Tài liệu Báo cáo khoa học: Binding of N- and C-terminal anti-prion protein antibodies generates distinct phenotypes of cellular prion proteins (PrPC) obtained from human, sheep, cattle and mouse doc

Binding of N- and C-terminal anti-prion protein antibodies generates distinct phenotypes of cellular prion proteins (PrP C ) obtained from human, sheep, cattle and mouse Thorsten Kuczius 1 , Jacques Grassi 2 , Helge Karch 1 and Martin H. Groschup 3 1 Institute for Hygiene, University Hospital Muenster, Muenster, Germany 2 CEA, Service de Pharmacologie et d’Immunologie, CEA ⁄ Saclay, Gif sur Yvette, France 3 Institute for Novel and Emerging Infectious Diseases, Friedrich Loeffler-Institute, Federal Research Centre for Virus Diseases of Health, Greifswald – Isle of Riems, Germany Prion diseases, also known as transmissible spongi- form encephalopathies, are a group of neurodegener- ative disorders affecting both humans and animals. The human forms encompass sporadic and familiar Creutzfeldt–Jakob disease and the new variant Cre- utzfeldt–Jakob disease (vCJD), which has been linked to BSE, the bovine spongiform encephalopathy of cattle [1,2]. Scrapie is the prion disease in sheep and goats. The main characteristic of the disease is the accumu- lation of an abnormal prion protein (PrP Sc ), thought to be the only infectious agent associated with prion neurodegeneration [3]. The pathogenic mechanism is assumed to involve conversion of physiological cellular prion protein (PrP C ) to a pathological isoform (PrP Sc ) accompanied by a conformational change from a largely a-helical form into a b-sheet structure [4]. In contrast to PrP C , the infectious PrP Sc protein is deter- gent-insoluble. PrP C and PrP Sc protein samples can be differentiated by pretreatment with proteinase K (PK), which completely hydrolyses PrP C but only removes 55–70 amino acid residues in the N-terminal region of PrP Sc resulting in a molecular reduction of 6–8 kDa. The western blot method is a useful in vitro assay for the characterization of PrP Sc and PrP C , in which fully glycosylated mouse PrP migrates at 33–35 kDa Keywords antibody; glycotyping; prion protein; PrP C ; signal intensity Correspondence T. Kuczius, Institute for Hygiene, University Hospital Mu ¨ nster, Robert Koch Strasse 41, 48149 Mu ¨ nster, Germany Fax: +49 251 9802868 Tel: +49 251 9802897 E-mail: tkuczius@uni-muenster.de Website: http://www.hygiene.uni-muenster.de (Received 14 July 2006, revised 20 Decem- ber 2006, accepted 12 January 2007) doi:10.1111/j.1742-4658.2007.05691.x Prion diseases are neurodegenerative disorders which cause Creutzfeldt– Jakob disease in humans, scrapie in sheep and bovine spongiform encephalopathy in cattle. The infectious agent is a protease resistant iso- form (PrP Sc ) of a host encoded prion protein (PrP C ). PrP Sc proteins are characterized according to size and glycoform pattern. We analyzed the glycoform patterns of PrP C obtained from humans, sheep, cattle and mice to find interspecies variability for distinct differentiation among species. To obtain reliable results, the imaging technique was used for measurement of the staining band intensities and reproducible profiles were achieved by many repeated immunoblot analysis. With a set of antibodies, we discov- ered two distinct patterns which were not species-dependent. One pattern is characterized by high signal intensity for the di-glycosylated isoform using antibodies that bind to the N-terminal region, whereas the other exhibits high intensity for protein bands at the size of the nonglycosylated isoform using antibodies recognizing the C-terminal region. This pattern is the result of an overlap of the nonglycosylated full-length and the glycosylated N-terminal truncated PrP C isoforms. Our data demonstrate the importance of antibody selection in characterization of PrP C . Abbreviations BSE, bovine spongiform encephalopathy; PK, proteinase K; PrP, prion protein; SAF, scrapie-associated fibril; vCJD, variant Creutzfeldt–Jakob disease. 1492 FEBS Journal 274 (2007) 1492–1502 ª 2007 The Authors Journal compilation ª 2007 FEBS and the nonglycosylated form at 27 kDa on SDS ⁄ PAGE [5]. PrP Sc strains and isolates are distin- guished by the size of their PK resistant core protein because differences in the PK cleavage sites in PrP Sc have been observed in scrapie, experimental scrapie and ruminant BSE [6,7]. PrP Sc exhibit different band- ing patterns following quantitative immunoblotting by densitometry, which reflects differences in the ratios of the di-, mono- and nonglycosylated PrP. In sporadic cases of human Creutzfeldt–Jakob disease, PrP Sc shows a characteristic glycopattern with high signal intensity of the mono-glycosylated isoform which dif- fers from that in ruminant BSE and scrapie PrP Sc .In addition to vCJD, the occurrence of