Biomedical subjects
Publications and source records attributed to M Schachner.
Altering or removing neural recognition molecules by blocking antibodies or genetic deletion has led to a flurry of predictions concerning their function. Each experimental approach has advantages and disadvantages that have to be considered when interpreting the resulting phenotypes.
We have recently characterized a chondroitin sulfate proteoglycan from the murine central nervous system which is expressed by astrocytes in vitro and carries the L2/HNK-1 and L5 carbohydrate structures. In the present study, we provide evidence that its three core proteins of different size are similar in their proteolytic peptide maps and thus designate this group of structurally related molecules astrochondrin. During development, astrochondrin and the L5 carbohydrate were hardly detectable in the brain of 14-d-old mouse embryos by Western blot analysis. Expression of astrochondrin and the L5 epitope was highest at postnatal day 8, the peak of cerebellar granule cell migration and Bergmann glial process formation, and decreased to weakly detectable levels in the adult. Immunocytochemical localization of astrochondrin in the cerebellar cortex of 6-d-old mice showed association of immunoreactivity with the cell surface of astrocytes, including Bergmann glial processes and astrocytes in the internal granular layer or prospective white matter. Endfeet of astrocytes contacting the basal lamina of endothelial and meningeal cells and contact sites between Bergmann glial processes and granule cells also showed detectable levels of astrochondrin. Furthermore, granule cell axons in the molecular layer were astrochondrin immunoreactive. In the adult, astrochondrin immunoreactivity was weakly present in the internal granular layer and white matter. Both Fab fragments of polyclonal antibodies to astrochondrin and monovalent fragments of the L5 monoclonal antibody reduced the formation of processes of mature GFAP-positive astrocytes on laminin and collagen type IV, but not on fibronectin as substrata. Interestingly, the initial attachment of astrocytic cell bodies was not disturbed by these antibodies. Antibodies to astrochondrin also reduced the migration of granule cells in the early postnatal mouse cerebellar cortex. In a solid phase radioligand binding assay, astrochondrin was shown to bind to the extracellular matrix components laminin and collagen type IV, being enhanced in the presence of Ca2+, but not to fibronectin, J1/tenascin or other neural recognition molecules. Furthermore, astrochondrin interacted with collagen types III and V, less strongly with collagen types I, II, and IX, but not with collagen type VI. The interaction of astrochondrin with collagen types III and V was saturable and susceptible to increasing ionic strength, and could be competed by chondroitin sulfate, heparin, and dextran sulfate, but not by hyaluronic acid, glucose-6-phosphate, or neuraminic acid.(ABSTRACT TRUNCATED AT 400 WORDS)
The oligodendrocyte-derived extracellular matrix glycoprotein J1-160/180 is a recognition molecule expressed exclusively in the central nervous system. J1-160/180 has been shown to be adhesive for astrocytes and repellent towards neurons and growth cones. We report here the complete nucleotide sequence of J1-160/180 in the rat. The predicted amino acid sequence showed a structural architecture very similar to tenascin: a cysteine-rich amino terminal region is followed by 4.5 epidermal growth factor-like repeats, 9 fibronectin type III homologous repeats and a domain homologous to fibrinogen. Sequence comparison analysis revealed highest homology of rat J1-160/180 to mouse tenascin and chicken restrictin with a similarity of 66% and 85%, respectively. The J1-160/180-coding mRNA is derived from a single copy gene. Using the polymerase chain reaction we could show that two J1-160/180 isoforms are generated by alternative splicing of the sixth fibronectin type III homologous repeat. Localization of J1-160/180 mRNA by in situ hybridization in the cerebellum, hippocampus and olfactory bulb confirmed the expression of J1-160/180 by oligodendrocytes with a peak of transcription at 7-14 d after birth, indicating a functional role during myelination. In addition, J1-160/180-specific RNA was found in a small subset of neurons in all three structures of the CNS analyzed. These neurons continue to express J1-160/180 in the adult.
