The L2/HNK-1 carbohydrate of neural cell adhesion molecules is involved in cell interactions.

We investigated whether the L2/HNK-1 carbohydrate epitope, expressed by two unusual glycolipids and several neural adhesion molecules, including L1, neural cell adhesion molecule, J1, and the myelin-associated glycoprotein, is involved in adhesion. Monoclonal L2 antibodies, the L2/HNK-1-reactive, sulfate-3-glucuronyl residue carrying glycolipids (L2 glycolipid) and a tetrasaccharide derived from the L2 glycolipid (L2 tetrasaccharide) were added to microexplant cultures of early postnatal mouse cerebellum, and cell migration and process extension were monitored. On the substrate poly-D-lysine, Fab fragments of L2 antibodies, L2 glycolipid, and L2 tetrasaccharide inhibited outgrowth of astrocytic processes and migration of cell bodies, but only L2 glycolipid and L2 tetrasaccharide reduced neurite outgrowth. On laminin, L2 antibodies, L2 glycolipid, and L2 tetrasaccharide inhibited outgrowth of astrocytic processes. Additionally, L2 glycolipid and L2 tetrasaccharide inhibited cell migration and neurite outgrowth. Several negatively charged glycolipids, lipids, and saccharides were tested for control and found to have no effect on outgrowth patterns, except for sulfatide and heparin, which modified outgrowth patterns in a similar fashion as L2 glycolipid and L2 tetrasaccharide. On astrocytes none of the tested compounds interfered with explant outgrowth. In short-term adhesion assays L2 glycolipid, sulfatide, and heparin inhibited adhesion of neural cells to laminin. L2 glycolipid and sulfatide interfered with neuron to astrocyte and astrocyte to astrocyte adhesion, but not with neuron-neuron adhesion. The most straightforward interpretation of these observations is that the L2/HNK-1 carbohydrate and the sulfated carbohydrates, sulfatide and heparin, act as ligands in cell adhesion.

Anti-peripheral nerve myelin antibodies in Guillain-Barre syndrome bind a neutral glycolipid of peripheral myelin and cross-react with Forssman antigen.

During acute-phase illness, serum of patients with Guillain-Barre syndrome (GBS) contain complement-fixing antibodies (Ab) to peripheral nerve myelin (PNM). We investigated PNM lipids as putative antigens for these Ab since GBS serum retained significant reactivity to PNM treated with protease. Ab binding to specific lipids was studied with a C1 fixation and transfer (C1FT) assay using fractions of PNM lipid reincorporated into liposomes as antigen targets or to lipids on HPTLC plates with peroxidase-labeled goat Ab to human IgM. Reactivity was detected to a neutral glycolipid (NGL) of human PNM with a similar number of carbohydrates residues to that of Forssman hapten (Forss). Anti-NGL Ab titers in GBS patients (50-220 U/ml) were significantly elevated over disease and normal controls (0-5 and 0-6 U/ml). We studied possible antigenic cross-reactivity of these Ab with Forss by first quantitating Ab activity with C1FT assay and liposomes containing Forss. All 12 GBS sera tested showed titers (54-272 U/ml) significantly elevated over 11 disease controls (0-22 U/ml) and 25 normal controls (0-11 U/ml). GBS serum Ab reacted with Forss isolated from dog nerve or sheep erythrocytes on HPTLC plates. Further, absorption of 80-100% of anti-NGL Ab activity and 17-97% of anti-PNM Ab activity from eight GBS patient serums was accomplished with liposomes containing Forss but not with control liposomes. In seven GBS patients anti-NGL Ab activity represented only a portion of anti-PNM Ab activity. These results suggest that a glycolipid with antigenic cross-reactivity to Forssman hapten may be responsible for some of the anti-PNM Ab activity in GBS.

Expression of HNK-1 carbohydrate and its binding protein, SBP-1, in apposing cell surfaces in cerebral cortex and cerebellum.