other Creutzfeldt– Jakob disease subtypes with differing glycoprofiles and molecular masses has been postulated [8–10]. The ability of prions to cross species barriers is lar- gely dependent on the PrP C sequence homology of the donor and recipient species [11,12]. In addition to spe- cies-specific characteristics of PrP Sc , there are also notable variations in the glycoform patterns, but the importance of these is not well understood. PrP C is expressed ubiquitously and in a highly conserved form in mammalian species [13,14]. Highest levels were found in neurons and the central nervous system [15,16]. Following expression, PrP C undergoes post- translational modification involving removal of an N-terminal signal peptide and C-terminal residues in the polypeptide chain and attachment of a glycosyl- phosphatidylinositol group for cytoplasmic membrane anchorage [17]. The structure is characterized by an N-terminal domain including octapeptide repeats, a central hydrophobic domain and a C-terminal region with two asparagine-linked glycosylation sites and a disulphide bond between cysteine residues [18]. The role of PrP C in cell function is not known, but it has been associated with synaptic, enzymatic and signaling functions, copper binding and transport [19–21]. Cop- per and heparan sulfate binding have been mediated through its N-terminal domain [22,23]. However, under physiological conditions the N-terminal region can be lost by cleavage [23–28]. From endogenous pro- teolysis, cleavage sites in human PrP C were mapped at amino acids 110–112 and at residues 80–100 generating N-truncated forms; these are referred to as C1 and C2, respectively. The nonglycosylated forms migrate at 18 and 21–22 kDa, respectively [25]. In the past, little attention was given to the banding patterns and different glycoforms of PrP C . In this study, we have analyzed the glycoform patterns of PrP C of human, sheep, cattle and mice and compared them. Variable immunoreactivity of anti-PrP antibod- ies determining different PrP C banding patterns is a feature used especially to find heterogeneity based on protein conformation in one species [29]. Independent of individual brain regions, in this study, we focused our analysis on PrP C glycoform patterns derived from different species which arose from binding of various antibodies recognizing sites in the amino, central or C-terminal PrP C sequence. The aim of the study was therefore to find imposing interspecies variations among human PrP C and PrP C derived from different species in order to find a first onset on the basis of PrP C expression why PrP Sc of human differed from other species. Using a panel of monoclonal antibod- ies we systematically analyzed the formed signal inten- sities of the di-, mono- and nonglycosylated PrP C and the N-truncated isoforms. We found that the mouse PrP C glycoforms differed from human when C-ter- minal PrP binding antibodies were used. This observa- tion was attributed to the proportion of full-length PrP and the truncated isoform, which was predominant in human, sheep and cattle brains. C-ter- minal binding antibodies detect full-length nonglyco- sylated PrP C as well as truncated glycosylated isoforms at the same size. Taken together, first, the banding pattern is largely dependent on the antibody used and, secondly, there are antibodies by which interspecies variations of glycoform ratios are detectable. The findings are important for studies of PrP C function, regulation and expression, as full-length and truncated isoforms of di-, mono- and nonglycosylated proteins are only detect- able with antibodies recognizing the C-terminal region and produce altered expression profiles. Results Proteins of brain homogenates derived from different species were separated on SDS ⁄ PAGE and the specific PrP C signals were detected by the western blot tech- nique. The PrP C banding patterns were analyzed using a set of monoclonal antibodies which recognize various epitopes within the prion protein sequence (Fig. 1 and Table 1). The two bands of higher molecular masses are the di- and mono-glycosylated isoforms and the band with the lowest molecular mass is nonglyco- sylated PrP C . Quantification of the three protein bands was always carried out in the linear range determined using serial dilutions of samples (Fig. 2). Linearity consisting of continuous signal increase and of repro- ducible glycoprotein patterns was determined in the range between 4 and 10 lL of brain homogenate con- firmed by repeated gel runs. Signal intensities therefore were analyzed within continuous, optimal and repro- ducible glycoform patterns. T. Kuczius et al. Glycotyping of PrP C FEBS Journal 274 (2007) 1492–1502 ª 2007 The Authors Journal compilation ª 2007 FEBS 1493 Brain PrP C from humans were detected as two dis- tinct main