From the
The BM88 antigen is a neuron-specific molecule widely
distributed in the mammalian nervous system. It is a 22-kDa, apparently
not glycosylated, integral membrane protein, which appears early during
brain development and remains at high levels in the mature animal.
Here, we describe the cDNA cloning of the porcine BM88 antigen and
present evidence that this protein is involved in neuroblastoma cell
differentiation. The deduced protein is a novel molecule consisting of
140 amino acids and bears a putative transmembrane domain at the
COOH-terminal region. The mRNA of this protein is expressed only in
neural tissues, where it is restricted to neurons. Stably transfected
Neuro-2a cells overexpressing the BM88 antigen exhibited a significant
change in morphology, reflected by enhanced process outgrowth, and a
slower rate of division. Moreover, in the presence of differentiation
agents, such as sucrose and retinoic acid, an accelerated
differentiation of the transfected Neuro-2a cells was observed.
Especially in the presence of sucrose, the consequent overexpression of
the BM88 antigen in the transfected cells resulted in their enhanced
morphological differentiation accompanied by the induction of
neurofilament protein expression. Our results suggest that the BM88
antigen plays a role in the differentiation of neuroblastoma cells.
The development of the nervous system requires several active
gene-regulated processes, including cell proliferation and
differentiation. During these complex processes, a large number of
genes are expressed in a predetermined and coordinated manner (Jessel,
1988; Wilkinson et al., 1989; Wright et al., 1989).
In the mammalian central nervous system, neurogenesis is largely
restricted to the ventricular zone along the inner surface of the
neural tube (McKay, 1989) and the external germinal layer of the
cerebellar cortex (Altman, 1972a, 1972b), while in the peripheral
nervous system precursor cells undergo proliferation and
differentiation as they migrate through the tissue (Anderson, 1989). In
the early embryonic neural tube, neural precursor cells undergo rapid
proliferation to form a multilayer system, from which post-mitotic
cells differentiate to generate the neurons (Sidman and Rakic, 1973).
At this point, neural precursor cells commit to specific
differentiation pathways and show a tightly regulated inverse
relationship between cell proliferation and differentiation (Cattaneo
and McKay, 1991). The molecular mechanisms that control these two
interrelated developmental events remain still largely undefined.
Previous studies have suggested that cell adhesion molecules
expressed on the surface of neural cells may function in development
and cell differentiation through both homophilic and heterophilic
interactions with molecules on the surface of opposing cells (Edelman,
1985; Jessel, 1988; Takeichi, 1991). In addition, members of the
different families of cell adhesion molecules, such as those of the
neural cell adhesion molecules and the cadherin families, have been
shown to trigger specific intracellular signal transduction pathways
when cell-cell interactions occur through these molecules (Schuch
et al., 1989; Walsh and Doherty, 1992). Moreover, recent
studies using neuronal or fibroblast cell lines have implicated a
number of intracellular neural molecules, such as the growth-associated
protein GAP-43 (Zuber et al., 1989; Morton and Buss, 1992;
Kumagai-Tohda et al., 1993), synaptotagmin (Feany and Buckley,
1993), and the microtubule-associated protein MAP 2C (LeClerk et
al., 1993), in the differentiation of these cells. Tumor
suppressor genes, like the depleted in colorectal cancer DCC gene, have
also been shown to stimulate neurite outgrowth in rat PC12
pheochromocytoma cells (Pierceall et al., 1994). These
observations suggest that a large number of molecules, some of which
may be still unknown, act independently or in concert in the same or
alternative pathways and are responsible for the complex mechanisms
that lead to neuronal differentiation. Indeed, the discovery of such
molecules has emphasized the important inverse relationship between
neuronal differentiation and tumorigenesis (Hedrick et al.,
1994; Kumar et al., 1994).
We have previously reported the
identification and characterization of a neuron-specific molecule, the
BM88 antigen, which is widely distributed in the central and peripheral
nervous system of mammals, including that of mouse, rat, rabbit, pig,
and human (Patsavoudi et al., 1989, 1995). The BM88 antigen,
which was first identified by means of a monoclonal antibody
(Patsavoudi et al., 1989), is an integral membrane protein,
composed of two 22-23-kDa polypeptide chains, depending on the
species tested. These polypeptide chains are apparently not
glycosylated and are linked together by disulfide bridges (Patsavoudi
et al., 1991). Recent electron microscopic observations in the
adult rat brain have shown that the BM88 antigen is mainly associated
with the limiting membrane of a number of intracellular organelles,
such as the endoplasmic reticulum, small electron-lucent vesicles, and
the outer membrane of mitochondria, but is also present at the plasma
membrane, especially at the level of synaptic densities (Patsavoudi
et al., 1995). Developmental studies have demonstrated that
the molecule is detected at the onset of neurogenesis in the rat brain
while its expression increases with age and remains at high levels in
the mature animal. In the developing nervous system, it is present in
both the neuroepithelium and in differentiated cellular areas as well
as in developing fiber tracts (Patsavoudi et al., 1995).
