©1995 by The American Society for Biochemistry and Molecular Biology, Inc.
The BM88 Antigen, a Novel Neuron-specific Molecule, Enhances the Differentiation of Mouse Neuroblastoma Cells (*)

Avgi Mamalaki , Effrossini Boutou (§) , Catherine Hurel , Evangelia Patsavoudi , Socrates Tzartos , Rebecca Matsas (¶)

From the (1) Department of Biochemistry, Hellenic Pasteur Institute, 127 Vassilissis Sofias Avenue, 115 21 Athens, Greece

ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
FOOTNOTES
ACKNOWLEDGEMENTS
REFERENCES

ABSTRACT

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.


INTRODUCTION

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.


EXPERIMENTAL PROCEDURES

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.

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.

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.

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, 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).

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 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.


RESULTS

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.

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.

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).

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.

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.

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).


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.

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.

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 ().

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 ().


DISCUSSION

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-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).

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.

  
Table: Effect of retinoic acid (20 µM) on the differentiation of Neuro 2a-BM88 transfectants and control cells after 4 days in culture

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.



FOOTNOTES

*
This work was supported by the Greek General Secretariat of Research and Technology and the European Union Human Capital and Mobility Program Grant CRH-CT930286, Biotechnology Program Grant BIO2-CT930326, and Biomedicine and Health Program Grant BMH-CT941378. The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore by 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 GenBank/EMBL Data Bank with accession number(s) X82027.

§
Supported by a doctorate fellowship from the State Scholarships Foundation.

To whom correspondence should be addressed. Tel.: 30-1-6430044; Fax: 30-1-6423498.

The abbreviations used are: PAGE, polyacrylamide gel electrophoresis; bp, base pair(s); kb, kilobase(s).


ACKNOWLEDGEMENTS

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.


