Richard H. Quarles

EVIDENCE FOR THE CLOSE ASSOCIATION OF A GLYCOPROTEIN WITH MYELIN IN RAT BRAIN

Abstract— Myelin was purified from rats which had been injected intracerebrally with radioactive fucose in order to label specifically the glycoproteins. Myelin contained a small amount of fucose‐labelled glycoproteins in comparison to that in other subcellular fractions, but polyacrylamide gel electrophoresis in the presence of sodium dodecyl sulphate revealed a unique pattern of radioactive glycoproteins dominated by a major peak. The same glycoprotein was not prominent in the other subcellular fractions which were examined. This major glycoprotein in the myelin fraction was also labelled after injection with [3H]glucosamine or N‐[3H]acetylmannosamine. It was the most intensely staining myelin protein when gels were treated with periodic acid‐Schiff reagents, an indication that, in terms of protein‐bound carbohydrate, it is the major glycoprotein in the myelin fraction. The glycoprotein was present in myelin purified from rats ranging in age from 14 days to 14 months. Extensive recycling of the myelin through the purification procedures did not significantly reduce the amount of glycoprotein in the myelin. Double label experiments with [3H]fucose and [14C]fucose were used to compare glycoproteins in myelin purified from white and grey matter, respectively, and from mixed homogenates of myelinated and unmyelinated brain. The results obtained from these experiments suggested that the glycoprotein is closely associated with myelin and that it is not in an unrelated contaminating structure. Possible locations of the glycoprotein are discussed. They include the myelin membrane itself, the oligodendroglial plasma membrane, and the axolemma of myelinated axons.

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.

Labeling of the oligosaccharide moieties of immunoglobulins

The ability to elicit an immune response to an antigen, and more particularly the advent of monoclonal antibody technology, has led to a proliferation of immunological techniques and diagnostic tests. Immunoglobuhns, or antibodies, are a diverse class of glycoproteins that bind specifically with antigen and initiate a variety of secondary responses such as complement fixation and release of histamine from mast cells. Antibodies have been divided into classes and sub-classes, some of the properties of which are listed in Table I for human immunoglobulins. The composition of immunoglobulins has been well documented (e.g., Clamp and Johnson, 1972) and will not be discussed here. However, it is important to note that they consist of 82-96% polypeptide and 4-18% carbohydrate consisting of both N-linked and O-linked oligosaccharides (Baenziger and Kornfeld, 1974a,b; Mellis and Baenziger, 1983a,b; Winkelhake et al., 1984). Many of the uses of immunoglobulins in basic research, diagnostics or immunotherapy, rely on the ability to label the antibody with a reporter molecule. Reporter molecules include enzymes, radiolabels, fluorochromes, biotin, toxins and drugs. Most of the techniques described for the covalent

Myelin-associated glycoprotein (MAG): past, present and beyond

The myelin‐associated glycoprotein (MAG) is a type I transmembrane glycoprotein localized in periaxonal Schwann cell and oligodendroglial membranes of myelin sheaths where it functions in glia–axon interactions. It contains five immunoglobulin (Ig)‐like domains and is in the sialic acid‐binding subgroup of the Ig superfamily. It appears to function both as a ligand for an axonal receptor that is needed for the maintenance of myelinated axons and as a receptor for an axonal signal that promotes the differentiation, maintenance and survival of oligodendrocytes. Its function in the maintenance of myelinated axons may be related to its role as one of the white matter inhibitors of neurite outgrowth acting through a receptor complex involving the Nogo receptor and/or gangliosides containing 2,3‐linked sialic acid. MAG is expressed as two developmentally regulated isoforms with different cytoplasmic domains that may activate different signal transduction pathways in myelin‐forming cells. MAG contains a carbohydrate epitope shared with other glycoconjugates that is a target antigen in autoimmune peripheral neuropathy associated with IgM gammopathy and has been implicated in a dying back oligodendrogliopathy in multiple sclerosis.

Susceptibility of the Myelin-Associated Glycoprotein and Basic Protein to a Neutral Protease in Highly Purified Myelin from Human and Rat Brain

Abstract: Incubation of highly purified human myelin at 25° and pH 8 in ammonium bicarbonate buffer resulted in the conversion of the myelin‐associated glycoprotein (MAG) to a smaller derivative (dMAG) with an apparent molecular weight about 10,000 less. dMAG was stable and was not degraded to lower‐molecular‐weight breakdown products. Incubation of myelin under these conditions also resulted in the degradation of basic protein, but at a much slower rate. Half of the MAG was converted to dMAG in about 30 min, whereas degradation of half of the basic protein required 18 h of incubation. There was no significant loss of proteolipid, the Wolfgram doublet, or other myelin proteins during incubation for up to 18 h under these conditions. The formation of dMAG and the degradation of basic protein appear to be mediated by similar enzymatic activities; both processes exhibited broad pH optima in the neutral range, were prevented by briefly heating the myelin to 70° before incubation, and were stimulated by ammonium bicarbonate and other salts. Incubation of purified rat myelin also resulted in the formation of dMAG and the degradation of basic protein, but the conversion to dMAG occurred much more slowly than in human myelin preparations. In the rat, the percentage decreases in intact MAG and in basic protein were similar to each other and proceeded at rates comparable to the loss of basic protein in human myelin. These studies confirm and extend earlier demonstrations of neutral protease activity in purified myelin, and show that cleavage of MAG is one of the effects of this activity. The proteolytic activity affecting MAG and basic protein was not significantly reduced by further purification of the myelin on sucrose or CsCl gradients, suggesting that the neutral protease may be a myelin‐related enzyme. The very high susceptibility of human MAG to this enzyme indicates that the effect of neutral protease on this glycoprotein should be considered in connection with demyelinating diseases.

