To identify mRNAs whose expression is enriched at embryonic day
(E) (
)17 or E20 in the embryonic striatum, we have
previously performed a differential screening of a cDNA library with
two radiolabeled probes corresponding to striatal mRNAs extracted at
E17 and E20(1) . The 8.5 mRNA was selected since it was
enriched at E20. The sequence of a partial cDNA clone showed a 60%
similarity between the translation product of its main open reading
frame and the protein encoded by the neuronal p1A75 cDNA whose
organelle-like location within cells suggests an involvement in
secretory, transport, or mitochondrial functions(2) . As a
first step in the study of this new protein family, the structural and
functional characterization of the 8.5 mRNA was undertaken. We describe
here the complete nucleotide sequence of the 8.5 mRNA whose expression
was detected only in neural and neuroendocrine tissues. Antibodies
directed against the recombinant 8.5 protein demonstrated the existence
of the natural protein, which was mainly localized in the Golgi
apparatus. Furthermore, using transfected COS cells, the exogenous
protein was also detected within the Golgi area, demonstrating
therefore that its primary sequence contains the information for this
specific subcellular compartmentalization.
MATERIALS AND METHODS
RNA Isolation and Northern Blotting
Total
cellular RNA was extracted from fresh tissue or cells by the guanidium
thiocyanate/phenol-chloroform extraction method(3) . Timed
pregnant OFA rats (Iffa-Credo) provided a source of fetal and neonatal
brains of precise gestational or post-natal ages. Other brain
structures or non-neural tissues were dissected from adult
Sprague-Dawley (Charles River) male rats. Dissociated neurons from
cerebral hemispheres of E17 rat embryos were plated at high density (6
10
/cm
) according to Di Porzio et
al. (4) and cultured for 2-4 days; astrocyte
cultures were prepared from the same cerebral areas as described by
Denis-Donini et al.(5) . Total cellular RNA (5
µg/lane) was fractionated on 1.2% agarose gel containing 3.7% (w/v)
formaldehyde, using standard procedures(6) . Gels were blotted
onto Hybond N (Amersham Corp.) nylon membranes. The 18 and 28 S
ribosomal RNAs observed on the filter by UV light were used to check
that equal amounts of RNA were loaded on each lane. Hybridization
conditions were: 65 °C, 16 h in the presence of 2
10
cpm/ml of probe in the following solution: 5
SSC, 5
Denhardt's solution, 50 mM sodium phosphate, pH
6.5, and 0.4% SDS. Final washes were done in 0.2
SSC and 0.1%
SDS at 65 °C. The 8.5 full-length cDNA, labeled with
[
-
P]dCTP to a specific activity of 10
cpm/µg by random priming(7) , was used as a probe.
In Situ Hybridization
In situ hybridization experiments were performed as described
previously(1) . Briefly, single-stranded RNA transcripts were
labeled with [
-
S]UTP (1000 Ci/mmol;
Amersham) as the only source of this nucleotide. Whole embryos from
Sprague-Dawley rats were sectioned on a cryostat and the sections
(10-20 µm thick) were thaw-mounted on silanized glass slides,
fixed with 4% paraformaldehyde, dehydrated, and stocked at -20
°C until use. Frozen sections were thawed, fixed with
paraformaldehyde, permeabilized with proteinase K, and again fixed with
paraformaldehyde. Prehybridization and hybridization were performed at
50 °C in a medium containing 50% formamide. Sections were then
washed at 50 °C in a medium containing 50% formamide and subjected
to RNase A digestion. Finally, sections were dehydrated and exposed to
-Max films (Amersham) for 1-3 weeks. Autoradiograms were
transformed into digitized images with an image analyzer (IMSTAR).