Sulfoglucuronyl carbohydrate is the terminal moiety of neolacto-oligosaccharides, expressed on several glycoproteins of the immunoglobulin superfamily involved in cell-cell recognition and on two glycolipids. Sulfoglucuronyl carbohydrate is temporally and spatially regulated in the developing nervous system. It appears to be involved in neural cell recognition and in cell adhesion processes through its interaction with specific proteins on cell surfaces. Previously we have characterized a specific sulfoglucuronyl carbohydrate-binding protein in rat brain. Sulfoglucuronyl carbohydrate binding protein-1 is structurally similar to a 30,000 mol. wt adhesive and neurite outgrowth promoting protein amphoterin [Rauvala and Pihlaskari (1987) J. biol. Chem. 262, p. 16,625]. The pattern of expression of sulfoglucuronyl carbohydrate binding protein-1 in developing rat nervous system was studied to understand the significance of its interaction with sulfoglucuronyl carbohydrate-bearing molecules. Biochemical analyses showed that the expression of sulfoglucuronyl carbohydrate binding protein-1 was developmentally regulated similarly to sulfoglucuronyl carbohydrate. Immunocytochemical localization of sulfoglucuronyl carbohydrate binding protein-1 and sulfoglucuronyl carbohydrate was performed by bright-field and fluorescent confocal laser scanning microscopy. In postnatal day 7 rat cerebellum, sulfoglucuronyl carbohydrate binding protein-1 was primarily associated with neurons of the external and internal granule cell layers. The sulfoglucuronyl carbohydrate binding protein-1 immunoreactivity was absent in Purkinje cell bodies and their dendrites in the molecular layer, as well as in Bergmann glial fibres and in white matter. In contrast, sulfoglucuronyl carbohydrate (reactive with HNK-1 antibody) was localized in processes surrounding granule neurons in the internal granule cell layer. Sulfoglucuronyl carbohydrate was also expressed in Purkinje neurons and their dendrites in the molecular layer and their axonal processes in the white matter. To a lesser extent Bergmann glial fibres were also positive for sulfoglucuronyl carbohydrate. In the cerebral cortex, at embryonic day 21, sulfoglucuronyl carbohydrate binding protein-1 was mainly observed in immature neurons of the cortical plate and subplate and dividing cells near the ventricular zone. Whereas, sulfoglucuronyl carbohydrate was strongly expressed in the fibres of the subplate and marginal zone. Sulfoglucuronyl carbohydrate was also found in the processes surrounding the sulfoglucuronyl carbohydrate binding protein-1-expressing neuronal cell bodies in the cortical plate and in ventricular zone. The specific localization of sulfoglucuronyl carbohydrate binding protein- in cerebellar granule neurons and neurons of the cerebral cortex was also confirmed by immunocytochemistry of the dissociated tissue cell cultures. The complementary localization of sulfoglucuronyl carbohydrate and sulfoglucuronyl carbohydrate binding protein-1, both in cerebral cortex and cerebellum, in apposing cellular structures indicate possible interaction between the two and signalling during the process of cell migration and arrest of migration.

Expression of sulfoglucuronyl (HNK-1) carbohydrate and its binding protein (SBP-1) in developing rat cerebellum.

Sulfoglucuronyl carbohydrate (SGC) is expressed on several glycoproteins of the immunoglobulin superfamily of cell-adhesion molecules. Developmental expression of SGC and its binding protein, SBP-1, was studied in the rat cerebellum by immunocytochemistry to understand the function of SBP-1 and the significance of its interaction with SGC. During early postnatal development (postnatal day (PD) 3-10) SBP-1 was strongly expressed in the granule neurons of the external and internal granule cell layers (EGCL and IGCL). This expression declined by PD 15, and disappeared in the adult. Between PD 3 and 15, SGC was expressed in cellular processes surrounding the granule neurons in the IGCL, and it also declined and disappeared with development. SGC expression, however, continued in Purkinje cells and their dendrites in the molecular layer in adults. The expressions of SBP-1 and SGC were developmentally regulated and appeared to be chronologically co-ordinated with granule neuron migration from EGCL to IGCL. High magnification confocal microscopy showed that SBP-1 was primarily localized in nuclei and plasma membranes of granule neurons, whereas SGC in the IGCL was localized on neuronal plasma membranes, dendrites and glial processes, but not in cell soma. The relative localization of SBP and SGC was confirmed by cellular and subcellular markers in vivo and with dissociated cerebellar cells in culture. It is proposed that SBP-1 on plasma membranes of granule neurons interacts with SGC on the surrounding processes and membranes and this interaction could provide a potential mechanism for guidance and cell signaling, in the processes of granule neuron migration and differentiation.

Characterization and developmental expression of lactotriosylceramide:galactosyltransferase for the synthesis of neolactotetraosylceramide in the nervous system.

