Sulfoglucuronyl Glycolipids Research Articles

HNK-1 sulfotransferase null mice express glucuronyl glycoconjugates and show normal cerebellar granule neuron migration in vivo and in vitro.

Sulfoglucuronyl carbohydrate (SGC), reactive with antibody against human natural killer cell antigen, is expressed in several glycolipids, glycoproteins and proteoglycans of the nervous system and has been implicated in cell-cell recognition, neurite outgrowth and neuronal migration during development, through its interaction with SGC-binding protein (SBP) 1. However, sulfotransferase (ST) null mutant mice, which lack SGC, were shown to have normal development with usual gross anatomy of the nervous system and other organs. Failure to observe a severe phenotype in the ST null mice prompted us to determine the compensatory molecular replacement of SGC by analyzing the carbohydrate of glycolipids and glycoproteins of the ST mutant nervous system. In the ST null mice, SGC-containing molecules were absent; instead the precursor glucuronyl carbohydrate (GC)-containing molecules accumulated. Other relevant glycolipids and proteins were not affected. The GC molecules in the mutant were localized at the same anatomical sites in the nervous system as the SGC molecules in the wild type. In vitro binding studies showed that, similar to sulfoglucuronyl glycolipids, glucuronyl glycolipids interacted with SBP-1, but with a lower binding capacity. In vitro studies with explant cultures of cerebellum indicated that neurite outgrowth and cell migration were not significantly affected in the mutant, possibly owing to interaction of SBP-1 with GC molecules. The results suggested that in vivo SBP-1-GC interaction was sufficient to allow normal neurite outgrowth and cell migration in the mutant, giving rise to a wild-type phenotype. However, the role of other compensatory molecules involved in these processes cannot be completely ruled out.

Poster Sessions CP06: Cell Surface, Cell Membrane. Glucuronyl glycoconjugates replace sulfoglucuronyl glycoconjugates in HNK‐1 knockout mice

Sulfoglucuronyl carbohydrate (SGC), reactive with HNK‐1 antibody, is expressed in several glycolipids, glycoproteins and proteoglycans of the nervous system. The interaction of SGC with SGC‐binding protein, SBP‐1 has been implicated in cell‐cell recognition, neurite outgrowth and neuronal migration during development. In sulfotransferase (ST) null mutant mice, which lack SGC, synaptic transmission in pyramidal cells of the hippocampus was increased and long‐term potentiation was reduced. However, ST null mice are viable, fertile and have wild type anatomy of all major brain areas and many non‐neural organs. Failure to observe severe phenotype in the ST null mice prompted us to determine the compensatory molecular replacement of SGC by analyzing the carbohydrate of glycolipids and glycoprotefins of the mutant nervous system. In the ST null mice, SGC containing molecules were absent and they were replaced by the precursor glucuronyl carbohydrate (GC) containing molecules. Other relevant glycolipids and proteins were not affected. The GC molecules in the mutant were localized at the same anatomical sites as the SGC molecules in the wild type. In vitro binding studies showed that similar to sulfoglucuronyl glycolipids, glucuronyl glycolipids interacted with SBP‐1, but with a lower binding capacity. In vitro studies with explant cultures of cerebellum indicated that neurite outgrowth and cell migration were not significantly affected, possibly due to interaction of SBP‐1 with the GC molecules. The results indicated that in vivo SBP‐1–GC interaction was sufficient enough for normal neurite outgrowth and cell migration in the mutant and thus having a minimal abnormal phenotype.

Minimal requirements for the binding of selectin ligands to a C-type carbohydrate-recognition domain.