Characterization of the 8.5 cDNA
The cDNA library
was constructed, as described previously(1) , from
poly(A)
RNA prepared from cerebral hemispheres of
newborn BALB/c mice. The original, 1500-base pairs long, 8.5 cDNA was
identified by differential screening of the library plated in the
pBluescript (Stratagene) plasmid vector(1) . This cDNA was used
as a probe to screen 2
10
cDNA clones from the same
library plated in the
gt10 phage vector. The insert of the longest
hybridizing cDNA was excised by partial EcoRI restriction and
was subcloned in both orientations in the pBluescript plasmid vector
and in the M13mp19 phage vector. Both strands of the 8.5 cDNA were
sequenced in M13 using the deletion method described by Dale et al.(8) coupled with the dideoxynucleotide chain termination
technique (9) and the modified T7 polymerase(10) .
Recombinant 8.5 Protein and Antibody
Production
The plasmid containing the 8.5 cDNA was amplified
with the Taq DNA polymerase (11) using the two
following primers: A, (5`-GCGGATCCATCGAAGGTAGAATGGTGAAGCTGAACAGCAA-3`);
and B, (5`-ACCCACCCGGATCCCAGCTG-3`). Primer A was designed to add a BamHI restriction site and a factor Xa (IEGR) cleavage site
fused to the initiating methionine of the 8.5 protein. Primer B was
designed to create, by site-directed mutagenesis, a BamHI
restriction site at the position 692 of the sequence shown in Fig. 1. The amplified DNA fragment was restricted using the BamHI endonuclease and cloned in the BamHI site of
the pQE-8 (Qiagen) expression vector which produces a recombinant
protein with a 6xHis tag on the N terminus. The construction was
sequenced in order to verify the absence of mutations, and the
synthesis of the recombinant protein was demonstrated by immunoblot
using an antibody directed against a synthetic peptide (residues
138-155). The recombinant 8.5 protein was produced and purified
using nickel-chelate affinity chromatography (12) in denaturing
conditions according to the protocols provided by Qiagen. The very low
solubility of the 8.5 recombinant protein in non-denaturing buffers
precludes its cleavage in bulk quantities with the factor Xa and
therefore the whole fusion protein was used to immunize rabbits. To
this end, 0.5 mg of the recombinant protein solubilized in 8 M urea were loaded onto a 14% preparative SDS-PAGE. The gel was
stained in 0.25 M KCl, and the band corresponding to the 8.5
protein was cut and washed in phosphate-buffered saline. New Zealand
white rabbits were immunized three times subcutaneously with a piece of
gel containing approximately 100 µg of protein. Human keratin
(Sigma) coupled to a CH-Sepharose 4B (Pharmacia Biotech Inc.) column
was used to get rid of a contaminating anti-keratin immunoreactivity of
the serum(13) . Antibodies to the 8.5 protein were
affinity-purified by passage through a column of CH-Sepharose 4B
coupled in 8 M urea to the purified recombinant protein and
subsequent elution in 0.1 M glycine buffer, pH 2.2.
Figure 1:
A, nucleotide sequence of the murine
8.5 cDNA and translation of its longest open reading frame. B,
alignment of the 79 C-terminal amino acids of the 8.5 protein (top) with the partial published (2) sequence of the
product of the p1A75 cDNA (bottom). A double bar indicates an identity and a single bar indicates a
replacement by an isofunctional amino acid.
Subcellular Fractionation and Western Blot
Analysis
Subcellular fractions were prepared from rat post-natal
day 1 brain according to Huttner et al.(14) . Briefly,
neonatal rat brains were homogenized in buffered sucrose (320 mM sucrose, 4 mM Hepes, pH 7.4) using a glass-Teflon
homogenizer. This homogenate (H) was centrifuged for 10 min at 800
g; the pellet was discarded, and the supernatant was
centrifuged for 15 min at 10,200
g to yield a pellet
(P2) and a supernatant (S2). The S2 fraction was then centrifuged for 1
h at 165,000
g to yield a pellet (P3) fraction and a
cytosolic fraction (S3). Equivalent fractions of each preparation,
corresponding to 150 µg of homogenate, were run on a 14% SDS-PAGE
and blotted onto poly(vinylidene fluroide) membranes
(Bio-Rad)(15) . Carbonate extractability was tested on the P2
and P3 fractions as described previously(16) . Briefly, pellets
were resuspended in a large volume of ice-cold 100 mM Na
CO
buffer, pH 11.5 (or
phosphate-buffered saline for the control experiment), incubated for 1
h on ice, and centrifuged for 1 h at 200,000
g. After
saturation with 5% nonfat dry milk and 0.05% Tween-20, membranes were
incubated with a 1/200 dilution of the affinity-purified antibody
(corresponding to a 1/200 dilution of the rabbit serum) followed by a
goat anti-rabbit IgG coupled to alkaline phosphatase. Enzymatic
activity was detected with nitro blue tetrazolium and
5-bromo-4-chloro-3-indolyl-phosphate substrates.
