J. Biomedical Science and Engineering, 2009, 2, 70-75
Published Online February 2009 in SciRes. http://www.scirp.org/journal/jbise JBiSE
1
Computer-Assisted analysis of subcellular
localization signals and post-translational
modifications of human prion proteins
Fatemeh Moosawi, Hassan Mohabatkar*
Department of Biology, College of Sciences, Shiraz University, Shiraz, Iran, *Corresponding author to Fatemeh Moosawi ([e-mail]), Telephone:
[phone], Mobile: [phone], Fax: [phone].
Received August 26th, 2008; revised December 19th, 2008; accepted December 7th, 2008
ABSTRACT
In the present work, computational analyses
were applied to study the subcellular localiza-
tion and posttranslational modifications of hu-
man prion proteins (PrPs). The tentative location
of prion protein was determined to be in the nu-
cleolus inside the nucleus by the following bio-
informatics tools: Hum-PLoc, Euk-PLoc and
Nuc-PLoc. Based on our results signal peptides
with average of 22 base pairs in N-terminal were
identified in human PrPs. This theoretical study
demonstrates that PrP is post-translationally
modified by: 1) attachment of two N-linked
complex carbohydrate moieties (N181 and N197),
2) attachmet of glycosylphosphatidylinositol
(GPI) at serine 230 and 3) formation of two di-
sulfide bonds between “6–22” and “179–214”
cysteines. Furthermore, ten protein kinase
phosphorylation sites were predicted in human
PrP. The above-noted phosphorylation was car-
ried out by PKC and CK2. By using bioinfor-
matics tools, we have shown that computation-
ally human PrPs locate particularly into the nu-
cleolus.
Keywords: Prion protein; Subcellular localiza-
tion; Signal peptides; Post-translational Modifi-
cations; Bioinformatics
1. INTRODUCTION
Prion diseases or transmissible spongiform encephalo-
pathies (TSE) are fatal neurodegenerative conditions in
humans and animals that originate spontaneously, ge-
netically or by infection [1]. Human TSE diseases include
sporadic, genetic, iatrogenic and variant Creutzfeldt-
Jakob disease (CJD) and sporadic or familial fatal in-
somnia. Animal counterparts are scrapie in sheep and
goats, bovine spongiform encephalopathy, and chronic
wasting disease of mule deer and elk [2,3]. The critical
pathogenetic events in TSE diseases are conformational
changes of the physiological host prion protein (PrPc)
into an insoluble form (PrPsc) [4].
Prions are devoid of nucleic acid and seem to be com-
posed exclusively of protein. The normal, cellular PrP is
converted into PrPsc through a process whereby a por-
tion of its α-helical and coil structure is refolded into
β-sheet [5,6]. This structural transition is accompanied
by profound changes in the physicochemical properties
of the PrP [7]. At the molecular level, PrP is a sialogly-
coprotein of 253 amino acids in human [8]. The
C-terminal end is a signal peptide allowing the anchoring
of the protein to the plasma membrane via a glycosyl-
phosphatidylinositol residue in the early steps of matura-
tion in the endoplasmic reticulum [9]. The ultimate des-
tination is then the plasma membrane where PrP can be
released by phospholipase or protease treatment [10].
Finally, PrP cycles between the plasma membrane and
the endocytic pathway. During infection, PrPsc is
thought to derive from PrPc after exposure to the plasma
membrane [11]. It has been suggested that PrPc can bind
a putative ‘‘protein X’’ that may function as a molecular
chaperone in the formation of PrPsc [12].
The cellular role of the normal host protein PrP is still
unknown. Oesch has characterized membrane associated
proteins that interact with PrP [13,14]. Immunocyto-
chemical studies reveal that PrPsc and PrPc are present,
not only at the plasma membrane or in cytoplasmic
compartments [15,16,17], but also in the nuclear com-
partment, particularly into the nucleolus [18]. The incon-
sistency in the nuclear localization of PrP between non-
infected and infected cells may account for important
pathogenetical mechanisms [19].
The posttranslational modification of amino acids ex-
pands the range of functions of the protein by attaching it
to other biochemical functional groups such as acetate,
phosphate lipids and carbohydrates. This occurs by al-
tering the chemical nature of an amino acid or by making
structural changes, like the formation of disulfide bridges.
