Modulation of NCAM expression by transforming growth factor-beta, serum, and autocrine factors.

The expression of NCAM (neural cell adhesion molecule) is precisely regulated in terms of cell type specificity and developmental control. We searched for extracellular factors that may be involved in this regulation using N2A neuroblastoma and NIH 3T3 fibroblastic cells. Factors contained in FBS promoted a two- to threefold increase in NCAM protein and mRNA abundance in both cell lines. This increase in NCAM expression in high serum could be entirely attributed to enhanced levels of the NCAM-140 message. Modulation of NCAM synthesis via an autocrine mechanism is suggested by the observation that medium conditioned by N2A cells stimulated NCAM mRNA expression by 3T3 and N2A cells. Among the pure factors tested, transforming growth factor-beta (TGF beta) was found to act as an inducer of NCAM expression in 3T3 but not in N2A cells. 3T3 cells responded to exposure to TGF beta with a two- to threefold increase in NCAM protein and mRNA. Exposure of early-passage embryonic cells to TGF beta resulted in four- and twofold increases in NCAM protein and mRNA abundance, respectively, suggesting a role for TGF beta in modulating NCAM expression in the embryo. TGF beta seems to act by stimulating the transcriptional activity of the NCAM gene because it did not affect transcript stability and stimulated transcription from a proximal promoter element of the NCAM gene.

Identification of positive and negative regulatory elements governing cell-type-specific expression of the neural cell adhesion molecule gene.

The neural cell adhesion molecule (NCAM) is one of the most prevalent cell adhesion molecules in vertebrates. Its expression is subject to complex cell-type- and developmental-stage-dependent regulation. To study this regulation at the level of transcription, we analyzed the promoter region of the mouse NCAM gene. The NCAM promoter did not contain a typical TATA box. Transcription started at several sites that were used indiscriminately by different cell types, implying that the different NCAM isoforms are expressed from a single promoter. Sequences responsible for both promotion and inhibition of transcription resided within 840 base pairs upstream of the main transcriptional start site. The sequence from positions -645 to -37 relative to the translation initiation site directed high levels of expression in NCAM-expressing N2A cells. The same fragment was six times less active but still significantly active in L cells, but this activity was repressed by inclusion of an additional upstream segment. We mapped eight domains of interactions with nuclear proteins within the 840-base-pair region. The segment with maximum promoter activity contained two adjacent footprints, the occupation of which appeared to be mutually exclusive. One of them corresponded to an Sp1-factor-binding consensus site, the other one bound a factor with nuclear factor I activity. The single protected domain in the fragment harboring a repressor activity consisted of a GGA repeat resembling negative regulatory elements in other promoters. Three adjacent binding sites occupied an A + T-rich segment and contained ATTA motifs also found in the recognition elements of homeodomain proteins. These results show that negative and positive elements interact to regulate the tissue-specific patterns of expression of the NCAM gene and indicate that a factor related to nuclear factor I is involved in its transcriptional control.

An upstream regulatory element of the NCAM promoter contains a binding site for homeodomains.

In the present study, we have analyzed an upstream regulatory element of the neural cell adhesion molecule (NCAM) promoter which is required for full promoter activity. It contains an ATTATTA motif that resembles the core recognition sequence of homeodomain (HD) proteins of the Antennapedia (Antp) and related types. Electrophoretic mobility shift (EMSA) and DNase I footprinting analyses revealed that the Drosophila HDs coded by the Antp and the zerknüllt (zen) genes bind this site in vitro. In contrast, the engrailed (en) protein did not produce a detectable footprint. The functional relevance of the ATTATTA motif was demonstrated by showing that a two-nucleotide exchange curtailed stimulation of an heterologous promoter. An oligonucleotide known to be recognized with high affinity by Antp-like HDs efficiently competed for endogenous factor binding. These results suggest that the NCAM gene may be a target for HD proteins.

A new brain cell surface glycoprotein identified by monoclonal antibody.