Cell Cultures, Transfection, and
Immunofluorescence
Dissociated neurons from cerebral hemispheres
of E17 rat embryos were plated in 35-mm culture dishes at high density
(6
10
/cm
) in serum-free medium
according to Di Porzio et al.(4) and cultured for 2
days. For transient transfection experiments the 8.5 cDNA was amplified (11) using the primer B described above and primer C
(5`-CCGCACAGTCTCGAGTAAAA-3`) which creates a XhoI site at
position 141. The amplified DNA fragment was restricted using the BamHI and XhoI endonucleases and inserted in the pSVL
(Pharmacia) expression vector. Transfections of COS-7 cells with this
construction were performed in 35-mm culture dishes using the
DEAE-dextran protocol and reagents provided in the Stratagene mammalian
transfection kit. For immunofluorescence experiments, dissociated
neurons or COS cells were seeded on glass coverslips and fixed with
periodate-lysine-paraformaldehyde (17) for 2 h at room
temperature, permeabilized with 0.005% saponin, and
immunocytochemically stained using the rabbit affinity-purified
specific antibody and goat immunoglobulins (IgG) against rabbit IgG
labeled with tetramethylrhodamine (Biosys,
Compiègne, France).
Immunoperoxidase Electron Microscopy
The
immunoperoxidase procedure was performed on dissociated neurons using a
preembedding approach, in situ in Petri dishes, as described
previously (18) . Briefly, cells were fixed with
periodate-lysine-paraformaldehyde (as described above) and
permeabilized with 0.005% saponin before incubation with the rabbit
affinity-purified specific antibody and then with sheep IgG against
rabbit IgG labeled with peroxidase (Institut Pasteur, Paris). After
postfixation in 1% glutaraldehyde, detection of peroxidase activity,
postfixation in 1% osmium tetroxide, cells were embedded in situ in Epon according to Brinkley et al.(19) . After
observation at the light microscopic level, selected areas of
immunoreactive cells were sectioned and ultrathin sections were
examined under the electron microscope without further staining.
RESULTS
Cloning and Sequence Analysis
We used a
previously described screen (1) in order to identify mRNAs
whose expression is enriched at E17 or E20 in the embryonic striatum.
The original 8.5 plasmid was selected due to its strong hybridization
with the E20 mRNA probe. Since its 1500-base pair long cDNA insert was
smaller than the guessed size of the corresponding mRNA (approximately
2500 bases; see above), it was used to screen a mouse embryonic brain
cDNA library constructed in the
gt10 phage vector. This procedure
led to the isolation of an apparently full-length cDNA whose size (2260
base pairs) corresponds to the estimated length of the mRNA if one
takes into account the poly(A) tract. Its insert was subcloned in the
M13mp19 phage vector and sequenced on both strands (Fig. 1A) using the deletion technique described by
Dale et al.(8) . This cDNA terminates at its 3` end by
a poly(A) tract preceded by a typical polyadenylation signal (AATAAA)
at position 2223. The longest open reading frame encodes a polypeptide
of 171 residues with a calculated molecular mass of 19 kDa. This open
reading frame is initiated by the first ATG (position 160), surrounded
by a favorable consensus sequence for the initiation of
translation(20) , and preceded by a stop codon (TGA, position
106) in the same reading frame.Study of hydropathy of this
polypeptide by the method of Kyte and Doolittle (21) indicated
that it is moderately hydrophilic with the exception of two adjoining
22-residue long stretches, being, respectively, strongly hydrophilic
(residues 47-69) or hydrophobic (residues 70-92) (Fig. 2). The N terminus of the polypeptide does not fit the
outline proposed for signal sequences (22) .
