RESUMEN
The differentiation and morphogenesis of neural tissues involves a diversity of interactions between neural cells and their environment. Many potentially important interactions occur with the extracellular matrix (ECM), a complex association of extracellular molecules organised into aggregates and polymers. The large modular glycoprotein, Tenascin-C, and the chondroitin sulphate proteoglycan, DSD-1-PG/Phosphacan, have complex and frequently overlapping expression patterns in the developing CNS. Their presence in zones of cell proliferation, migration, and differentiation, as well as in boundary structures, suggest that they may be involved in the modulation of an extensive range of cellular processes. They are both strongly up-regulated in a range of CNS lesions and pathologies, being components of the glial scar, and expressed by gliomas. Functional roles in many cellular processes are possible through their extensive molecular interaction sites, both with each other, and with many of the same cell surface receptors, adhesion molecules, growth factors and other matrix proteins. These multiple interactions involve sites on both their protein domains and on the heterogeneous carbohydrate groups with which they are post-translationally modified. In vitro assays demonstrate cell-type specific effects on adhesion, migration and the formation and extension of cellular processes, including neurites and axons.
Asunto(s)
Sulfatos de Condroitina/metabolismo , Matriz Extracelular/química , Matriz Extracelular/fisiología , Regulación del Desarrollo de la Expresión Génica/fisiología , Tenascina/fisiología , Animales , Proteoglicanos Tipo Condroitín Sulfato/biosíntesis , Proteoglicanos Tipo Condroitín Sulfato/química , Proteoglicanos Tipo Condroitín Sulfato/metabolismo , Proteoglicanos Tipo Condroitín Sulfato/fisiología , Sulfatos de Condroitina/biosíntesis , Sulfatos de Condroitina/química , Sulfatos de Condroitina/fisiología , Matriz Extracelular/metabolismo , Humanos , Ligandos , Proteínas Tirosina Fosfatasas Clase 5 Similares a Receptores , Tenascina/biosíntesis , Tenascina/químicaRESUMEN
DSD-1-PG is a chondroitin sulfate proteoglycan (CSPG) expressed by glial cells that can promote neurite outgrowth from rat embryonic mesencephalic (E14) and hippocampal (E18) neurons, an activity that is associated with the CS glycosaminoglycans (GAGs). Further characterization of DSD-1-PG has included sequencing of peptides from the core protein and the cloning of the corresponding cDNA using polyclonal antisera against DSD-1-PG to screen phage expression libraries. On the basis of these studies we have identified DSD-1-PG as the mouse homolog of phosphacan, a neural rat CSPG. Monoclonal antibodies 3H1 and 3F8 against carbohydrate residues on rat phosphacan recognize these epitopes on DSD-1-PG. The epitopes of the antibodies, L2/HNK-1 and L5/Lewis-X, which have been implicated in functional interactions, are also found on DSD-1-PG. Although DSD-1-PG has previously been shown to promote neurite outgrowth, its upregulation after stab wounding of the CNS and its localization in regions that are considered boundaries to axonal extension suggested that it may also have inhibitory functions. Neonatal dorsal root ganglion (DRG) explants grown on a rich supportive substrate (laminin) with and without DSD-1-PG were strikingly inhibited by the proteoglycan. The inhibitory effects of DSD-1-PG on the DRG explants were not relieved by removal of the CS GAGs, indicating that this activity is associated with the core glycoprotein. The neurite outgrowth from embryonic hippocampal neurons on laminin was not affected by the addition of DSD-1-PG. This indicates that DSD-1-PG/mouse phosphacan can have opposing effects on the process of neurite outgrowth dependent on neuronal lineage.