glycoform patterns, depending on the monoclonal antibody used (Fig. 3). The di-glycosylated isoform was most abundant using antibodies directed against epitopes within the octapeptide or an interme- diary region (i.e. amino acids 59–120, designated here as the N-terminal region), but was much less abundant using antibodies binding to the core region (i.e. amino acids 121–166; C-terminal region). The di-glycosylated band of human PrP C showed the heaviest staining with antibodies binding to the N-terminal region (Fig. 3A,B). For example, mAb SAF34, which recogni- zes the octapeptide sequence, gave a high (50%), an intermediate (29%) and a low (21%) intensity signal with di-, mono- and nonglycosylated PrP C , respect- ively. Similar ratios were obtained with human PrP C and the antibody mAb 8G8 which binds to the inter- mediary region at amino acids 97–102 of human PrP C (Fig. 3A,B). In contrast, deviant profiles were found with antibodies binding to the central region of PrP C, as the signal intensities at the size of the nonglycosylat- ed full-length PrP C (at 27 kDa) were high with monoclonal antibodies 6H4, SAF60 and SAF70 while signals for the di-glycosylated PrP C were low. In these experiments the mono-glycosylated forms of human PrP C were almost invisible and not detectable. Heterogeneity of PrP C proteins is enhanced by endogenous proteolytic modifications, which occurs in vivo [25–27]. PrP C from non-infected brains consists in addition to full-length PrP to a significant amount of an N-terminal truncated PrP C fragment termed C1. Glycosylated C1 protein fragments migrate to a posi- tion around the nonglycosylated full-length PrP C . The degree of truncated PrP C to full-length PrP was ana- lyzed after deglycosylation. While N-terminal binding antibodies as SAF34 detected only full-length PrP C , C-terminal binding antibodies recognized two bands comprising full-length PrP C and an 18–19 kDa pro- tein band corresponding to the N-terminally truncated form. The distribution of the signal intensities of the Fig. 1. Sequence alignment of prion proteins of humans, sheep, cattle and mice. Recognition sites of the antibodies SAF34, P4, 8G8, SAF60 and SAF70 are indicated. Sequences of the species are recognizable by the antibodies are marked in bold letters. Table 1. Monoclonal antibodies for PrP detection. Antibody Isotype Region a Linear epitope Source Species recognized SAF34 IgG2a Octapeptide region (N-terminal region) 59–89 Hamster scrapie Human, sheep, cattle, mouse P4 IgG1 Intermediary region (N-terminal region) 93–99 b Ovine peptide Sheep, cattle 8G8 IgG2a Intermediary region (N-terminal region) 97–102 c Human peptide Human, sheep 6H4 IgG1 Central region (C-terminal region) 144–152 Human peptide Human, sheep, cattle, mouse SAF60 IgG2b Central region (C-terminal region) 157–161 Hamster scrapie Human, sheep, cattle, mouse SAF70 IgG2b Central region (C-terminal region) 156–162 Hamster scrapie Human, sheep, cattle, mouse SAF84 IgG2b Central region (C-terminal region) 126–164 d Hamster scrapie Sheep, cattle, mouse a N-terminal region (N terminus; N), C-terminal region (C terminus; C). b Linear epitope of ovine PrP. c Linear epitope of human PrP. d Recog- nized solid-phase immobilized peptide 126–164, but failed to bind peptide 142–160 [50]. Glycotyping of PrP C T. Kuczius et al. 1494 FEBS Journal 274 (2007) 1492–1502 ª 2007 The Authors Journal compilation ª 2007 FEBS two bands demonstrated a higher intensity of the C1 fragment than intensity of full-length PrP C . Human C1 fragments revealed high signal intensities with antibod- ies SAF60, SAF70 and 6H4 compared with full-length PrP C (Fig. 3C). Observations similar to human PrP C were also observed with PrP C from cattle, sheep and mice. High signal ratios were determined for di-glycosylated ovine PrP C with antibodies SAF34, 8G8 and P4 and lower intensities for mono- and nonglycosylated ovine PrP C (Fig. 4A,B). However, antibodies 6H4, SAF60, SAF70 and SAF84 gave rather low signal intensities for the di-glycosylated isoforms and highest intensities for pro- teins at 27 kDa, which comprise full-length non- glycosylated PrP C and glycosylated N-terminal truncated isoforms. However, the intensity of the mono- glycosylated band was not dependent on the choice of antibody. After deglycosylation, high signal intensity was determined for the truncated isoform and low inten- sity of deglycosylated full-length proteins (Fig. 4C). Results similar to these were obtained with PrP C from cattle where the antibodies SAF34 and P4 strongly stained the di-glycosylated band, and mAbs 6H4, SAF60, SAF70 and SAF84 showed the highest staining with the overlapping bands of nonglycosylated full-length PrP C and glycosylated truncated isoforms (Fig. 5A–C). In the case of murine PrP C , N-terminal antibodies showed less pronounced