Especially in the cerebellum, the expression of the BM88 antigen is
particularly prominent in the Purkinje cells during the period of
development of their dendritic arbors. These data have suggested to us
that the BM88 antigen, which participates in an activity common to all
neurons and is associated with these cells and their precursors during
the period of their differentiation and maturation, may directly
contribute to these processes.
In the present study, we describe the
cDNA cloning and functional characterization of the porcine BM88
antigen, which is a novel protein. We present evidence which indicates
that overexpression of the BM88 cDNA in mouse neuroblastoma cells
enhances their morphological and molecular differentiation. This
biological activity of the BM88 antigen suggests that it may play a
role in the differentiation of neuronal cells.
The BM88 cDNA was subcloned into
pBluescript KS plasmid at the EcoRI site. Isolation of DNA
fragments, preparation of plasmid DNA, and other standard techniques
were performed as described by Sambrook et al.(1989).
Double-stranded pBluescript plasmids containing cDNA subclones were
used to determine the sequence on both strands (Sanger et al.,
1977) using Sequenase version 2.0 (U. S. Biochemical Corp.), as
described by the manufacturer. Sequence analysis was carried out using
the PCGENE software.
Solubilization of membrane proteins and phase separation with Triton
X-114 was performed according to Bordier(1981). Membrane stripping was
done by extraction with 0.1 M NaHCO
Scoring of neurite outgrowth in
differentiation experiments with retinoic acid was performed with the
use of the Pro Plus Media Cybernetics Image Analysis System. In this
case, cells were plated at an even lower density (10
No
significant sequence homology with any known nucleotide or amino acid
sequences was identified by searching the EMBL and GenBank data bases.
To resolve the discrepancy between the molecular mass of the BM88
antigen predicted from our cDNA sequence (14 kDa) and the apparent
molecular mass of the native protein from porcine brain estimated from
reducing SDS gels (22 kDa), we performed in vitro translation
analysis. For this purpose, we constructed a full-length cDNA by
cutting the two overlapping clones with appropriate restriction enzymes
and ligating them into the pBluescript vector (Stratagene), which
permits in vitro translation from each strand of the insert.
Subsequently, in vitro transcription and translation were
carried out by using two different constructs, containing either this
full-length cDNA or only the predicted coding region (610-bp
EcoRI-SphI fragment). In each case, a 22-kDa
immunoprecipitable polypeptide identical with the native pig brain
protein was produced, as identified after reducing SDS-electrophoresis
and autoradiography (data not shown). No polypeptide was obtained with
the RNAs transcribed from the antisense strands. These data suggest
that the apparent discrepancy between the predicted and the
electrophoretically estimated molecular weight of the BM88 antigen
should arise from the molecule's anomalous migration in SDS gels.
Southern blot analysis of pig DNA digested with several
restriction enzymes showed a simple hybridization pattern. In all
cases, the BM88 cDNA probe revealed one restriction fragment
(Fig. 2c). This is consistent with the BM88 antigen
being encoded by a single gene.
Western blot
analysis of whole cell extracts confirmed the expression of the BM88
cDNA in the transfected cells. A single polypeptide of 22 kDa was
detected in the transfected cells under reducing SDS-electrophoresis
(Fig. 3a). This polypeptide comigrated with the native
BM88 antigen from porcine brain (Fig. 3a). Under
non-reducing electrophoresis of either pig brain membranes or whole
cell extracts prepared from the transfected cells, a 42-kDa band was
visible in addition to the 22-kDa polypeptide (Fig. 3a).
This is in accordance with previously reported data for the endogenous
BM88 antigen from brain (Patsavoudi et al., 1989, 1991, 1995)
and demonstrates that the two polypeptide chains of the BM88 antigen
are identical. On the other hand, no polypeptide was detected in the
control non-transfected (Fig. 3a) or vector-transfected
(not shown) cells, presumably because of the very low expression of the
BM88 antigen in these cells. As previously demonstrated for the
endogenous protein from porcine brain (Patsavoudi et al.,
1989, 1991), solubilization and phase separation with Triton X-114
resulted in predominant partitioning of the transfected molecule in the
detergent phase (Fig. 3a).
Immunofluorescence labeling with either the
monoclonal or polyclonal antibodies to the BM88 antigen clearly
demonstrated overexpression of this molecule in the transfected cells
(Fig. 3g) when compared with controls that exhibited
only background levels of immunoreactivity (Fig. 3d). To
investigate whether the alterations observed in the shape of the Neuro
2a-BM88 cells were indicative of a differentiation process leading to a
neuronal phenotype, we immunostained these cells for neurofilament
protein, which was used as a molecular marker of neuronal
differentiation. We found that, in this case, the BM88 antigen-induced
processes did not contain neurofilament protein after 4 or 7 days in
culture (not shown). This suggests that the transfected BM88 antigen in
Neuro 2a cells is capable of converting the morphology of these cells
by inducing the formation of filopodial extensions but is not
sufficient by itself to trigger their differentiation to a neuronal
phenotype.