Asunto(s)
Proteoglicanos Tipo Condroitín Sulfato/farmacología , Sulfatos de Condroitina/farmacología , Ganglios Espinales/efectos de los fármacos , Neuritas/efectos de los fármacos , Neuronas/efectos de los fármacos , Secuencia de Aminoácidos , Animales , Secuencia de Bases , Linaje de la Célula , Proteoglicanos Tipo Condroitín Sulfato/química , Sulfatos de Condroitina/química , Clonación Molecular , ADN Complementario/genética , Ganglios Espinales/citología , Glicosilación , Hipocampo/efectos de los fármacos , Hipocampo/ultraestructura , Ratones , Datos de Secuencia Molecular , Neuronas/ultraestructura , Ratas , Proteínas Tirosina Fosfatasas Clase 5 Similares a Receptores , Homología de Secuencia de Aminoácido , Homología de Secuencia de Ácido NucleicoRESUMEN
Neurocan is a member of the aggrecan family of proteoglycans which are characterized by NH2-terminal domains binding hyaluronan, and COOH-terminal domains containing C-type lectin-like modules. To detect and enhance the affinity for complementary ligands of neurocan, the COOH-terminal neurocan domain was fused with the NH2-terminal region of tenascin-C, which contains the hexamerization domain of this extracellular matrix glycoprotein. The fusion protein was designed to contain the last downstream glycosaminoglycan attachment site and was expressed as a proteoglycan. In ligand overlay blots carried out with brain extracts, it recognized tenascin-C. The interaction was abolished by the addition of EDTA, or TNfn4,5, a bacterially expressed tenascin-C fragment comprising the fourth and fifth fibronectin type III module. The fusion protein directly reacted with this fragment in ligand blot and enzyme-linked immunosorbent assay procedures. Both tenascin-C and TNfn4,5 were retained on Sepharose 4B-linked carboxyl-terminal neurocan domains, which in BIAcore binding studies yielded a KD value of 17 nM for purified tenascin-C. We conclude that a divalent cation-dependent interaction between the COOH-terminal domain of neurocan and those fibronectin type III repeats is substantially involved in the binding of neurocan to tenascin-C.
Asunto(s)
Proteoglicanos Tipo Condroitín Sulfato/química , Proteoglicanos Tipo Condroitín Sulfato/metabolismo , Proteínas del Tejido Nervioso/química , Proteínas del Tejido Nervioso/metabolismo , Tenascina/química , Tenascina/metabolismo , Secuencia de Aminoácidos , Animales , Anticuerpos Monoclonales , Sitios de Unión , Unión Competitiva , Encéfalo/metabolismo , Línea Celular , Pollos , Cromatografía de Afinidad , Ácido Edético/farmacología , Humanos , Immunoblotting , Lectinas Tipo C , Ligandos , Ratones , Modelos Estructurales , Datos de Secuencia Molecular , Neurocano , Fragmentos de Péptidos/química , Fragmentos de Péptidos/aislamiento & purificación , Fragmentos de Péptidos/farmacología , Conformación Proteica , Ratas , Proteínas Recombinantes de Fusión/química , Proteínas Recombinantes de Fusión/metabolismo , Proteínas Recombinantes/química , Proteínas Recombinantes/metabolismoRESUMEN
Glia and neurons of the hypothalamoneurohypophysial system (HNS) undergo reversible morphological changes, which are concomitant with the remodelling of afferents onto the neurons, under different conditions of neurohormone secretion. Here, we show that the adult rat HNS contains high levels of tenascin-C (TN-C), which is an extracellular matrix glycoprotein whose expression is usually associated with neuronal-glial interactions in the developing and lesioned central nervous system (CNS). By using light and electron microscopic immunocytochemical procedures, we visualized TN-C immunoreactivity in the hypothalamic supraoptic (SON) and paraventricular nuclei, where somata of the neurons are localized; in the median eminence, where their axons transit; and in the neurohypophysis, where they terminate. Hypothalamic areas adjacent to the magnocellular nuclei were devoid of immunoreactivity. Electron microscopy of the neurohypophysis showed immunolabelling of perivascular spaces, glial (pituicyte) and axonal surfaces, a type of labelling that also characterized the median eminence. In the hypothalamic nuclei, there was labelling of extracellular spaces and astrocytic surfaces. In normal animals, we detected no cytoplasmic reaction in glia somata, neurons, or endothelial cells. However, in animals treated with the intracellular transport blocker colchicine, there was intracytoplasmic labelling of all HNS glial cells, indicating a glial source for TN-C. Immunoblot analysis revealed TN-C isoforms of apparent high molecular weight (225, 240, and 260 kD) in the SON and median eminence, whereas lower MW forms (190/200 kD) predominated in the neurohypophysis. By using immunocytochemistry and immunoblot analysis, we found no visible differences in TN-C expression in relation to age, sex, or differing neurohypophysial secretion, which suggests that the expression of TN-C is a permanent feature of the HNS.