Firoze B. Jungalwala

Structure of a glycolipid reacting with monoclonal IgM in neuropathy and with HNK-1

An acidic glycolipid antigen that reacts with monoclonal IgM in patients with demyelinating neuropathy and with the mouse monoclonal antibody, HNK-1, was purified from human peripheral nerves. This lipid sharing antigenic determinants with the myelin-associated glycoprotein was shown to be an unusual glucuronic acid-containing sulfated glycosphingolipid with five sugars, but without sialic acid. Mild acid methanolysis converted the GlcUA to its methyl ester, removed the acidic sulfate group and abolished the antigenicity. Results from chemical, enzymatic, infrared, and mass spectral analysis suggested the following structure with a sulfate in a position that remains to be determined: GlcUA beta 1----3Gal beta 1----4GlcNAc beta 1----3Gal beta 1----4Glc beta 1----1 ceramide.

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.

Demonstration of glucuronic acid on brain glycoproteins which react with HNK-1 antibody

Ependymins, a family of extracellular glycoproteins of goldfish and mammalian brain, were shown to contain N-linked complex glycan chains. These glycoproteins reacted with a monoclonal antibody, HNK-1 which recognizes a membrane antigen on a subset of human lymphocytes, myelin-associated glycoprotein glycoprotein epitope reacting with HNK-1 antibody was previously shown to include a terminal 3-sulfoglucuronosyl residue present in certain glycolipids of the nervous tissue (Chou et al., Biochem. Biophys. Res. Commun. 1985, 128, 383-388). In this report, the presence of glucuronic acid in ependymins was demonstrated by gas-liquid chromatography and mass spectrometry. We suggest that a 3-sulfoglucuronosyl residue may be the common epitope on HNK-1-reactive glycoproteins.

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.

Monoclonal Antibody to Embryonic CNS Antigen A2B5 Provides Evidence for the Involvement of Membrane Components at Sites of Alzheimer Degeneration and Detects Sulfatides as Well as Gangliosides

Immunohistological and biochemical studies were initiated to determine whether or not neural membrane components were associated with degenerative changes characteristic of Alzheimers disease (AD). Monoclonal antibody A2B5, developed against embryonic chick retinal cells and previously shown to react with neural surface gangliosides, was applied to formalin‐fixed sections of control and AD brain tissue. Frontal cortex and hippocampus of AD cases exhibited high levels of A2B5 immunoreactivity within those neurons undergoing neurofibrillary degeneration. Neuritic processes associated with senile plaques were also highly reactive with the A2B5 antibody. The amount of gangliosides and their pattern after HPTLC were the same in control and AD cases. However, the unexpected observation was made that the A2B5 antibody reacted with human brain sulfatides in addition to the expected reactivity with minor gangliosides. The average level of sulfatides in AD brain was significantly higher than in normal controls. The data support the involvement of one or more membrane components with neu‐rodegeneration in the Alzheimer brain.

Composition and Metabolism of Gangliosides in Rat Peripheral Nervous System During Development

Abstract: Ganglioside composition of rat trigeminal nerve was studied during development in order to understand the changes that occur as a result of cellular differentiation in the nerve. The ganglioside composition of the trigeminal nerve was entirely different from that of brain. The major gangliosides in adult trigeminal nerve were GM3, GD3, and LM1 (sialosyl‐lactoneotetraosylceramide or sialosylparagloboside). The structure of LM1 and other gangliosides was established by enzymatic degradation and by analysis of the products of acid hydrolysis. At 2 days after birth, when the Schwann cells were immature, GM3 and GD3 were the major gangliosides in the nerve, 50 and 18 mol %, respectively. As the nerve developed and Schwann cells proliferated and myelinated the axons, the mol % of GM3 and GD3 reduced and that of LM1 steadily increased. Polysialogangliosides did not change drastically with nerve development. The rate of deposition of LM1 in the nerve with age was very similar to that of myelin marker lipids, cerebrosides, and sulfatides; thus, deposition appears to be localized mainly in the rat nerve myelin. LM1 also had long‐chain fatty acids 22:0 and 24:0, which are not usually found in CNS gangliosides. The ganglioside pattern of the rat trigeminal nerve was very similar to that of rat sciatic nerve, but was different from that of rabbit and chicken sciatic nerve. The activity of the two key enzymes involved in the metabolism of GM3, viz., CMP‐N‐acetylneuraminic acid:lactosylceramide sialyltransferase and UDP‐N‐acetylgalactosamine:GM3‐N‐acetylgalactosaminyltransferase, was also studied during development of the nerve and brain. The developmental profiles of both enzymes were consistent with the amounts of GM3 present in the nerve.

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.

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.