As it
was observed for the cells grown in the absence of differentiation
agents or those cultured in the presence of sucrose, a growth arrest
phenomenon in the transfected versus control cells was also
noted in the presence of retinoic acid ().
In this study, we report the molecular cloning and functional
characterization of the porcine BM88 antigen, a novel neuron-specific
molecule that, according to the data presented here, appears to be
involved in the differentiation of mouse neuroblastoma cells.
The
BM88 cDNA was isolated from a pig brain expression library using
specific monoclonal (Patsavoudi et al., 1989) and polyclonal
(Patsavoudi et al., 1995) antibodies.
Analysis of the
nucleotide and the deduced amino acid sequences revealed that the BM88
antigen bears no similarities with any of the known proteins and may
therefore belong to a novel family of neural molecules. Our sequence
data suggest that the BM88 antigen is an integral membrane protein
anchored to the membrane via a putative transmembrane domain located in
the COOH-terminal region of the protein. This is in accordance with
previous biochemical data derived from either the purified molecule or
crude membrane preparations from brain (Patsavoudi et al.,
1989, 1991, 1995). Moreover, our recent electron microscopic studies
showed that the BM88 antigen is preferentially associated with the
limiting membrane of a number of intracellular organelles such as the
endoplasmic reticulum, mitochondria, and small electron-lucent
vesicles, but is also present in the plasma membrane, especially at the
level of synaptic densities (Patsavoudi et al., 1995). Since
the predicted protein lacks an NH
The tissue distribution of the BM88
mRNA and protein expression revealed that the molecule is found only in
neural tissues. Moreover, the neuron-specific expression of the BM88
mRNA was demonstrated by in situ hybridization and verified
previously obtained immunocytochemical data (Patsavoudi et
al., 1989, 1995) on the BM88 protein expression.
To analyze the
functional characteristics of the BM88 antigen, we transfected a
full-length BM88 cDNA into mouse neuroblastoma (Neuro 2a) cells, which
express background levels of endogenous BM88 antigen. The transfected
molecule was found to be identical with the native antigen from porcine
brain according to the following criteria. 1) Both proteins comigrated
in SDS-electrophoresis, exhibiting the same apparent molecular mass of
22 and 42 kDa under reducing and non-reducing conditions, respectively.
2) Both proteins behaved identically upon high salt extraction or
solubilization and phase separation with Triton X-114, thereby
demonstrating that the transfected molecule is an integral membrane
protein of the Neuro 2a cells.
By overexpressing this molecule into
a cell-line that is inherently capable of extending neurites given the
appropriate conditions, we have uncovered an ability of the BM88
antigen to influence cell morphology by inducing process outgrowth
accompanied by a slower rate of division. Although in the absence of
differentiation agents the BM88-induced processes did not possess a
neuronal phenotype, as judged by the lack of neurofilament protein
expression, our results suggest that the BM88 antigen is involved in
the initial stages of the morphological differentiation of the cell. It
is well known that this process is tightly associated with growth
arrest (Hedrick et al., 1994; Kumar et al., 1994), a
phenomenon that was also observed in the transfected Neuro 2a-BM88
cells. We do not know how the BM88 antigen stimulates process formation
in transfected neuroblasts, but it is likely that the molecule
interacts with the machinery that mediates normal neuroblast process
outgrowth.
As discussed above, the BM88 antigen is an integral
membrane protein present in the limiting membrane of a number of
intracellular organelles and to a lesser extent at the plasma membrane.
Such a localization implies that the primary interactions of the BM88
antigen should occur intracellularly. Therefore, its mode of action
should be distinct from that of the members of the different cell
adhesion molecules that are known to mediate neurite outgrowth and
contribute to neuronal differentiation mainly through cell surface
interactions (Edelman, 1985; Jessel, 1988; Takeichi, 1991).
Apart
from the BM88 antigen, previous reports have implicated three other
intracellular molecules in neuronal differentiation. In particular, the
neural proteins GAP-43, synaptotagmin, and the microtubule-associated
protein 2C have been shown to induce changes in the morphology of
transfected fibroblasts and to promote the formation of filopodial
processes in these cells (Zuber et al., 1989, Feany and
Buckley, 1993, LeClerk et al., 1993). In addition,
overexpression of GAP-43 in transfected Neuro 2a cells (Morton and
Buss, 1992) resulted, in the absence of differentiation agents, in a
similar effect to what we observed in this study with the BM88 antigen.
Whether these molecules act in the same or different pathways, in a
synergistic or not manner, or to assist in cell-shape formation remains
to be established and represents a point of particular interest.
However, it should be noted in this respect that it is still unclear
whether the mechanisms that mediate the extension of filopodia in
non-neuronal cells are similar to or distinct from those that lead to
process outgrowth in cells capable of differentiating to a neuronal
phenotype (Smith, 1988; Bray and White, 1988).
Even more noteworthy
was the influence of the BM88 antigen on the phenotype of the
transfected cells in the presence of differentiation agents.