Neolactoglycolipids are derived from neolactotetraosylceramide (nLcOse4Cer). They are found during the embryonic and neonatal developmental periods in the rat cerebral cortex and disappear shortly after birth. These glycolipids are, however, abundant in the adult cerebellum. Lactotriosylceramide (LcOse3Cer):galactosyltransferase (GT), which catalyzes the terminal step in the biosynthesis of nLcOse4Cer, was characterized in mammalian brain. The enzyme was highly specific for LcOse3Cer, with a terminal GlcNAc beta 1-3Gal-residue, and it did not catalyze the transfer of galactose to other glycolipids studied with alternate carbohydrate residues. The microsomal membrane enzyme required Mn2+ and a detergent for in vitro activity. The optimal pH was 7.4, and the Km value for LcOse3Cer was 34 microM (Vmax = approximately 2 nmol/mg/h). The LcOse3Cer:GT was shown to be different from the GM2:GT and the soluble enzyme lactose synthase A. The specific activity of LcOse3Cer:GT was enriched fivefold higher in the white matter than in the gray matter of young adult rat brain, whereas GM2:GT was enriched only about 1.5-fold higher in the white matter. The developmental expression of LcOse3Cer:GT in the cerebral cortex and cerebellum was not correlative with the levels of nLcOse4Cer in these neural areas. Despite the complete absence of nLcOse4Cer in the cerebral cortex of animals older than 5 days, significant activity of the LcOse3Cer:GT was found even in the adult cortex. In cerebellum, the levels of nLcOse4Cer increased with development, but the specific activity of the enzyme was reduced by 50% soon after birth and then remained practically the same with development.(ABSTRACT TRUNCATED AT 250 WORDS)

Characterization and developmental expression of a novel sulfotransferase for the biosynthesis of sulfoglucuronyl glycolipids in the nervous system.

Sulfoglucuronyl glycolipids (SGGLs) are temporally and spatially regulated molecules in the developing nervous system. A novel sulfotransferase (ST) from rat brain which catalyzes the terminal step in the biosynthesis in vitro of SGGLs is described. The enzyme catalyzes a transfer of sulfate from 3'-phosphoadenosine 5'-phosphosulfate to a hydroxyl group on carbon 3 of the terminal glucuronyl residue in IV3 beta-glucuronyl neolactotetraosylceramide (GlcAnLcOse4Cer) and VI3 beta-glucuronyl neolactohexaosylceramide (GlcAnLcOse6Cer) to form 3-sulfated glucuronyl glycolipids. The enzyme is highly specific for glucuronylglycolipids (GGLs) and requires the free-COOH group of the terminal glucuronic acid for reactivity. GGL:ST present in the microsomal membranes requires Mn2+ ions and a nonionic detergent, Triton X-100 for activity. The optimal pH is 7.2 with Tris-HCl buffer and Km values were 7 microM for 3'-phosphoadenosine 5'-phosphosulfate and 29 microM for GlcAnLcOse4Cer. GGL:ST was shown to be different from previously well studied galactocerebroside:sulfotransferase for the synthesis of myelin membrane-specific lipid sulfatide. This conclusion was based upon several criteria, i.e. including different requirements of incubation conditions for maximal activity, substrate competition experiments, different effects of heat, dithiothreitol, NaCl, and pyridoxal phosphate, as well as different profiles of expression of activity during development of the nervous tissues. The two enzymes were also partially resolved on a pyridoxal phosphate-ligated agarose column. Studies on the developmental expression of the GGL:ST in the rat cerebral cortex and cerebellum showed that it is not a regulatory enzyme controlling the expression of SGGLs in these neural tissues.

N-acetylglucosaminyltransferase regulates the expression of neolactoglycolipids including sulfoglucuronylglycolipids in the developing nervous system.

Lactosylceramide N-acetylglucosaminyltransferase (GlcNac-Tr) in the synthesis of lactotriosylceramide (LcOse3Cer) was characterized in the nervous system. The microsomal membrane GlcNAc-Tr required a divalent metal ion, preferably Mn2+, and a nonionic detergent. The pH optimum was around 7.0. The enzyme also transferred GlcNAc to neolactotetraosylceramide (nLcOse4Cer), GM1, and asialo-GM1, but not to other glycolipids. The Km value for lactosylceramide was 21 microM (Vmax = 91 pmol/mg/h), and that for nLcOse4Cer was 35 microM (Vmax = 112 pmol/mg/h). The GlcNAc-Tr for the glycolipids appears to be separate from that for oligosaccharides. The developmental expression of GlcNAc-Tr, both in the cerebral cortex and cerebellum, correlated well with the tissue levels of LcOse3Cer, nLcOse4Cer, sulfoglucuronylglycolipids (SGGLs), and other neolacto series glycolipids (nLSGs). In the cerebral cortex, the specific activity of GlcNAc-Tr decreased sharply from a maximum level at embryonic day 15, and by postnatal day 10 onward, it was undetectable. In the adult cerebral cortex, although significant activities of other glycosyltransferases involved in the subsequent steps of the synthesis of SGGLs were present, the absence of GlcNAc-Tr stymied the formation of LcOse3Cer and therefore the synthesis of nLSGs, including SGGLs. In the cerebellum, the GlcNAc-Tr specific activity declined from the day of birth to postnatal day 3, but later, the activity increased and reached a maximum at postnatal day 15, which correlated with the increasing synthesis of nLSGs. The results indicate that lactosylceramide GlcNAc-Tr is the key regulatory enzyme controlling the differential expression of all nLSGs in the developing nervous system.

