Olfactory neurons express a unique glycosylated form of the neural cell adhesion molecule (N-CAM)

mAb-based approaches were used to identify cell surface components involved in the development and function of the frog olfactory system. We describe here a 205-kD cell surface glycoprotein on olfactory receptor neurons that was detected with three mAbs: 9-OE, 5-OE, and 13-OE. mAb 9-OE immunoreactivity, unlike mAbs 5-OE and 13-OE, was restricted to only the axons and terminations of the primary sensory olfactory neurons in the frog nervous system. The 9-OE polypeptide(s) were immunoprecipitated and tested for cross-reactivity with known neural cell surface components including HNK-1, the cell adhesion molecule L1, and the neural cell adhesion molecule (N-CAM). These experiments revealed that 9-OE-reactive molecules were not L1 related but were a subset of the 200-kD isoforms of N-CAM. mAb 9-OE recognized epitopes associated with N-linked carbohydrate residues that were distinct from the polysialic acid chains present on the embryonic form of N-CAM. Moreover, 9-OE N-CAM was a heterogeneous population consisting of subsets both with and without the HNK-1 epitope. Thus, combined immunohistochemical and immunoprecipitation experiments have revealed a new glycosylated form of N-CAM unique to the olfactory system. The restricted spatial expression pattern of this N-CAM glycoform suggests a possible role in the unusual regenerative properties of this sensory system.     

Tissue specific O-linked glycosylation of the neural cell adhesion molecule (N-CAM)

We have shown previously that the predominant N-CAM isoform in skeletal muscle myotubes contains as a result of alternative splicing a novel domain (MSD1) in its extracellular region. Here we show that this region represents a site for O-linked carbohydrate attachment. The lipid tailed N-CAM in myotubes was found to bind peanut lectin while the transmembrane isoform from myoblasts lacking MSD1 did not. In addition, N-CAM from a variety of neural sources failed to bind the lectin. Analysis of 3T3 fibroblasts transfected with various N-CAM cDNAs, showed that peanut lectin binding was correlated specifically with the expression of the MSD1 region. The oligosaccharides isolated from a purified preparation of myotube N-CAM were shown to contain an O-linked oligosaccharide whose core structure was a sialylated version of Gal beta 1----3GalNac which is the structure recognized specifically by peanut lectin. These data provide the first evidence for the expression of O-linked carbohydrate on any N-CAM isoform and more specifically target this oligosaccharide to the MSD1 region of myotube N-CAM.     

Localization of neural cell adhesion molecule (N-CAM) immunoreactivity in adult rat tissues

Neural cell adhesion molecule (N-CAM) mediates homophilic adhesion between cells and heterophilic adhesion between cells and extracellular matrix in a Ca²⁺-independent manner. N-CAM is widely expressed during development and plays a crucial role in cell division, migration, and differentiation, but its expression is restricted in adults. The distribution of N-CAM immunoreactivity in adult rat tissues was investigated in the present study. N-CAM immunoreactivity was present in the nervous system in the molecular layer of the cerebellum, ependymal cells surrounding the central canal, axons of the white matter, and in Lamina X of the gray matter of the spinal cord. N-CAM immunoreactivity also was found in autonomic nerves. In the digestive system, N-CAM immunoreactivity was found in the stratified squamous epithelium and nerve plexus of the esophagus, glandular cells of the stomach and pylorus, lamina propria, and epithelium of the villi of the duodenum, jejunum, and ileum. N-CAM immunoreactivity was demonstrated in the secretory cells of the adenohypophysis, islets of Langerhans, and acinar cells of the exocrine pancreas. Alveolar cells of the lung were also N-CAM immunoreactive. In the urinary system, N-CAM immunoreactivity was seen in the proximal convoluted tubules of the kidney. In the male reproductive system, N-CAM immunoreactivity was demonstrated in the nerve plexus around the urethral epithelium and in the nerve fibers around the smooth muscle cells of the corpus cavernosum penis. In the visual system, N-CAM immunoreactivity was seen in the epithelial cells of the corpus ciliaris. Cornea and lens epithelium also showed positive immunoreactivity. Our results suggest that cells in many tissues and organs of the adult rat synthesize N-CAM.     

