J Biol Chem, Vol. 273, Issue 29, 18015-18018, July 17, 1998
COMMUNICATION
Characterization of the Human Analogue of a Scrapie-responsive
Gene*
Michel
Dron
,
Françoise
Dandoy-Dron,
Frédéric
Guillo,
Louisa
Benboudjema§,
Jean-Jacques
Hauw¶,
Pierre
Lebon
,
Dominique
Dormont**, and
Michael G.
Tovey
From the Laboratory of Viral Oncology CNRS UPR 9045, IFC1, 94801 Villejuif cedex, France, the ¶ Laboratoire de
Neuropathologie Raymond Escourolle, Groupe Hospitalier
Pitié-Salpêtrière, INSERM U360, Association Claude
Bernard, 75651 Paris cedex 13, France, the Laboratoire de
Virologie, Hôpital Saint Vincent de Paul, 75674 Paris cedex 14, France, and the ** Laboratoire de Neurovirologie CEA, 92265 Fontenay aux
Roses cedex, France
 |
ABSTRACT |
We have recently described a novel mRNA
denominated ScRG-1, the level of which is increased in the
brains of Scrapie-infected mice (Dandoy-Dron, F., Guillo, F.,
Benboudjema, L., Deslys, J.-P., Lasmézas, C., Dormont, D., Tovey,
M. G., and Dron, M. (1998) J. Biol. Chem. 273, 7691-7697). The increase in ScRG-1 mRNA in the brain
follows the accumulation of PrPSc, the proteinase
K-resistant form of the prion protein (PrP), and precedes the
widespread neuronal death that occurs in late stage disease. In the
present study, we have isolated a cDNA encoding the human
counterpart of ScRG-1. Comparison of the human and mouse transcripts firmly established that both sequences encode a highly conserved protein of 98 amino acids that contains a signal peptide, suggesting that the protein may be secreted. Examination of the distribution of human ScRG-1 mRNA in adult and fetal
tissues revealed that the gene was expressed primarily in the central
nervous system as a 0.7-kilobase message and was under strict
developmental control.
| |
INTRODUCTION |
The transmissible spongiform encephalopathies
(TSE)1 are in a group of
progressive neurodegenerative diseases that includes human pathologies
such as Creutzfeldt-Jakob disease (CJD),
Gerstman-Sträussler-Scheinker syndrome and Kuru, and animal
diseases such as scrapie and bovine spongiform encephalopathy (1).
To identify the genes the altered expression of which is associated
with or may even be responsible for the neurodegenerative changes
observed in TSE, we have systematically analyzed modifications of gene
expression in scrapie-infected mouse brain using "mRNA differential display" (2). This approach has led to the detection of
an increased level of expression of eight cellular genes. One of these
genes, denominated scrapie-responsive gene 1 (ScRG-1), previously unrecognized, is expressed principally in the brain. Enhanced expression of ScRG-1 in the brain of
scrapie-infected mice occurs concomitantly with increased expression of
GFAP mRNA, a marker of astrocytosis (3). Moreover,
ScRG-1 mRNA was found to be preferentially expressed in
cells of glial origin and to encode a protein with a putative signal
peptide (2). These observations suggest that ScRG-1 may play
a role in the host response to prion-associated infections. Previous
reports have suggested that certain molecules enhanced in TSE may be
detrimental to neurone survival (4-6). However, the role of
overexpressed proteins (7-9), including ScRG-1, in the
pathogenesis of TSE remains to be determined. We report herein the
nucleotide sequence, size characterization, and tissue distribution of
human ScRG-1 mRNA.