Of 207 monoclonal antibodies produced against cultured mouse cerebellar cells, 16 reacted with cerebellar cell surfaces and 4 reacted with glycoproteins. One of them, called an anti-BSP-3 (Brain cell Surface Protein-3) defines a 48,000 molecular weight protein which can be iodinated at the surface of cultured cerebellar cells. Lectin-binding and sugar incorporation studies established the glycoprotein nature of the antigen. Astroglia (glial fibrillary acidic protein-positive cells) in primary cerebellar cultures were labelled intensely for this antigen by the indirect immunofluorescence method while neuronal cells and their processes were more weakly labelled. Fibronectin-positive cells were negative for BSP-3. In cerebellar sections using the immunoperoxidase method at both the optical and electron microscope levels, the difference in staining intensity between astrocytes and neuronal cells was not significant: in Purkinje cells and in the large neurones present in the deep cerebellar nuclei the immunoperoxidase percipitate was confined to the plasma, membrane while in both astrocytes and granule cells cytoplasmic labelling was also observed. Oligodendrocytes do not appear to react with the anti-BSP-3 monoclonal antibody; neither do endothelial or leptomeningeal cells. The availability of a monoclonal antibody produced by a stable hybridoma line will be a powerful tool in attempts to purify the BSP-3 antigen and to elucidate its function.

Molecular heterogeneity and structural evolution during cerebellar ontogeny detected by monoclonal antibody of the mouse cell surface antigen BSP-2.

The monoclonal antibody anti-BSP-2 defines a set of glycoproteins present on the neuronal cell surface in dissociated mouse cerebellar cultures and on neurons and astrocytes in sections of the mouse cerebellum. This antibody was used in the present study to characterize the antigens recognized in cerebellar cultures and in the developing and adult mouse cerebellum in vivo. In extracts from cerebellar cultures and from late postnatal or adult cerebellum, the anti-BSP-2 antibody reacted with a triplet of glycosylated polypeptide chains of 180,000, 140,000 and 120,000 mol. wt. Early postnatal cerebellum contained a different form of BSP-2 antigen which migrated as one broad or several closely spaced diffuse bands in the 190,000-250,000 mol. wt. region of SDS polyacrylamide gels. During cerebellar ontogeny, the adult pattern emerged gradually between postnatal days 5 and 13. The cellular expression of the BSP-2 antigen was studied by immunohistochemistry on sections of the developing cerebellum. At postnatal day 3, the antigen was found mainly on cell bodies and fibers of the Bergmann glia and on astrocytes of the granular layer. Immature granule cells of the outer zone of the external granular layer lacked the antigen, but they appeared to acquire the antigen during their migration to the internal granular layer. At postnatal day 13, the immunofluorescence pattern was not different from the one seen in the adult. These results suggest that the neonatal 190,000-250,000 mol. wt. form of BSP-2 may at least in part be expressed by astroglial cells and they show a close correlation between the emergence of the adult forms of the antigen and the appearance of labeled granule cells in the internal granular layer. In vitro degradation implying cleavage of sialic acid residues, but probably also proteolysis and/or cleavage of different glycans converted the neonatal form of BSP-2 into the triplet pattern and ultimately into a p120 component. Neuraminidase digestion of the adult antigens produced small molecular weight shifts without converting one band into the other, but endogenous enzyme activities were capable of degrading the p180 and p140 bands by converting them into the p120 protein. Our findings support the idea that distinct, but structurally similar surface glycoproteins created by post-translational modifications from a common precursor molecule may be expressed by different cell types or during different developmental stages. As shown by sequential immunoprecipitation experiments, BSP-2 and the rat neuronal membrane protein D2 may belong to the same family of surface glycoproteins.

Monoclonal antibody against cell surface glycoprotein of neurons.

A monoclonal antibody, named BSP-2, has been produced against glycoproteins extracted from neonatal mouse brain. Its reactivity with live cells established the surface location of the antigen. In primary cultures of dissociated cerebellar cells, the antibody bound to neuronal cell types, but not to astrocytes nor to fibroblasts. Immunoprecipitates prepared with the BSP-2 antibody contained a triplet of high-molecular weight glycoproteins with apparent molecular weights of 180,000, 140,000 and 120,000.