Figure 2:
Hydropathy plot of the amino acid
sequence of the murine 8.5 protein. Negative values indicate
hydrophobic regions.
Comparison of
this predicted polypeptide with protein data base (23) (release
37 of NBRF) revealed a highly significant similarity of its C-terminal
part with the C-terminal, 83-residue long, partial sequence of the
p1A75 cDNA product (2) (Fig. 1B). Indeed, 35
residues of the putative 8.5 protein were found to be identical to the
partial sequence of the product of the p1A75 cDNA, and 12 were replaced
by an amino acid belonging to the same biochemical group. One should
note that the similarity of these two polypeptides does not include the
15 most C-terminal residues which are completely divergent.
The 8.5
protein contains also a cysteine-rich segment (residues 85-112)
remotely related to the epidermal growth factor (EGF)-like
regions(24) . This segment, even if it lacks cysteine 1, 2, and
4 of the type 2 sequence(24) , displays a similarity with six
EGF-like regions belonging to types 2 and 3 described by Appella et
al.(24) (Fig. 3).
Figure 3:
Alignment of the atypical EGF-like region
of the 8.5 protein (amino acids 85-112) with the partial sequence
of the product of the p1A75 cDNA (2) and with the following
sequences: Agrin (amino acids 1727-1747)(30) , Notch (amino acids 270-290)(31) , TGF-binding
protein (TGF-bp) (amino acids 850-874)(32) ,
coagulation factor IX (F.IX) (amino acids
108-126)(33) ; low density lipoprotein receptor (LDL-R) (amino acids 330-353)(34) . Identical or
isofunctional residues are boxed.
Expression of the 8.5 mRNA
The 8.5 mRNA was
further characterized by Northern blot analysis as well as by in
situ hybridization. The pattern of hybridization of the 8.5 cDNA
probe to RNA from a variety of rat tissues (Fig. 4A)
revealed an absence of expression in liver, spleen, testis, kidney, and
heart and a strong but variable signal in all brain and spinal cord
samples tested. The 8.5 mRNA was also strongly expressed in the
pituitary and to a much lesser extent in the adrenal gland. The 8.5
probe recognized an apparently unique mRNA with a migration rate
slightly slower than the 18 S ribosomal RNA. Comparison of the 8.5
hybridization signal in RNA samples extracted from cultured astrocytes
or neurons suggested a neuronal origin for this mRNA whose faint
expression in the astrocyte culture can be attributed to a neuronal
contamination. As shown in Fig. 4B, the 8.5 mRNA was
present as early as E14 in cerebral hemispheres, where, as expected,
its expression increased during embryogenesis. The maximal abundance of
this mRNA was found around E20, and its expression faded afterward to
reach adult levels at P15.
Figure 4:
A, neural expression of the 8.5 mRNA
Northern blot hybridization of total RNA samples from various adult rat
organs, brain structures, or cultured cells using the full-length 8.5
cDNA as a probe. At 4 days of exposure of the autoradiogram, the
migration of the 18 and 28 S ribosomal RNAs is indicated. B,
Northern blot hybridization of total RNA samples from rat cerebral
hemispheres from various embryonic (E) or post-natal ages (P). Overnight exposure of the
autoradiogram.
In situ hybridization to 8.5
mRNA, performed on an oblique section of the whole E17 head (Fig. 5A), showed an intense labeling in several brain
areas, this mRNA being mostly expressed in the striatal rudiment, the
cerebral cortical plate, and in germinal zones containing proliferating
cells. The spinal cord and the nearby dorsal root ganglia were faintly
labeled. This is in contrast with in situ hybridization
experiments performed on a similar section with the neuron-specific
SCG10 probe indicating a very intense staining of these
structures(1) . A frontal section of the anterior part of the
whole E20 head (Fig. 5B) revealed a strong labeling of
the retinas and of the brain but not of the rest of the head.