Differentiation of the Neuro 2a cells in the presence of sucrose or
retinoic acid presumably occurs via different mechanisms triggered,
respectively, by increased osmolarity (Ross et al., 1975) or
the activation of retinoic acid receptor (He and Rosenfeld, 1991). In
the presence of 0.2 M sucrose, which is optimal for the
differentiation of the parental Neuro 2a cells, the Neuro 2a-BM88
transfectants exhibited low levels of viability and, comparatively,
poor differentiation. However, in the presence of suboptimal sucrose
concentrations, insufficient by themselves to induce the
differentiation of the parental cell line, the Neuro 2a-BM88
transfectants showed a dramatic change in their morphology and
displayed the formation of an extensive network of long processes. This
time, the effect was accompanied by the appearance of a molecular
neuronal phenotype, suggesting that the BM88 antigen induced the
formation of neurites in the transfected cells. Thus, the combination
of increased osmolarity, together with the overexpression of the BM88
antigen, resulted in the accelerated morphological and molecular
differentiation of the Neuro 2a cells.
A marked acceleration of
neuritogenesis in the Neuro 2a-BM88 transfectants was also observed in
response to retinoic acid, which represents a recognized
differentiation signal (He and Rosenfeld, 1991). This effect again
resembles the action of GAP-43 in transfected neuroblasts (Morton and
Buss, 1992; Kumagai-Tohda et al., 1993). It has been
established that upon activation of retinoic acid receptor, multiple
genes become activated, and neuritogenesis proceeds in a controlled,
concentration-dependent manner (He and Rosenfeld, 1991). It is possible
that under normal circumstances, the concentration of the BM88 antigen
is limited, but its presence at high levels in the transfected cells,
in concert with the action of other so far unknown proteins, speeds up
dramatically the differentiation of Neuro 2a cells.
Taken together,
our results support the idea that a novel molecule, the BM88 antigen,
may play a role in the differentiation of neuronal cells. This idea is
consistent with the developmental profile of expression of the BM88
antigen, which is already present in neural precursor cells at the
onset of neurogenesis and is up-regulated during the period of neuronal
differentiation, axonal growth, and synapse formation (Patsavoudi
et al., 1995). Moreover, the abundant presence of the BM88
antigen in mature neurons (Patsavoudi et al., 1995) suggests
that besides a role in neuronal development, the molecule could also
contribute to neuronal plasticity in the adult nervous system. The
mechanism by which the BM88 antigen exerts its function awaits and
merits clarification. In this respect, the transfected cell lines we
have created should prove to be invaluable tools.
Values represent total cells counted in two
independent experiments, except for Neuro 2a-BM88 clone 2, which was
measured only in one experiment. The mean density, the mean percentage
of cells bearing at least one process >20 µM in length,
and the mean process length (±S.E.) was determined from these
two independent experiments (>100 cells were analyzed per
experiment). All measurements were blinded with respect to the cell
lines before assessment of differentiation.
The
nucleotide sequence(s) reported in this paper has been submitted to the
GenBank
We thank L. Denoroy for amino acid microsequencing, G.
Kollias for introducing us to the in situ hybridization
method, L. Margaritis and M. Issidoridou for access to the Pro Plus
Media Cybernetics Image Analysis System, C. Lolitsas for help with
computing and statistical analysis, and D. Thanos, E. Kouvelas, and K.
Soteriadou for discussions.
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
FOOTNOTES
ACKNOWLEDGEMENTS
REFERENCES
Protein Sequence
Determination
Purification of the detergent-solubilized
BM88 antigen from pig brain by immunoadsorption chromatography was
performed on an affinity column of monoclonal BM88 antibody coupled to
Sepharose beads, as described by Patsavoudi et al.(1991). The
purified protein was apparently homogenous (purity greater than 95%) as
estimated by SDS-PAGE.(
)
For determination of the
amino-terminal sequence of the BM88 antigen, the purified protein (50
µg) was subjected to SDS-PAGE and transferred onto polyvinylidene
difluoride membranes (Immobilon, Millipore). Protein was detected by
Coomassie Blue staining, and the 22-kDa band containing the BM88
antigen was cut and sequenced (Service Central d'Analyze,
Vernaison, France) on a protein microsequencer (Applied Biosystems,
model 470A) with an on-line high pressure liquid chromatography for
analysis of phenylthiohydantoin amino acid derivatives.
cDNA Library Screening
A specific
polyclonal antibody to the BM88 antigen (Patsavoudi et al.,
1995) was used for cDNA library screening. This antibody was affinity
purified on the isolated BM88 antigen that had been immobilized on
nitrocellulose filter after SDS-electrophoresis (Patsavoudi et
al., 1995). The specificity of this antibody for the BM88 antigen
has been previously documented (Patsavoudi et al., 1995). A
pig brain cDNA gt11 library (Clontech) was screened in duplicates
according to standard protocols (Sambrook et al., 1989) with
this affinity-purified polyclonal antibody, and selected clones were
tested for BM88 monoclonal antibody (Patsavoudi et al., 1989)
binding by lysogen analysis. Lysogens were prepared as described by the
manufacturer (Stratagene), and total protein was subjected to SDS-PAGE
and immunoblotting. Fusion proteins were detected by using a monoclonal
antibody to
-galactosidase, and bands of interest were selected on
the basis of their reactivity with both the monoclonal and polyclonal
anti-BM88 antigen antibodies.