staining with the di-glycosylated PrP C than those recognizing the central region. Antibodies 6H4, SAF60, SAF70 and SAF84 gave strong signals for full-length PrP C and less intense signals for the truncated fragments (Fig. 6A–C). Taken together, these findings indicate that the sig- nal intensities of PrP C glycoform patterns strongly depend on the choice of the antibody which was used and to a lesser extent on the species from which the PrP C was obtained (Fig. 7). The di-glycosylated PrP protein bands of humans, sheep, cattle and mice were always predominant, with antibodies binding to the N-terminal region. These patterns changed when PrP C 0 20 40 60 80 100 A B µl 12 108 6 4 2 1 0.5 kDa 36 27 1 024681012 10 100 1000 10000 100000 1000000 10000000 homogenate suspension (µl) 0246810 12 homogenate suspension (µl) units glycosylation (%) C Fig. 2. Western blot analysis and determin- ation of the linear range for signal increase and consistently reproducible glycoprotein banding patterns. (A) Immunodetection of PrP C derived from pooled cattle brain homo- genates (10%; 0.5, 1.0, 2.0, 4.0, 6.0, 8.0, 10.0 and 12.0 lL). Antibody p4 was used for detection. (B) PrP proteins were meas- ured by densitometry and quantified using QUANTITY ONE software. The combined PrP signals are given as computer internal units to determine the linear range of reaction. (C) For glycotyping, the combined PrP signals for the di- (d), mono- (j) and non- glycosylated (m) isoform were defined as 100% and the contribution of each band was calculated as percentage. Linearity in the range of 4–10 lL of brain homogenates was confirmed by repeated separate gel runs. T. Kuczius et al. Glycotyping of PrP C FEBS Journal 274 (2007) 1492–1502 ª 2007 The Authors Journal compilation ª 2007 FEBS 1495 was detected by C-terminal binding antibodies. A pro- tein band with highest signal intensity at the size of the nonglycosylated PrP C was determined for humans, sheep and cattle. This high signal intensity resulted from an overlay of nonglycosylated full-length and gly- cosylated truncated PrP C . However, mouse PrP C ,in most cases, showed highest intensities for the di-glycos- SAF34 6H4 SAF60 SAF708G8 A N-terminal C-terminal human PrP binding antibodies B C SAF34 6H4 SAF60 SAF70 kDa 36 27 kDa 27 20 0 20 40 60 80 100 SAF34 6H4 SAF60 SAF708G8 )%(noitalysocylg Fig. 3. (A) Western blot analysis of human PrP C . Proteins of brain homogenates were separated by SDS ⁄ PAGE followed by immuno- blotting. PrP C signals were detected using the antibodies indicated. (B) The glycoforms of the protein bands were analyzed by calcula- tion of the percentages of the di- (d), mono- (j) and nonglycosy- lated (m) isoform as arithmetic means of separate gel runs. The number of gel runs for the analyses are given for each antibody. Accounting for differences among gel runs, SE values were calcula- ted according to antibody used for PrP detection. Calculation of the banding patterns of 10 gels using antibody SAF34 gave an SE value of 2.1 for the di-glycosylated isoform, 1.6 for the mono-glycosylated band and 3.2 for the nonglycosylated protein; six gels using anti- body 8G8 (SE 1.1; 0.6; 1.1); seven gels with antibody 6H4 (SE 0.4; 0; 0,4); seven gels with antibody SAF60 (SE 1.2; 0; 1.2); and 13 gels with antibody SAF70 (SE 0.8; 0.4; 1.0). (C) Electrophoretic pat- tern of deglycosylated PrP C . Brain homogenates were treated with PNGase F and proteins were separated by SDS ⁄ PAGE. Deglycosyl- ated full-length PrP C and the N-terminal truncated forms (C1) were detected using the antibodies indicated. Results were confirmed by repeated separate gel runs per antibody. 0 20 40 60 80 SAF84 SAF34 6H4 SAF60 SAF708G8 P4 SAF84 SAF34 6H4 SAF60 SAF70 8G8 P4 A N-terminal C-terminal sheep PrP binding antibodies B C SAF84 SAF34 6H4 SAF60 SAF70 kDa 36 27 kDa 27 20 ) % ( n o i t a l y s o c y l g Fig. 4. (A) Immunoblotting of proteins derived from sheep brain homogenates. PrP C signals were specifically detected using the antibodies indicated. (B) Signal intensities of the di- (d), mono- (j) and nonglycosylated isoform (m) of PrP C were quantified and calcu- lated as percentages of the total signal. The glycoforms of the pro- tein bands were analyzed as arithmetic means of separate gel runs. The number of gel runs are given for each antibody, and, accounting for differences among gel runs, SE values were calcula- ted according to antibody used for PrP detection. Calculation of the banding patterns of 17 gels using antibody SAF34 gave an SE of 2.1 for the di-glycosylated isoform, 1.0 for the mono-glycosylated band and 1.6 for the nonglycosylated protein; five gels using anti- body 8G8 (SE 1.5; 1.1; 0.5); 30 gels with antibody P4 (SE 0.9; 0.7; 1.2); five gels with antibody 6H4 (SE 4.6; 3.9; 4.7); seven gels with antibody SAF60 (SE 1.1; 1.0; 1.4); 