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:
Website:
(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. Kuczius et al.
1500 FEBS Journal 274 (2007) 1492–1502 ª 2007 The Authors Journal compilation ª 2007 FEBS
506579) and the Bundesministerium fu
¨
r Bildung und
Forschung (BMBF; project 0312733).
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Glycotyping of PrP
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T. Kuczius et al.
1502 FEBS Journal 274 (2007) 1492–1502 ª 2007 The Authors Journal compilation ª 2007 FEBS