The C-type carbohydrate-recognition domains of E-selectin and rat serum mannose-binding protein have similar structures. Selectin/mannose-binding protein chimeras created by transfer of key sequences from E-selectin into mannose-binding protein have previously been shown to bind the selectin ligand sialyl-Lewis(X) through a Ca(2+)-dependent subsite, common to many C-type lectins, and an accessory site containing positively charged amino acid residues. Further characterization of these chimeras as well as analysis of novel constructs containing additional regions of E-selectin demonstrate that selectin-like interaction with sialyl-Lewis(X) can be faithfully reproduced even though structural evidence indicates that the mechanisms of binding to E-selectin and the chimeras are different. Selectin-like binding to the nonfucosylated sulfatide and sulfoglucuronyl glycolipids can also be reproduced with selectin/mannose-binding protein chimeras that contain the two subsites involved in sialyl-Lewis(X) binding. These results indicate that binding of structurally distinct anionic glycans to C-type carbohydrate-recognition domains can be mediated by the Ca(2+)-dependent subsite in combination with a positively charged region that forms an ionic strength-sensitive subsite.

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.

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).

Carbohydrate-protein interactions between HNK-1-reactive sulfoglucuronyl glycolipids and the proteoglycan lectin domain mediate neuronal cell adhesion and neurite outgrowth.

Lecticans, a family of chondroitin sulfate proteoglycans, represent the largest group of proteoglycans expressed in the nervous system. We previously showed that the C-type lectin domains of lecticans bind two classes of sulfated cell surface glycolipids, sulfatides and HNK-1-reactive sulfoglucuronylglycolipids (SGGLs). In this paper, we demonstrate that the interaction between the lectin domain of brevican, a nervous system-specific lectican, and cell surface SGGLs acts as a novel cell recognition system that promotes neuronal adhesion and neurite outgrowth. The Ig chimera of the brevican lectin domain bind to the surface of SGGL-expressing rat hippocampal neurons. The substrate of the brevican chimera promotes adhesion and neurite outgrowth of hippocampal neurons. The authentic, full-length brevican also promotes neuronal cell adhesion and neurite outgrowth. These activities of brevican substrates are neutralized by preincubation of cells with HNK-1 monoclonal antibodies and by pretreatment of the brevican substrates with purified SGGLs. Brevican and HNK-1 carbohydrates are coexpressed in specific layers of the developing hippocampus where axons from entorhinal neurons elongate. Our observations suggest that cell surface SGGLs and extracellular lecticans comprise a novel cell-substrate recognition system operating in the developing nervous system.

The Proteoglycan Lectin Domain Binds Sulfated Cell Surface Glycolipids and Promotes Cell Adhesion

The lecticans are a group of chondroitin sulfate proteoglycans characterized by the presence of C-type lectin domains. Despite the suggestion that their lectin domains interact with carbohydrate ligands, the identity of such ligands has not been elucidated. We previously showed that brevican, a nervous system-specific lectican, binds the surface of B28 glial cells (Yamada, H., Fredette, B., Shitara, K., Hagihara, K., Miura, R., Ranscht, B., Stallcup, W. B., and Yamaguchi, Y. (1997) J. Neurosci. 17, 7784-7795). In this paper, we demonstrate that two classes of sulfated glycolipids, sulfatides and HNK-1-reactive sulfoglucuronylglycolipids (SGGLs), act as cell surface receptors for brevican. The lectin domain of brevican binds sulfatides and SGGLs in a calcium-dependent manner as expected of a C-type lectin domain. Intact, full-length brevican also binds both sulfatides and SGGLs. The lectin domain immobilized as a substrate supports adhesion of cells expressing SGGLs or sulfatides, which was inhibited by monoclonal antibodies against these glycolipids or by treatment of the substrate with SGGLs or sulfatides. Our findings demonstrate that the interaction between the lectin domains of lecticans and sulfated glycolipids comprises a novel cell substrate recognition system, and suggest that lecticans in extracellular matrices serve as substrate for adhesion and migration of cells expressing these glycolipids in vivo.

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.

Characterization of a sulfoglucuronyl carbohydrate binding protein in the developing nervous system.