Figure 5:
In situ hybridization to 8.5
mRNA. A, oblique section of a rat E17 head; B,
anterior frontal section of a rat E20 head.
Expression of the 8.5 Protein
Western blot
analysis (Fig. 6A) of 8.5 expression using an
affinity-purified antibody raised against the recombinant 8.5 protein
showed the existence of one predominant band in post-natal day 1 rat
brain. The apparent molecular mass of the corresponding protein (19
kDa) is very similar to that of the recombinant 8.5 protein after
removal of its added polyhistidine tail by proteolytic cleavage with
coagulation factor Xa (data not shown). The 8.5 protein was absent from
the soluble cytosolic fraction (S3) and was found at
comparable levels in the post nuclear pellet (P2) and the
supernatant (S2) in which it was fully associated with the
pellet (P3) obtained after ultracentrifugation. We also
performed (Fig. 6B), on the P2 and P3 fractions, a
carbonate treatment which allows to discriminate between integral and
peripheral membrane proteins(16) . Fig. 6B shows that the 8.5 protein is not released from the P2 and P3
fractions after carbonate treatment. Control experiments, performed
with an anti-synapsin antibody indicated that this peripheral protein
is, as expected, completely extracted from the P2 and P3 pellets by the
carbonate treatment (data not shown). Another protein of apparent
molecular mass 4O kDa was also weakly recognized by the antibody. The
presence of this protein in tissues in which the 8.5 mRNA was not
expressed suggests that it does not correspond to a dimer of the 8.5
protein (data not shown). Moreover, the molecular mass of this protein,
very different from that of the p1A75 cDNA product (28 kDa) (2) indicates that it must be different from this latter
protein.
Figure 6:
Immunoblot detection of the 8.5 protein
in subcellular fractions of a post-natal day 1 rat brain homogenate. A, fractions equivalent to 150-µg homogenate were loaded
on each lane. H, homogenate; S2, post-nuclear
supernatant; P2, post-nuclear pellet; S3, cytosol; P3, ultracentrifugation pellet. The migration of the molecular
mass markers is indicated. B, carbonate extractability of the
8.5 protein. P2 and P3 fractions equivalent to 100 µg of the S3
fraction were treated with sodium carbonate (+) or with
phosphate-buffered saline as a control(-) as described under
``Materials and Methods.''
Cellular and Subcellular Localization of the 8.5
Protein
Immunocytochemical analysis of primary cultures from the
embryonic striatum and cerebral cortex demonstrated the presence of a
prominent juxtanuclear Golgi-like labeling in the majority of neurons (Fig. 7A). This signal can be exclusively attributed to
the 8.5 protein immunoreactivity since Western blot experiments
indicated an extremely low expression of the 40-kDa protein in these
primary cultures (data not shown). In contrast, the few contaminating
glial cells were devoid of immunolabeling (Fig. 7A).
Analysis of the same preparation by immunoelectron microscopy showed
that most of the specific labeling is on the membrane of all the Golgi
saccules and on some vesicles in the Golgi zone (Fig. 8, A and B). Some dispersed small vacuolar structures and
multivesicular bodies were also observed in the cell body or in
neurites (Fig. 8C).
Figure 7:
A, immunofluorescence of cultured rat
neurons using affinity-purified anti 8.5 antibody. The juxtanuclear
area of the majority of neurons is conspicuously immunolabeled. One can
notice the immunostaining of a neurite (arrowhead) but the
majority of the neurites and a contaminating glial cell (arrow) are devoid of labeling. The bar represents 10
µm. B, immunofluorescence, using affinity-purified anti
8.5-antibody, of COS cells transfected with the 8.5 cDNA inserted in
the pSVL expression vector. Note the strong juxtanuclear
immunolabeling. The bar represents 10
µm.
Figure 8:
A
and B, ultrastructural localization of the 8.5 protein in the
Golgi apparatus (G) of cultured neurons. The stack of all
Golgi saccules is labeled. Note the strong labeling of two Golgi
vesicles (arrowheads). C, a labeled multivesicular
body (arrowhead) located within a neurite. N,
nucleus. The bar represents 0.5
µm.