| |
EXPERIMENTAL PROCEDURES |
RNA Extraction and Northern Blot Hybridization--
RNA was
extracted from the brains of either mock infected C57Bl/6 mice or mice
infected with the C506M3 strain of scrapie, as described previously
(2). Total RNA was extracted by the method of Chirgwin et
al. (10) from the frontal cortex obtained at autopsy from a
patient free from any neurological disease (patient 941005, 46 years
old) and from a patient with typical neuropathological findings of
sporadic Creutzfeldt-Jakob disease (patient 93005, 59 years old). The
diagnosis was confirmed by the presence of the proteinase K-resistant
form of PrP in the sample (data not shown). Samples of normal human
brain and of the brain from a patient diagnosed with CJD were obtained
by informed consent, under the auspices of the Program de Recherche sur
les Encéphalopathies Spongiformes Sub-aigües Transmissibles
et les Prions (CNRS, France). Human poly(A)+ mRNA was
obtained as described previously (2). Northern blots were performed
using glyoxal denaturation, and the blotted membranes were hybridized
using probes radiolabeled to a specific activity of at least 1 × 109 cpm/mg, as described previously (2). The blots were
first exposed to autoradiography and then quantified using a
PhosphorImager (Molecular Dynamics). The multiple human tissue Northern
blot and the membrane containing the RNA dots from different human tissues were from CLONTECH laboratories.
Cloning and Sequencing of the Human cDNA--
1 µg of
poly(A)+ mRNA from the human control sample was primed
with oligo(dT) and converted into double strand cDNA using standard procedures. One-twentieth of the cDNA synthesized was amplified by
polymerase chain reaction using specific forward
(5'-TAAGGGAAAATCACGCTGTG-3') and reverse
(5'-CTTTTATTACTACTTGTTTAACAC-3') primers and Taq DNA polymerase. The amplified product was purified, sequenced using the
Thermo Sequenase cycle sequencing kit (Amersham Pharmacia Biotech), and
further cloned in the pCR2.1 Topo plasmid vector from Invitrogen.
Cloning and Sequencing of Mouse ScRG-1 cDNA--
Mouse
ScRG-1 cDNA was isolated by screening a library of whole
BALB/c adult brain cDNA cloned in 
gt11, with the ScRG-1 cDNA clone 24 previously isolated by RACE (2) used as a probe. The cDNA
inserted in the selected lambda (clone 1) was isolated and further
sequenced.
| |
RESULTS AND DISCUSSION |
Isolation of Human ScRG-1 cDNA--
To isolate the human
counterpart of the mouse ScRG-1 cDNA, the murine
sequence (2) was compared with the randomly isolated human cDNA
sequences reported as expressed sequence tags. The sequences
potentially related to the mouse cDNA were combined in a contig and
used to establish a human consensus sequence corresponding to a
putative 902-bp cDNA. To ascertain the existence of the human ScRG-1 mRNA, primers derived from the 5' and 3' ends of the
consensus sequence were used to synthesize cDNA from human brain
mRNA by specific reverse transcription and polymerase chain
reaction amplification. An unique cDNA fragment of the predicted
size was obtained, cloned, and sequenced (Fig.
1A). Mapping using expressed
sequence tag cDNA related to ScRG-1 indicated that the
human gene was located on the long arm of chromosome four
(4q31-4q32).
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Fig. 1.
Comparison of the human and murine
ScRG-1 nucleotide sequences. The nucleic acid
sequences (A) of the human and murine ScRG-1
cDNAs were aligned for comparison. The beginning of the human
cDNA sequence amplified from the brain of patient 941005 is
indicated by an arrow. Position 1 of the mouse sequence is
the 5' end of the longest brain cDNA obtained by 5'-RACE (2). Most
of the mouse cDNAs obtained previously were shorter than the clone
24 cDNA, and their 5' ends start at a position between 143 and 148. The mouse cDNA clone 1, isolated from the lambda cDNA library
starts also at position 148, indicated by an arrow. The
coding sequences of both the human and murine cDNA as well as the
two potential polyadenylation sites were underlined or
overlined. The translation products of the human and murine
coding sequences (B) were aligned without introduction of
any gap in the sequence. For both sequences the first 20 amino acids
corresponding to a potential cleavable signal peptide were
underlined.
|
|
The sequence of the human cDNA was compared with the 700-bp murine
ScRG-1 cDNA. The two sequences were found to be very
similar with an homology of 75.8% in an overlap of 720 nucleotides
(Fig. 1A). The human cDNA contained an open reading
frame (ORF) at positions 414-710 encoding a protein exhibiting strong
homology with the protein predicted from the murine ScRG-1
cDNA (Fig. 1B).