30 gels with antibody SAF70 (SE 1.9; 1.9; 2.9) and nine gels with antibody SAF84 (SE 1.8; 2.3; 3.4). (C) Brain homogenates were treated with PNGase F for deglyco- sylation of the proteins and subjected to immunoblotting. Full length PrP C and the N-terminal truncated forms were detected using antibodies indicated. The proportion of full-length PrP C and truncated isoforms was confirmed by repeated separate gel runs. Glycotyping of PrP C T. Kuczius et al. 1496 FEBS Journal 274 (2007) 1492–1502 ª 2007 The Authors Journal compilation ª 2007 FEBS ylated band. A differentiation of mouse PrP C to other species is feasible by antibodies recognizing the C-ter- minal region. The comparison of PrP C patterns from brains of humans, sheep, cattle and mouse demonstra- ted consistent differences in the proportion of the C1 fragment. According to these results, PrP C banding patterns seem to depend strongly on the choice of the antibody used for detection and also, albeit to a lesser extent, on the species of origin from which PrP C derived. As PrP Sc and PrP C glycoform patterns in humans have previously been reported to vary considerably in the 0 20 40 60 80 SAF84 SAF34 6H4 SAF60 SAF70 P4 SAF84 SAF34 6H4 SAF60 SAF70 P4 A N-terminal C-terminal cattle PrP binding antibodies B C SAF84 SAF34 6H4 SAF60 SAF70 kDa 36 27 kDa 27 20 )%(noitalysocylg Fig. 5. (A) Western blot analysis of brain tissues obtained from cat- tle. After immunoblotting, PrP C signals were detected using the antibodies indicated. (B) The protein banding pattern of the three PrP C protein bands, the di- (d), mono- (j) and nonglycosylated iso- form (m), was analyzed using densitometry. The percentages of each band regarding to the total signal of PrP C were calculated as arithmetic means of separate gel runs. The number of gel runs for the analyses are given for each antibody. Considering differences among gel runs, SE values were calculated according to antibody used for PrP detection. Calculation of the banding patterns of six gels using antibody SAF34 gave an SE of 1.7 for the di-glycosylated isoform, 0.3 for the mono-glycosylated band and 0.9 for the nongly- cosylated protein; 17 gels using antibody P4 (SE 1.3; 1.2; 0.5); six gels with antibody 6H4 (SE 1.6; 1.0; 0.9); 13 gels with antibody SAF60 (SE 3.1; 1.5; 3.1); 21 gels with antibody SAF70 (SE 1.1; 1.1; 1.3); and eight gels with antibody SAF84 (SE 1.1; 1.7; 1.1). (C) Pro- teins of cattle brain homogenates were deglycosylated using PNGase F followed by immunoblotting. Signals of full-length PrP C and truncated PrP C were detected using the antibodies indicated and the patterns were confirmed by repeated gel runs. SAF84 SAF34 6H4 SAF60 SAF70 SAF84 SAF34 6H4 SAF60 SAF70 A N-terminal C-terminal mouse PrP binding antibodies B C SAF84 SAF34 6H4 SAF60 SAF70 kDa 36 27 kDa 27 20 0 20 40 60 80 )%(noitalysocylg Fig. 6. (A) Detection of mouse PrP C by western blotting. Proteins of brain homogenates were immunoblotted and PrP C signals were detected using the antibodies indicated. (B) Signals of each of the three PrP C protein bands, the di- (d), mono- (j) and nonglycosylat- ed isoform (m), were quantified. The number of gel runs for the analyses are given for each antibody. Following differences among gel runs, many gel runs were analyzed. The percentages of the PrP C bands were calculated as arithmetic means and SE according to the antibody used for PrP detection. Calculation of the banding patterns of 16 gels using antibody SAF34 gave an SE of 1.2 for the di-glycosylated isoform, 0.9 for the mono-glycosylated band and 0.5 for the nonglycosylated protein; six gels with antibody 6H4 (SE 1.9; 1.1; 1.1); four gels with antibody SAF60 (SE 0.7; 0.6; 0.7); 17 gels with antibody SAF70 (SE 1.4; 0.6; 1.6); and nine gels with antibody SAF84 (SE 2.5; 1.0; 2.1). (C) Brain homogenates were treated with PNGase F. After immunoblotting, membranes were probed with the antibodies indicated. Repeated gel runs confirmed the propor- tion of full-length and truncated PrP C . T. Kuczius et al. Glycotyping of PrP C FEBS Journal 274 (2007) 1492–1502 ª 2007 The Authors Journal compilation ª 2007 FEBS 1497 same individual depending on the kind of tissue sam- ples that were analyzed and even between different brain regions [29], we have examined whether this is also reflected in the PrP C glycoform patterns of ovine PrP C which originated from different brain regions such as cortex, cerebellum and brain stem. To give evi- dence that the banding profile is mostly the result of the antibody recognizing the N- or C-terminal PrP sequence, we analyzed three different brain regions pooled from three individual sheep. Interestingly, we found only small regional independent differences on the antibody used (Fig. 8A.B). Only brain stem seems to contain a slightly smaller di-glycosylated PrP C frac- tion