Plasmid Construction
To generate the
full-length cDNA of BM88, we inserted the EcoRI-SfiI
fragment from clone 2 (which contains the 5`-end and the first 431 bp
of the cDNA) into clone 3 after digestion with the same enzymes
(SfiI and partial digestion with EcoRI) and isolated
the fragment that contains the pBluescript vector and the cDNA of BM88
from the 431 bp to the 3`-end. The expression vector for BM88 was
constructed by digesting the above construct with the enzymes
HindIII and XbaI and ligating the fragment containing
the full-length cDNA into the HindIII-XbaI
restriction sites of the pcDNA I vector (Invitrogen).
In Vitro Transcription and
Translation
RNAs were transcribed in vitro using
bacteriophage T7 or T3 RNA polymerase (Stratagene) according to the
manufacturer's suggested protocols. For antisense or sense RNA
probes, the plasmid DNAs were linearized in the HindIII or
BamHI sites, respectively. In vitro protein synthesis
was carried out with the Reticulocyte Lysate System (Promega) in the
presence of S-labeled methionine. For immunoprecipitation
(Merkouri and Matsas, 1992) with the monoclonal antibody BM88,
translation products were diluted 10-fold, and a mixture of protease
inhibitors was added. The mixture was then absorbed for 2 h at 4 °C
with bovine serum albumin coupled to Sepharose followed by overnight
incubation at 4 °C with monoclonal antibody BM88 coupled to
Sepharose (50 µl of gel, 10 mg of antibody/ml of gel). Immune
complexes were collected by centrifugation and washed. Proteins were
recovered by boiling in SDS loading buffer and were analyzed by
SDS-electrophoresis and autoradiography.
DNA and RNA Transfer Analysis
Genomic DNA
and total RNA were isolated from newborn pig tissues as described
(Sambrook et al., 1989, Brown and Kafatos, 1988), separated by
electrophoresis on agarose gel, and then transferred to Zeta probe
nylon membrane (Bio-Rad). A full-length BM88 P-cDNA probe
labeled with the random priming kit (Amersham Corp.) was used for
hybridization as described (Church and Gilbert, 1984). The blots were
autoradiographed at -80 °C.
Preparation of Membrane Fractions and Western
Blotting
For the immunoblots, membrane protein fractions
were prepared, and Western blotting was performed as described
(Merkouri and Matsas, 1992). Whole cell extracts were prepared from
10 cells by brief sonication in 60 µl of 10 mM
Tris-HCl, pH 7.4, and were immediately mixed with SDS-PAGE loading
buffer. Half of the total cellular protein was loaded per lane.
, pH 11.0.
Briefly, membrane fractions were resuspended in the pH 11.0 buffer and
rotated for 1 h at 4 °C, after which time they were centrifuged at
30.000
g for 1 h. Both supernatant and pelleted
fractions were collected and analyzed by SDS-PAGE and immunoblotting.
In Situ Hybridization
Tissues from
newborn pigs were appropriately dissected and fixed by overnight
immersion at 4 °C in 4% paraformaldehyde in phosphate-buffered
saline. After fixation, tissues were embedded in paraffin (Paraplast
Plus), and serial sections (5-µm thick) were cut and collected on
gelatin/potassium chrome-sulfate-coated glass slides. Antisense or
sense RNA probes were labeled by S-dATP. Protocols for
hybridization and post-hybridization treatments were according to
Wilkinson et al.(1987) and Lyons et al.(1990).
Cell Culture and Transfections for Stable
Expression
Mouse neuroblastoma Neuro 2a cells (ATCC) were
maintained in Dulbecco's modified Eagle's medium
supplemented with 10% heat-inactivated fetal calf serum and
antibiotics. Cells were cotransfected with pcDNA I constructs and
pSVneo plasmid using DNA-calcium phosphate coprecipitates
as described by Graham and Van der Eb(1973). Selection with G-418 was
started 48 h later and was maintained for 5 weeks. For cell cloning,
the cells were aliquotted into 96-well tissue culture plates in
different dilutions, and stable transformants were selected. Lines were
subcloned until all cells were found to express the BM88 antigen by
immunofluorescence.
Immunofluorescence Analysis
For
immunofluorescence analysis, cells were plated at low density
(3-5 10
cells/well) and were grown on
poly-L-lysine-coated sterile glass coverslips in 48-well
plates. For immunofluorescence labeling, cells were fixed in 4%
paraformaldehyde and incubated with BM88 monoclonal antibody (overnight
at 4 °C) followed by 2 h of incubation with fluorescein
isothiocyanate-labeled rabbit anti-mouse secondary antibody (Kioussi
et al., 1992). An identical staining pattern was seen using
the rabbit polyclonal anti-BM88 antigen antibody (Patsavoudi et
al., 1995) followed by fluorescein isothiocyanate-conjugated
anti-rabbit secondary antibody.
