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

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 p

generates distinct phenotypes of cellular prion proteins

Thorsten Kuczius1, Jacques Grassi2, Helge Karch1and Martin H Groschup3

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 (PrPSc), 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 (PrPC) to a pathological isoform (PrPSc) accompanied by a conformational change from a largely a-helical form into a b-sheet structure [4] In contrast to PrPC, the infectious PrPScprotein is deter-gent-insoluble PrPCand PrPScprotein samples can be differentiated by pretreatment with proteinase K (PK), which completely hydrolyses PrPC but only removes 55–70 amino acid residues in the N-terminal region of PrPScresulting in a molecular reduction of 6–8 kDa The western blot method is a useful in vitro assay for the characterization of PrPSc and PrPC, in which fully glycosylated mouse PrP migrates at 33–35 kDa

Keywords

antibody; glycotyping; prion protein; PrPC;

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 (PrPSc) of a host encoded prion protein (PrPC) PrPSc proteins are characterized according to size and glycoform pattern We analyzed the glycoform patterns of PrPC 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 PrPCisoforms Our data demonstrate the importance

of antibody selection in characterization of PrPC

Abbreviations

BSE, bovine spongiform encephalopathy; PK, proteinase K; PrP, prion protein; SAF, scrapie-associated fibril; vCJD, variant Creutzfeldt–Jakob disease.

and the nonglycosylated form at 27 kDa on

SDS⁄ PAGE [5] PrPSc strains and isolates are

distin-guished by the size of their PK resistant core protein

because differences in the PK cleavage sites in PrPSc

have been observed in scrapie, experimental scrapie

and ruminant BSE [6,7] PrPSc exhibit different

band-ing patterns followband-ing quantitative immunoblottband-ing by

densitometry, which reflects differences in the ratios of

the di-, mono- and nonglycosylated PrP In sporadic

cases of human Creutzfeldt–Jakob disease, PrPSc

shows a characteristic glycopattern with high signal

intensity of the mono-glycosylated isoform which

dif-fers from that in ruminant BSE and scrapie PrPSc 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 PrPCsequence homology of the

donor and recipient species [11,12] In addition to

spe-cies-specific characteristics of PrPSc, there are also

notable variations in the glycoform patterns, but the

importance of these is not well understood PrPC 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, PrPC 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 PrPC 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 PrPCwere 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 PrPC In this

study, we have analyzed the glycoform patterns of

PrPC of human, sheep, cattle and mice and compared

them Variable immunoreactivity of anti-PrP

antibod-ies determining different PrPC 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 PrPCglycoform patterns derived from different species which arose from binding of various antibodies recognizing sites in the amino, central or C-terminal PrPC sequence The aim of the study was therefore to find imposing interspecies variations among human PrPC and PrPC derived from different species in order to find a first onset on the basis of PrPC expression why PrPSc 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 PrPCand the N-truncated isoforms We found that the mouse PrPC glycoforms differed from human when C-ter-minal PrP binding antibodies were used This observa-tion was attributed to the proporobserva-tion 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 PrPCas 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 PrPCfunction, 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 PrPC signals were detected by the western blot tech-nique The PrPCbanding 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 PrPC 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

Brain PrPCfrom 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 PrPC 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 PrPC,

respect-ively Similar ratios were obtained with human PrPC

and the antibody mAb 8G8 which binds to the

inter-mediary region at amino acids 97–102 of human PrPC

(Fig 3A,B) In contrast, deviant profiles were found

with antibodies binding to the central region of PrPC,

as the signal intensities at the size of the

nonglycosylat-ed full-length PrPC (at 27 kDa) were high with monoclonal antibodies 6H4, SAF60 and SAF70 while signals for the di-glycosylated PrPCwere low In these experiments the mono-glycosylated forms of human PrPCwere almost invisible and not detectable

Heterogeneity of PrPC proteins is enhanced by endogenous proteolytic modifications, which occurs

in vivo [25–27] PrPCfrom non-infected brains consists

in addition to full-length PrP to a significant amount

of an N-terminal truncated PrPC fragment termed C1 Glycosylated C1 protein fragments migrate to a posi-tion around the nonglycosylated full-length PrPC The degree of truncated PrPC to full-length PrP was ana-lyzed after deglycosylation While N-terminal binding antibodies as SAF34 detected only full-length PrPC, C-terminal binding antibodies recognized two bands comprising full-length PrPC 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.

(N-terminal region)

(N-terminal region)

(N-terminal region)

(C-terminal region)

(C-terminal region)

157–161 Hamster scrapie Human, sheep, cattle, mouse

(C-terminal region)

156–162 Hamster scrapie Human, sheep, cattle, mouse

(C-terminal region)

126–164d 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].

two bands demonstrated a higher intensity of the C1

fragment than intensity of full-length PrPC Human C1

fragments revealed high signal intensities with

antibod-ies SAF60, SAF70 and 6H4 compared with full-length

PrPC(Fig 3C)