The molecular mechanism by which the PrPsc is formed
and causes infectivity or neurodegeneration is not known.
In an emerging view, post-translational modifications
play roles in the transformation of PrPc to PrPsc [20].
Moreover, Post-translational modification of the scrapie
prion protein is thought to account for the unusual fea-
SciRes Copyright © 2009

F. Moosawi et al. / J. Biomedical Science and Engineering 2 (2009) 70-75 71
SciRes Copyright © 2009 JBiSE
tures of this protein [21].
All eukaryotic cells are compartmentalized into sepa-
rate membrane-bound organelles and require tightly
regulated transport of proteins and lipids between these
compartments. The function of a protein is closely cor-
related with its subcellular location. With the rapid in-
crease in new protein sequences entering into data banks,
we are confronted with a challenge. Proteins are classi-
fied, according to their subcellular locations, into the
following 18 groups: cell wall, centriole, chloroplast,
cyanelle, cytoplasm, cytoskeleton, endoplasmic reticulum,
extracell, Golgi apparatus, hydrogenosome, lysosome,
mitochondria, nucleus, peroxisome, plasma membrane,
plastid, spindle pole body, and vacuole [22].
Determination of subcellular location of a protein is
essential for understanding its biochemical function.
These data are hard to obtain experimentally but have
become especially significant since many protein se-
quences are still lacking detailed functional information.
To address this rarity of data, many computational
analysis methods have been developed. However, these
methods have varying levels of accuracy and perform
differently based on the sequences that are presented to
the underlying algorithm. Giving the huge number of
uncharacterized protein sequences, computer-aided analy-
sis of posttranslational modifications and translocation
signals from amino acid sequence becomes a necessity.
In this study, we have analyzed subcellular localiza-
tion, signals peptides and posttranslational modifications
of human PrPs.
2. MATERIALS AND METHODS
2.1. Amino Acids Sequence
The sequences of human PrPs were obtained from
http://www.ncbi.nlm.nih.gov/sites/Entrez and http//beta.
uniprot.org. Accession numbers of human PrPs are
shown in Table 1.
2.2. Consensus Sequence and Percentage
of Different Amino Acids
The consensus sequence was achieved by using Multalin
5.4.1 server and the percentage of different amino acids
was calculated by expasy server.
Table 1. Accession numbers of human PrPs
AAD46098 AAG21693 AAO83636 AAC05365
AAC78725 AAV38303 AAB59442 AAB59443
AAA60182 AAO83635 A2A2V1 AAR21603
AAH12844 AAH22532 AAS80162 AAA19664
A1YVW6 ABM85428 ABD63004 ABM82244
ABL75508 BAA00011 CAG46836 CAD62016
CAB75503 CAM27320 CAA56283 CAI19053
CAI19053 CAA58442 CAG46869 EAX10449
EAX10450 NP_000302 NP_001073592 NP_001073591
NP_898902 NP_001073590 O75942 P04156
P23907 Q6FGR8 Q5QPB4 Q53YK7
Q6FGN5 Q540C4 Q6SES1 Q5U0K3
Q86XR1
2.3. Prediction of Signal Peptides
Signal-CF server was employed to study the signal pep-
tides. The web interface to the Signal-CF tool was acces-
sible at http://www.chou.med.harvard.edu/shen. This server
is called Signal-CF, where C stands for “coupling” and F
for “fusion”, meaning that Signal-CF is formed by in-
corporating the subsite coupling effects along a protein
sequence and by fusing the results derived from many
width-different scaled windows through a voting system.
Signal-CF is featured by high success prediction rates
with short computational time, and hence is particularly
useful for the analysis of large-scale datasets [23].
2.4. Prediction of Subcellular Localization
Several computational tools for predicting the subcellu-
lar localization of a protein are available. In this study,
Hum-PLoc, Euk-PLoc and Nuc-PLoc have been utilized
to study the localization of prion protein sequences.
Hum-PLoc is a server that analyzes the subcellular lo-
calization of human proteins among the following 12 loca-
tions: centriole, cytoplasm, cytoskeleton, endoplasmic re-
ticulum, extracell, Golgi apparatus, lysosome, microsome,
mitochondrion, nucleus, peroxisome, and plasma mem-
brane [24]. The web interface to this tool is present at
http://www URL: http://www.chou.med.harvard.edu/shen.