The developmentally regulated and stage-specifically expressed HNK-1 carbohydrate found on sulfoglucuronylglycolipids (SGGLs) and certain glycoproteins has been proposed to be involved in neural cell adhesion and recognition processes through its interaction with protein "receptors." We have isolated and purified a approximately 30-kDa SGGL-binding protein (SBP-1) from neonatal rat brain. SBP-1 specifically bound to SGGLs and sulfatide both in solid-phase immunobinding and high-performance thin-layer chromatography-immunooverlay assays. N-terminal sequence analysis showed that SBP-1 is similar to an adhesive neurite outgrowth promoting protein amphoterin. Desulfation of SGGLs resulted in abolition of SBP-1 binding. However, chemical modification of glucuronic acid moiety by either esterification or reduction of the carboxyl group had no effect, suggesting requirement of the carbohydrate-linked sulfate group for SBP-1 binding. The binding of SBP-1 to SGGLs was specifically inhibited by HNK-1 antibody but not by other IgM antibodies. The binding of SBP-1 to sulfatide, however, was not inhibited by HNK-1 antibody. Heparin, fucoidan, and dextran sulfate (50K) also inhibited the binding of SBP-1 to SGGLs. During development of the rat cerebral cortex, the level of SBP-1 decreased after embryonic day 18 to an almost undetectable level by postnatal day 10, whereas in the cerebellum, the expression of SBP-1 was maximal at postnatal day 7. SBP-1 also bound specifically to the HNK-1 glycoproteins isolated from rat brain by HNK-1 immunoaffinity chromatography. Proteins without HNK-1 carbohydrate did not bind SBP-1. The binding to HNK-1 glycoproteins was inhibited by HNK-1 antibody, but not by other IgM antibodies, indicating that the binding was mediated through the HNK-1 carbohydrate moiety of the proteins. The interaction and coexpression of SBP-1 with SGGLs and HNK-1 glycoproteins, during the perinatal brain development, suggest a functional role for this protein.

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.

Expression and biological functions of sulfoglucuronyl glycolipids (SGGLs) in the nervous system--a review.

Sulfoglucuronyl carbohydrate linked to neolactotetraose reacts with HNK-1 antibody. The HNK-1 carbohydrate epitope is found in two major glycolipids, several glycoproteins and in some proteoglycans of the nervous system. Most of the HNK-1 reactive glycoproteins so far identified are neural cell adhesion molecules and/or are involved in cell-cell interactions. HNK-1 carbohydrate is highly immunogenic. Several HNK-1-like antibodies, including IgM of some patients with plasma cell abnormalities and having peripheral neuropathy, have been described. This article summarizes published work mainly on sulfoglucuronyl glycolipids, SGGLs and covers: structural requirements of the carbohydrate epitope for binding to HNK-1 and human antibodies, expression of the lipids in various neural areas, stage and region specific developmental expression in CNS and PNS, immunocytochemical localization, loss of expression in Purkinje cell abnormality murine mutations, biosynthetic regulation of expression by a single enzyme N-acetylglucosaminyl transferase, identification of receptor-like carbohydrate binding neural proteins (lectins), and perceived role of the carbohydrate in physiological functions. The latter includes role in: pathogenesis of certain peripheral neuropathies, in migration of neural crest cells, as a ligand in cell-cell adhesion/interaction and as a promoter of neurite outgrowth for motor neurons. Multiple expression of HNK-1 carbohydrate in several molecules and in various neural cell types at specific stages of nervous system development has puzzled investigators as to its specific biological function, but this may also suggest its importance in multiple systems during cell differentiation and migration processes.

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.

Rostrocaudal expression of antibody HNK-1-reactive glycolipids in mouse cerebellum: relationship to developmental compartments and leaner mutation.