Autoantibodies associated with peripheral neuropathy

High titers of serum antibodies to neural antigens occur in several forms of neuropathy. These include neuropathies associated with monoclonal gammopathy, inflammatory polyneuropathies, and paraneoplastic neuropathies. The antibodies frequently react with glycosylated cell surface molecules, including glycolipids, glycoproteins, and glycosaminoglycans, but antibodies to intracellular proteins have also been described. There are several correlations between antibody specificity and clinical symptoms, such as anti‐MAG antibodies with demyelinating sensory or sensorimotor neuropathy, anti‐GM1 ganglioside antibodies with motor nerve disorders, antibodies to gangliosides containing disialosyl moieties with sensory ataxic neuropathy and Miller–Fisher syndrome, and antibodies to the neuronal nuclear Hu antigens with paraneoplastic sensory neuronopathy. These correlations suggest that the neuropathies may be caused by the antibodies, but evidence for a causal relationship is stronger in some examples than others. In this review, we discuss the origins of the antibodies, evidence for and against their involvement in pathogenic mechanisms, and the implications of these findings for therapy.

Cell-mediated immunity to myelin-associated glycoprotein, proteolipid protein, and myelin basic protein in multiple sclerosis.

Peripheral blood lymphocytes (PBL) from active and stable multiple sclerosis (MS) patients, patients with other neurologic diseases (OND), and control subjects were tested for sensitization to two myelin antigens not previously examined in multiple sclerosis, using a [3H]thymidine incorporation assay. The antigens investigated were myelin-associated glycoprotein (MAG) and proteolipid protein (PLP). In addition, sensitization to myelin basic protein (MBP) was also tested. Lymphocyte stimulation indices in active MS patients that were greater than 2 standard deviations above controls were as follows: 9/30 for MAG, 0/17 for PLP, and 8/81 for MBP. No control subjects responded to MAG or PLP, and only 1/29 control subjects responded to MBP. Three of the patients that responded to MAG also responded to MBP. Although the mean proliferative response to MAG and to MBP was greater in the population of active MS patients than in stable MS, ONDs, or controls, the difference was not statistically significant. The OND group was the only population which proliferated to PLP (6/16). The only statistically significant differences among the groups for all myelin antigens tested were the proportion of individuals with active MS vs. controls that responded to MAG (P less than 0.05), and OND vs. controls and active MS that responded to PLP (P less than 0.025). The greatest individual responses to the three antigens tested were to MBP in active MS patients. Elimination of the T8 (cytotoxic/suppressor) subset amplified the responses to myelin antigens in some patients and ONDs studied. These studies have demonstrated reactivity to MAG but not PLP in some patients with active MS, and reactivity to PLP in some patients with other neurologic diseases.

EVIDENCE THAT THE MAJOR PROTEIN IN RAT SCIATIC NERVE MYELIN IS A GLYCOPROTEIN

Evidence is presented that the major protein of rat sciatic nerve myelin is a glycoprotein. When myelin proteins were separated by polyacrylamide gel electrophoresis, the major band which was stained with amido black–Coomassie blue was also stained with periodic acid‐Schiff reagents for carbohydrate. Radioactive labelling of myelin in vivo with [3H]leucine and [14C]fucose, followed by electrophoresis of the proteins, indicated that with both isotopes the major labelled peak corresponded to the major stained band. In addition, a second smaller peak of [14C]fucose migrated ahead of the major peak. Delipidated myelin contained galactose, mannose, fucose and sialic acid.

A novel procedure for labeling immunoglobulins by conjugation to oligosaccharide moieties

A novel method is described for the biotinylation of immunoglobulins. The procedure relies on the generation of reactive aldehydes on the carbohydrate moieties of the immunoglobulin by oxidation with sodium periodate and subsequent reaction with biotin hydrazide. The method is simple and specific and results in stable conjugates retaining full immunologic activity. It has been applied successfully to a number of mouse monoclonal antibodies of both IgG and IgM classes, and to human IgM preparations. The procedure may also be applied to conjugation of immunoglobulins with fluorescent dyes.

Variation of proteins, enzyme markers and gangliosides in myelin subfractions

A discontinuous sucrose gradient was used to separate adult rat brain myelin into light, medium and heavy subfractions. Basic proteins decreased sharply, proteolipid potein changed very little, and high molecular weight proteins increased from the light to the heavy fraction. The concentration of monosialoganglioside GM1 was the highest in the middle fraction. The amount of carbohydrate in the major myelin-associated glycoprotein per mg total myelin protein increased 3.5-fold from the light to the heavy fraction. 2′,3′-Cyclic nucleotide 3′-phosphohydrolase, which is related to myelin or the oligodendroglial membrane, and acetylcholinesterase, which is in neural membranes such as the axolemma, both increased between the light and the heavy fraction, although their relative distributions among the three fractions were different. The glycoprotein and 2′,3′-cyclic nucleotide 3′-phosphohydrolase had similar distributions suggesting that they were concentrated in similar locations, possibly in the loose myelin and oligodendroglial plasma membrane. Electron microscopic examination of the subfractions was consistent with this interpretation.