Euk-PLoc is available as a web-server at http://202.
120.37.186/bioinf/euk. A new benchmark dataset is con-
structed that covers the following 18 localizations: cell
wall, centriole, chloroplast, cyanelle, cytoplasm, cy-
toskeleton, endoplasmic reticulum, extracell, Golgi ap-
paratus, hydrogenosome, lysosome, mitochondria, nu-
cleus, peroxisome, plasma membrane, plastid, spindle
pole body, and vacuole [25].
A new classifier, called Nuc-PLoc, has been devel-
oped that can be exploited to recognize nuclear proteins
among the following nine subnuclear locations: chroma-
tin, heterochromatin, nuclear envelope, nuclear matrix,
nuclear pore complex, nuclear speckle, nucleolus, nucleo-
plasm and nuclear promyelocytic leukemia (PML) body.
As a user-friendly web-server, Nuc-PLoc is accessible at
http://chou.med.harvard.edu/ bioinf/Nuc-PLoc [25].
2.5. Analysis of Posttranslational Modifica-
tions
N-myristoylation, N-glycosylation, protein kinase C,
casein kinase II and Serine, threonine, tyrosine phos-
phorylation sites were predicted. Expasy which is avail-
able at www.expasy.ch/tools was applied for this purpose.
Big-PI server was utilized to study the glycosylphos-
phatidylinositol (GPI) anchor signal [26]. The web server
http://clavius.bc.edu /~clotel ab/DiANNA was chosen for
prediction of disulfide bonds [27].
3. RESULTS
3.1. Sequences and Signal Peptides
Number of amino acids and molecular weight of human
consensus sequence of prion protein were 253 and
27661.1 respectively. The consensus sequence of human
PrPs is shown in Figure 1.

72 F. Moosawi et al. / J. Biomedical Science and Engineering 2 (2009) 70-75
SciRes Copyright © 2009 JBiSE
Figure 1. Consensus sequence of the human prion protein
Percentage of different amino acids in the protein was
calculated (Table 2). The most prevalent amino acid was
glycine (45 residues), and the least one was cysteine (4
residues).
To identify functional signal peptides in the human PrP,
49 FASTA format sequences of prion protein input in Sig-
nal-CF sever. Signal peptide sequences of human PrPs
were sorted in 4 groups based on their length (Table 3).
3.2. Prediction of Subcellular Localization
The subcellular distribution of PrP proteins was verified
by Euk-Ploc, Hum-Ploc and Nuc-ploc. These results
showed a strong tendency of the protein to nucleus and
especially to nucleolus.
3.3. Prediction of Post-translation Modifica-
tions
It is interesting that the post-translational modifications
alone, or in combination with amino acid changes, play
dominant roles in the pathogenic transformation of
Table 2. Residue composition for consensus sequence of human
PrPs
A %4 10 C %1.6 4 D %2.46 E %3.6 9 F%2.87
G %17.8 45 H %4.0 10 I %3.69 K %4.0 10 L%4.7 12
M %4.7 12 N %4.7 12 P %6.7 17 Q %5.9 15 R %4.311
S %5.9 15 T %5.1 13 V %5.514 W %3.6 9 Y%5.113
Table 3. Categories of signal peptides of human PrPs
Position of signal peptides 1-14 1-15 1-221-24
Number of signal peptides 1 8 38 2
PrP(C) to PrP (SC). According to our analysis 2 aspara-
gines in positions 181 and 197 were predicted to be gly-
cosylated. Results also showed that threonine in posi-
tions 107, 183, 190, 191, 192, and 193 and serine in po-
sition 132 were predicted to be kinase C phosphorylated.
Serine in position 143, and threonines in positions 201
and 206 were expected to be casein kinase II phosphory-
lated and no glycine was predicted to be myristoylated.
Disulfide bridges in cysteine residues at positions 6–22
and 179–214 were predicted.