Sulfoglucuronyl neolactoglycolipids in adult cerebellum: specific absence in murine mutants with Purkinje cell abnormality.

It is shown here that glycolipids of the sulfoglucuronyl neolacto series (SGGLs) are present in the adult rodent cerebellum. SGGLs were not detected in the cerebellar murine mutants lurcher, Purkinje cell degeneration, and staggerer, in which Purkinje cell loss is the primary defect. SGGLs were present, however, in normal amounts in weaver and reeler mutants, in which there is a major and relatively specific loss of granule cells without obvious deficiency in Purkinje cells. In the myelin-deficient quaking mutant, the expression of SGGLs also was nearly normal. The loss of SGGLs in Purkinje cell-deficient mutants was specific, since most of the major lipids were not affected significantly and only the percentage composition of other lipids, such as sulfatides and gangliosides, was altered in the mutants. These and other results strongly suggest that SGGLs and other glycolipids of the paragloboside family are localized specifically in Purkinje cells and their arbors in the adult cerebellum. This is the first demonstration of the localization of a specific glycolipid and its analogs in a specific cell type in the nervous system.

N-Acetylglucosaminyl transferase regulates the expression of the sulfoglucuronyl glycolipids in specific cell types in cerebellum during development.

In the adult cerebellum, sulfoglucuronyl glycolipids (SGGLs) are specifically localized in Purkinje cells and their dendrites in the molecular layer. Other major cell types such as granule neurons and glial cells lack SGGLs. To explain the cell specific localization and the known biphasic expression of SGGLs, enzymic activities of four glycosyltransferases involved in the biosynthesis of SGGLs were studied in murine cerebellar mutants, in distinct cellular layers of rat cerebellum, and in isolated granule neurons during development. The enzymes studied were lactosylceramide: N-acetylglucosaminyl transferase (GlcNAc-Tr), lactotriaosylceramide:galactosyltransferase, neolactotetraosylceramide:glucuronyltransferase, and glucuronylglycolipid:sulfotransferase. In the cerebellum of Purkinje cell-deficient mutants, such as (pcd/pcd) and lurcher (Lc/+) where Purkinje cells are lost, GlcNAc-Tr was absent, but the other three glycosyltransferase were not severely affected. This indicated that the latter three enzymes were localized in other cell types, such as in mature granule neurons and glial cells, in addition to that in Purkinje cells, and the lack of SGGLs in these mutants was due to absence of GlcNAc-Tr. Analyses of the enzymes in the specific micro-dissected cellular layers also showed that Purkinje cell layer and molecular layer (where Purkinje cell dendrites are localized) contained all four enzymes. However, granule neurons and glial cells in the white matter lacked GlcNAc-Tr, but expressed the other three enzymes. It was concluded that the absence of SGGLs in adult granule neurons and glial cells was due to specific deficiency of the GlcNAc-Tr. Although adult granule neurons lacked GlcNAc-Tr and therefore SGGLs, isolated granule neurons from the neonatal cerebellum contained all four enzymes necessary for the synthesis of SGGLs. With development, the activity of GlcNAc-Tr in the isolated granule neurons declined but the other enzymes were not as affected, indicating that immature granule neurons were capable of synthesizing SGGLs and with maturation the synthesis was down-regulated. This also explains the biphasic expression of SGGLs in the developing cerebellum.

Regulation of sulfoglucuronyl glycolipid synthesis in the developing rat sciatic nerve.