The human ORF was found to encode a protein of 98 amino acids, which is
51 amino acids more than previously predicted for the murine protein
(2). Comparison of human and murine cDNA sequences indicated that
introduction of a one-nucleotide gap in the murine ORF would generate
an open reading frame corresponding to a protein of 98 amino acids the
sequence of which would be almost identical to that encoded by the
human ScRG-1 cDNA. This prompted us to clone a new mouse
brain ScRG-1 cDNA from a lambda library, using as a
probe the ScRG-1 cDNA clone 24 previously isolated by
RACE (2). The sequence of clone 1 obtained from the library was then
determined (Fig. 1A). The nucleotide sequences of the
different cDNAs previously obtained by RACE (2) were also
re-examined. The mouse ORF, which was reported to contain two
guanosines at positions 345 and 346, was found in fact to contain three
guanosines at positions 345, 346, and 347, immediately following a
sequence consisting of a succession of nucleotides repeated twice,
thereby constructing an imperfect palindrome. The additional guanosine
signal (position 345) was very weak in intensity and abnormally close
to the following nucleotide. The presence of the "missing G" was
confirmed by sequencing a recently isolated murine genomic DNA clone of
ScRG-1 (data not shown). The corrected nucleotide sequence
of the mouse brain ScRG-1 cDNA and derived amino acid sequence
(Fig. 1, A and B) have been reported in the data
bases.
Comparison of the human and mouse ScRG-1 coding sequences
showed that 82.5% of the amino acids of the two predicted proteins are
identical, with a stretch of 40 identical contiguous amino acids in the
carboxyl-terminal region of the protein (Fig. 1B). The
ScRG-1 proteins exhibit no apparent homology with other
known proteins. Both the human and murine proteins contain a cleavable signal peptide of 20 amino acids in length. Furthermore, the
probability that ScRG-1 is external to the plasma membrane
is 56% for the murine protein and 48% for human protein according to
the PSORT II program of protein localization site prediction. The
predicted molecular mass of the mature protein for the two species is
approximately 9 kDa, which is in the range of the molecular weight of
most cytokines and neurotransmitters. A N-glycosylation site
was detected at positions 72-75 of the protein for both species so
that the molecular weight of the ScRG-1 protein could be
substantially higher in vivo. A tyrosine kinase
phosphorylation site was also detected at positions 63-70 in both
proteins.
Characterization of Human Brain ScRG-1 mRNA--
Northern blot
analysis was carried out using RNA from both human and mouse brain to
determine the size of the transcripts in the two species. 14 individual
RNA species were used as molecular weight markers. The blot was split
in two parts to separate the mouse and human samples, and each part was
hybridized under stringent conditions with a radiolabeled probe derived
from murine and human ScRG-1 cDNA, respectively (Fig.
2, A and B). One
band corresponding to 0.7 kb in size was detected in the human samples
and as expected, two bands of 2.6 and 0.7 kb were detected in the
murine samples. A very faint band of 2.6 kb was also revealed upon
overexposure of the autoradiograms of the human blot (data not shown).
The faster migrating band was relatively broad, with an estimated size
of between 0.66 and 0.82 kb (mean size of 0.74 kb) in both species.
Quantification by PhosphorImager indicated that the 0.7-kb message
represented at least 75-80% of the ScRG-1 transcripts in murine brain
and about 98% of the transcripts in human brain. The relative
abundance of the ScRG-1 mRNA in this organ was about 40 times less than the level of 
-actin mRNA in both species.
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Fig. 2.