Sulfoglucuronylglycolipids (SGGLs) and glycoproteins, reacting with monoclonal antibody HNK-1, are developmentally and spatially regulated in the mammalian cortex and cerebellum. It has been proposed that the HNK-1 carbohydrate epitope is involved in intercellular adhesion and cell-cell interactions. Biochemical analysis and immunocytochemical localization of SGGLs and other neolacto series glycolipids were studied in the leaner mutant mouse cerebellum, where a slow and progressive rostral to caudal degeneration occurs with a gradual loss of both granule cells and Purkinje cells. Biochemical analyses showed that SGGLs and other neolacto series of glycolipids were significantly decreased in the adult leaner cerebellum; however, HNK-1-reactive glycoproteins were not affected. By an immunocytochemical method which selectively localizes the lipid antigens, it is shown that SGGLs are primarily associated with Purkinje cell bodies and their dendrites in the molecular layer and in cerebellar nuclei where Purkinje cell axons terminate. At postnatal day 30 (P30), SGGL immunoreactivity (SGGL-ir) in the leaner cerebellum was reduced moderately compared to normal littermates, which correlated with the minimal degree of Purkinje cell degeneration at this age in leaner and with the biochemical data. At P67 and P90, the SGGL-ir was significantly more reduced in the leaner as Purkinje cell degeneration proceeded. There was a direct correlation between loss of Purkinje cells and SGGL-ir in the cerebellar molecular layer. In both normal and young leaner cerebella, the SGGL-ir in different lobules was not uniform; there were distinct rostrocaudal and mediolateral differences. SGGL-ir was markedly more intense in rostral than in caudal lobules in the vermis, the dividing line being the region immediately caudal to the primary fissure and rostral to the declival sulcus. In the lateral cerebellum, the SGGL-ir was less intense than in the vermis and the rostrocaudal difference was not as pronounced. There was also nonuniformity in the intensity of staining in different folia. The rostrocaudal as well as mediolateral differences in the intensity of SGGL-ir were confirmed independently by biochemical analysis. The differential phenotypic expression of SGGLs and the selective susceptibility to Purkinje cell death in leaner mutant are discussed in relation to the known embryologic and ontogenetic compartmentation of cerebellum.

The HNK-1 reactive sulfoglucuronyl glycolipids are ligands for L-selectin and P-selectin but not E-selectin.

E-selectin, L-selectin, and P-selectin are related cell adhesion molecules that bind via their lectin domains to sialyl Lewis x and related carbohydrate determinants. Reports have indicated that sulfated glycolipids and polysaccharides also bind selectins. To extend these findings, we compared binding of selectin-IgG chimeras to immobilized sulfated and sialylated glycosphingolipids. E-, L-, and P-selectin chimeras all bound to surfaces absorbed with 2,3-sialyl Lewis x glycolipid or sulfatide (galactosylceramide I3-sulfate) but not to surfaces adsorbed with control sulfated lipids (octadecyl sulfate, sphingosine sulfate). Notably, the L- and P-selectin chimeras but not E-selectin chimera bound to surfaces adsorbed with sulfoglucuronyl glycosphingolipids (SGNL lipids; e.g., IV3 glucuronylneolactotetraosylceramide V3-sulfate). These unusual lipids have been reported as antigenic determinants for monoclonal IgM antibodies produced in patients with neuropathy associated with paraproteinemia and react with the mouse monoclonal antibody HNK-1. Binding of L- and P-selectin chimeras to SGNL lipids was specifically inhibited by appropriate anti-selectin antibodies. While binding of all three selectin chimeras to sialyl Lewis x was blocked by removal of calcium, binding to SGNL lipid was only modestly reduced by EDTA. Chemically desulfated SGNL lipid retained binding activity for L- and P-selectin chimeras, while methyl esterification of the glucuronic acid eliminated binding. We conclude that SGNL lipids, unlike sialyl Lewis x and sulfatides, selectively support L- and P-selectin but not E-selectin chimera binding. The presence of SGNL lipids on brain microvascular endothelium (and other endothelia) may implicate these molecules in leukocyte trafficking to the nervous system and elsewhere.

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.

Characterization of a glucuronyltransferase: neolactotetraosylceramide glucuronyltransferase from rat brain.

The properties of a rat brain glucuronyltransferase, which is presumed to be associated with the biosynthesis of the HNK-1 epitope on sulfoglucuronyl glycolipids, are described. The enzyme required divalent cations for reaction, with maximal activity at 10 mM Mn2+, and exhibited a dual optimum at pH 4-5 and pH 6 depending upon the buffer used, with the highest activity at pH 4.5 in MES buffer. This enzyme strictly recognized the Gal beta 1-4GlcNAc terminal structure, and was highly specific for neolacto (type 2) glycolipids as acceptor. The enzyme was localized specifically in the brain, and was barely detected in other issues, including the thymus, spleen, liver, kidney, lung, and sciatic nerve fibres. Phosphatidylinositol and phosphatidylserine increased the enzymatic reaction 4.4- and 2.3-fold, respectively, whereas phosphatidylcholine slightly decreased the rate.