We have previously shown that the neural adhesion molecules L1 and NCAM interact with each other to form a complex which binds more avidly to L1 than L1 to L1 alone (Kadmon, G., A. Kowitz, P. Altevogt, and M. Schachner. 1990a. J. Cell Biol. 110:193-208). This cis-association between L1 and NCAM is carbohydrate-dependent (Kadmon, G., A. Kowitz, P. Altevogt, and M. Schachner. 1990b. J. Cell Biol. 110:209-218). In the present study, we report that L1 and NCAM bind to each other via oligomannosidic carbohydrates expressed by L1, but not by NCAM, as shown in several experiments: (a) complex formation between L1 and NCAM is inhibited by a mAb to oligomannosidic carbohydrates and by the oligosaccharides themselves; (b) NCAM binds to oligomannosidic carbohydrates; (c) within the L1/NCAM complex, the oligomannosidic carbohydrates are hidden from accessibility to a mAb against oligomannosidic carbohydrates; (d) the recombinant protein fragment of NCAM containing the immunoglobulin-like domains and not the fragment containing the fibronectin type III homologous repeats binds to oligomannosidic glycans. Furthermore, the fourth immunoglobulin-like domain of NCAM shows sequence homology with carbohydrate recognition domains of animal C-type lectins and, surprisingly, also with plant lectins. A peptide comprising part of the C-type lectin consensus sequence in the fourth immunoglobulin-like domain of NCAM interferes with the association between L1 and NCAM. The functional importance of oligomannosidic glycans at the cell surface was shown for neurite outgrowth in vitro. When neurons from early postnatal mouse cerebellum were maintained on laminin or poly-L-lysine, neurite outgrowth was inhibited by oligomannosidic glycans, by glycopeptides, glycoproteins, or neoglycolipids containing oligomannosidic glycans, but not by nonrelated oligosaccharides or oligosaccharide derivates. Neurite outgrowth was also inhibited by the peptide comprising part of the C-type lectin consensus sequence in the fourth immunoglobulin-like domain of NCAM. The combined results suggest that carbohydrate-mediated cis-associations between adhesion molecules at the cell surface modulate their functional properties.
Monoclonal L3 and L4 antibodies have been shown to recognize carbohydrate epitopes on several neural cell adhesion molecules; these epitopes can be released by treatment with endoglycosidase H. In the present study, we have identified the oligosaccharides released by endoglycosidase H from the cell adhesion molecules AMOG and L1 by fast-atom bombardment mass spectrometry as being solely of the oligomannosidic type. Using neoglycolipids of oligomannosidic glycans, we also report that both antibodies show the highest reactivity with the alpha-manno-pentaose Man alpha 1-3-[Man alpha 1-6(Man alpha 1-3)Man alpha 1-6]-Man, but decreasing reactivity with the alpha-manno-hexaose, heptaose, octaose and nonaose glycans. Thus, to our knowledge, we describe here for the first time monoclonal antibodies recognizing N-glycosidically linked oligomannosidic glycans.
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The extracellular matrix molecules tenascin, laminin, and fibronectin, the cell adhesion molecule L1, and the lectin concanavalin A (ConA) were tested for their effects on neuritogenesis in cultures of hippocampal neurons. We analyzed neurite outgrowth between 3 and 21 hr after plating and found that, on polyornithine as control substrate, lengths of axon-like major neurites and dendrite-like minor neurites increased continuously with time in culture. Moreover, growth of minor neurites was faster than growth of major neurites. When the extracellular matrix molecules tenascin, laminin, and fibronectin were coated on polyornithine substrates, growth of all neurites was faster than on control substrates during the first hours of culture. After this initial phase of enhanced neurite outgrowth, elongation of major neurites continued at a higher rate than on the control substrate and growth of minor neurites ceased after 12 hr. Correspondingly, neuronal polarity was strongly increased on the extracellular matrix substrates during later phases of culture. In contrast, lengths of both major and minor neurites were increased over control values on L1 and ConA substrates at all time points investigated. Thus, neuronal polarity was similar for control, L1, and ConA substrates. Spreading of neuronal cell bodies was reduced by about 50% on tenascin, laminin, and fibronectin and by less than 20% on L1 and ConA substrates after 21 hr of culture, when compared to the control substrate. Neuron-to-substrate adhesion was reduced on all three extracellular matrix substrates but not affected on L1 or ConA substrates, after 3 and 21 hr of culture. These observations indicate that induction of neuronal polarity is not a general feature of neurite outgrowth-promoting molecules, such as L1 or ConA, but a distinctive property of the three extracellular matrix glycoproteins studied, and may suggest that enhancement of polarity is correlated with decreased strength of adhesion.