Northern blot analysis of human and mouse
ScRG-1 mRNA. A Northern blot containing 2 µg of
poly(A)+ RNA from the frontal cortex of patient 941005 (normal) and patient 930005 (CJD) (A)
were transferred to a membrane that was hybridized under stringent
conditions with radiolabeled human ScRG-1 cDNA. The blot
was further dehybridized and rehybridized successively with probes
derived from cDNA of the human cathepsin S, hamster GFAP, and
murine -actin. Similarly, a Northern blot (B) containing
10 µg of total brain RNA from C57Bl/6 mice, mock infected or infected
with the C506M3 strain of scrapie and sacrificed 170 days post
inoculation, was hybridized with the murine ScRG-1 cDNA
clone 24 isolated by RACE (2). The third part of figure (C)
shows the autoradiogram of a membrane containing 2 µg of
poly(A)+ mRNA from different adult human tissues,
probed with the human ScRG-1 cDNA. The different
ScRG-1 transcripts shown in the three panels are indicated
by arrows, and the corresponding sizes are stated.
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|
We have reported previously that the 0.7-kb ScRG-1
transcript is overexpressed 2-3-fold in scrapie-infected mouse brain
(Ref. 2 and Fig. 2). As shown in Fig. 2B, the level of the
2.6-kb transcript is also increased 2-3-fold in this experimental
model of TSE disease. It was of interest to determine whether the level of human ScRG-1 mRNA is increased in samples of brain
from patients with CJD. Poly(A)+ mRNA was isolated from
normal human brain and from the brain (frontal cortex) of a patient
diagnosed with CJD. The expression of ScRG-1 mRNA was
examined by Northern blot analysis using human ScRG-1
cDNA as a probe (Fig. 2A). ScRG-1 mRNA
was found to be 3-fold more abundant in the brain of a patient with CJD
than in the brain of a normal individual. In contrast, -actin
mRNA levels were similar in both samples (Fig. 2A). As
expected an increased expression of GFAP transcripts was also detected
in the CJD mRNA, indicating that a glial reaction had occurred in
the brain tissue examined (Fig. 2A), and the presence of
proteinase K-resistant PrP was also clearly detected in this sample
(data not shown). Although these results are highly suggestive, only
two individuals have been compared and any definitive conclusion
concerning the expression of the ScRG-1 gene will have to
await more extensive studies, particularly those employing the
techniques of in situ hybridization and/or
immunocytochemistry in addition to Northern blot analysis. It is quite
possible that the increased expression observed results from the
activation of a particular subset of cells in the brain. Thus, the
increase in ScRG-1 mRNA detected using Northern blot
analysis may underestimate an increased expression in a particular cell
population, because all brain cells contribute to the mRNA analyzed
by this method. Interestingly, the level of cathepsin S transcripts was
also found to be higher in the CJD RNA sample examined (Fig.
2A) in agreement with the 3-8-fold increase in the
cathepsin S mRNA, which has been reported in scrapie-infected mouse
brain (2). The gliosis that precedes the spongiosis and neuronal death
in TSE consists of both an astrocytosis and a microglial activation.
The increased expression of ScRG-1 and cathepsin S transcripts that we observed in CJD brain tissue may result from this
double cellular activation. In Alzeihmer's disease an increased expression of cathepsin S has been reported to reflect the activation of microglial cells (11).
The laminin receptor, which has recently been shown to interact with
PrP, may constitute a receptor for this protein (12). The laminin
receptor is increased 2-fold in extracts of brain from scrapie-infected
mice (12), indicating that even a modest increase in the level of
expression of a gene may be of considerable biological importance in
such disorders.
Tissue Distribution and Developmental Expression of Human ScRG-1
mRNA--
Although mouse ScRG-1 mRNA was found to
be expressed preferentially in brain tissue (2), the expressed sequence
tags related to ScRG-1 and used to define primers to isolate the human
cDNA (see above) were recovered from various tissues including
brain, testis, aorta, and pregnant uterus. It was of interest therefore to determine the specificity of expression of the gene in different tissues, using a membrane to which poly(A)+ RNA from 50 human tissues have been immobilized in separate dots (Fig.