Sulfoglucuronyl glycolipids (SGGLs) have been considered as target antigens in demyelinating peripheral neuropathies associated with IgM monoclonal gammopathy. The regulation of expression of SGGLs in the rat sciatic nerve during development was studied by assaying the levels of SGGLs and activities of four glycosyltransferases sequentially involved in their synthesis from lactosylceramide. The levels of SGGLs in the sciatic nerve increased with development and reached a maximum at sixty days after birth. The rate of increase in the level of SGGLs between day 5 to 20 was similar to rate of deposition of myelin in the nerve. Analysis of the activities of the glycosyltransferases showed that only lactotriosylceramide galactosyltransferase (LcOse3Cer-GalTr) increased in parallel with the levels of SGGLs during development. The other three enzymes were not co-relative with the synthesis of SGGLs. The product of LcOse3Cer-GalTr reaction, nLcOse4Cer is the key intermediate for all neolactoglycolipids, particularly NeuAc alpha2-3nLcOse4Cer or nLM1, which is the major ganglioside (60%) of myelin in rat sciatic nerve. The results suggest that in the sciatic nerve SGGLs are mostly associated with Schwann cell myelin and their synthesis is regulated by LcOse3Cer-GalTr, unlike in the cerebral cortex and cerebellum where SGGLs are associated with the neuronal membranes and their synthesis is regulated by lactosylceramide N-acetylglucosaminyltransferase (LcOse2Cer-GlcNAcTr).

Expression and regulation of UDP-glucuronate: neolactotetraosylceramide glucuronyltransferase in the nervous system.

Sulfoglucuronyl glycolipids (SGGLs) are temporally and spatially regulated molecules present in the nervous system during its development. The characteristics of the rat brain enzyme glucuronyltransferase involved in the biosynthesis of SGGLs have been described. The enzyme catalyzes the transfer of glucuronic acid (GlcA) from UDP-GlcA to terminal galactose of the neolacto (type 2) series of glycolipids to form beta 1-3-linked glucuronyl neolacto glycolipids. The enzyme was highly specific for the neolacto series of acceptor glycolipids, neolactotetraosylceramide (nLcOse4Cer), neolactohexaosylceramide (nLcOse6-Cer), and neolactooctaosylceramide (nLcOse8Cer) and was different from the drug-inducible phenol:GlcA transferase. Considerable activity of GlcA transferase was present in the adult rat cerebral cortex, even though SGGLs almost completely disappeared from the cortex by postnatal day 15. In the cerebellum, although levels of SGGLs increased with development, the specific activity of GlcA transferase declined. The results indicated that GlcA transferase was not a regulatory enzyme controlling the expression of SGGLs. Measurements of the levels of nLcOse4Cer and nLcOse6Cer in these neural tissues indicated that the availability of these precursors may regulate the differential expression of SGGLs seen previously. GlcA transferase was significantly reduced in the cerebellar Purkinje cell degenerating murine mutant (pcd/pcd), which is consistent with the loss of SGGLs in the cerebellum of this mutant and specific association of these glycolipids with Purkinje cells.

Interaction of sulfoglucuronyl (HNK-1) carbohydrate and its binding protein, SBP-1, in microexplant cultures of rat cerebellum.

Sulfoglucuronyl carbohydrate (SGC) is expressed on several neural cell-adhesion molecules and on glycolipids. SGC and its binding protein, SBP-1 are developmentally regulated in the nervous system and have been implicated in regulating neurite outgrowth and cell-cell recognition during neuronal cell migration. To elucidate the role of interaction between SGC and SBP-1, microexplant cultures of postnatal day 5 rat cerebellum were employed. In explant cultures, SGC was localized primarily in the neuronal cell processes, neurofilaments, and dendrites that emerge from the core of the explants up to 90 microm, after 24 hr in culture. SGC was also present in the short astrocytic processes near the core of the explant. SBP-1 was localized mainly in the granule neuron cell bodies and faintly on cell plasma membranes and processes. Granule neurons, expressing SBP-1, migrated outward in close contact with the SGC bearing neuronal processes, suggesting interaction between SGC and SBP-1. The neurite outgrowth and cell migration were specifically and severely reduced, in dose-dependent manners, by anti-SGC (HNK-1) and anti-SBP-1 antibodies and sulfoglucuronyl glycolipid (SGGL). Other irrelevant antibodies and glycolipids had little effect. The results showed that SBP-1 was required for neurite outgrowth and that SGC-SBP-1 interaction was important for cell-cell recognition and cell migration.

Restoration of synthesis of sulfoglucuronylglycolipids in cerebellar granule neurons promotes dedifferentiation and neurite outgrowth.