as compared with that found in the two other regions. However, a major antibody-associated effect was once again observed for PrP C glycoprotein pat- terns for all three regions: di-glycosylated PrP C bands were heavily stained by N-terminally binding antibody SAF34. Lower intensities were recorded for mono- than for nonglycosylated PrP C . However, the glyco- form pattern was remarkably different again, when PrP C was detected by SAF70: there was a high signal intensity of proteins at the size of full-length nongly- cosylated PrP C , a low intensity for the di-glycosylated isoform and the mono-glycosylated isoform was only just undetectable. A protein band at 27 kDa was most abundant, resulting in an overlay of the full-length 0 20 40 60 80 100 cattlehuman sheep mouse % N- C- N- C- N- C- N- C- -terminal binding antibodies Fig. 7. Comparison of the PrP C banding patterns of various species detected by amino- and carboxyl-binding antibodies. After immuno- blotting, PrP C proteins were detected using N- or C-terminal binding antibodies. The signal intensity of each of the three protein bands was quantified by densitometry. The mean values of the calculated signal intensities were analyzed for each of the N- or C-terminal binding antibodies. The banding pattern of the di- (d), mono- (j) and nonglycosylated isoform (m) is shown for human, sheep, cattle and mouse. The calculation is composed of signals from the N-ter- minal binding antibodies SAF34, P4 and 8G8 or the C-terminal bind- ing antibodies 6H4, SAF60, SAF70 and SAF84 in consideration of species recognition. Values are calculated for the N-terminal anti- bodies SAF34 and 8G8 for humans, SAF34, 8G8 and P4 for sheep, SAF34 and P4 for cattle, and SAF34 for mice; and for the C-ter- minal antibodies 6H4, SAF60 and SAF70 for humans, and mAbs 6H4, SAF60, SAF70 and SAF84 for sheep, cattle and mouse. cbc bs cbc bs cbc bs )ydobitnagnidniblanimret-N(43FAS % 0 52 0 5 57 0 01 % 0 52 0 5 57 001 B A C aDk 63 7 2 aDk 72 02 FesaGNP + ++ )y d o b itn ag ni d n i bl a n i m re t - C ( 0 7 FAS aD k 6 3 72 aDk 72 02 FesaGNP + + + Fig. 8. Immunoblot analysis and diagrammatic presentation of PrP C bands obtained from three different regions of sheep brains. (A) Immu- nodetection of PrP C derived from cortex (c), cerebellum (cb) and brain stem (bs) of sheep detected by antibodies SAF34 and SAF70, respect- ively. (B) PrP C signals of cortex (c), cerebellum (cb) and brain stem (bs) were quantified and the percentages of the di- (d), mono- (j) and nonglycosylated isoform (m) were calculated as arithmetic means of separate gel runs. The calculation represents seven, nine and nine gels for cortex, cerebellum and brain stem samples, respectively, detected by SAF34, and nine gels each for the different regions detected by SAF70. To account for differences among gel runs, SE values were calculated. SE values of PrP C of cerebrum, cerebellum and brain stem detected by SAF34 were determined for the di- (3.5; 2.1; 1.2), mono- (1.4; 0.9; 0.5) and nonglycosylated isoform (2.2; 1.5; 1.0); and detected by SAF70 were determined for the di- (1.8; 3.1; 1.2), the mono- (0.3; 3.3; 3.6) and the nonglycosylated isoforms (1.8; 5.8; 2.8), respectively. (C) Deglycosylation of PrP C from cortex (c), cerebellum (cb) and brain stem (bs). Proteins were incubated with PNGase F before electrophor- esis and transfer to membranes. PrP proteins were detected using antibodies SAF34 or SAF70 as indicated. Glycotyping of PrP C T. Kuczius et al. 1498 FEBS Journal 274 (2007) 1492–1502 ª 2007 The Authors Journal compilation ª 2007 FEBS nonglycosylated PrP C and the N-terminal truncated isoform shown after deglycosylation (Fig. 8C). The truncated C1 fragment exhibited higher signal intensity than the full-length PrP C , indicating a predominance of the truncated isoforms in cortex, cerebellum and brain stem. Discussion The western blotting technique is frequently used for the diagnostic confirmation of prion diseases and to distinguish between the various prion strains. How- ever, the sensitivity of PrP Sc to treatment with PK and the glycotyping pattern obtained depend on the prion strain [1,6–9,30–35]. PK treatment reflects in the molecular mass of the initial PK-resistant cleavage product and the reaction kinetics under high proteo- lytic conditions. The PK cleavage sites have been shown to differ between species, e.g. residue N96 (and Q97 as minor site) in PrP Sc from BSE while in scrapie, cleavage is at G81, G85 and G89 (or mainly G89 under different PK concentrations) [36]. In different cases of Creutzfeldt–Jakob disease, two primary clea- vage sites at residues 82 and 97 for types 1 and 2, respectively, have been identified; minor cleavage points are present at residues 74–102 [37]. Differences in the glycoprotein pattern are due to differences in the relative