Cloning of a mouse beta 1,3 N-acetylglucosaminyltransferase GlcNAc(beta 1,3)Gal(beta 1,4)Glc-ceramide synthase gene encoding the key regulator of lacto-series glycolipid biosynthesis.

The distinction between the different classes of glycolipids is conditioned by the action of specific core transferases. The entry point for lacto-series glycolipids is catalyzed by the beta1,3 N-acetylglucosaminyltransferase GlcNAc(beta1,3)Gal(beta1,4)Glc-ceramide (Lc3) synthase enzyme. The Lc3 synthase activity has been shown to be regulated during development, especially during brain morphogenesis. Here, we report the molecular cloning of a mouse gene encoding an Lc3 synthase enzyme. The mouse cDNA included an open reading frame of 1131 base pairs encoding a protein of 376 amino acids. The Lc3 synthase protein shared several structural motifs previously identified in the members of the beta1,3 glycosyltransferase superfamily. The Lc3 synthase enzyme efficiently utilized the lactosyl ceramide glycolipid acceptor. The identity of the reaction products of Lc3 synthase-transfected CHOP2/1 cells was confirmed by thin-layer chromatography immunostaining using antibodies TE-8 and 1B2 that recognize Lc3 and Gal(beta1,4)GlcNAc(beta1,3)Gal(beta1,4)Glc-ceramide (nLc4) structures, respectively. In addition to the initiating activity for lacto-chain synthesis, the Lc3 synthase could extend the terminal N-acetyllactosamine unit of nLc4 and also had a broad specificity for gangliosides GA1, GM1, and GD1b to generate neolacto-ganglio hybrid structures. The mouse Lc3 synthase gene was mainly expressed during embryonic development. In situ hybridization analysis revealed that that the Lc3 synthase was expressed in most tissues at embryonic day 11 with elevated expression in the developing central nervous system. Postnatally, the expression was restricted to splenic B-cells, the placenta, and cerebellar Purkinje cells where it colocalized with HNK-1 reactivity. These data support a key role for the Lc3 synthase in regulating neolacto-series glycolipid synthesis during embryonic development.

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).

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

Developmental expression of sulfoglucuronyl carbohydrate (SGC) and its binding protein, SBP-1 was studied in the rat cerebral cortex to understand their function. Between embryonic day (ED) 14-19, SBP-1 was strongly expressed in neurons of the ventricular zone and migrating neurons throughout the cortex. SBP-1 declined at birth and by postnatal day (PD) 3 only the latest arriving neurons in the most superficial segment of the cortical plate expressed SBP-1. Between ED 14-16, SGC was expressed in a thin row of glial cells near the ventricles and on their radial processes. Between ED 16-PD 3, SGC was not in neuronal cell soma, but was in neuronal plasma membranes and processes surrounding the neuronal perikarya. The expression of SGC declined similar to SBP-1 and both of them disappeared by PD 7. The expression of SBP-1 and SGC was chronologically coordinated with neuronal migration. SBP-1 was specifically expressed in immature neuronal nuclei and plasma membranes. SBP-1 and SGC were colocalized and were available for interaction with each other on neuronal cell membranes and processes. This was confirmed with isolated neurons in culture. As in vivo, the expression of SBP-1 in neurons declined with time in culture. The dissociated cortical neurons when plated on SBP-1 as a substratum produced extensive neuritic outgrowth. HNK-1, anti-SBP-1 antibodies and sulfoglucuronyl glycolipid, SGGL specifically and severely reduced neurite outgrowth. SBP-1-SGC interactions provide a potential mechanism for guidance and cell signaling, in the processes of neuronal migration and terminal differentiation.

The beta 1,3-galactosyltransferase beta 3GalT-V is a stage-specific embryonic antigen-3 (SSEA-3) synthase.

We have previously reported the molecular cloning of beta1, 3-galactosyltransferase-V (beta3GalT-V), which catalyzes the transfer of Gal to GlcNAc-based acceptors with a preference for the core3 O-linked glycan GlcNAc(beta1,3)GalNAc structure. Further characterization indicated that the recombinant beta3GalT-V enzyme expressed in Sf9 insect cells also utilized the glycolipid Lc3Cer as an efficient acceptor. Surprisingly, we also found that beta3GalT-V catalyzes the transfer of Gal to the terminal GalNAc unit of the globoside Gb4, thereby synthesizing the glycolipid Gb5, also known as the stage-specific embryonic antigen-3 (SSEA-3). The SSEA-3 synthase activity of beta3GalT-V was confirmed in vivo by stable expression of the human beta3GalT-V gene in F9 mouse teratocarcinoma cells, as detected with the monoclonal antibody MC-631 by flow cytometry analysis and immunostaining of extracted glycolipids. The biological relation between SSEA-3 formation and beta3GalT-V was further documented by showing that F9 cells treated with the differentiation-inducing agent retinoic acid induced the expression of both the SSEA-3 epitope and the endogenous mouse beta3GalT-V gene. This study represents the first example of a glycosyltransferase, which utilizes two kinds of sugar acceptor substrates without requiring any additional modifier molecule.