Sulfoglucuronyl carbohydrate (SGC) linked to the terminal moiety of neolacto-oligosaccharides is expressed in several glycoproteins of the immunoglobulin superfamily involved in neural cell-cell recognition as well as in two sulfoglucuronylglycolipids (SGGLs) of the nervous system. SGGLs and SGC-containing glycoproteins are temporally and spatially regulated during development of the nervous system. In the cerebellum, the expression of SGC, particularly that of SGGLs, is biphasic. Several studies have suggested that the initial rise and decline in the levels of SGGLs and SGC-containing proteins correlated with the migration of granule neurons from the external granule cell layer to the internal granule cell layer and their subsequent maturation, whereas the later rise and continued expression of SGGLs in the adult was associated with their localization in the Purkinje neurons and their dendrites in the molecular layer. Here it is shown by immunocytochemical methods that the expression of SGC declined progressively in granule neurons isolated from cerebella of increasing age. The decline in the expression of SGC in granule neurons was also shown with time in culture. These results correlated with the previously shown declining activity of the regulatory enzyme lactosylceramide N-acetylglucosaminyltransferase (GlcNAc-Tr) with age in vivo and in isolated granule neurons in culture. GlcNAc-Tr synthesizes a key precursor, lactotriosylceramide, involved in the biosynthesis of SGGL-1. The down-regulated synthesis of SGGLs in the mature granule neurons was shown by immunocytochemical and biochemical methods to be restored when a precursor, glucuronylneolactotetraosylceramide (GGL-1), which is beyond the GlcNAc-Tr step, was exogenously provided to these cells. The biological effect of such restoration of the synthesis of SGGLs in the mature granule neurons leads to cell aggregation and enhanced proliferation of neurites, amounting to dedifferentiation.

Expression of neolactoglycolipids: sialosyl-, disialosyl-, O-acetyldisialosyl- and fucosyl- derivatives of neolactotetraosyl ceramide and neolactohexaosyl ceramide in the developing cerebral cortex and cerebellum.

The following neolacto glycolipids were identified and their developmental expression was studied in the rat cerebral cortex and cerebellum: Fuc alpha 1-3IIInLcOse4Cer,Fuc alpha 1-3VnLcOse6Cer and (Fuc)2 alpha 1-3III,3VnLcOse6Cer, as well as acidic glycolipids, NeuAc alpha 2-3IVnLcOse4Cer [nLM1], (NeuAc)2 alpha 2-3IVnLcOse4Cer [nLD1], O-acetyl (NeuAc)2 alpha 2-3IVnLcOse4Cer [OAc-nLD1] and their higher neolactosaminyl homologues NeuAc alpha 2-3VlnLcOse6Cer [nHM1] and (NeuAc)2 alpha 2-3VlnLcOse6Cer [nHD1]. These glycolipids were expressed in the cerebral cortex only during embryonic stages and disappeared postnatally. This loss was ascribed to the down regulation of the synthesis of the key precursor LcOse3Cer which is synthesized by the enzyme lactosylceramide: N-acetylglucosaminyl transferase. On the other hand in the cerebellum, these glycolipids increased with postnatal development due to increasing availability of LcOse3Cer. In the cerebellum, only nLM1 and fucosyl-neolactoglycolipids declined after postnatal day 10-15, perhaps due to regulation by other glycosyltransferases. Also, in the cerebellum, nLD1 and nHD1 were shown to be specifically associated with Purkinje cells and their dendrites in the molecular layer and with their axon terminals in the deep cerebellar nuclei, similar to other neolactoglycolipids shown previously.

Sulfoglucuronyl glycolipids bind laminin.

Previous studies have shown that HNK-1 antibody reactive glycoconjugates, including the glycolipids 3-sulfoglucuronylneolactotetraosylceramide (SGGL-1) and 3-sulfoglucuronylneolactohexaosylceramide (SGGL-2), are temporally and spatially regulated antigens in the developing mammalian cortex. Extracellular matrix glycoprotein laminin is involved in cell adhesion by interacting with cell surface components and also promotes neurite outgrowth. Laminin has been shown to bind sulfatide. The interaction of sulfated glycolipids SGGL-1 and SGGL-2 with laminin was studied by employing a solid-phase radioimmunoassay and by HPTLC-immunoblotting. Laminin binding was detected with anti-laminin antibodies followed by 125I-labelled Protein A and autoradiography. Laminin binds SGGL-1 and SGGL-2, besides sulfatide, but does not bind significantly gangliosides and neutral glycolipids. The binding of SGGLs to laminin was two to three times less compared to sulfatide when compared on a molar basis. Desulfation of SGGLs and sulfatide by mild acid treatment resulted in abolition of laminin binding. On the other hand, chemical modification of glucuronic acid moiety by either esterification or reduction of the carboxyl group had no effect. This showed that the sulfate group was essential for laminin binding. Of the various glycosaminoglycans tested, only heparin inhibited the binding of laminin to SGGLs and sulfatide in a dose-dependent manner. This indicated that SGGLs and sulfatide bind to the heparin binding site present in the laminin molecule. The availability of HNK-1 reactive glycolipids and glycoproteins such as SGGLs and several neural cell adhesion molecules to bind laminin at critical stages of neural development may serve as important physiological signals.