Observations similar to human PrPC were also

observed with PrPCfrom cattle, sheep and mice High

signal ratios were determined for di-glycosylated ovine

PrPC with antibodies SAF34, 8G8 and P4 and lower

intensities for mono- and nonglycosylated ovine PrPC

(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 PrPC 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 PrPC 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 PrPC and glycosylated truncated isoforms (Fig 5A–C) In the case of murine PrPC, N-terminal antibodies showed less pronounced staining with the di-glycosylated PrPCthan those recognizing the central region Antibodies 6H4, SAF60, SAF70 and SAF84 gave strong signals for full-length PrPCand less intense signals for the truncated fragments (Fig 6A–C) Taken together, these findings indicate that the sig-nal intensities of PrPC glycoform patterns strongly depend on the choice of the antibody which was used and to a lesser extent on the species from which the PrPC 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 PrPC

0 20 40 60 80 100

A

B

µl 0.5 1 2 4 6 8 10 12 kDa

36 27

1

10 100 1000 10000 100000 1000000 10000000

homogenate suspension (µl)

homogenate suspension (µl)

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.

was detected by C-terminal binding antibodies A

pro-tein band with highest signal intensity at the size of

the nonglycosylated PrPC was determined for humans,

sheep and cattle This high signal intensity resulted

from an overlay of nonglycosylated full-length and gly-cosylated truncated PrPC However, mouse PrPC, in most cases, showed highest intensities for the

di-glycos-SAF34 8G8 6H4 SAF60 SAF70

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 8G8 6H4 SAF60 SAF70

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 antianti-body 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 PrPCand 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 8G8 P4 6H4 SAF60 SAF70

SAF84 SAF34 8G8 P4 6H4 SAF60 SAF70

A

sheep PrP binding antibodies

B

C

SAF84

kDa

36

27

kDa

27

20

Fig 4 (A) Immunoblotting of proteins derived from sheep brain homogenates PrPC 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 antianti-body 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.

ylated band A differentiation of mouse PrPCto other

species is feasible by antibodies recognizing the

C-ter-minal region The comparison of PrPC patterns from

brains of humans, sheep, cattle and mouse

demonstra-ted consistent differences in the proportion of the C1

fragment

According to these results, PrPC 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 PrPC derived As PrPSc and PrPC glycoform patterns in humans have previously been reported to vary considerably in the

0

20

40

60

80

SAF84

SAF84

A

N-terminal C-terminal

cattle PrP binding antibodies

B

C

SAF84

kDa

36

27

kDa

27

20

Fig 5 (A) Western blot analysis of brain tissues obtained from

cat-tle After immunoblotting, PrPC 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 PrPC

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

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 quantifinonglycosylat-ed 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

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 PrPCglycoform patterns of ovine PrPC 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 PrPC frac-tion as compared with that found in the two other regions However, a major antibody-associated effect was once again observed for PrPC glycoprotein pat-terns for all three regions: di-glycosylated PrPC bands were heavily stained by N-terminally binding antibody SAF34 Lower intensities were recorded for mono-than for nonglycosylated PrPC However, the glyco-form pattern was remarkably different again, when PrPC was detected by SAF70: there was a high signal intensity of proteins at the size of full-length nongly-cosylated PrPC, 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

cattle

%

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-terN-ter-minal

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.

cb

) y d b i t n a g i d i b l a n i m r e t N ( 4 F A S

%

0 5 0 5 0 1

%

0 5 0 5 0 1

B

a D k 6 7

a D k 7 0

F e a G N

) y d b i t n a g i d i b l a n i m r e t C ( 0 F A S

a D k 6 7

a D k 7 0

F e a G N

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) PrPCsignals 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 PrPCof 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 PrPCfrom 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.

nonglycosylated PrPC and the N-terminal truncated

isoform shown after deglycosylation (Fig 8C) The

truncated C1 fragment exhibited higher signal intensity

than the full-length PrPC, 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 PrPScto 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 PrPScfrom 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 PrPSc 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 PrPC serves as the substrate for the

PrPScconversion reaction

However, little is known about the glycoprotein

pat-terns found in PrPCof animal and human origin, and

about the effect which the detection antibody might

have on these Brain regional variability of PrPC has

been described [29,38] We systematically analyzed the

PrPC 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 PrPC

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 PrPCthan did antibodies to

the structured core region However, the glycoform

patterns of mouse PrPCalways 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 PrPCof 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 PrPCas 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 PrPC, 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 PrPCresulted in higher signal intensities than their full-length proteins However, this observation is different to the mouse PrPC banding pattern On the basis of differences in the proportions

of the signal intensities of full-length and truncated isoforms, we suggest that PrPCmetabolism and regula-tion varies among the different species The N-terminal cleavage of PrPCin 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 PrPC As shown by NMR (13C, 15N, 1H) and⁄ or X-ray studies, PrPC 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] PrPC 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 PrPC 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 PrPCin 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 PrPC glycoprotein patterns than antibodies recognizing the N-terminal PrP region

Experimental procedures

Antibodies

The monoclonal Ig61, Ig62a and Ig62b antibodies (mAbs)

used in this study, SAF34, SAF60, SAF70 and SAF84,

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

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

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

order to avoid effects of prolonged storage on the stability

Deglycosylation

For enzymatic deglycosylation, SDS was added to the

protein samples were diluted 2.5-fold in incubation buffer

consisting of Tris-buffered saline (20 mm Tris and 150 mm

unit of N-glycosidase F (PNGase F; Roche, Mannheim,

were treated in the same way, but were incubated without the addition of PNGase F Finally, SDS-loading buffer was

Immunoblot analysis

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

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

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–

506579) and the Bundesministerium fu¨r Bildung und

Forschung (BMBF; project 0312733)

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