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.

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.

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.

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.

Pathogenesis of corneal infection: binding of Pseudomonas aeruginosa to specific phospholipids.

Clinical isolates of Pseudomonas aeruginosa were examined for binding interactions with phospholipids of corneal epithelium. Thin-layer chromatography (TLC) of lipids extracted from corneal epithelia followed by staining with an ammonium molybdate spray reagent revealed three phospholipid components, PL1, PL2, and PL3. The chromatographic mobility of PL1 was similar to that of the phospholipid standards phosphatidylinositol (PI) and phosphatidylserine (PS), which were not well resolved from one other; PL2 and PL3 comigrated with the standards phosphatidylcholine and phosphatidylethanolamine, respectively. By use of a TLC-bacterial overlay procedure, 35S-labeled P. aeruginosa organisms were shown to bind to PL1 but not to PL2 or PL3. P. aeruginosa binding to PL1 was concentration dependent. Alkaline methanolysis abolished the binding. PL1 was separated into two components, PL1-I and PL1-S, by chromatography on borate-treated TLC plates. Both PL1-I and PL1-S contained binding sites for P. aeruginosa. Mass spectral analysis identified PL1-I and PL1-S as PI and PS, respectively. Radiolabeled P. aeruginosa organisms were subsequently shown to bind to commercially available bovine PI and PS and synthetic dipalmitoyl-PS but not to other phospholipid standards, including bovine SM and PC or synthetic dioleoyl- and distearoyl-PC. A control Escherichia coli strain did not bind to either PS or PI. Tetramethylurea, a disrupter of hydrophobic associations, did not influence the binding of P. aeruginosa to PS or PI. P. aeruginosa bound to the monolayers of corneal epithelial cells. P. aeruginosa binding to the monolayer cultures as well as to rabbit corneas pretreated with exogenous PS and PI was significantly higher than that to those preincubated with PC or medium alone. The data suggest that phospholipids PS and PI present in mucus or on the cell surface may function as P. aeruginosa receptors and contribute to selective bacterium-host interactions responsible for initial colonization.

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.

Neolactoglycosphingolipids, potential mediators of corneal epithelial cell migration.

Cell migration is a fundamental process of wound repair in biological systems. In an attempt to identify plasma membrane glycoconjugates which mediate cell migration, migrating and nonmigrating rabbit corneal epithelia were analyzed for reactivity with monoclonal antibodies (mAbs) specific for unsubstituted N-acetyl-lactosamine (mAb 1B2), Le(x) (mAbs 7A and MMA), and sialyl Le(x) (mAb CSLEX1) carbohydrate chains of neolactoglycoconjugates. Immunohistochemical analysis indicated that regardless of whether the epithelia analyzed were from corneas of animals in vivo, corneas in organ culture, or cells in tissue culture, migrating cells stained intensely with mAb 1B2, whereas nonmigrating cells either did not stain or stained only weakly. mAbs MMA and 7A stained migrating epithelium as well as basal and middle cell layers of normal, nonmigrating epithelium. mAb CSLEX1 did not stain wounded corneas but stained the superficial cell layer of normal corneal epithelium. Biochemical analyses by TLC immunostaining revealed the presence of three mAb 1B2-reactive glycosphingolipids (GSL), neolactotetraosyl-(nLc4, paragloboside), neolactohexaosyl- (nLc6), and neolacto-octaosylceramide (nLc8) in migrating epithelia. In contrast, nonmigrating epithelia contained only trace amounts of these glycolipids. Exogenous addition of nLc4, but not various other GSLs including a Le(x)-GSL (SSEA-1), stimulated re-epithelialization of wounds in an experimental model of corneal epithelial wound healing. Moreover, re-epithelialization of wounds was significantly inhibited by mAb 1B2 but not by mAb MMA. The data suggest that neolacto-GSLs of corneal epithelium may be among the molecules which mediate healing of corneal epithelial wounds by influencing cell migration.

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.

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)

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.

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.