The neural cell adhesion molecule L1 is a multidomain protein that plays important roles in cell adhesion, migration, and neurite outgrowth. To analyze structure-function relationships of L1 in neurite outgrowth and cell body adhesion, we have expressed and purified a set of different fragments of the extracellular part of this glycoprotein in CHO cells and in Escherichia coli. When neurite outgrowth from small cerebellar neurons was measured on substrate-coated L1 or L1 fragments, neurite outgrowth was promoted by the immunoglobulin-like domains I-II, III-IV, and V-VI, and by the fibronectin type III homologous repeats 1-2, while the fibronectin type III homologous repeats 3-5 were ineffective. In contrast, cell bodies of small cerebellar neurons adhered mostly to the immunoglobulin-like domains I-II and V-VI, and to the fibronectin type III homologous repeats 3-5, but less to the immunoglobulin-like domains III-IV and fibronectin type III homologous repeats 1-2. In both assays, the neuronal cell surface receptor for all active protein fragments was identified as L1. No significant differences in functional activities were found between fragments with and without carbohydrate structures. These findings indicate that L1 uses several domains for homophilic interactions overlapping for the two functions analyzed here, but also showing some regional specialization. Furthermore, we show that a homophilic molecule uses several domains in one function, with neurite outgrowth requiring more domains than adhesion for maximal activity.
We have used homologous recombination in embryonic stem cells to generate mice carrying a mutation in the gene encoding P0, an immunoglobulin-related recognition molecule and the major protein of peripheral nervous system myelin. These mice are deficient in normal motor coordination and exhibit tremors and occasional convulsions. Axons in their peripheral nerves are severely hypomyelinated and a subset of myelin-like figures and axons degenerate. The mutation leads to an abnormal regulation of some, but not all, molecules involved in myelination. These results demonstrate that P0 is essential for the normal spiraling, compaction, and maintenance of the peripheral myelin sheath and the continued integrity of associated axons. They further suggest that this protein conveys a signal that regulates Schwann cell gene expression.
The adhesion molecule on glia, AMOG, an integral cell surface glycoprotein highly expressed by cerebellar astrocytes and involved in neuron to astrocyte adhesion and granule neuron migration (Antonicek, H., Persohn, E., and Schachner, M. (1987) J. Cell Biol. 104, 1587-1595) has been identified as a beta 2 subunit isoform of the mouse sodium pump (Gloor, S., Antonicek, H., Sweadner, K.J., Pagliusi, S., Frank, R., Moos, M., and Schachner, M. (1990) J. Cell Biol. 110, 165-174). Here we demonstrate that AMOG/beta 2 expressed by cRNA injection in Xenopus oocytes is capable of combining with endogenous Xenopus alpha 1 subunits or coexpressed Torpedo alpha 1 subunits to yield a functional alpha 1/AMOG sodium pump isozyme. Determinations of the number of ouabain binding sites and ouabain-sensitive 86Rb+ uptake suggest that the alpha 1/AMOG isozyme has slightly lower maximum transport rate and apparent affinity for external K+ than the alpha 1/beta 1 isozyme. Immunoprecipitation of alpha 1/AMOG complexes from digitonin extracts of [35S]methionine-labeled oocytes with a monoclonal anti-AMOG antibody provides direct evidence for a stable association between AMOG and the alpha 1 subunits of Xenopus and Torpedo.
Tenascin is an extracellular matrix molecule synthesized and released by young astrocytes during embryonic and early postnatal development of the nervous system, and it is concentrated in boundaries around emerging functional neuronal units. In the adult nervous system, tenascin can be detected only in very low levels. Distinct spatial and temporal distributions of tenascin during developmental events suggest a role in the guidance and/or segregation of neurons and their processes within incipient functional patterns. We show here, using in situ hybridization and immunocytochemistry, that stab wounds of the adult mouse cerebellar and cerebral cortices result in an enhanced expression of tenascin in a discrete region around the lesion site that is associated with a subset of glial fibrillary acidic protein-positive astrocytes. Tenascin up-regulation in the lesioned adult brain may be directly involved in failed regeneration or indirectly involved through its interactions with other glycoconjugates that either inhibit or facilitate neurite growth.