Differentiation Experiments
Cells for
differentiation studies in the presence of sucrose (2 10
cells/well) or retinoic acid (3-5
10
cells/well) were plated onto poly-L-lysine-coated
sterile coverslips in 48-well plates. Differentiation was initiated
either at the time of plating by the addition of sucrose or the day
after by the addition of retinoic acid. Cells were monitored up to 7
days in culture in the presence of differentiation agents.
Immunofluorescence labeling was performed as described above in sister
cultures of paraformaldehyde-fixed cells with the BM88 monoclonal or
polyclonal antibodies or a monoclonal antibody to neurofilament protein
(Wood and Anderton, 1981). In the latter case, cells were permeabilized
with 0.1% Triton X-100 prior to application of the primary antibody
(Kioussi et al., 1992).
cells/well) to restrict cluster formation and allow cells to grow
separately. Neurite length was measured as the distance between the
edge of the soma and the tip of its longest neurite. Neurites were only
measured if 1) the neurite emerged from a cell in isolation (not in a
cluster of cells), 2) the neurite did not contact other cells or
neurites, and 3) the neurite was greater than 20 µm in length.
These criteria excluded the majority of cells; thus, quantification was
performed on relatively few cells that fulfilled all the above
criteria.
Isolation and Characterization of the BM88-cDNA
Clones
To isolate the BM88 cDNA, we screened 1
10
independent plaques from a pig brain
gt11 cDNA
library using a specific polyclonal anti-BM88 antibody (Patsavoudi
et al., 1995). Several positive clones were isolated and were
subsequently subjected to lysogen analysis for further identification.
Three of these, which were also recognized by the anti-BM88 monoclonal
antibody (Patsavoudi et al., 1989, 1991), were selected for
further study. Two of them, designated 1 and 2, were of the same length
containing a 1.0-kb cDNA insert, and the third one, designated 3,
contained a longer insert of 1.4 kb (Fig. 1a). Sequence
analysis showed that the two clones with the same length are identical
containing the 5`-end of the BM88 cDNA and are overlapping with the
third one that contains the 3`-end of the BM88 cDNA
(Fig. 1a). The cDNA sequence predicts a putative AUG
initiation codon followed by a 420-nucleotide open reading frame and a
TGA termination codon at position 544. The coding region is followed by
a long 3`-untranslated sequence containing a poly(A)+ addition
site at position 1505 preceded by a polyadenylation signal. This open
reading frame encodes a polypeptide of 140 amino acids with a predicted
molecular mass of 14 kDa (Fig. 1b). Direct protein
microsequencing of the BM88 antigen purified from porcine brain
revealed the following amino acid sequence at the amino-terminal region
of the protein: SANSPKADAKA. The open reading frame obtained from the
cDNA clones contains this partial amino acid sequence
(Fig. 1b), thus confirming the identity of our clones.
Figure 1:
Structure of the BM88 antigen.
a, restriction map of full-length BM88 cDNA with the following
restriction sites: X, XbaI; E,
EcoRI; S, SfiI; P, PstI;
Sp, SphI; Xh, XhoI; H,
HindIII. Three positive clones were isolated. Two of them
(1 and 2) were identical with a length of 1007 bp,
and the third one (3), 1406 bp long, was overlapping with the
first two. The wave line denotes pBluescript vector DNA. The thickshadedsection represents the location of the
coding region for the BM88 antigen. The 5`- and 3`-untranslated regions
with the poly(A) signal (A) are indicated. b,
complete nucleotide sequence of the pig BM88 cDNA and the deduced
protein sequence. The translated amino acid sequence is shown in
three-letter code below the nucleotide sequence; both are
numbered on the right. The NH-terminal
amino acid sequence determined from the purified protein
(underlined) matches the cDNA deduced amino acid sequence. The
putative transmembrane region (in italics) is also
underlined, and the asterisk denotes a stop codon.
The polyadenylation signal is underlined. Symbols indicate potential sites as follows:
, protein kinase C
phosphorylation;
, cAMP- and cGMP-dependent protein kinase
phosphorylation; X, N-linked glycosylation;
,
casein kinase II phosphorylation;
,
myristoylation.
Hydrophobicity analysis suggested that the BM88 antigen is an
integral membrane protein consisting of a large hydrophilic part (115
amino acids) followed by a putative transmembrane domain (21 amino
acids) and a short hydrophilic tail (4 amino acids)
(Fig. 1b). However, no candidate for a signal sequence
was identified in the NH-terminal portion of the predicted
molecule. The deduced protein is rich in alanine and proline (20.7 and
15% of the residues, respectively). Several consensus sequences for
post-translational modifications have been identified by the Prosite
analysis program as described in the legend to Fig. 1.
