Denise K. H. Chou

Sulfated glucuronic acid-containing glycoconjugates are temporally and spatially regulated antigens in the developing mammalian nervous system☆

Monoclonal antibody 4F4, which was raised against a cell suspension of embryonic rat forebrain, reacts with acidic glycolipids and several high-molecular-weight glycoproteins in rodent brain. The major reactive glycolipid is maximally expressed at Embryonic Day 15 (E15) and is no longer detectable at Postnatal Day 14 (P14) in the rat. 4F4 antibody reacts with a glucuronic acid- and sulfate-containing lipid isolated from human sciatic nerve as well as with lipids from mouse and rat embryonic brain tissue. Although the glycolipid disappears postnatally, the immunoreactive glycoproteins continue to be expressed in brain until adulthood. Both sciatic nerve and embryonic brain glycolipids are hydrolyzed by glucuronidase/sulfatase treatment but are insensitive to all other glycosidases tested. In addition, the observed 4F4 reactivity with extracted glycolipids, glycoproteins, and tissue sections of embryonic brain is identical to the reactivity demonstrated by HNK-1 antibodies. Immunocytochemical studies in developing brain showed stage-specific distribution of this carbohydrate antigen. At E10 in the mouse, immunoreactivity is associated with the mantle layer of the neural tube. At E15 in the cortex, the most intense staining is associated with the molecular layer and the subplate, and weaker staining is seen in the intermediate zone and cortical plate, suggesting that the antigen is highly concentrated on postmigratory cells in the embryonic nervous system.

Variability in the Structural Requirements for Binding of Human Monoclonal Anti‐Myelin‐Associated Glycoprotein Immunoglobulin M Antibodies and HNK‐1 to Sphingoglycolipid Antigens

A high proportion of patients with neuropathy have immunoglobulin M (IgM) paraproteins that react with carbohydrate determinants on the myelin‐associated glycoprotein (MAG) and two sphingoglycolipids, 3‐sulfoglucuronyl paragloboside (SGPG) and 3‐sulfoglucuronyl lactosaminyl paragloboside. In order to characterize the fine specificities of these human antibodies further, the binding of 10 anti‐MAG paraproteins to several chemically modified derivatives of SGPG was compared with the binding to intact SGPG by both TLC‐overlay and enzyme‐linked immunosorbent assay. The following derivatives were tested: the desulfated lipid, glucuronyl paragloboside (GPG); the methyl ester of GPG (MeGPG); the methyl ester of SGPG, 3‐sulfomethylglucuronyl paragloboside (SMeGPG); and 3‐sulfoglucosyl paragloboside (SGlcPG) produced by reduction of the carboxyl group of the glucuronic acid with sodium borohydride. All 10 IgM paraproteins and the related mouse IgM antibody, HNK‐1, reacted most strongly with intact SGPG, but variations in the reactivity with the derivatives revealed striking differences in the structural requirements for binding between the antibodies. Five distinct patterns of reactivity were observed: (a) three of the human antibodies and HNK‐1 exhibited partial reactivity with the sulfated derivatives, SMeGPG and SGlcPG, but not with GPG or MeGPG, indicating an absolute requirement for the sulfate group; (b) two of the human antibodies reacted only with GPG, demonstrating a requirement for the free carboxyl group on the glucuronic acid; (c) three of the antibodies bound to SMeGPG, SGlcPG, and GPG, but not to MeGPG, suggesting that at least one negative charge was needed for binding; (d) one antibody bound to SMeGPG and GPG, but not to SGlcPG or MeGPG, suggesting that both the carbonyl group and at least one negative charge were required; and (e) another antibody bound to MeGPG, SMeGPG, and SGlcPG, but not to GPG, a pattern that is difficult to explain simply based on the chemical structures. Interestingly, only those antibodies that exhibited reactivity with GPG bound to a third, minor, unidentified glycolipid of normal peripheral nerve that chromatographs faster than SGPG. The results clearly demonstrate heterogeneity in the fine specificities of the anti‐MAG antibodies that may affect their pathogenic properties.

Sulfoglucuronyl glycolipids bind laminin.

Abstract: 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 treatent 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.

