Functional characterization of chondroitin sulfate proteoglycans of brain: interactions with neurons and neural cell adhesion molecules

Ng-CAM and N-CAM are cell adhesion molecules (CAMs), and each CAM can bind homophilically as demonstrated by the ability of CAM-coated beads (Covaspheres) to self-aggregate. We have found that the extent of aggregation of Covaspheres coated with either Ng-CAM or N-CAM was strongly inhibited by the intact 1D1 and 3F8 chondroitin sulfate proteoglycans of rat brain, and by the core glycoproteins resulting from chondroitinase treatment of the proteoglycans. Much higher concentrations of rat chondrosarcoma chondroitin sulfate proteoglycan (aggrecan) core proteins had no significant effect in these assays. The 1D1 and 3F8 proteoglycans also inhibited binding of neurons to Ng-CAM when mixtures of these proteins were adsorbed to polystyrene dishes. Direct binding of neurons to the proteoglycan core glycoproteins from brain but not from chondrosarcoma was demonstrated using an assay in which cell-substrate contact was initiated by centrifugation, and neuronal binding to the 1D1 proteoglycans was specifically inhibited by the 1D1 monoclonal antibody. Different forms of the 1D1 proteoglycan have been identified in developing and adult brain. The early postnatal form (neurocan) was found to bind neurons more effectively than the adult proteoglycan, which represents the C-terminal half of the larger neurocan core protein. Our results therefore indicate that certain brain proteoglycans can bind to neurons, and that Ng-CAM and N-CAM may be heterophilic ligands for neurocan and the 3F8 proteoglycan. The ability of these brain proteoglycans to inhibit adhesion of cells to CAMs may be one mechanism to modulate cell adhesion and migration in the nervous system.     

Conserved regulatory elements in the promoter region of the N-CAM gene.

Genomic clones containing 5'-flanking sequences, the first exon, and the entire first intron from the chicken N-CAM gene were characterized by restriction mapping and DNA sequencing. A > 600-bp segment that includes the first exon is very G + C-rich and contains a large proportion of CpG dinucleotides, suggesting that it represents a CpG island. SP-1 and AP-1 consensus elements are present, but no TATA- or CCAAT-like elements were found within 300 bp upstream of the first exon. Comparison of the chicken promoter region sequence with similar regions of the human, rat, and mouse N-CAM genes revealed that some potential regulatory elements including a "purine box" seen in mouse and rat N-CAM genes, one of two homeodomain binding regions seen in mammalian N-CAM genes, and several potential SP-1 sites are not conserved within this region. In contrast, high CpG content, a homeodomain binding sequence, an SP-1 element, an octomer element, and an AP-1 element are conserved in all four genes. The first intron of the chicken gene is 38 kb, substantially smaller than the corresponding intron from mammalian N-CAM genes. Together with previous studies, this work completes the cloning of the chicken N-CAM gene, which contains at least 26 exons distributed over 85 kb.     

Differential contributions of Ng-CAM and N-CAM to cell adhesion in different neural regions

Individual neurons can express both the neural cell adhesion molecule (N-CAM) and the neuron-glia cell adhesion molecule (Ng-CAM) at their cell surfaces. To determine how the functions of the two molecules may be differentially controlled, we have used specific antibodies to each cell adhesion molecule (CAM) to perturb its function, first in brain membrane vesicle aggregation and then in tissue culture assays testing the fasciculation of neurite outgrowths from cultured dorsal root ganglia, the migration of granule cells in cerebellar explants, and the formation of histological layers in the developing retina. Our strategy was initially to delineate further the binding mechanisms for each CAM. Antibodies to Ng-CAM and N-CAM each inhibited brain membrane vesicle aggregation but the binding mechanisms of the two CAMs differed. As expected from the known homophilic binding mechanism of N-CAM, anti-N- CAM-coated vesicles did not co-aggregate with uncoated vesicles. Anti- Ng-CAM-coated vesicles readily co-aggregated with uncoated vesicles in accord with a postulated heterophilic binding mechanism. It was also shown that N-CAM was not a ligand for Ng-CAM. In contrast to assays with brain membrane vesicles, cellular systems can reveal functional differences for each CAM reflecting its relative amount (prevalence modulation) and location (polarity modulation). Consistent with this, each of the three cellular processes examined in vitro was preferentially inhibited only by anti-N-CAM or by anti-Ng-CAM antibodies. Both neurite fasciculation and the migration of cerebellar granule cells were preferentially inhibited by anti-Ng-CAM antibodies. Anti-N-CAM antibodies inhibited the formation of histological layers in the retina. The data on perturbation by antibodies were correlated with the relative levels of expression of Ng-CAM and N-CAM in each of these different neural regions. Quantitative immunoblotting experiments indicated that the relative Ng-CAM/N-CAM ratios in comparable extracts of brain, dorsal root ganglia, and retina were respectively 0.32, 0.81, and 0.04. During culture of dorsal root ganglia in the presence of nerve growth factor, the Ng-CAM/N-CAM ratio rose to 4.95 in neurite outgrowths and 1.99 in the ganglion proper, reflecting both polarity and prevalence modulation. These results suggest that the relative ability of anti-Ng-CAM and anti-N-CAM antibodies to inhibit cell-cell interactions in different neural tissues is strongly correlated with the local Ng-CAM/N-CAM ratio.(ABSTRACT TRUNCATED AT 400 WORDS)     