The type beta transforming growth factors (TGF) are potent regulators of the growth and functions of lymphocytes and macrophages. Recently the human glioblastoma cell line 308 was shown to produce TGF-beta 2. The relevance of this finding was evaluated further by comparing human glioblastoma cells with their nontransformed animal counterpart, astrocytes, with regard to the production of the three TGF-beta isoforms observed so far in mammals. In this report astrocytes are demonstrated to secrete also TGF-beta 2 and to express TGF-beta 1, -beta 2, and -beta 3 mRNA in vitro. In contrast, cultured murine brain macrophages release TGF-beta 1 and are positive for TGF-beta 1 mRNA only. Glia cell-derived TGF-beta 1 and -beta 2 are detected in latent form whereas both latent and active TGF-beta are identified in the supernatant of three human glioblastoma cell lines tested. These cell lines, however, show heterogeneity in regard to the isoform of TGF-beta expressed but share with astrocytes the inability to release TGF-beta 3. Provided production and activation of latent TGF-beta occur in vivo, astrocytes and microglia may then be expected to exert regulatory influences on immune mediated diseases of the central nervous system.
The caudate-putamen (neostriatum) of the mammalian basal ganglia is composed of two neurochemically distinct compartments termed patch (island, striosome) and matrix that overall contribute to a mosaic organization. In the present study, the distribution of the developmentally regulated extracellular matrix molecule tenascin, as well as several other neural cell adhesion molecules, was examined in the neostriatal mosaic of the early postnatal mouse and compared with tyrosine hydroxylase distribution following partial destruction of the dopaminergic nigrostriatal projection. During normal neostriatal development, tenascin is most dense within the matrix compartment and highly concentrated in boundaries around patches. This pattern is apparent on embryonic day 18, and for the most part disappears by postnatal day 12. Tenascin immunoreactivity is altered in the neostriatum following lesions of the nigrostriatal pathway in the first postnatal week revealed by an overall reduced expression of this molecule and a marked reduction in tenascin staining of boundaries at the interface of tyrosine hydroxylase-rich patch and tyrosine hydroxylase-poor matrix compartments. When compared to tyrosine hydroxylase immunoreactivity, other cell adhesion molecules tested failed to show altered intensities and patterns of immunoreactivity within the neostriatum after similar lesions. Reduced levels of tenascin in the lesioned neostriatum, in register with altered levels of tyrosine hydroxylase immunostaining of dopaminergic inputs, suggests that axons may affect the expression of particular recognition molecules in their target structures. The fact that boundaries are malleable can be related to afferent-induced plastic events in the differentiation of cellular elements in the developing nigrostriatal system.
The neural cell adhesion molecule L1 is a cell surface glycoprotein of the immunoglobulin superfamily which mediates adhesion between neural cells. The possibility that similar cell-cell recognition mechanisms may be shared by the nervous and immune systems prompted us to study the expression and function of L1 in cells of the hematopoietic system. Immunofluorescence analysis using monoclonal L1 antibody revealed that the molecule is expressed in the bone marrow, spleen, and thymus of the mouse. This observation was confirmed by amplifying cDNA derived from these organs by the polymerase chain reaction with L1-specific oligonucleotide primers. Two-color fluorescence analysis indicated that bone marrow lymphoid and granulocyte precursor cells express low and high levels of L1, respectively. In the thymus L1 is primarily expressed by mature cells that have a strong expression of CD3 and in the spleen both B cells and T cells express L1. The possible function of L1 in lymphoid cells was studied using subcloned ESb-MP lymphoma cells having high or low densities of L1 on the cell surface as well as activated splenic B lymphoblasts. Parental and subcloned ESb-MP cells that strongly expressed L1 could form homotypic aggregates in the presence of low Ca2+ levels, whereas subcloned ESb-MP cells with a weak expression of L1 did not aggregate, suggesting that L1 mediates the Ca(2+)-independent aggregation of the parental ESb-MP cells. Furthermore, the aggregation of activated B lymphoblasts under physiological concentrations of Ca2+ and Mg2+ was inhibited by 30% in the presence of Fab fragments of polyclonal L1 antibodies, implying that L1 also mediates adhesion among normal lymphoid cells. A possible role of L1 on lymphocytes in stimulating the innervation of lymphoid organs is discussed.