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.

Sulfoglucuronyl-Neolacto Series of Glycolipids in Peripheral Nerves Reacting with HNK-1 Antibody

Abstract: Novel sulfoglucuronyl‐neolacto series of glycolipids were detected in peripheral nerves of various species by TLC followed by immunostaining with HNK‐1 antibody. The major antigenic glycolipid, sulfoglucuronylneolactote‐traosylceramide, previously described in human nerves, was shown to be also present in the sciatic nerves of various species including rodents. A second slower migrating antigenic glycolipid present in the sciatic nerves of human and dog was isolated and purified. It was characterized by chemical and enzymatic degradation, sugar analysis after permethylation, and gas liquid chromatography‐mass spectrometry techniques as well as by fast atom bombardment‐mass spectrometry, as 3‐sulfogIucuronylneolactohexaosylceramide. During postnatal development of the rat sciatic and trigeminal nerves the concentration of these antigenic glycolipids increased with age.

Identification of Disialosyl Paragloboside and O-Acetyldisialosyl Paragloboside in Cerebellum and Embryonic Cerebrum

Abstract: The lacto series of glycolipids are only minor constituents in mammalian CNS and are found mostly during development. Expression of a significant amount (70 μg 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‐NeuAcα2‐3Galβ1‐4GlcNAcβ1‐3Galβ1‐4Glcβ1‐1‐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.

Developmental Expression of HNK‐1‐Reactive Antigens in the Rat Cerebellum and Localization of Sulfoglucuronyl Glycolipids in Molecular Layer and Deep Cerebellar Nuclei

Abstract: Monoclonal antibody HNK‐1‐reactive carbohydrate epitope is expressed on proteins, proteoglycans, and sulfoglucuronyl glycolipids (SGGLs). The developmental expression of these HNK‐1‐reactive antigens was studied in rat cerebellum. The expression of sulfoglucuronyl lacto‐N‐neotetraosylceramide (SGGL‐1) was biphasic with an initial maximum at postnatal day one (PD 1), followed by a second rise in the level at PD 20. The level of sulfoglucuronyl lacto‐N‐norhexaosyl ceramide (SGGL‐2) in cerebellum was low until PD 15 and then increased to a plateau at PD 20. The levels of SGGLs increased during postnatal development of the cerebellum, contrary to their diminishing expression in the cerebral cortex. The expression of HNK‐1‐reactive glycoproteins decreased with development of the rat cerebellum from PD 1. Several HNK‐1‐reactive glycoproteins with apparent molecular masses between 150 and 325 kDa were visualized between PD 1 and PD 10. However, beyond PD 10, only two HNK‐1‐reactive bands at 160 and 180 kDa remained. The latter appeared to be neural cell adhesion molecule, N‐CAM‐180. A diffuse HNK‐1‐reactive band seen at the top of polyacrylamide electrophoretic gels was due mostly to proteoglycans. This band increased in its reactivity to HNK‐1 between PD 15 and PD 25 and then decreased in the adult cerebellum. The lipid antigens were shown by two complementary methodologies to be localized primarily in the molecular layer and deep cerebellar nuclei as opposed to the granular layer and white matter. A fixation procedure which eliminates HNK‐1‐reactive epitope on glycoproteins and proteoglycans, but does not affect glycolipids, allowed selective immunoreactivity in the molecular layer and deep cerebellar nuclei. In order to confirm this localization, SGGLs were analyzed by HPTLC‐immunoverlay method in micro‐dissected cerebellar layers from freeze‐dried cryocut sections: they were found primarily in the molecular layer and deep cerebellar nuclei and were undetectable in the granule cell layer and white matter. These results, along with the lack of SGGLs and disialosyl lacto‐N‐neotetraosylceramide (LD1) in several Purkinje cell‐deficient murine mutants reported previously, indicate that these glycolipids are associated specifically with Purkinje cell dendrites in the molecular layer and Purkinje cell axon synapses in deep cerebellar nuclei.

Sulfoglucuronyl Neolactoglycolipids in Adult Cerebellum: Specific Absence in Murine Mutants with Purkinje Cell Abnormality

Abstract: 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.

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.

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.