Sulfoglycolipids bind to adhesive protein amphoterin (P30) in the nervous system

HNK-1 antibody reactive carbohydrate epitope carried by glycolipids and glycoproteins has been shown to be involved in cell to cell interactions. It has been proposed that the HNK-1 reactive 3-sulfoglucuronyl carbohydrate epitope in glycolipids may interact with a cell surface receptor to promote the biological response in the developing nervous system. The possible occurrence of such a receptor was examined in rat nervous system. A specific binding of sulfoglycolipids to a 30 kD protein from adult rat cerebellum is described. Little binding was found with neutral glycolipids and gangliosides. The 30 kD protein from cerebellum was partially purified on a sulfatide-octyl-Sepharose affinity column. Binding of sulfoglucuronyl glycolipids to a similar 30 kD protein from forebrain previously identified as amphoterin is also shown. Amphoterin is developmentally regulated and is involved in neural cell adhesion and neurite extension.

Developmental Expression of HNK‐1‐Reactive Antigens in Rat Cerebral Cortex and Molecular Heterogeneity of Sulfoglucuronylneolactotetraosylceramide in CNS Versus PNS

Monoclonal antibody HNK-1 reacts with a carbohydrate epitope present in proteins, proteoglycans, and sulfoglucuronylglycolipids (SGGLs). On high-performance TLC plates, SGGLs of the CNS from several species migrated consistently slower than those from the PNS, a result indicating possible differences in the structures. The structural characteristics of the major SGGL, sulfoglucuronylneolactotetraosylceramide (SGGL-1), from CNS was compared with those of SGGL-1 from PNS. Although the composition, sequence, and linkages of the carbohydrate moiety of the SGGL-1 species were identical, SGGL-1 from CNS contained mainly short-chain fatty acids, 16:0, 18:0, and 18:1, amounting to 85% of the total fatty acids, whereas SGGL-1 from PNS contained large proportions (59%) of long-chain fatty acids (greater than 18:0). These differences in the fatty acid composition accounted for the different migration pattern observed. The developmental expression of SGGLs and HNK-1-reactive proteins was studied in rat cerebral cortex between embryonic day (ED) 15 to adulthood. SGGLs in the rat cortex were maximally expressed around ED 19 and almost completely disappeared by postnatal day (PD) 20. This expression was contrary to their increasing expression in the cerebellum and sciatic nerve with postnatal development. Six to eight protein bands with a molecular mass of greater than 160 kDa were HNK-1 reactive in the rat cerebral cortex at different ages. The major HNK-1 reactivity to the 160-kDa protein band seen in ED 19 to PD 10 cortex decreased and completely disappeared from the adult cortex, whereas several other proteins remained HNK-1 reactive even in the adult. Western blot analyses of the neural cell adhesion molecules (N-CAMs) during development of the rat cortex with a polyclonal anti-N-CAM antibody showed that the major HNK-1-reactive protein bands were not N-CAMs. Between PD 1 and 10, 190-200-kDa N-CAM was the major N-CAM, and between PD 15 to adulthood, 180-kDa N-CAM was the only N-CAM present in the rat cortex.

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.

Induction of demyelination by intraneural injection of antibodies against sulfoglucuronyl paragloboside

Sulfoglucuronyl glycolipids (SGGLs) carry the glucuronyl 3-sulfate (HNK-1) epitope which is recognized by monoclonal IgM paraproteins from patients with demyelinating polyneuropathy. We report that intraneural injections of rat anti-SGGL antibodies induce demyelination in rat sciatic nerve, along with mild to moderate clinical symptoms. Morphologically, vesiculation and loosening of the myelin sheath were observed 3 h postinjection, followed by extensive demyelination and macrophage infiltration after 4 days. Since the antiSGGL antibodies showed no cross-reactivity with other components in rat sciatic nerve, these results indicate that SGGLs alone can serve as the target antigens in demyelinating neuropathy.