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

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

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

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

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

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-

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

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

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-

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-

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).

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

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

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

-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

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
+
+

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

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

. 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.

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
.

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
¨

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Glycotyping of PrP
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