Occurrence of alpha 2-8 linked polysialosyl units in a neural cell adhesion molecule.

A brain cell surface protein (BSP-2) was isolated from mice of different ages by affinity chromatography using a monoclonal antibody. Analysis of glycopeptides obtained after pronase digestion revealed that the embryonal and neonatal forms of the antigen contained an unusually high proportion of sialic acid, which decreased during development. Methylation analysis of native and neuraminidase treated glycopeptides indicated that the sialic acid occurred as alpha 2-8 bound polysialosyl units, similar to those of the recently described developmentally regulated polysialosyl glycopeptides of rat brain. Furthermore, the carbohydrate and amino acid composition, and electrophoretic mobility of BSP-2 antigen correspond to those reported for a neural cell adhesion molecule (N-CAM).

Studies on the transmembrane disposition of the neural cell adhesion molecule N-CAM. The use of liposome-inserted radioiodinated N-CAM to study its transbilayer orientation.

The transmembrane orientation of the polypeptide chains present in preparations of adult and neonatal mouse N-CAM was studied using, as a model system, liposome-inserted purified N-CAM preparations. N-CAM purified from adult or neonatal mouse brain was 125I-labeled and reconstituted into artificial lipid vesicles. After trypsin digestion, the peptides that remained associated with the liposomes were isolated by floatation of the vesicles on sucrose gradients. In control experiments the liposomes were lysed before trypsin treatment. Large, overlapping peptides were obtained after this treatment, several of which were protected by the liposome membrane. Sialic-acid-bearing peptides were revealed by their sensitivity to neuraminidase. To distinguish between peptides corresponding to intracellular or extracellular domains use was made of the P61 and H28.123 monoclonal antibodies, which recognize determinants located on the cytoplasmic and the extracellular part of the molecules respectively. There was no indication that the N-CAM chains were inserted in an inside-out configuration. Peptides protected from trypsin attack by the liposomes and recognized only by P61 had Mr values of 92 000, 42 000 and 35 000. The H28.123 determinant could be mapped to a 32 000-Mr peptide located close to the membrane at the vesicle's exterior. The bulk of the sialic acid seemed to be carried by a rather short sequence distal to the H28.123-reactive peptide but at some distance from the N terminus. Fragments of very similar Mr were generated from young and adult material. However, a 45 000-Mr peptide from neonatal N-CAM appeared to migrate in the higher-Mr region of sodium dodecyl sulfate/polyacrylamide gels in its fully sialylated form. It is concluded that (a) identical polypeptide chains are present in young and adult preparation, (b) the 180 000-Mr, 140 000-Mr and 120 000-Mr chains differ by the length of their cytoplasmic extensions and (c) the largest cytoplasmic sequences have a Mr close to 90 000. A tentative linear model of the transmembrane topography of the N-CAM polypeptides is presented.

Antennapedia homeobox peptide regulates neural morphogenesis.

We synthesized the 60-amino acid polypeptide corresponding to the sequence of the Drosophila antennapedia gene homeobox. This peptide (pAntp) recognized the consensus motif for binding to the promoter region of Hox-1.3. pAntp mechanically introduced into mammalian nerve cells provoked a dramatic morphological differentiation of the neuronal cultures. Moreover, pAntp directly added to already differentiated neuronal cultures penetrated the cells and further augmented their morphological differentiation. Examination of live and fixed neurons in classical and confocal fluorescence microscopy demonstrated that pAntp was captured at all regions of the nerve cells and accumulated in the nuclei. In addition, the effect of pAntp on neurite extension was blocked in the presence of the protein synthesis inhibitor cycloheximide. Thus, our results demonstrate that neurons possess an efficient uptake system for the antennapedia homeobox peptide and suggest that binding of pAntp to consensus motifs present in nerve cell nuclei influences neuronal morphogenetic programs.