The 180-kD component of the neural cell adhesion molecule N-CAM is involved in cell-cell contacts and cytoskeleton-membrane interactions

Summary N-CAM₁₈₀, the molecular form of the three neural cell adhesion molecules (N-CAM) with the largest cytoplasmic domain, is accumulated at sites of cell-cell contact (cell bodies, neurites, growth cones) in cultures of neuroblastoma and cerebellum. At these sites the cytoskeletonmembrane linker protein brain spectrin and actin are also accumulated. Brain spectrin copurifies with N-CAM₁₈₀ by immunoaffinity chromatography and binds specifically to N-CAM₁₈₀ but not to N-CAM₁₄₀ or N-CAM₁₂₀ in a solid-phase binding test. These observations indicate an association of N-CAM₁₈₀ with the cytoskeleton in vivo. This association may underlie the reduced lateral mobility of N-CAM₁₈₀ in the surface membrane compared to N-CAM₁₄₀ (Pollerberg et al. 1986). Together with the fact that N-CAM₁₈₀ is only expressed after termination of neuron migration in vivo (Persohn and Schachner, unpublished) these results suggest a role for N-CAM₁₈₀ in stabilization of cell contacts.     

Association between the first two immunoglobulin-like domains of the neural cell adhesion molecule N-CAM.

The extracellular domain of N-CAM contains five immunoglobulin-like (Ig) and two fibronectin type III-like domains and facilitates cell-cell binding through multiple, weak interdomain interactions. NMR spectroscopy indicated that the two N-terminal Ig-like domains from chicken N-CAM (Ig I and Ig II) interact with millimolar affinity. Physico-chemical studies show that this interaction is significantly amplified when the domains are covalently linked, consistent with an antiparallel domain arrangement. The binding of the two individual domains and the dimerization of the concatenated protein were essentially independent of salt, up to a concentration of 200 mM. The residues in Ig I involved in the interaction map to the BED strands of the beta sandwich, and delineate a largely hydrophobic patch.     

Expression of neural cell adhesion molecule (N-CAM) in perisinusoidal stellate cells of the human liver

Neural cell adhesion molecule (N-CAM) is distributed in most nerve cells and some non-neural tissues. The present immunohistochemical study has revealed, for the first time, the expression of N-CAM in perisinusoidal stellate cells of the human liver. Liver specimens were stained with monoclonal antibody against human Leu19 (N-CAM) by a streptoavidin-biotin-peroxidase-complex method. Light- and electron-microscopic analyses have shown that N-CAM-positive nerve fibers are distributed in the periportal and intermediate zones of the liver lobule. Perisinusoidal stellate cells in these zones are also positive for N-CAM. N-CAM is expressed on the surface of the cell, including cytoplasmic projections. Close contact of N-CAM-positive nerve endings with N-CAM-positive stellate cells has been observed. On the other hand, stellate cells in the centrilobular zone exhibit weak or no reaction for N-CAM. Perivascular smooth muscle cells and fibroblasts in the portal area and myofibroblasts around the central veins are negative for N-CAM. The present results indicate that the perisinusoidal stellate cells in the periportal and intermediate zones of the liver lobule characteristically express N-CAM, unlike other related mesenchymal cells, and suggest that the intralobular heterogeneity of N-CAM expression by stellate cells is related to the different maturational stages of these cells.     

Topographic localization of the heparin-binding domain of the neural cell adhesion molecule N-CAM

Previous studies have reported that the cell-binding region of the neural cell adhesion molecule (N-CAM) resides in a 65,000-D amino-terminal fragment designated Frl (Cunningham, B. A., S. Hoffman, U. Rutishauser, J. J. Hemperly, and G. M. Edelman, 1983, Proc. Natl. Acad. Sci. USA, 80:3116-3120). We have reported the presence of two functional domains in N-CAM, each identified by a specific mAb, that are required for cell-cell or cell-substratum adhesion (Cole, G. J., and L. Glaser, 1986, J. Cell Biol., 102:403-412). One of these domains is a heparin (heparan sulfate)-binding domain. In the present study we have determined the topographic localization of the heparin-binding fragment from N-CAM, which has been identified by our laboratory. The B1A3 mAb recognizes a 25,000-D heparin-binding fragment derived from chicken N-CAM, and also binds to a 65,000-D fragment, presumably Frl, produced by digestion of N-CAM with Staphylococcus aureus V8 protease. Amino-terminal sequence analysis of the isolated 25,000-D heparin-binding domain of N-CAM yielded the sequence: Leu-Gln-Val-Asp-Ile-Val-Pro-Ser-Gln-Gly. This sequence is identical to the previously reported amino-terminal sequence for murine and bovine N-CAM. Thus, the 25,000-D polypeptide fragment is the amino-terminal region of the N-CAM molecule. We have also shown that the B1A3 mAb recognizes not only chicken N-CAM but also rat and mouse N-CAM, indicating that the heparin-binding domain of N-CAM is evolutionarily conserved among different N-CAM forms. Additional peptide-mapping studies indicate that the second cell-binding site of N-CAM is located in a polypeptide region at least 65,000 D from the amino-terminal region. We conclude that the adhesion domains on N-CAM identified by these antibodies are physically distinct, and that the previously identified cell-binding domain on Frl is the heparin-binding domain.     