Expression of cell-adhesion molecules in embryonic induction. I. Morphogenesis of nestling feathers

The potential relationship of cell adhesion to embryonic induction during feather formation was examined by immunohistochemical analysis of the spatiotemporal distribution of three cell-adhesion molecules (CAMs), neural CAM (N-CAM), liver CAM (L-CAM), and neuron-glia CAM (Ng-CAM), and of substrate molecules (laminin and fibronectin) in embryonic chicken skin. The N-CAM found at sites of embryonic induction in the feather was found to be similar to brain N-CAM as judged by immuno-cross-reactivity, migratory position in PAGE, and the presence of embryonic to adult conversion. In contrast to the N-CAM found in the brain, however, only one polypeptide of Mr 140,000 was seen. N-CAM-positive dermal condensations were distributed periodically under L-CAM-positive feather placodes at those sites where basement membranes are known to be disrupted. After initiation of induction, L-CAM-positive placode cells became transiently N-CAM-positive. N-CAM was asymmetrically concentrated in the dorsal region of the feather bud, while fibronectin was concentrated in the ventral region. During feather follicle formation, N-CAM was expressed in the dermal papilla and was closely apposed to the L-CAM-positive papillar ectoderm, while the dermal papilla showed no evidence of laminin or fibronectin. The collar epithelium was both N-CAM- and L-CAM-positive. During the formation of the feather filament, N-CAM appeared periodically and asymmetrically on basilar cells located in the valleys between adjacent barb ridges. In contrast to the two primary CAMs, Ng-CAM was found only on nerves supplying the feather and the skin. These studies indicate that at each site of induction during feather morphogenesis, a general pattern is repeated in which an epithelial structure linked by L-CAM is confronted with periodically propagating condensations of cells linked by N-CAM.     

Expression of the homeobox gene barx2 in the gut.

Barx2 is a member of the Bar class of homeobox genes and has been shown to regulate specific cell adhesion molecules, L1, Ng-CAM, N-CAM, and cadherin 6. By Northern blotting and in situ hybridization, we show that Barx2 is expressed throughout the gut and is located in epithelial cells of the proliferative and differentiative regions of the stomach, esophagus, and intestine. Barx2 was expressed in muscle cells of the muscularis externa and also showed a graded pattern of expression in intestinal enterocytes, decreasing in a crypt-to-villous direction. We speculate that Barx2 may regulate cell adhesion molecules in epithelial cells of the gut.     

Expression of cell adhesion molecules during embryonic induction. III. Development of the otic placode