Expression of the BM88 Antigen Is Limited to the
Nervous System
The tissue distribution of the BM88 mRNA was
examined by performing Northern blot analysis. Total RNA isolated from
different pig tissues (15 µg/lane) was electrophoresed,
transferred, and hybridized with the BM88 full-length cDNA probe. A
single band of 1.5 kb was apparent only in neural tissues
(cerebrum, cerebellum, and spinal cord) and not in leukocytes, muscle,
kidney, adrenals pancreas, spleen, liver, lung, and aorta
(Fig. 2a). Glyceraldehyde-3-phosphate dehydrogenase
hybridization, which served as a control for loaded RNA, was apparent
in all tissues tested (data not shown). Similarly, the BM88 protein
expression was investigated by Western blot analysis. Membrane proteins
from several pig tissues (30 µg/lane) were analyzed by reducing
SDS-PAGE and immunoblotting. The blot was probed with the BM88
monoclonal antibody, and a prominent 22-kDa band was visible only in
brain (Fig. 2b). Identical results were obtained when
the blot was probed with the polyclonal anti-BM88 antibody (data not
shown).
Figure 2:
a, tissue distribution of BM88 mRNA
determined by Northern blot analysis. 28 and 18 S show the positions of
these rRNA species on the blot. b, examination of BM88 protein
expression by Western blot analysis. Membrane proteins from the
indicated pig tissues (30 µg/lane) were analyzed by reducing
SDS-PAGE and immunoblotting. A prominent 22-kDa band is visible only in
brain. Positions of molecular weight standards are indicated on the
right. c, Southern blot analysis of pig liver DNA
digested with the indicated restriction enzymes shows a simple
hybridization pattern. The numbers at the right represent DNA size markers in kilobases. d and
e, localization of BM88 mRNA in paraffin-embedded sections of
newborn pig striatum (d) and dorsal root ganglia (e).
Hybridization with the antisense RNA probe is detected in neurons.
Scalebars, 100 µm.
The site of BM88 mRNA synthesis in the nervous system was
examined by in situ hybridization using a
S-labeled antisense RNA probe. In paraffin-embedded
sections from newborn pig striatum (Fig. 2d) and dorsal
root ganglia (Fig. 2e), intense grains were observed
only in neurons. No significant autoradiographic grains were observed
in the remaining parts of the tissues. BM88 mRNA could not be detected
when control tissues were hybridized with the sense RNA probe (data not
shown).
Overexpression of the BM88 Antigen in Neuro 2a-BM88
Cell Clones
To analyze the functional characteristics of
the BM88 antigen, we transfected a full-length cDNA into mouse
neuroblastoma (Neuro 2a) cells that normally express very low to
undetectable levels of endogenous BM88 antigen. The full-length BM88
cDNA was inserted to the pcDNA I vector adjacent to the 3`-end of the
cytomegalovirus promoter. The resulting plasmid was cotransfected into
Neuro 2a cells with a selectable plasmid containing the
neomycin-resistant gene. Numerous stable cell lines were isolated
expressing the BM88 antigen as determined by immunofluorescence
screening using the BM88 monoclonal antibody and a
fluorescein-conjugated secondary antibody. Three of these with the
highest BM88 antigen expression (Neuro 2a-BM88 cells), were selected
for further studies, and all three stable cell lines produced identical
results. As controls, we used either the parental non-transfected Neuro
2a cells or cells transfected with the vectors alone.
Figure 3:
Expression of the BM88 antigen in
transfected Neuro 2a cells. a, Western blot analysis using the
BM88 monoclonal antibody. The BM88 antigen is detected in pig brain
membranes (30 µg/lane) and whole cell extracts prepared from the
Neuro 2a-BM88 transfectants, as a 22- or 42-kDa polypeptide band under
reducing and non-reducing conditions, respectively. The 22-kDa band is
also visible under non-reducing electrophoresis. The BM88 antigen is
not detected by this method in the parental Neuro 2a cells because of
its very low expression. Triton X-114 treatment of whole cell extracts
from Neuro 2a-BM88 transfectants results in predominant partitioning of
the BM88 antigen in the detergent phase. b-g,
phase-contrast imaging of Neuro-2a (b, c) and Neuro
2a-BM88 (e, f) cells cultured for 4 or 7 days in the
absence of differentiation agents. Neuro 2a-BM88 transfectants extend
elaborate processes, while the non-transfected cells are essentially
round. This phenomenon is more pronounced after 7 days in culture.
d and g, immunofluorescent detection of the BM88
antigen in the parental (d) and BM88-transfected Neuro 2a
cells (g), after 7 days in culture. Micrographs (d and g) correspond to phase contrast images in c and f, respectively. Overexpression of the BM88 antigen
in the Neuro2a-BM88 transfectants is apparent. Scalebars, 25 µm.
To exclude the possibility
that the transfected BM88 antigen may be strongly associated with a
transmembrane protein, thereby partitioning in the detergent phase of
Triton X-114 without being in itself an integral membrane protein of
the Neuro 2a cells, we carried out membrane stripping at pH 11.0,
followed by immunoblotting. Again, we found that the BM88 antigen
remained associated with the membrane fraction (not shown). These
results demonstrate that the transfected molecule is an integral
membrane protein of the Neuro 2a cells.