GPI anchors, which allow the attachment of proteins
to the extracellular leaflet of the plasma membrane, were
also analyzed. Glycosylphosphoatidylinositol (GPI) lipid
anchoring is a common posttranslational modification
known mainly in extracellular eukaryotic proteins. At-
tachment of the GPI moiety to the carboxyl terminus
(omega site) of a polypeptide happens following prote-
olytic cleavage of a C-terminal propeptide (Figure 2).
The best predicted site was G229 and the second best
was S230 (underlined). Furthermore, potential phos-
phorylation sites of serine, threonine and tyrosine in the
human PrPs were determined (Table 4).
Table 4. Predicted phosphorylation sites of consensus sequence
of human PrPs
Amino acidPhosphorylation position
Ser
Thr
Tyr
43
191
145
143
192
149
23
163
169
231
-
225
MANLGCWMLVLFVAT WSDLGLCKKRPKPGGW NTGGSRYPGQGSPGGN RYPPQ GG
GGWGQPHGGGWGQPH GGGWG QPHG GGWG QPHG GGWGQGG GTHSQWNK PSKP
KTNMKHMAGAAAAGAVVGGL GGY MLGSAMSRPIIH FGSDYE DRYYRENMHRYP N
QVYYRPMDEYSNQNNF VHDCVNITIKQHTVTTT TKGENFTET DVKMMERVVEQM
CITQYERESQAYYQRGS SMVLFSSPPV ILLISFLIFL IVG
Figure 2. GPI lipid anchoring signals sequence
10203040506070
MANLGCWMLVLFVATWSDLGLCKKRPKPGGWNTGGSRYPGQGSPGGNRYPPQGGGGWGQPHGGGWGQPHG
8090100110120130140
GGWGQPHGGGWGQPHG GGWGQGG GTHS QWNKPSKPKTNMKHMAGAAAAGAVVGGLGGYMLGSAMSRPIIH
150160170180190200210
FGSDYEDRYYRENMHRYPNQVYYRPMDEYSNQNNFVHDCVNITIKQHTVT TTTKGENFTETDVKMMERVV
220230240250
EQM C ITQYE R E S QAYYQRGSSMVLFSSPPVILLISFLIFLIVG

F. Moosawi et al. / J. Biomedical Science and Engineering 2 (2009) 70-75 73
SciRes Copyright © 2009 JBiSE
4. DISCUSION
The goal of this investigation was to apply bioinformat-
ics methods to study the subcellular localizations, signal
peptides and posttranslational modifications of human
PrPs.
4.1. Identification of Signal Peptides in PrP
Based on our results, there were signal peptides with
average 22 bp in N-terminal of human PrPs. According
to a survey, conducted by Alexandre and his colleagues,
PrP does not contain a nuclear localization signal and
that, in normal conditions, PrP cannot be released in the
cytosolic compartment remaining membrane bound till
its degradation and in infected cells, PrP can interact
with a molecule to form a complex able to be released in
the cytosol and then targeted enters the nucleus [18].
However in another investigation, the presence of two
independent nuclear localization signals in the N-termi-
nal region of PrP was observed. The first signal included
residues 23–28, and the second one included residues
101–106 of PrP [29].
Protein signals have become crucial tools for re-
searchers to construct new drugs which are expected to
enter a particular organelle to correct a specific defect.
For example, by adding a specific tag to a desired protein,
one can tag it for excretion, making it much easier to
harvest. To use such a tool successfully, first one has to
identify the signal sequences. Since the number of nas-
cent protein sequences entering databanks is rapidly in-
creasing, it is time consuming and expensive to identify
the signal peptides entirely by experiments [28].
4.2. Determinants of Subcellular localization
of PrP
Protein subcellular localization prediction has been
widely studied (reviewed in [30,31]). Available servers
differ in many aspects including the computational
method, the type and diversity of protein characteristics,
the localization coverage, the target organism(s) and the
reliability. Servers can be grouped into 4 general classes
based upon the protein characteristics that are considered:
amino acid composition and order based predictors
[32,33,34], sorting signal predictors [35,36], homology
based predictors [37,38] and hybrid methods that inte-
grate several sources of information to predict localiza-
tion [39,40]. Nowadays, the importance of developing a
powerful high-throughput tool to predict protein subcel-
lular location has become obvious [41].