During embryonic development, the inner ear develops from a placode into a richly differentiated structure with defined borders between neural and non-neural elements. In an effort to define the origin of such differentiation boundaries from the time of appearance of the placode, immunocytochemical methods have been used to map the developmental distributions of the cell adhesion molecules, N-CAM, L-CAM, and Ng-CAM, and the extracellular matrix molecules, cytotactin and fibronectin, in the cochlea of the chicken embryo. As the otic placode was induced by the underlying N-CAM-containing rhombencephalon and mesoderm, the placode expressed both N-CAM and L-CAM. During the period when the otic vesicle differentiated to give rise to the acoustic ganglion and to the differentiated structures of the cochlea, N-CAM increased in the innervated sensory regions while L-CAM increased in the non-sensory areas of the auditory epithelium adjacent to the sensory regions. During subsequent development, the differential expression of N-CAM and L-CAM again formed striking borders within the epithelium between the five morphologically and functionally distinct regions of the cochlea. This pattern of CAM expression is consistent with previous observations suggesting that primary CAMs of different binding specificities are expressed in two different modes to form borders at all sites of embryonic induction and at sites of further cytodifferentiation (K. L. Crossin, C -M. Chuong, and G. M. Edelman, 1985, Proc. Natl. Acad. Sci. USA 82, 6942-6946). Unlike inductive sites involving mesenchyme, however, the placode showed only changes in which an epithelium containing both CAMs loses one or the other or remains unchanged. As differentiation occurred during innervation of the sensory region, the secondary Ng-CAM appeared. Ng-CAM-positive fibers penetrated into the basilar papilla and Ng-CAM and the matrix protein cytotactin appeared within the epithelium in a radial pattern that was consistent with the previously described roles of these molecules in neurite movement. Immunoblot analyses confirmed the identity and biochemical properties of the CAMs and also revealed that N-CAM underwent embryonic to adult conversion during inner ear formation. These studies support the idea that CAMs are expressed in specific modal patterns in the cell collectives participating in inductive events, and strongly suggest that cellular regulation of these patterns is correlated with border formation.(ABSTRACT TRUNCATED AT 400 WORDS)     

Expression of cell-adhesion molecules in embryonic induction. II. Morphogenesis of adult feathers

The developmental appearance of cell-adhesion molecules (CAMs) was mapped during the morphogenesis of the adult chicken feather. Neural CAM (N-CAM), liver CAM (L-CAM), and neuron-glia CAM (Ng-CAM), as well as substrate molecules (laminin and fibronectin), were compared in newborn chicken skin by immunohistochemical means. N-CAM was found to be enriched in the dermal papilla, which was closely apposed to L-CAM-positive papillar ectoderm. The two CAMs were then co-expressed in cells of the collar epithelium. Subsequently generated barb epithelia expressed only L-CAM, but N-CAM reappeared periodically on cells between developing barbs and barbules. N-CAM first appeared on a single L-CAM-positive basilar cell located in each valley flanked by two adjacent barb ridges. Subsequently, the expression of N-CAM extended one cell after another to include the whole basilar layer. N-CAM also appeared in the L-CAM-positive axial-plate epithelia, beginning in a single cell located at the ridge base. The two collectives of N-CAM-positive epithelia constituting the marginal and axial plates then disintegrated, leaving interdigitating spaces between keratinized structures that had previously expressed L-CAM. The morphological transformation from an epithelial cylinder to a three-level branched feather pattern is thus achieved by coupling alternating CAM expression in linked cell collectives with specific differentiation events, such as keratinization. During all of these morphogenetic processes, laminin and fibronectin formed a continuous basement membrane separating pulp from feather epithelia, and were excluded from the sites involved in periodic appearances of N-CAM. The same staining pattern described for developing chickens persisted in the feather follicles of adult chicken tissue that have gone through several cycles of molting. Cyclic expression of the two different CAMs underlies each of the different morphological events that are generated epigenetically during feather morphogenesis.     

Neuronal cell adhesion molecules and cytotactin are colocalized at the node of Ranvier

Immunocytochemical methods were used to show that Ng-CAM (the neuron-glia cell adhesion molecule), N-CAM (the neural cell adhesion molecule), and the extracellular matrix protein cytotactin are highly concentrated at nodes of Ranvier of the adult chicken and mouse. In contrast, unmyelinated axonal fibers were uniformly stained by specific antibodies to both CAMs but not by antibodies to cytotactin. Ultrastructural immunogold techniques indicated that both N-CAM and Ng-CAM were enriched in the nodal axoplasm and axolemma of myelinated fibers as well as within the nodal regions of the myelinating Schwann cell. At embryonic day 14, before myelination had occurred, small-caliber fibers of chick embryos showed periodic coincident accumulations of the two CAMs but not of cytotactin, with faint labeling in the axonal regions between accumulations. Cytotactin was found on Schwann cells and in connective tissue. By embryonic day 18, nodal accumulations of CAMs were first observed in a few medium- and large-caliber fibers. Immunoblot analyses indicated that embryonic to adult conversion of N-CAM and a progressive decrease in the amount of Ng-CAM and N-CAM occurred while nodes were forming. Sciatic nerves of mouse mutants with defects in cell interactions showed abnormalities in the distribution patterns and amount of Ng-CAM, N-CAM, and cytotactin that were consistent with the known morphological nodal disorders. In trembler (+/Tr), intense staining for both CAMs appeared all along the fibers and the amounts of N-CAM in the sciatic nerve were found to be increased. In mice with motor endplate disease (med/med), Ng-CAM and N-CAM, but not cytotactin, were localized in the widened nodes. Both trembler and med/med Schwann cells stained intensely for cytotactin, in contrast to normal Schwann cells which stained only slightly. All of these findings are consistent with the hypothesis that surface modulation of neuronal CAMs mediated by signals shared between neurons and glia may be necessary for establishing and maintaining the nodes of Ranvier.     