3). ScRG-1 is abundantly
expressed in the central nervous system of the adult, in all the areas
of brain investigated, and in spinal cord but is poorly or not
expressed at all in fetal brain, indicating marked developmental
regulation of the gene. A high level of ScRG-1 transcripts
was also observed in testis and aorta, organs with low specificity of
expression, which is in agreement with the report of expressed sequence
tags from these tissues. We also analyzed, by Northern blotting,
mRNA from various human tissues, heart, brain, placenta, lung,
liver, skeletal muscle, kidney, and pancreas for the presence of
ScRG-1 mRNA (Fig. 2C). The ScRG-1
message was highly expressed in the brain as expected and also to a
6-fold lesser extent in the heart. A faint signal was also detected
upon overexposure of the blot in almost all the organs examined,
indicating that the low level of hybridization observed in most of the
human organs and shown in Fig. 3 corresponds to a real albeit low level
of expression (data not shown).
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Fig. 3.
Tissue distribution of the human ScRG-1
mRNA. A membrane to which poly(A)+ RNA from 50 human tissues had been immobilized in separate dots was hybridized with
the human ScRG-1 cDNA according to the instructions of
the supplier, and the membrane was subjected to PhosphorImager
quantification. A1-A8: whole brain, amygdala, caudate
nucleus, cerebellum, cerebral cortex, frontal lobe, hippocampus, and
medulla oblongata. B1-B7, occipital lobe, putamen,
substantia nigra, temporal lobe, thalamus, subthalamic nucleus, and
spinal cord. C1-C8, heart, aorta, skeletal muscle, colon,
bladder, uterus, prostate, and stomach. D1-D8, testis,
ovary, pancreas, pituitary gland, adrenal gland, thyroid gland,
salivary gland, and mammary gland. E1-E8, kidney, liver,
small intestine, spleen, thymus, peripheral leukocyte, lymph node, and
bone marrow. F1-F4, appendix, lung, trachea, and placenta.
G1-G7, fetal brain, fetal heart, fetal kidney, fetal liver,
fetal spleen, fetal thymus, and fetal lung. Most of the tissues are
pools of several individuals except for the cerebral cortex and adult
liver. The fetal mRNA are pools from at least 14 embryos of ages
between 17 and 25 weeks. The mRNA samples dotted on the membrane
have been normalized to the mRNA expression level of eight
different housekeeping genes.
|
|
Conclusions--
The increased level of expression of
ScRG-1 transcripts in scrapie-infected mouse brain (2) and
probably also in the brain of CJD-infected individuals suggests that
ScRG-1 may be involved in the neurodegenerative process in
TSE. Indeed, the ScRG-1 protein may play an important physiological
role in the central nervous system, as indicated by the high degree of
conservation of its amino acid sequence in both man and mouse, the
presence of a cleavable signal peptide indicating that the ScRG-1
protein is secreted outside the cell, and its high level of expression
in the central nervous system. The potential importance of the gene is
also emphasized by the observation that its expression is under
developmental regulation.
| |
FOOTNOTES |
*
This work was supported by grants from CNRS (Programme de
Recherche sur les Encéphalopathies Spongiformes Sub-aigües
Transmissibles et les Prions, Action Concertée Coordonnée
Number 2), from INSERM, and from the Association Nouvelles Recherches
Biomédicales.The costs of publication of this
article were defrayed in part by the
payment of page charges. The article
must therefore be hereby marked
"advertisement" in
accordance with 18 U.S.C. Section
1734 solely to indicate this fact.
The nucleotide sequence(s) reported in this paper has been submitted to the GenBankTM/EMBL Data Bank with accession number(s) AJ223206 and AJ224677.
§
Recipient of a fellowship from the Association Recherche et Partage
(Paris, France).
To whom correspondence should be addressed: Lab. of Viral
Oncology, 7 rue Guy Moquet, BP8, 94801 Villejuif, France. Tel.: 33-1-49-58-34-22; Fax: 33-1-49-58-34-44; E-mail:
mdron{at}infobiogen.fr.
1
The abbreviations used are: TSE, transmissible
spongiform encephalopathies; CJD, Creutzfeldt-Jakob disease; PrP, prion
protein; RACE, rapid amplification of cDNA ends; ORF, open reading
frame; kb, kilobase(s); contig, group of overlapping clones; bp, base pair(s).
| |
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Copyright © 1998 by The American Society for Biochemistry and Molecular Biology, Inc.
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Abstract
Introduction