The BM88 Antigen Affects the Morphology of the
Transfected Neuro 2a-BM88 Cells
The transfected Neuro
2a-BM88 cells showed a significant change in their basic morphology
when compared with controls as judged by phase-contrast microscopy. The
transfected cells displayed enhanced process outgrowth (Fig. 3,
e-g), while control cells consisting of either
non-transfected Neuro 2a cells or Neuro 2a cells transfected with the
vectors alone were found to be essentially round (Fig. 3,
b-d). This change in morphology was accompanied by a
slower rate of division in the transfected cells. The density of the
transfected and control cells was determined in two separate
experiments, in which more than 100 cells/experiment were counted. We
found that the average cell density of the non-transfected Neuro 2a
cells was 50 cells/field versus only 23 cells/field for the
transfected cells. This shows a growth arrest of >50% in the
transfected cells.
The BM88 Antigen Accelerates the Differentiation of
Neuro 2a-BM88 Cells in the Presence of Sucrose
To further
investigate the influence of the BM88 antigen in the transfected Neuro
2a cells, we performed differentiation studies. It is well established
that the Neuro 2a cell line can be induced to differentiate to a
neuronal phenotype when cultured in medium that has been made
hypertonic with sucrose (Ross et al., 1975) or in the presence
of various agents, including retinoic acid (Morton and Buss, 1992). For
differentiation studies, control and Neuro 2a-BM88 cells were
differentiated in parallel and were monitored between 24 h and 7 days
after plating. Markedly different responses were noted between the
transfected and control cells. In the presence of 0.2 M
sucrose, which is optimal for the differentiation of the wild-type or
the vector-transfected cells (Ross et al., 1975), the Neuro
2a-BM88 transfectants exhibited low levels of viability and,
comparatively, poor differentiation (data not shown). However, in the
presence of suboptimal sucrose concentration (0.1 M),
insufficient by itself to induce the differentiation of control cells,
the Neuro 2a-BM88 transfectants showed a dramatic change in their
morphology elaborated by an extensive network of long neurite-like
processes involving the majority of cells (Fig. 4, a and
b). A similar but less pronounced effect was obtained in the
presence of 0.05 M sucrose (data not shown). To further
examine the processes induced by the BM88 antigen, we stained by
immunofluorescence sister cultures of the transfected cells for either
the BM88 antigen or neurofilament protein (Fig. 4,
a-d). We found that the BM88 antigen-induced processes
contained neurofilament protein, demonstrating that the morphological
differentiation of the Neuro 2a-BM88 cells was accompanied by the
appearance of a molecular neuronal phenotype (Fig. 4,
c-d). In contrast, under the same conditions, control
cells exhibited minimal process outgrowth and very low levels of
neurofilament protein expression (Fig. 4, e and
f). The previously observed slower rate of division of the
Neuro 2a-BM88 transfectants as compared with the controls was also
noted in the presence of sucrose.
Figure 4:
Upper panel, effect of 0.1 M
sucrose (a-f) on the morphological and molecular
differentiation of Neuro 2a-BM88 transfectants (a-d) and
control non-transfected cells (e, f) after 6 days in
culture. Phase contrast imaging (left) and immunofluorescence
labeling (right) for the BM88 antigen (a, b)
or neurofilament protein (c-f) is shown. An induction of
neurofilament protein (NF) is apparent in the well
differentiated process-bearing Neuro 2a-BM88 transfectants when
compared with the wild-type Neuro 2a cells. Lower panel, phase
contrast imaging of Neuro 2a-BM88 transfectants (g) and
control vector-transfected cells (h) reveals an accelerated
differentiation of the BM88 transfectants after 4 days in culture in
the presence of retinoic acid. Scalebar, 50
µm.
The BM88 Antigen Enhances Neurite Formation in Neuro
2a-BM88 Cells in Response to Retinoic Acid
A marked
acceleration of neurite formation by the Neuro 2a-BM88 cells was also
observed in response to 20 µM retinoic acid (Fig. 4,
g and h). Within 24 h of addition of retinoic acid,
the Neuro 2a-BM88 transfectants had started to extend visible
processes. To quantitate the BM88 antigen-induced effect, we measured
the number of process-bearing cells and their process length in two
independently derived Neuro 2a-BM88 cell lines and in control cells. We
found that by 4 days, more than 80% of the Neuro 2a-BM88 cells were
well differentiated versus only 50% of the
vector-transfected cells or
60% of the wild-type Neuro 2a cells.
Moreover, the average process length of the transfected cells measured
at least 30% longer than that of control cells ().
-terminal signal sequence,
it is possible to assume that it may be membrane-anchored with its
NH
-terminal on the cytoplasmic side of the bilayer. Similar
observations have led to analogous predictions for the orientation of
the synaptic proteins VAMP (Trimble et al., 1988) and syntaxin
(Bennett et al., 1992).
Table:
Effect of retinoic acid (20 µM) on
the differentiation of Neuro 2a-BM88 transfectants and control cells
after 4 days in culture
/EMBL Data Bank with accession number(s) X82027.
©1995 by The American Society for Biochemistry and Molecular Biology, Inc.