Altered expression of neuronal cell adhesion molecules induced by nerve injury and repair

Peripheral nerve injury results in short-term and long-term changes in both neurons and glia. In the present study, immunohistological and immunoblot analyses were used to examine the expression of the neural cell adhesion molecule (N-CAM) and the neuron-glia cell adhesion molecule (Ng-CAM) within different parts of a functionally linked neuromuscular system extending from skeletal muscle to the spinal cord after peripheral nerve injury. Histological samples were taken from 3 to 150 d after crushing or transecting the sciatic nerve in adult chickens and mice. In unperturbed tissues, both N-CAM and Ng-CAM were found on nonmyelinated axons, and to a lesser extent on Schwann cells and myelinated axons. Only N-CAM was found on muscles. After denervation, the following changes were observed: The amount of N-CAM in muscle fibers increased transiently on the surface and in the cytoplasm, and in interstitial spaces between fibers. Restoration of normal N-CAM levels in muscle was dependent on reinnervation; in a chronically denervated state, N-CAM levels remained high. After crushing or cutting the nerve, the amount of both CAMs increased in the area surrounding the lesion, and the predominant form of N-CAM changed from a discrete Mr 140,000 component to the polydisperse high molecular weight embryonic form. Anti-N-CAM antibodies stained neurites, Schwann cells, and the perineurium of the regenerating sciatic nerve. Anti-Ng-CAM antibodies labeled neurites, Schwann cells and the endoneurial tubes in the distal stump. Changes in CAM distribution were observed in dorsal root ganglia and in the spinal cord only after the nerve was cut. The fibers within affected dorsal root ganglia were more intensely labeled for both CAMs, and the motor neurons in the ventral horn of the spinal cord of the affected segments were stained more intensely in a ring pattern by anti-N-CAM and anti-Ng-CAM than their counterparts on the side contralateral to the lesion. Taken together with the previous studies (Rieger, F., M. Grumet, and G. M. Edelman, J. Cell Biol. 101:285-293), these data suggest that local signals between neurons and glia may regulate CAM expression in the spinal cord and nerve during regeneration, and that activity may regulate N-CAM expression in muscle. Correlations of the present observations are made here with established events of nerve degeneration and suggest a number of roles for the CAMs in regenerative events.(ABSTRACT TRUNCATED AT 400 WORDS)     

The homeodomain protein Barx2 promotes myogenic differentiation and is regulated by myogenic regulatory factors

The homeobox protein Barx2 is expressed in both smooth and skeletal muscle and is up-regulated during differentiation of skeletal myotubes. Here we use antisense-oligonucleotide inhibition of Barx2 expression in limb bud cell culture to show that Barx2 is required for myotube formation. Moreover, overexpression of Barx2 accelerates the fusion of MyoD-positive limb bud cells and C2C12 myoblasts. However, overexpression of Barx2 does not induce ectopic MyoD expression in either limb bud cultures or in multipotent C3H10T1/2 mesenchymal cells, and does not induce fusion of C3H10T1/2 cells. These results suggest that Barx2 acts downstream of MyoD. To test this hypothesis, we isolated the Barx2 gene promoter and identified DNA regulatory elements that might control Barx2 expression during myogenesis. The proximal promoter of the Barx2 gene contained binding sites for several factors involved in myoblast differentiation including MyoD, myogenin, serum response factor, and myocyte enhancer factor 2. Co-transfection experiments showed that binding sites for both MyoD and serum response factor are necessary for activation of the promoter by MyoD and myogenin. Taken together, these studies indicate that Barx2 is a key regulator of myogenic differentiation that acts downstream of muscle regulatory factors.     