Tissue- and developmental stage-specific forms of a neural cell surface antigen linked to differences in glycosylation of a common polypeptide.

We have previously identified a cell surface glycoprotein of the mouse nervous system named brain cell surface protein-2 (BSP-2). Here we report that this antigen is not a single, discrete entity, but a family of antigenically and structurally related molecules. Three components of 180, 140, and 120 K were characteristic for more mature nervous tissues. Adult cerebral cortex contained the 140-K and 120-K antigens, adult spinal cord only the 120-K, and dorsal root ganglia from young mice mainly the 180-K component. Very different forms of the antigen that migrated as a diffuse zone from 180-250-K in SDS-polyacrylamide gels were found in immature nervous tissues. A molecule different from the previous ones was found in a neuroblastoma line. Evidence is presented that the structural diversity of BSP-2 is due to differences in glycosylation. This result indicates that cell type- and developmental stage-specific glycoprotein patterns previously found in the nervous system may in part be due to different glycosylation of identical polypeptides. The finding that a neural cell surface protein may be glycosylated in different ways has important implications for the generation of cell surface specificity.

Adult and embryonic mouse neural cell adhesion molecules have different binding properties.

Interactions between neural cell surfaces seem to be of prime importance during neuroontogenesis, and responsible for the guidance of migrating neuroblasts and growing axons and for the formation of synapses. Little is known about the underlying molecular mechanisms, but most hypotheses imply the existence of cell-surface molecules that mediate the formation of transient or permanent bonds between neural cells. Recently, a membrane glycoprotein called neural cell adhesion molecule (N-CAM) has been characterized in chick and rodent nervous tissue that appears to act as a ligand in adhesion among neural cell bodies or neurites. We have identified a mouse neural surface glycoprotein, named BSP-2 (ref. 7), which by criteriaof electrophoretic migration, developmental changes, amino acid and sugar composition seems to be closely related or identical to N-CAM. Both BSP-2 (refs 8, 9) and N-CAM undergo conversion from an embryonic to an adult form during brain development and it has been suggested that this transition changes the adhesive properties or the binding specificity of the molecule. Using a neuroblastoma line to study functional differences between embryonic and adult BSP-2/N-CAM molecules, we show here that liposomes bearing adult BSP-2 but not those bearing the embryonic form adhere to neuroblastoma cells, demonstrating that the two forms do indeed possess different binding properties.

Studies on the transmembrane disposition of the neural cell adhesion molecule N-CAM. A monoclonal antibody recognizing a cytoplasmic domain and evidence for the presence of phosphoserine residues.

The N-CAMs are a group of surface glycoproteins involved in adhesive interactions of neurones. Related molecules of the mouse nervous system, identified in our laboratory, have been called BSP-2 and shown to act as ligands in adhesion of neuroblastoma cells. Results presented in this report show that they are immunochemically identical with N-CAM. A monoclonal anti-(N-CAM) antibody, that recognized a determinant accessible only after permeabilization of intact cells, was used to define the mode of association of the N-CAMs with the plasma membrane. This antibody bound a 35 000-Mr fragment in lysates of trypsin-treated neuroblastoma cells. It is concluded that the antibody reacts with a transmembrane or cytoplasmic domain of the molecules. The same antibody recognized the Mr-180 000 and Mr-140 000 proteins but not the Mr-120 000 chain, which co-purify from adult mouse brain. The latter polypeptide was detected in the cytosol and could be partially released from brain membranes by osmotic shock. Part or all of the Mr-120 000 protein may thus lack a transmembrane segment. Our conclusion that the N-CAM forms of higher Mr are transmembrane proteins was further corroborated by our finding that they contain phosphoserine residues, which can be labeled with (32P)phosphate in intact neuroblastoma cells.

Characterization of neural cell adhesion molecules (NCAM) expressed by Ewing and neuroblastoma cell lines.