REFERENCES
  1. Altman, J. (1972a) J. Comp. Neurol. 145, 353-398 [Medline] [Order article via Infotrieve]
  2. Altman, J. (1972b) J. Comp. Neurol. 145, 399-464 [Medline] [Order article via Infotrieve]
  3. Anderson, D. J.(1989) Neuron 3, 1-12 [Medline] [Order article via Infotrieve]
  4. Bennett, M. K., Calakos, N., and Scheller R. H.(1992) Science 257, 255-259 [Medline] [Order article via Infotrieve]
  5. Bordier, C.(1981) J. Biol. Chem. 256, 1604-1607 [Abstract/Free Full Text]
  6. Bray, D., and White, J. G.(1988) Science 239, 883-888 [Medline] [Order article via Infotrieve]
  7. Brown N. A., and Kafatos, F. C.(1988) J. Mol. Biol. 203, 425-432 [Medline] [Order article via Infotrieve]
  8. Cattaneo, E., and McKay, R.(1991) Trends Neurosci. 14, 338-340 [Medline] [Order article via Infotrieve]
  9. Church, G. M., and Gilbert, W.(1984) Proc. Natl. Acad. Sci. U. S. A. 81, 1991-1995 [Abstract]
  10. Edelman, G. M.(1985) Annu. Rev. Biochem. 54, 135-169 [CrossRef][Medline] [Order article via Infotrieve]
  11. Feany, M. B., and Buckley, K. M.(1993) Nature 364, 537-540 [Medline] [Order article via Infotrieve]
  12. Graham, R., and Van der Eb, A.(1973) Virology 52, 452-467
  13. He, X., and Rosenfeld, M.(1991) Neuron 7, 183-196 [Medline] [Order article via Infotrieve]
  14. Hedrick, L., Cho, K. R., Fearon, E. R., Wu, T., Kinzler, K. W., and Vogelstein, B.(1994) Genes & Dev. 8, 1174-1183
  15. Jessel, T. M.(1988) Neuron 1, 3-13 [Medline] [Order article via Infotrieve]
  16. Kioussi, C., Crine, P., and Matsas, R.(1992) Neuroscience 50, 69-83 [Medline] [Order article via Infotrieve]
  17. Kumagai-Tohda, C., Tohda, M., and Nomura, Y.(1993) J. Neurochem. 61, 526-532 [Medline] [Order article via Infotrieve]
  18. Kumar, S., Matsuzaki, T., Yoshida, Y., and Noda, M.(1994) J. Biol. Chem. 269, 11318-11326 [Abstract/Free Full Text]
  19. LeClerk, N., Kosik, K. S., Cowan, N., Pienkowski, T. P., and Baas, P. W.(1993) Proc. Natl. Acad. Sci. U. S. A. 90, 6223-6227 [Abstract]
  20. Lyons, G. E., Ontell, M., Cox, R., Sassoon, D., and Buckingham, M. (1990) J. Cell Biol. 111, 1465-1476 [Abstract]
  21. McKay, R. D. G.(1989) Cell 58, 815-821 [Medline] [Order article via Infotrieve]
  22. Merkouri, E., and Matsas, R.(1992) Neuroscience 50, 53-68 [CrossRef][Medline] [Order article via Infotrieve]
  23. Morton, A. J., and Buss, T. N.(1992) Eur. J. Neurosci. 4, 910-916 [Medline] [Order article via Infotrieve]
  24. Patsavoudi, E., Hurel, C., and Matsas, R.(1989) Neuroscience 30, 463-478 [CrossRef][Medline] [Order article via Infotrieve]
  25. Patsavoudi, E., Hurel, C., and Matsas, R.(1991) J. Neurochem. 56, 782-788 [Medline] [Order article via Infotrieve]
  26. Patsavoudi, E., Merkouri, E., Thomaidou, D., Sandillon, F., Alonso, G., and Matsas, R.(1995) J. Neurosci. Res. 40, 506-518 [Medline] [Order article via Infotrieve]
  27. Pierceall, W. E., Cho, K. R., Getzenberg, R. H., Reale, M. A., Hedrick L., Vogelstein, B., and Fearon, E. R.(1994) J. Cell Biol. 124, 1017-1027 [Abstract]
  28. Ross, J., Olmsted, J. B., and Rosenbaum, J. L.(1975) Tissue & Cell 7, 107-136
  29. Sambrook, J., Fritsch, E. F., and Maniatis, T.(1989) Molecular Cloning: A Laboratory Manual, Cold Spring Harbor Laboratory Press, Cold Spring Harbor, NY
  30. Sanger, F., Nicklen, S., and Coulson, A. R.(1977) Proc. Natl. Acad. Sci. U. S. A. 82, 5463-5467
  31. Schuch, U., Lohse, M. J., and Schachner, M.(1989) Neuron 3, 13-20 [Medline] [Order article via Infotrieve]
  32. Sidman, R. L., and Rakic, P.(1973) Brain Res. 62, 1-35 [CrossRef][Medline] [Order article via Infotrieve]
  33. Smith, S. J.(1988) Science 242, 708-715 [Medline] [Order article via Infotrieve]
  34. Takeichi, M.(1991) Science 251, 1451-1455 [Medline] [Order article via Infotrieve]
  35. Trimble, W. S., Cowan, D. M., and Scheller, R. H.(1988) Proc. Natl. Acad. Sci. U. S. A. 85, 4538-4542 [Abstract]
  36. Walsh, F. S., and Doherty, P.(1992) Cold Spring Harbor Symp. Quant. Biol. 57, 431-440 [Medline] [Order article via Infotrieve]
  37. Wilkinson, D. G., Bailes, J. A., Champion, J. E., McMahon, A. P.(1987) Development 99, 493-500 [Abstract]
  38. Wilkinson, D. G., Bhatt, S., Chavrier, P., Bravo, R., and Charnay, P. (1989) Nature 337, 461-464 [CrossRef][Medline] [Order article via Infotrieve]
  39. Wood, J. N., and Anderton, B.(1981) Biosci. Rep. 1, 263-268 [Medline] [Order article via Infotrieve]
  40. Wright, C. V. E., Cho, K. W. Y., Oliver, G., and De Robertis, E. M. (1989) Trends Biochem. Sci. 14, 52-56 [CrossRef][Medline] [Order article via Infotrieve]
  41. Zuber, M. X., Goodman, D. W., Karns, L. R., and Fishman, M. C.(1989) Science 244, 1193-1195 [Medline] [Order article via Infotrieve]

©1995 by The American Society for Biochemistry and Molecular Biology, Inc.