staining intensities of the di-, mono- and nonglycosylated isoforms of PrP Sc . BSE and human vCJD, the latter presumably being linked to the con- sumption of BSE-contaminated meat, have a similar glycoprotein profile [1] that can be distinguished from that found in sporadic Creutzfeldt–Jakob disease and sheep scrapie. PrP C serves as the substrate for the PrP Sc conversion reaction. However, little is known about the glycoprotein pat- terns found in PrP C of animal and human origin, and about the effect which the detection antibody might have on these. Brain regional variability of PrP C has been described [29,38]. We systematically analyzed the PrP C glycoform patterns in human, sheep, cattle and mouse brains using a set of antibodies recognizing sev- eral epitopes within various regions of the PrP sequence in order to find imposed interspecies varia- tions. Irrespective of the species and of pooled sheep brain regions analyzed, two representative PrP C glyco- form patterns were observed depending on the antibody used. Antibodies to the nonstructured N-terminus gave significantly stronger signals with the di-glycosylated isoform of PrP C than did antibodies to the structured core region. However, the glycoform patterns of mouse PrP C always showed the highest sig- nal intensity of the di-glycosylated isoform, independ- ently if an N- or C-terminal binding antibody was used. In contrast, a protein band at the size of the nonglycosylated full-length PrP C of humans, sheep and cattle was highly abundant when using C-terminal binding antibodies. Our data show that the high signal intensity corres- ponding to the size of the nonglycosylated full-length protein indicated antibody binding at the structured core region of PrP C as the result of an overlap of two proteins, the nonglycosylated full-length form and the glycosylated N-truncated fragments. From endogenous proteolysis, two amino truncated isoforms termed C1 and C2 are described migrating at 18 and 21–22 kDa with human PrP C , respectively [23–28]. A separation of both protein isoforms, full-length and N-truncated, could clearly be demonstrated after enzymatic deglyco- sylation. Interestingly, truncated C1 fragments of human, sheep and cattle PrP C resulted in higher signal intensities than their full-length proteins. However, this observation is different to the mouse PrP C banding pattern. On the basis of differences in the proportions of the signal intensities of full-length and truncated isoforms, we suggest that PrP C metabolism and regula- tion varies among the different species. The N-terminal cleavage of PrP C in vivo may be the result of a down- regulation of functions arranged by the N-terminal region [26]. The occurrence of two distinct glycoform patterns demonstrated by antibodies binding to the N- or C-terminal region is most likely to be due to differ- ences in epitope and protein fragment accessibility rather than to differences in the glycosylation of PrP C . As shown by NMR ( 13 C, 15 N, 1 H) and ⁄ or X-ray studies, PrP C in all species contains a flexible N-terminus (amino acids 23–120) [39–41] and a struc- tured core and C-terminal region (amino acids 121– 231). This folded domain contains three helices and two short antiparallel b-sheets [41]. PrP C has two linked glycosylation sites at asparagines 180 and 196 (calculated here for murine PrP) [18]. Taken together, the results of various signal intensi- ties of the three PrP C bands are accredited to the development of the truncated isoforms, to the epitope recognition of the antibodies and in part to the protein structure. These data illustrate that emergent truncated fragments must be taken into account when studying the expression and regulation of PrP C in consideration of the di-, mono- and nonglycosylated protein bands. For distinct discrimination among various species, such as mouse, sheep, cattle and humans, C-terminal binding antibodies will provide more detailed varia- tions in PrP C glycoprotein patterns than antibodies recognizing the N-terminal PrP region. T. Kuczius et al. Glycotyping of PrP C FEBS Journal 274 (2007) 1492–1502 ª 2007 The Authors Journal compilation ª 2007 FEBS 1499 Experimental procedures Antibodies The monoclonal Ig61, Ig62a and Ig62b antibodies (mAbs) used in this study, SAF34, SAF60, SAF70 and SAF84, have been raised in PrP° ⁄ ° mice by immunizing with formic acid-denatured, SAF obtained from an infected hamster brain (263K) [42]. The linear epitopes recognized by these antibodies were identified by pepscan analysis as described [43]. All antibodies were applied as ascetic fluids obtained in mice and used in this study from one charge in each case. mAbs 8G8 and 6H4 (Prionics, Schlieren, Switzerland) were raised against recombinant human PrP [44–46]. A syn- thetic peptide based on the amino acid sequence