Loss of sulfoglucuronyl and other neolactoglycolipids in Purkinje cell abnormality murine mutants.

A significant reduction in the content of two members of the sulfoglucuronyl-neolacto series of glycolipids (SGGLs), 3-sulfoglucuronyl-lacto-N-neotetraosylceramide (SGGL-1) and 3-sulfoglucuronyl lacto-N-norhexaosylceramide (SGGL-2), in the cerebellum of the Purkinje cell abnormality mutants, Purkinje cell degeneration (pcd/pcd), lurcher (Lc/+), and staggerer (sg/sg), was also confirmed in the mildly affected nervous (nr/nr) mutant. The expression of SGGLs was studied during development of the pcd/pcd mutant cerebellum, and it was shown that the rate of decline in the level of SGGLs practically coincided with the loss of Purkinje cell perikarya. This indicated that SGGLs are primarily localized in Purkinje cells and that initially, at least, there is no genetic defect in the biosynthesis of SGGLs in the mutant. The precursors of SGGLs, viz., lacto-N-neotetraosylceramide (paragloboside) and lacto-N-norhexaosylceramide, as well as other glycolipids derived from these precursors, such as X-determinant fucoglycolipids and disialosyllacto-N-neotetraosylceramide, were also present in normal cerebellum. Levels of paragloboside and its other derivatives, similar to SGGLs, were also significantly reduced in the Purkinje cell abnormality mutants pcd/pcd, sg/sg, Lc/+, and nr/nr but were normal in other cerebellar mutants, such as quaking (qk/qk), weaver (wv/wv), and reeler (rl/rl), where Purkinje cells are not involved. Thus, the entire paragloboside family of glycolipids is primarily associated with Purkinje cells in the cerebellum. Although levels of monoclonal antibody HNK-1-reactive glycolipids were reduced in the Purkinje cell abnormality mutants, HNK-1-reactive glycoproteins were not affected in these mutants.

Identification of disialosyl paragloboside and O-acetyldisialosyl paragloboside in cerebellum and embryonic cerebrum.

The lacto series of glycolipids are only minor constituents in mammalian CNS and are found mostly during development. Expression of a significant amount (70 micrograms of neuraminic acid/g dry weight) of disialosyl-lacto-N-neotetraosylceramide (LD1) in adult mouse cerebellum is reported for the first time in the nervous system. The structure of this ganglioside was determined by hydrolysis with various glycosidases, immunochemical tests, sugar and fatty acid analyses after permethylation and capillary GLC-mass spectrometry, sugar linkage analysis of permethylated alditol acetates, and fast-atom bombardment-mass spectrometry of the native ganglioside. The structure of LD1 was determined to be NeuAc-NeuAc alpha 2-3Gal beta 1-4GlcNAc beta 1-3Gal beta 1-4Glc beta 1-1-ceramide. The major fatty acid was 18:0, and the long-chain base was C18-sphingenine. Mouse cerebellum also contained O-acetyl-LD1 and several other O-acetylated gangliosides as recognized by monoclonal antibodies ME311 and 3G5. The levels of LD1 and O-acetyl-LD1 in cerebellum increased during postnatal development. During development of the Purkinje cell degeneration mutant, pcd/pcd, the levels of both of these gangliosides in the cerebellum declined with the loss of Purkinje cells, a finding indicating that these gangliosides are primarily associated with Purkinje cells. In the cortex, LD1, O-acetyl-LD1, and O-acetyl GD3, like GD3, are developmentally regulated antigens and are only expressed in the fetal cortex and not to any significant extent in the adult.

Identity of nuclear high-mobility-group protein, HMG-1, and sulfoglucuronyl carbohydrate-binding protein, SBP-1, in brain.