Interactions of the chondroitin sulfate proteoglycan phosphacan, the extracellular domain of a receptor-type protein tyrosine phosphatase, with neurons, glia, and neural cell adhesion molecules

Phosphacan is a chondroitin sulfate proteoglycan produced by glial cells in the central nervous system, and represents the extracellular domain of a receptor-type protein tyrosine phosphatase (RPTP zeta/beta). We previously demonstrated that soluble phosphacan inhibited the aggregation of microbeads coated with N-CAM or Ng-CAM, and have now found that soluble 125I-phosphacan bound reversibly to these neural cell adhesion molecules, but not to a number of other cell surface and extracellular matrix proteins. The binding was saturable, and Scatchard plots indicated a single high affinity binding site with a Kd of approximately 0.1 nM. Binding was reduced by approximately 15% after chondroitinase treatment, and free chondroitin sulfate was only moderately inhibitory, indicating that the phosphacan core glycoprotein accounts for most of the binding activity. Immunocytochemical studies of embryonic rat spinal phosphacan, Ng-CAM, and N-CAM have overlapping distributions. When dissociated neurons were incubated on dishes coated with combinations of phosphacan and Ng-CAM, neuronal adhesion and neurite growth were inhibited. 125I-phosphacan bound to neurons, and the binding was inhibited by antibodies against Ng-CAM and N-CAM, suggesting that these CAMs are major receptors for phosphacan on neurons. C6 glioma cells, which express phosphacan, adhered to dishes coated with Ng-CAM, and low concentrations of phosphacan inhibited adhesion to Ng-CAM but not to laminin and fibronectin. Our studies suggest that by binding to neural cell adhesion molecules, and possibly also by competing for ligands of the transmembrane phosphatase, phosphacan may play a major role in modulating neuronal and glial adhesion, neurite growth, and signal transduction during the development of the central nervous system.     

Cell Adhesion Molecules and the Migration of LHRH Neurons during Development

During embryogenesis, LHRH neurons arise in the olfactory epithelium, migrate along the olfactory nerve, and enter the forebrain. We have examined the distribution of several cell adhesion molecules (CAMs) in the developing chick olfactory system and brain to determine whether differential distributions of these adhesion molecules might be important in pathway choices made by migrating LHRH neurons. Single- and double-label immunocytochemical studies indicated that high levels of N-CAM and N-cadherin were expressed throughout the olfactory epithelium and not restricted to the medial half of the olfactory epithelium where most of the LHRH neurons originate. Further, high levels of N-CAM, Ng-CAM, and N-cadherin were uniformly expressed throughout the entire olfactory nerve while migrating LHRH neurons were confined to the medial half of the nerve. However, once LHRH neurons reach the brain, they migrate dorsally and caudally, tangential to the medial surface of the forebrain, along a region enriched in N-CAM and Ng-CAM. After this first stage of migration within the brain, LHRH neurons migrate laterally. At this stage, there is no correlation between the intensity of N-CAM and Ng-CAM immunostaining and the location of LHRH neurons. These results suggest that N-CAM, Ng-CAM, and N-cadherin do not play a guiding role in LHRH neuronal migration through the olfactory epithelium and olfactory nerve but that migrating LHRH neurons may follow a "CAM-trail" of N-CAM and Ng-CAM along the medial surface of the forebrain.     

The neuronal chondroitin sulfate proteoglycan neurocan binds to the neural cell adhesion molecules Ng-CAM/L1/NILE and N-CAM, and inhibits neuronal adhesion and neurite outgrowth

