections from sCJD, BSE, BASE andscrapie cases. In sCJD cases with type 1 PrPSc, the typi-cal synaptic-type pattern was observed (Fig. 3A), whereasplaque-like PrP deposition and kuru-type plaques were seenin sCJD cases with type 2 PrPSc (Fig. 3B).

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In BSE brainstem tissues, an intense synapticeposition and a glial pattern were observed (Fig. 3C),hile thalamic sections from cattle with BASE disclosedresence of kuru-type plaques (Fig. 3D). Brainstem section

rom scrapie-affected sheep and goats showed an instarry sky” pattern and PrP deposition around perivascpaces (Fig. 3E and F).

.5. Immunoelectron microscopy

The ultrastructural localization of prion protein in humnd animal TSEs provides valuable information on the paenesis of these disorders[8,9]. Over the course of this studAbs were also selected based on their suitability for pmbedding immunoelectron microscopy techniques. S

ngly, mAb SA65 revealed a high versatility, as evideny the immunostaining pattern obtained in semithin sec

rom a number of sCJD cases (Fig. 4A–C) and animal TSEltrathin sections of cerebral and cerebellar cortices ftaxic sCJD cases showed an expected intense immuneposition on PrP amyloid fibrils of kuru-type plaqueslaque-like deposits. In classical sCJD, PrP immunor

ivity was observed in synaptic terminals and pleomorescicular structures (Fig. 4D and E).

. Discussion

In the present study, we selected and characterizedonoclonal antibodies recognizing PrP from human, bov

vine and caprine brain tissues. One of these antibo


Fig. 3. Immunocytochemistry with mAb SA65 carried out on human and animal brain tissues. Brain areas include cerebellum in classical sCJD (A) andataxic sCJD (B) cases, brainstem in BSE (C), BASE (D), scrapie in sheep (E) and goats (F). Note that PrP is intensely stained by mAb SA65 according todisease-associated deposition patterns. Bar = 50�m.

mAb SA65, showed a high sensitivity for PrPSc by Westernblot, and resulted in intense immunoreactivity of PrP depositsby immunohistochemistry on paraffin- and plastic-embeddedbrain tissues from human and animal TSEs. As expected,mAb SA65 specifically labeled PrP also at the ultrastructurallevel when post-embedding immunodetection was achievedwith colloidal gold staining. We can anticipate that this speci-ficity is currently being used to explore the subcellular local-ization of PrP in the olfactory mucosa of patients undergoingdiagnostic evaluation for sCJD, and to answer key-questionson the peripheral localization of PrP in extraneural tissues.

Epitope mapping studies revealed that mAb SA65 recog-nizes a sequence located within the�-helix 1 region, whichhas been shown to be highly immunogenic when PrP0/0 miceare immunized with full-length PrP; in addition, this sequenceis a highly conserved PrP in humans, rodents and ungulates[1]. Recently, it has been reported that anti-PrP antibodies

recognizing the octarepeat region[19], the 90–109 segment[2], and residues 159–178[10] interfere with PrPSc replica-tion in infected cells. In addition, several lines of evidencessuggest that mAbs and Fab fragments directed against the�-helix 1 region are able to prevent PrPSc replication in vitroand in vivo[7,11]. Therefore, the targeting of helix 1 by mAbSA65, besides its efficient binding of PrPSc, encourages thefuture use of this antibody for prophylaxis or treatment ofprion diseases.

To date, PrPSc represents the only available marker ofprion diseases and the immunobiochemical or immunohis-tochemical demonstration of its presence in affected tissuesis required for a definite diagnosis of human and animal TSEs.Prion diseases occur as different strains which segregate withdistinct molecular types of PrP27–30, currently classifiedaccording to the electrophoretic migration of the protease-resistant unglycosylated PrP fragment and the degree of gly-


Fig. 4. Post-embedding immunoelectron microscopic study with mAbSA65. Toluidine blue (A) and PrP immunoperoxidase staining (B and C)showing the presence of plaque-like PrP deposition in the cerebellar cortexof a subject with ataxic sCJD. Bar = 50�m. Immunogold labelling of pleo-morphic vescicular structures and synaptic membranes in the frontal cortexof a subject with classical sCJD (D and E). Bar = 0.5�m.

cosylation[6,18,22,27]. Here, we showed that mAb SA65allows a direct comparison of PrPSc from different speciesmaking feasible, under strictly controlled conditions, a directmolecular comparison of different mammalian TSE forms.As shown in the present investigation, immunoblots withSA65 confirmed the presence of distinct PrPSc types in cattleBSE and BASE as well as in sheep and goat scrapie, whereasmolecular similarities between PrP molecules of BASE anda sCJD subtype were clearly evident. Moreover, the useful-ness of mAb SA65 in the molecular typing of human sCJDis underscored by the recognition of a newly identified 17-kDa band, which is selectively detected in classical sCJD[27]. Taken together, the foregoing properties make this anti-body particularly suitable for the study of human and animalTSE.

The other selected antibody, mAb SA21, targeted a PrPepitope located in the 165–185 sequence of the unglycosy-lated molecule. Interestingly, this mAb while recognizing PrPin all investigated species, failed to bind PrP with the first gly-cosylation site occupied by glycans.

As a consequence, immunoblot with SA21 exclusively la-beled unglycosylated PrP and its monoglycoform with thesecond site occupied, but not fully glycosylated species. Therecognition by SA21 of the slower migrating monoglycosy-lated PrP isotypes is consistent with a higher glycan amountaT im-i s ther pro-v yco-s rPi ionso yco-s ncm , bea ationf

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ttached to the second site, as opposed to the first site[16,23].he availability of an antibody such as SA21, which discr

nates distinct PrP glycoforms, is very valuable to assesole of glycosylation in prion diseases. mAb SA21 alsoides the unique opportunity to determine how each glylation site is involved in the differential conversion of Pn different TSEs. In prion diseases, strain-specific variatf PrP27–30 glycoforms are influenced by the host glylated PrPC and the PrPSc glycosylation profile, which ioncert, dictate the final glycosylation pattern of PrPSc [25].Ab SA21 and other similar antibodies, might thereforelso of interest to address the issue of PrP glycoform vari

ollowing experimental transmission of prion agents.Taken together, the present antibodies may provide

iagnostic tools for the detection of PrP and the typinrPSc in mammalian prion diseases.

cknowledgements

This work was supported in part by grant FISR 1999 “Puction of monoclonal antibodies for diagnosis and trent of human and animal neurogenerative disorders”ridente, and by grants from Fondazione Cariverona 2Neurodegenerative disorders” and from the Italian Minif Health in collaboration and with the contribution of

stituto Superiore di Sanita (“Search for infection-related eogenous or exogenous factors in the cerebrospinal fluatients with transmissible spongiform encephalopathy”Study of pathogenic mechanisms in neurodegenerativerders for the diagnosis and development of therapeuti


proaches”) to S. Monaco. R. Chiesa is an Assistant TelethonScientist (DTI, Fondazione Telethon).

The authors are grateful to BIOD-srl (Valenzano, Italy)for collaboration in obtaining monoclonal antibodies and toFloriana Gismondi for her help in the immunoelectron mi-croscopic study.

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Analysis of mammalian scrapie protein by novel monoclonal antibodies recognizing distinct prion...

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