To gain insight into the molecular mechanisms underlying the regulation of expression of the neural cell adhesion molecule L1 and into the exon-intron structure of the L1 gene, a genomic clone from the mouse was characterized. The clone was identified by screening an EMBL3 library with an L1-specific cDNA probe and comprises approximately 15 kb, in which the first 2,206 nucleotides of the coding region are included. Of the 5 of 6 immunoglobulin (Ig)-like domains sequenced, all are encoded by 2 exons, with the first exon being smaller than the second. The exon encoding the signal peptide is separated from a mini-exon containing 15 bp by a large intron, approximately 2.6 kb in length, whereas the other introns are smaller, with the coding information for the Ig-like domains 3-5 clustered in a 1,643-bp-long fragment with introns only 110-217 bp in length. The 5' upstream region of the clone comprises 5 kb, with the first 112 bp lying upstream to the coding sequence and containing a start site for transcription. No consensus sequence for a TATA box was found. Consensus DNA sequences for the binding of the gene products of Hox 1.3, engrailed and bicoid, are localized upstream to the transcription start site. A 1,262-bp fragment containing part of the first exon showed promoter activity in neuroblastoma cells, but hardly in L cells and not in CHO cells, indicating that this fragment is sufficient for neural cell directed promoter activity.
The neural adhesion molecule P0, the most abundant glycoprotein in peripheral myelin of mammals, is a member of the immunoglobulin superfamily and expresses the L2/HNK-1 and L3 oligosaccharides at a single N-glycosylation site. It acts in both homophilic and heterophilic binding mechanisms. To investigate the molecular requirements for homophilic interaction, we have used P0 from human sciatic nerve and the extracellular domain of P0 expressed in bacteria to determine binding of P0 to P0 in solid phase and bead aggregation assays. The binding of P0 to P0 could be partially inhibited in both assays by antibodies to the L2/HNK-1 epitope and by the L2/HNK-1 carbohydrate, but not by L3 antibodies or other carbohydrates. Inhibition of binding was also seen with polyclonal antibodies reacting with the protein backbone of P0. These observations indicate that both carbohydrate and protein structures are involved in the binding of P0 to P0 and that P0 acts as a presenter of and a receptor for a functionally important carbohydrate.
Monoclonal M6 antibody binds to the surface of murine central nervous system neurons as well as to apical surfaces of epithelial cells in the choroid plexus and proximal tubules of the kidney. M6 antigen is expressed in the central nervous system as early as embryonic day 10, most strongly in the marginal zone of the neural tube, and remains detectable in adulthood. IgG or Fab fragments of M6 antibody interfere with the extension of neurites by cultured cerebellar neurons. Effects of the antibody on neurite extension are readily detectable after 24 h. No reduction of cell viability is detected during the first 3 days of antibody treatment. Cultures maintained in the presence of antibody for longer than 5 days exhibit reduced viability of neurons. This reduction in long-term viability in the presence of M6 antibody is largely avoided when 25 mM KCl is included in the culture medium. The antibody-mediated perturbation of neurite outgrowth is not blocked by the presence of elevated KCl. The unusually short and flattened appearance of neurites in these cultures suggests that the M6 antibody selectively affects neurite extension. Time-lapse cinematography of anti-M6-treated neurons reveals no apparent effect on movement of lamellipodia and filopodia of growth cones. Only the overall extension of the neurite appears to be inhibited. M6 antigen is a 35 kD glycoprotein that can be isolated from a deoxycholate- (DOC) solubilized membrane fraction from adult mouse brain.
In this report, we describe the preparation of a library of N-linked glycans from whole murine brain obtained by the large-scale hydrazinolysis of an acetone powder of the tissue followed by chromatographic procedures. 84% of the characterized oligosaccharides were found to be anionic, the remainder neutral. The anionic species were successively neutralized by neuraminidase (29%), aq. hydrofluoric acid (30%), and methanolysis (26%), indicating that approximately equal portions were sensitive to desialylation, dephosphorylation and desulfation, respectively. The presence of the sulfated fraction was confirmed by direct 35SO4 metabolic labelling. A residual partially characterized fraction was found to be anionic through possession of carboxylic acid groups, unrelated to sialic acid. The purified oligosaccharides, in the absence of their original protein conjugates, were shown to retain those immunological characteristics essential for recognition by a specific monoclonal antibody, LS (412), that is known to recognize a carbohydrate epitope present on a number of neural adhesion molecules and functional in neural cell adhesion. These properties confirm the viability of scaling up the size of the hydrazinolysis procedure and adapting it to whole tissue for the production of glycan libraries and for the probing of structures of interest.