Immunocytochemical analysis of
transfected COS cells expressing the 8.5 cDNA revealed a conspicuous
immunolabeling of a juxtanuclear zone (Fig. 7B). No
specific immunoreactivity was detected in wild-type COS cells (data not
shown).
DISCUSSION
In this study, we have characterized the second member of a
new protein family. This new 19-kDa protein (p19) is specifically
expressed in neural and neuroendocrine cells where it is localized
mainly in the Golgi apparatus.
The similarity of the primary
sequences of p19 and of the p1A75 cDNA product (2) indicates
that these proteins belong to the same family. Moreover, these two
proteins are specifically expressed in the cytoplasm of neural cells.
In addition to the atypical epidermal growth factor-like motif, a
search in the protein data bases for conserved regions revealed that
these two proteins share a highly similar short segment with
secretogranin III (Fig. 9)(25) . p19, whose calculated
isoelectric point is basic (9.48), cannot belong to the granin family
of acidic secretory proteins(26) , even if its precocious
expression peak is reminiscent of this family(25) .
Nevertheless, this motif, shared by p19 and secretogranin III,
respectively, expressed in the Golgi apparatus and in intracellular
vesicles of neural cells, could characterize proteins involved in the
process of neurosecretion. The Golgi-like localization of transfected
p19 in COS cells indicated that its primary sequence contains a Golgi
targeting signal. Comparison of the p19 primary sequence with that of
other proteins known to be expressed in this cellular compartment did
not provide any significant similarity. However, immunofluorescence and
immunoelectron microscopy analyses clearly indicated that the Golgi
apparatus is indeed the main expression site of p19 in embryonic
neurons (Fig. 8A) which express high amounts of the
corresponding mRNA (Fig. 4).
Figure 9:
Alignment of residues 142-153 of the
8.5 protein with residues 51-62 of the partial sequence of the
product of the p1A75 cDNA (2) and with residues 137-146
of the product of the 1B1075 cDNA (secretogranin III)(25) .
Identical or isofunctional amino acids are boxed.
Our data do not allow the
determination of the topology of p19 with respect to the membrane of
the Golgi apparatus. Nevertheless, the presence in p19 of an highly
hydrophobic segment (residues 70-92, Fig. 2) and its
carbonate-resistant membrane association (Fig. 6B)
indicated that it is an integral membrane protein. The absence of a
characteristic sequence for signal peptides, on the one hand, and the
migration to the same apparent molecular mass of native and factor
Xa-cleaved recombinant p19 (data not shown), on the other hand,
suggests that p19 have no signal sequence and that it could therefore
belong to the class of membrane protein with a C-terminal
anchor(27) .
The recognition, by our affinity-purified
antiserum, of a 40-kDa protein could be due to a reactivity against the
non-8.5 added part (MRGSHHHHHHIEGR, see ``Materials and
Methods'') of the recombinant protein. However, another antiserum
directed against an unrelated recombinant protein which bears the same
N-terminal tail did not recognize the 40-kDa protein. (
)The
40-kDa band could therefore correspond to a cross-reacting protein,
possibly belonging to the same family of that of p19. Southern blot
analysis of rat and human DNA indicated the presence of a single 8.5
gene (data not shown). In addition, the chromosomal assignment, in
5q35, of the human 8.5 gene, that we determined in a complementary
study (28) in which we sought for a possible colocalization
with a disease gene, does not correspond to any known neurological
hereditary disease.
Although the Golgi apparatus is the main
expression site of p19 in cultured neurons, its presence in the P3
fraction of a neonatal rat brain homogenate (Fig. 6) suggests
that this protein is also expressed in small organelles. Indeed, the
labeling of some vacuolar structures and multivesicular bodies, which
could be involved in neuronal endocytosis(29) , indicates that
the function of p19 may be related to some intraneuronal vesicular
trafficking. These data provide a working hypothesis that should be
tested by functional studies.