In the present study, the tentative location of prion
protein was determined to be in the nucleolus inside the
nucleus by bioinformatics tools, Hum-PLoc, Euk-PLoc
and Nuc-PLoc. There are different opinions regarding
the subcellular localization of PrP. Stahl and his col-
leagues considered a signal peptide at the C-terminus of
prion protein allowing the anchoring of the protein to the
plasma membrane via a glycosylphosphatidylinositol
residue in the early steps of maturation in the endoplas-
mic reticulum [8]. Oesch has characterized membrane-
associated proteins that interact with PrP [12,13]. More
recently, it has been shown that the 37-kDA laminin re-
ceptor interacts with PrP [42].
A number of cellular proteins, among them the nuclear
lectin CBP35, was identified that bound to the predicted
RNA stem-loop structure of PrP RNA. CBP35 could also
be detected in purified infections prions, [43]. Moreover,
the presence of PrP in the nucleus and its subnuclear
location in the nucleolus has been reported [17,44].
In addition, Gu and his coworkers demonstrated that
nuclear accumulation of PrP fragments was mediated by
nuclear localization signals in the N-terminal domain of
PrP that became functional under certain conditions and
might contribute to the pathogenesis of certain prion
disorders [29].
4.3. Post-translational Modifications of
Consensus Sequence of PrP
Our analysis shows that PrP is post-translationally modi-
fied by the attachment of two N-linked complex carbo-
hydrate moieties (N181 and N197) and a GPI anchor at
serine 230 as well as by the formation of a disulfide bond
between 6-22 and 179-214 cysteins.
Glycosylation is one of the most complex and ubiqui-
tous post-translational modifications of proteins in eu-
karyotic cells. It is a dynamic enzymatic process in
which saccharides are attached to proteins or lipoproteins,
usually on serine (S), threonine, asparagine, and trypto-
phan residues. Glycosylation, like phosphorylation, is
clinically important because of its role in a wide variety
of cellular, developmental and immunological processes,
including protein folding, protein trafficking and local-
ization, cell-cell interactions, and epitope recognition
[45,46,47,48,49,50]. The number of glycosylation sites
in our work is in agreement with the results obtained by
molecular cloning of a PrP cDNA [20]. It has already
been shown also that addition of one or two N-glycans
causes retention of the N-terminal PrP fragment in the
endoplasmic reticulum in a partially aggregated form,
and a small amount is secreted into the medium. Pres-
ence of two glycans in the N-terminal fragment is more
conducive to proper folding and secretion into the medium
than one glycan, which largely remains in the ER [29].
In GPI anchors, a hydrophobic phosphatidylinositol
group is linked to a residue at or near the C-terminus of a
protein through a carbohydrate-containing linker. GPI
anchor addition is both structurally and functionally re-
lated to another important post-translational modification,
prenylation, in which hydrophobic farnesyl or geranyl-
geranyl moieties are added to C-terminal cysteine resi-
dues of target proteins. Additionally, GPI anchors pro-
teins to the cell membrane [51]. Although we determined
the nucleus as the tentative location for prion proteins,
this fact also should be taken in mind that according to a
previous study GPI anchor and the N-glycans function in
a complicated way to reduce the tendency of PrP for lo-
calization in nucleus [30].

74 F. Moosawi et al. / J. Biomedical Science and Engineering 2 (2009) 70-75
SciRes Copyright © 2009 JBiSE
In our study, 10 protein kinases phosphorylation sites
were predicted in the human PrPs. The addition of a
phosphate molecule to a polar R group of an amino acid
residue can turn a hydrophobic portion of a protein into a
polar and extremely hydrophilic part. In this way, it can
introduce a conformational change in the structure via
interaction with other hydrophobic and hydrophilic resi-
dues in the protein. Moreover, phosphorylation may
modulate PrP biological activity. Regarding the bonding
states of cysteine also, it has been found out that it plays
important functional and structural roles in proteins. Par-
ticularly, disulfide bond formation is one of the most
important factors influencing the three-dimensional fold
of proteins [52].
In conclusion, this study can help in better under-
standing of signal peptides of prion proteins. Generally,
our results indicate the role that bioinformatics can play
in analysis of proteins modification and localization.
ACKNOWLEDGMENT
Support of this study by Shiraz University is acknowledged.
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