of ovine PrP (amino acids 89–104) was used as antigen for produ- cing the monoclonal antibody P4 (r-biopharm, Darmstadt, Germany) [47]. Pepscan analysis revealed P4 peptides at the sequence 93–99 of ovine PrP [48]. The epitopes recognized by the various antibodies and the detection of PrP C derived from various species are listed in Table 1. Preparation of brain tissue Brain tissue was obtained from noninfected sheep, cattle, mice and humans. Homogenates of mice were prepared using pooled whole brains from four individuals. Human homogenates derived from pooled tissues obtained from several different brain regions of six subjects. The regions were not specified, but were comprised mostly of cortex and cerebellum. Brain homogenates of cattle were obtained from the brain stems of six animals. Pooled homogenates of sheep brains were prepared from tissues taken from var- ious regions of five animals. Furthermore, based on three individual sheep, brain tissues of cortex, cerebellum and brain stem were each pooled. The homogenates were prepared by homogenization in nine volumes of lysis buffer [0.32 m sucrose, 0.5% (w ⁄ v) igepal and 0.5% (w ⁄ v) SDS in Tris-buffered saline (20 mm Tris and 150 mm NaCl, pH 7.4; Sigma, Taufkirchen, Ger- many)] in glass homogenizers followed by intensive ultra- sonification as described [49]. After centrifugation at 900 g for 5 min (5415 R centrifuge, FA-45-24-11 rotor, Eppen- dorf, Hamburg, Germany), the supernatants were stored in aliquots at )70 °C. Aliquots mixed with SDS loading buffer were stored at )20 °C and were used within a few days in order to avoid effects of prolonged storage on the stability of PrP C . Deglycosylation For enzymatic deglycosylation, SDS was added to the homogenates to a final concentration of 1.5% (w ⁄ v). The protein samples were diluted 2.5-fold in incubation buffer consisting of Tris-buffered saline (20 mm Tris and 150 mm NaCl; pH 7.4) with 10 mm EDTA, 1% (w ⁄ v) igepal and 1.5% (v ⁄ v) 2-mercaptoethanol. Protein samples were dena- tured at 99 °C for 10 min followed by incubation with one unit of N-glycosidase F (PNGase F; Roche, Mannheim, Germany) for 16 h at 37 °C. Non-deglycosylated samples were treated in the same way, but were incubated without the addition of PNGase F. Finally, SDS-loading buffer was applied to the samples processing for SDS ⁄ PAGE. Immunoblot analysis Proteins were separated using SDS ⁄ PAGE. Samples were re- suspended in SDS-loading buffer, heated to 99 °C for 5 min and the proteins separated in a mini slab gel apparatus (Bio- Rad, Munich, Germany) using 13% polyacrylamide gels. After electroblotting onto Immobilon-P membranes (Roth, Karlsruhe, Germany) using a semi-dry blotting system (Roth), membranes were blocked in Tris-buffered saline con- taining 0.1% (w ⁄ w) Tween 20 (TBST) and 1% (w ⁄ v) nonfat dry milk powder for 60 min. Specific binding of antibodies to PrP proteins was determined by incubating membranes for at least 2 h with the antibodies indicated. Horseradish peroxidase-conjugated affinity purified goat (anti-mouse IgG) (Dianova, Hamburg, Germany) served as secondary antibody. Protein signals were visualized using a chemilumi- nescence enhancement kit (Pierce, Bonn, Germany). Glycotyping of prion proteins In order to analyze the PrP glycoform patterns, proteins were scanned on a chemiluminescence photo-imager (Bio- Rad, Munich, Germany). Densitometry was carried out using quantity one software (Bio-Rad, Munich, Ger- many), determining the signal intensities of the di-, mono- and nonglycosylated PrP isoforms. The combined signals with one sample were defined as 100% and each band was calculated as a percentage of the total signal. Protein pro- files were analyzed by calculation of the arithmetic means of the tissue samples after separation on SDS ⁄ PAGE. Vari- ations in separation in repeat SDS ⁄ PAGE runs were expressed as standard errors of the mean (se). Acknowledgements The authors thank O. Mantel and O. Bo ¨ hler for their excellent technical assistance. We are indebted to K. Keyvani, Institute for Neuropathology, Mu ¨ nster, for providing human brain samples, the Chemisches Landes- und Staatliches Veterina ¨ runtersuchungsamt (CVUA) Mu ¨ nster for providing sheep and cattle sam- ples and the Max Planck Institute, Department Vascu- lar Cell Biology, Mu ¨ nster, for providing mouse samples. This work was supported in part by grants from the EU Network Neuroprion (FOOD-CT-2004– Glycotyping of PrP C T. 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Binding of N- and C-terminal anti -prion protein antibodies generates distinct phenotypes of cellular prion proteins (PrP C ) obtained from human, sheep,. alignment of prion proteins of humans, sheep, cattle and mice. Recognition sites of the antibodies SAF34, P4, 8G8, SAF60 and SAF70 are indicated. Sequences of