We have previously shown that aggregation of microbeads coated with N- CAM and Ng-CAM is inhibited by incubation with soluble neurocan, a chondroitin sulfate proteoglycan of brain, suggesting that neurocan binds to these cell adhesion molecules (Grumet, M., A. Flaccus, and R. U. Margolis. 1993. J. Cell Biol. 120:815). To investigate these interactions more directly, we have tested binding of soluble 125I- neurocan to microwells coated with different glycoproteins. Neurocan bound at high levels to Ng-CAM and N-CAM, but little or no binding was detected to myelin-associated glycoprotein, EGF receptor, fibronectin, laminin, and collagen IV. The binding to Ng-CAM and N-CAM was saturable and in each case Scatchard plots indicated a high affinity binding site with a dissociation constant of approximately 1 nM. Binding was significantly reduced after treatment of neurocan with chondroitinase, and free chondroitin sulfate inhibited binding of neurocan to Ng-CAM and N-CAM. These results indicate a role for chondroitin sulfate in this process, although the core glycoprotein also has binding activity. The COOH-terminal half of neurocan was shown to have binding properties essentially identical to those of the full-length proteoglycan. To study the potential biological functions of neurocan, its effects on neuronal adhesion and neurite growth were analyzed. When neurons were incubated on dishes coated with different combinations of neurocan and Ng-CAM, neuronal adhesion and neurite extension were inhibited. Experiments using anti-Ng-CAM antibodies as a substrate also indicate that neurocan has a direct inhibitory effect on neuronal adhesion and neurite growth. Immunoperoxidase staining of tissue sections showed that neurocan, Ng-CAM, and N-CAM are all present at highest concentration in the molecular layer and fiber tracts of developing cerebellum. The overlapping localization in vivo, the molecular binding studies, and the striking effects on neuronal adhesion and neurite growth support the view that neurocan may modulate neuronal adhesion and neurite growth during development by binding to neural cell adhesion molecules.     

Heterotypic binding between neuronal membrane vesicles and glial cells is mediated by a specific cell adhesion molecule

By means of a multistage quantitative assay, we have identified a new kind of cell adhesion molecule (CAM) on neuronal cells of the chick embryo that is involved in their adhesion to glial cells. The assay used to identify the binding component (which we name neuron-glia CAM or Ng-CAM) was designed to distinguish between homotypic binding (e.g., neuron to neuron) and heterotypic binding (e.g., neuron to glia). This distinction was essential because a single neuron might simultaneously carry different CAMs separately mediating each of these interactions. The adhesion of neuronal cells to glial cells in vitro was previously found to be inhibited by Fab' fragments prepared from antisera against neuronal membranes but not by Fab' fragments against N-CAM, the neural cell adhesion molecule. This suggested that neuron-glia adhesion is mediated by specific cell surface molecules different from previously isolated CAMs . To verify that this was the case, neuronal membrane vesicles were labeled internally with 6-carboxyfluorescein and externally with 125I-labeled antibodies to N-CAM to block their homotypic binding. Labeled vesicles bound to glial cells but not to fibroblasts during a 30-min incubation period. The specific binding of the neuronal vesicles to glial cells was measured by fluorescence microscopy and gamma spectroscopy of the 125I label. Binding increased with increasing concentrations of both glial cells and neuronal vesicles. Fab' fragments prepared from anti-neuronal membrane sera that inhibited binding between neurons and glial cells were also found to inhibit neuronal vesicle binding to glial cells. The inhibitory activity of the Fab' fragments was depleted by preincubation with neuronal cells but not with glial cells. Trypsin treatment of neuronal membrane vesicles released material that neutralized Fab' fragment inhibition; after chromatography, neutralizing activity was enriched 50- fold. This fraction was injected into mice to produce monoclonal antibodies; an antibody was obtained that interacted with neurons, inhibited binding of neuronal membrane vesicles to glial cells, and recognized an Mr = 135,000 band in immunoblots of embryonic chick brain membranes. These results suggest that this molecule is present on the surfaces of neurons and that it directly or indirectly mediates adhesion between neurons and glial cells. Because the monoclonal antibody as well as the original polyspecific antibodies that were active in the assay did not bind to glial cells, we infer that neuron- glial interaction is heterophilic, i.e., it occurs between Ng-CAM on neurons and an as yet unidentified CAM present on glial cells.     