Growth cone localization of neural cell adhesion molecule on central nervous system neurons in vitro

Ultrastructural analysis of colloidal gold immunocytochemical staining and immunofluorescence microscopy has been used to study the presence of neural cell adhesion molecule (NCAM) on the surface of neuronal growth cones. The studies were carried out with cultures of rat hypothalamic and ventral mesencephalic cells, using morphology and expression of tyrosine hydroxylase, neurofilaments, and glial fibrillary acidic protein as differential markers for neurons and glia. NCAM was found on all plasmalemmal surfaces of neurons including perikarya and neurites. The density of NCAM varied for different neurons growing in the same culture dish, and neurons had at least 25 times more colloidal gold particles on their plasmalemmal membranes than astroglia. Of particular interest in the present study was a strong labeling for NCAM on all parts of neuritic growth cones, including the lamellar and filopodial processes that extend from the tip of the axon. The density of NCAM was similar on different filopodia of the same growth cone. Therefore, in situations where homophilic (NCAM-NCAM) binding might contribute to axon pathfinding, a choice in direction is more likely to reflect differences in the NCAM content of the environment, rather than the distribution of NCAM within a growth cone. On the other hand, the variation in NCAM levels between single neurons in culture was significant and could provide a basis for selective responses of growing neurites.     

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.     

Selective expression of the 180-kD component of the neural cell adhesion molecule N-CAM during development

The rodent neural cell adhesion molecule (N-CAM) consists of three glycoprotein chains of 180, 140, and 120 kD in their adult forms. Although the proportions of the three components are known to change during development and differ between brain regions, their individual distribution and function are unknown. Here we report studies carried out with a monoclonal antibody that specifically recognizes the 180-kD component of mouse N-CAM (N-CAM180) in its highly sialylated embryonic and less glycosylated adult forms. In primary cerebellar cell cultures, N-CAM180 antibody reacts intracellularly with all types of neural cells including astrocytes, oligodendrocytes, and neurons. During cerebellar, telencephalic, and retinal development N-CAM180 is detectable by indirect immunohistology in differentiated neural cells, but, in contrast to total N-CAM, not in their proliferating precursors in the ventricular zone and primordial and early postnatal external granular layer. In monolayer cultures of C1300 neuroblastoma cells, N-CAM180 appears by immunofluorescence more concentrated at contact points between adjacent cells, while N-CAM comprising the 180- and 140-kD component shows a more uniform distribution at the plasma membrane. Treatment of neuroblastoma cells with dimethylsulfoxide, which promotes differentiation, induces a shift toward the predominant expression of N- CAM180. These observations support the notion that N-CAM180 is expressed selectively in more differentiated neural cells and suggest a differential role of N-CAM180 in the stabilization of cell contacts.     

Identification of a heparin binding domain of the neural cell adhesion molecule N-CAM using synthetic peptides

The neural cell adhesion molecule (N-CAM) plays an integral role in cell interactions during neural development, with the binding of heparan sulfate proteoglycan to the amino-terminal region of N-CAM being required for N-CAM function. In the present study we have used synthetic peptides (HBD-1 and HBD-2), derived from the primary amino acid sequence of rat N-CAM, to identify the region of N-CAM that binds heparan sulfate. The 28 amino acid HBD-1 synthetic peptide was shown to bind both [3H]heparin and dissociated retinal cells. Retinal cells also attach to a substratum of HBD-2 peptide, but fail to bind to a control peptide containing a scrambled amino acid sequence of HBD-2. The HBD-2 peptide also inhibits retinal cell adhesion to N-CAM, demonstrating the physiological importance of the amino acid sequence encoded by the HBD peptide. These data therefore permit the localization of a heparin binding domain to a 17 amino acid region of immunoglobulin-like loop 2.