High-mobility-group (HMG) proteins are a family of non-histone chromosomal proteins which bind to DNA. They have been implicated in multiple aspects of gene regulation and cellular differentiation. Sulfoglucuronyl carbohydrate binding protein, SBP-1, which is also localized in the neuronal nuclei, was shown to be required for neurite outgrowth and neuronal migration during development of the nervous system. In order to establish relationship between SBP-1 and HMG family proteins, two HMG proteins were isolated and purified from developing rat cerebellum by heparin-sepharose and sulfatide-octyl-sepharose affinity column chromatography and their biochemical and biological properties were compared with those of SBP-1. Characterization by high performance liquid chromatography--mass spectrometry (HPLC-MS), partial peptide sequencing and western blot analysis showed the isolated HMG proteins to be HMG-1 and HMG-2. Isoelectric focusing, HPLC-MS and peptide sequencing data also suggested that HMG-1 and SBP-1 were identical. Similar to SBP-1, both HMG proteins bound specifically to sulfated glycolipids, sulfoglucuronylglycolipids (SGGLs), sulfatide and seminolipid in HPTLC-immuno-overlay and solid-phase binding assays. The HMG proteins promoted neurite outgrowth in dissociated cerebellar cells, which was inhibited by SGGLs, anti-Leu7 hybridoma (HNK-1) and anti-SBP-1 peptide antibodies, similar to SBP-1. The proteins also promoted neurite outgrowth in explant cultures of cerebellum. The results showed that the cerebellar HMG-1 and -2 proteins have similar biochemical and biological properties and HMG-1 is most likely identical to SBP-1.

Restriction of high CD15 expression to a subset of rat cerebellar astroglial cells can be overcome by transduction with adenoviral vectors expressing the rat alpha 1,3-fucosyltransferase IV gene.

Glycoconjugates bearing the epitope 3-fucosyl-N-acetyllactosamine (CD15) are believed to be involved in cell-cell interactions and are temporally and spatially regulated in the brain. In the rat postnatal cerebellum, CD15 is predominantly expressed in the molecular layer by Bergmann glial cells, but little CD15 expression is seen in other astroglia, and the basis for this restricted expression is not known. Adenoviral vectors were shown to efficiently deliver transgenes to cerebellar glial cells and were used to determine whether manipulation of glycosyltransferase activities could enhance the expression of CD15 in these cells. In dissociated cerebellar cell cultures, few glial cells normally express CD15. However, transduction of these cells with an adenoviral vector (AdGFPCMVFucT) that expressed both green fluorescent protein (GFP) and FLAG-tagged rat alpha 1, 3-fucosyltransferase IV (rFuc-TIV) resulted in high CD15 expression on the surface of all transduced glial cells. Likewise, infection of cerebellar slice cultures caused the appearance of CD15-positive transduced cells of glial cell morphology in the internal granule cell layer. Thus, enhancement of Fuc-T activity caused robust CD15 expression in cerebellar glial cells that normally show little expression of CD15, suggesting a role for Fuc-T levels in regulating CD15 expression in this cell type. The manipulation of levels of glycosyltransferases using adenoviral vectors may prove a useful tool to investigate questions of glycoconjugate regulation in glial cells in the developing rodent cerebellum.

Biosynthesis in vitro of GlcA beta 1-3nLcOse4Cer by a novel glucuronyltransferase (GlcAT-1) from embryonic chicken brain.

A novel glucuronyltransferase (GlcAT-1) has been detected in embryonic chicken brains. This enzyme catalyzes the biosynthesis in vitro of glucuronic acid containing glycolipids starting from neolactotetraosylceramide (nLcOse4Cer) and neolactohexaosylceramide (nLcOse6Cer). The activity is present primarily in the Golgi-rich membrane fraction and can be extracted (60%) from the membrane using a neutral detergent, Nonidet P-40, at pH 7.0. The detergent-solubilized GlcAT-1 is stable (70%) at -20 degrees C for at least 4 months. Both membrane-bound GlcAT-1 and solubilized GlcAT-1 show similar pH optima, 6.5-7.0, in HEPES buffer. The Km values were 15 and 200 microM with UDP-[14C] GlcA and nLcOse4Cer, respectively, when the detergent-solubilized supernatant fraction was used as enzyme source. The purified 14C radioactive product that comigrated with chemically characterized GlcA beta 1-3nLcOse4Cer (GlcA-nLc4) also yielded a positive immunostain with monoclonal antibody (human IgM-RI). The anomeric linkage was established as beta-linked GlcA to the terminal galactose of the substrate, as evidenced by 90-99% cleavage of the terminal [14C] GlcA by purified Helix pomatia and limpet glucuronidases. Permethylation studies of the radioactive product obtained from [6-3H]Gal beta 1-4LcOse3Cer and non-radioactive UDP-GlcA showed the presence of 2,4,6-tri-O-methylgalactose in the hydrolyzed enzymatic product. These studies established the structure of the biosynthesized product from nLcOse4Cer as GlcA beta 1-3Gal beta 1-4 GlcNAc beta 1-3Gal beta 1-4Glc-ceramide.