Molecular cloning and characterisation of two calmodulin isoforms of the Madagascar periwinkle Catharanthus roseus

Involvement of Ca(2+) signalling in regulation of the biosynthesis of monoterpene indole alkaloids (MIA) in Catharanthus roseus has been extensively studied in recent years, albeit no protein of this signalling pathway has been isolated. Using a PCR strategy, two C. roseus cDNAs encoding distinct calmodulin (CAM) isoforms were cloned and named CAM1 and CAM2. The deduced 149 amino acid sequences possess four Ca(2+) binding domains and exhibit a close identity with Arabidopsis CAM isoforms (>91%). The ability of CAM1 and CAM2 to bind Ca(2+) was demonstrated following expression of the corresponding recombinant proteins. Furthermore, transient expression of CAM1-GFP and CAM2-GFP in C. roseus cells showed a typical nucleo-cytoplasm localisation of both CAMs, in agreement with the wide distribution of CAM target proteins. Using RNA blot analysis, we showed that CAM1 and CAM2 genes had a broad pattern of expression in C. roseus organs and are constitutively expressed during a C. roseus cell culture cycle, with a slight inhibitory effect of auxin for CAM1. Using RNA in situ hybridisation, we also detected CAM1 and CAM2 mRNA in the vascular bundle region of young seedling cotyledons. Finally, using specific inhibitors, we also showed that CAMs are required for MIA biosynthesis in C. roseus cells by acting on regulation of expression of genes encoding enzymes that catalyse early steps of MIA biosynthesis, such as 1-deoxy-d-xylulose 5-phosphate reductoisomerase and geraniol 10-hydroxylase.     

Structure of the chicken neuron-glia cell adhesion molecule, Ng-CAM: origin of the polypeptides and relation to the Ig superfamily

The neuron-glia cell adhesion molecule (Ng-CAM) mediates both neuron-neuron and neuron-glia adhesion; it is detected on SDS-PAGE as a predominant 135-kD glycoprotein, with minor components of 80, 190, and 210 kD. We have isolated cDNA clones encoding the entire sequence of chicken Ng-CAM. The predicted extracellular region includes six immunoglobulin-like domains followed by five fibronectin-type III repeats, structural features that are characteristic of several neural CAMs of the N-CAM superfamily. The amino acid sequence of chicken Ng-CAM is most similar to that of mouse L1 but the overall identity is only 40% and Ng-CAM contains a short fibronectin-like segment with an RGD sequence that has no counterpart in L1. These findings suggest that Ng-CAM and L1 may not be equivalent molecules in chicken and mouse. The amino-terminal sequences of the 210-, 190-, and 135-kD components of Ng-CAM are all the same as the predicted amino terminus of the molecule, whereas the 80-kD component begins within the third fibronectin repeat. The cDNA sequence is continuous across the junction between the 135- and 80-kD components, and a single 170-kD Ng-CAM polypeptide was isolated from tunicamycin-treated cells. In addition, all cDNA probes hybridized on Northern blots to a 6-kb RNA, and most hybridized to single bands on Southern blots. These results indicate that the Ng-CAM components are derived from a single polypeptide encoded by a single gene, and that the 135- and 80-kD components are generated from the 210/190-kD species by proteolytic cleavage. The 135-kD component contains most of the extracellular region including all of the immunoglobulin-like domains. It has no transmembrane segment, but it is tightly associated with the membrane. The 80-kD component contains two and a half type III repeats plus the RGD-containing segment, as well as the single transmembrane and cytoplasmic domains. These structural features of Ng-CAM provide a framework for understanding its multiple functions in neuron-neuron interactions, neurite fasciculation, and neuron-glia interactions.     

The 5'-flanking sequence and regulatory elements of the cystatin S gene.

The gene encoding rat cystatin S (Cys S), a salivary gland-specific secretory protein, has CAAT and TATA boxes upstream of the inititation codon (Cox and Shaw, 1992), and contains regions that resemble those of other hormonally responsive eukaryotic genes. The 5'-flanking sequence of the rat Cys S gene has a potential CREB/AP-1 binding site (Rupp et al., 1990; Trejo et al., 1992), two potential glucocorticoid responsive elements (GREs, Drouin et al., 1989), and a possible GR/PR (glucocorticoid/progesterone) responsive element (Forman and Samuels, 1990). One of these potential GREs is adjacent to a potential AP-2 binding site, and another is typical of the glucocorticoid and progesterone receptor binding site. In this report, we have identified three regions in the 5'-flanking region of the Cys S gene that are found in salivary gland-specific genes (Ting et al., 1992) with a GT-rich region located between conserved elements II and III. Transfection experiments described in this paper suggest that a 281-bp DNA fragment from the Cys S gene promoter region with conserved elements II and III, the GT-rich region, and a possible GR/PR responsive element contains a negative regulatory element. In addition, our experiments suggest that the GT-rich region by itself is acting as a positive regulatory element.