The status of the neural cell adhesion molecule NCAM gene which is mapped to human chromosome 11q23-24 has been investigated in Ewing-tumor-derived cell lines which present the t(11;22)(q23-24;q12) translocation characteristic of this malignancy. No rearrangement was detected when 2 different non-overlapping probes to mouse NCAM were used. The expression of the NCAM gene was analysed at both the protein and messenger levels in material extracted from Ewing cell lines, human neuroblastoma cell line and fetal mouse brain. Immune blot and immunoprecipitation studies showed that the neuroblastoma cell line contained more NCAM material than the Ewing lines. In neuroblastoma but not in Ewing, the NCAM material had the electrophoretic characteristics of molecules with long polysialic acid chains. After treatment with endosialidase, the diffusely migrating neuroblastoma material was resolved into 3 discrete bands of 120, 140 and 180 kDa. In Ewing extract, high-molecular-weight NCAM species were also detected with a 3-band pattern more reminiscent of mature brain. Endoglycosidase F treatment of Ewing NCAM indicated that all 3 species were largely N-glycosylated. Northern blot analysis confirmed that NCAM was expressed more abundantly in neuroblastoma than in Ewing cell lines. Among the 4 NCAM messengers (7.0, 6.5, 4.3 and 4.1 kb) detected in the neuroblastoma, the 6.5 kb species was largely predominant. The Ewing messenger RNA pattern was clearly different as the largest 7.0-kb species was virtually absent and the other bands were of similar intensities.

Structural and functional studies on N-CAM neural cell adhesion molecules.

The neural cell adhesion molecules N-CAM are to date the best characterized adhesion molecules of the nervous system. They have a high content of sialic acid residues which are present in the form of unusual sialic acid polymers. During development, a 3 fold decrease in the sialic acid content is observed. These changes in the degree of sialylation profoundly affect the binding properties of the molecules. A subpopulation of mouse brain N-CAM bears a carbohydrate determinant shared with other brain cell surface proteins and with the HNK-1 antigen of natural killer cells. Not only the carbohydrate side chains but also the protein moieties of the N-CAMs are heterogeneous. Three polypeptides of 180 K, 140 K and 120 K have been characterized in mouse brain. The 180 K and 140 K chains span the membrane. They differ mainly by the length of their cytoplasmic extensions. These intracellular domains are unusually long and contain phosphorylated serine residues. The 120 K chain exists in two forms, one membrane-bound and one soluble. Earlier studies had shown the presence of N-CAM on neurones and astrocytes of the mouse central nervous system, whereas cultured astrocytes had been reported to be N-CAM-negative. Recent results show that N-CAM is also expressed on astrocytes in culture. To study expression and heterogeneity of N-CAM polypeptides at the mRNA and gene level, cDNA clones for mouse N-CAM have been isolated. They reveal multiple mRNA species in mouse brain. By contrast, the corresponding sequences seem to be present only a few times, perhaps only once, in the mouse genome.(ABSTRACT TRUNCATED AT 250 WORDS)

Isolation of mouse N-CAM-related cDNA: detection and cloning using monoclonal antibodies.

Clones coding for the mouse neural cell adhesion molecule (N-CAM) were isolated from a cDNA library prepared in the expression vector lambda gt 11 from mRNA extracted from a mouse neuroblastoma cell line. This library was screened with two anti-N-CAM monoclonal antibodies directed against different sites on the molecule and with rabbit anti-N-CAM serum. Two clones were identified with the first monoclonal antibody, three with the second one, none reacted with both. The relevance of these cDNA clones to N-CAM was confirmed by several observations. First, cDNA sequences detected with one monoclonal antibody cross-hybridized with those identified by the other antibody. Second, the different fusion proteins all bound the rabbit serum in addition to one monoclonal antibody. Finally, the probes hybridized to discrete mRNA species of sufficient lengths to code for the very large N-CAM polypeptides in RNA preparations from N-CAM-expressing, but not from N-CAM-negative cells. An additional mRNA species not seen in embryonic brain was expressed in adult mouse brain. Genomic blot experiments indicated that sequences corresponding to one of our probes are present only a few times in the mouse genome.