Rat Myelin-associated Glycoprotein Research Articles

S100β inhibits the phosphorylation of the L-MAG cytoplasmic domain by PKA

The myelin-associated glycoprotein (MAG) is a cell adhesion molecule expressed by myelinating glia, existing as two isoforms that differ only by their cytoplasmic domains. We have studied the in vitro phosphorylation of recombinant rat MAG cytoplasmic domains by three kinases for which consensus sequences exist within this domain, revealing phosphorylation of the L-MAG-specific domain by protein kinase A (PKA). Phosphorylation of the L-MAG cytoplasmic domain by PKA was decreased in the presence of S100β, providing a functional significance to the interaction between L-MAG and S100β, and further indicating that L-MAG may play a role in myelinating glial cell signalling processes.

Generation and characterization of anti-sulfoglucuronosyl paragloboside monoclonal antibody NGR50 and its immunoreactivity with peripheral nerve.

Sulfoglucuronosyl paragloboside (SGPG) is a member of the sulfated glucuronic acid-containing glycolipid (SGGL) family found primarily in peripheral nerves. These glycolipids contain the HNK-1 carbohydrate epitope and are recognized by monoclonal IgM from patients with chronic demyelinating neuropathy and paraproteinemia. Recent studies indicate that SGGLs may serve as ligands for selectins, amphoterin, and laminin, suggesting that these glycolipids may play an important role in cellular adhesion. To elucidate the biological function of these glycolipids, we produced a murine monoclonal antibody (mAb) and studied its antigenic specificity. Using an enzyme-linked immunosorbent assay (ELISA), we found that the mAb designated as NGR50 belonged to the IgG2a subclass, and that the minimal titer (2 SD above the mean optical density value of control) of this mAb was 1:640, with 20 ng of purified SGPG as the antigen. Thin-layer chromatography (TLC) immunoblotting revealed that this mAb reacted specifically with SGPG and sulfoglucuronosyl lactosaminyl paragloboside (SGLPG), which is a structural analogue of the former, but not with other glycolipids. Desulfated derivates of SGPG and SGLPG did not react with mAb NGR50. Western blot analysis showed crossreactivity with human myelin-associated glycoprotein (MAG), but not with rat MAG or rat glycoprotein P0. Unlike anti-HNK-1 monoclonal antibody, however, NGR50 reacted only weakly with several proteins in the 20-30-kD regions, including human P0, suggesting that mAb50 has a different fine specificity as an anti-HNK-1 antibody. Immunocytochemical study of rat sciatic nerve using mAb NGR50 revealed positive staining at the outer surface of the myelin sheath and Schwann cells, as well as in the intervening connective tissues. Faint staining was also visible at the axolemmal-myelin interface; however, compact myelin was not stained.

Sensitization of Lewis rats with sulfoglucuronosyl paragloboside: Electrophysiological and immunological studies of an animal model of peripheral neuropathy

Antibodies against sulfoglucuronosyl glycosphingolipids (SGGLs) are known to be present in sera of patients with chronic polyneuropathy associated with IgM paraproteinemia. We recently studied rats sensitized with sulfoglucuronosyl paragloboside (SGPG), a major SGGL species, emulsified with keyhold limpet hemocyanin and Freund's adjuvant. The titer of the IgM class antibodies against SGPG increased up to 1:1,600, while that of the IgG class increased up to 1:800 2 weeks after sensitization. The antibodies showed a high degree of antigenic specificity; no cross-reactivity with other brain glycolipids could be detected. They, however, reacted with human myelin-associated glycoprotein (MAG) by Western blot analysis, but not with rat MAG. These animal models showed minor but clear clinical signs of neuropathy, consisting of mild tail muscle tone loss and walking disabilities. Electrophysiological examination of the sciatic nerves revealed nerve conduction abnormalities which consisted of conduction block and mild decrease in conduction velocity. Thus, our results support the concept that anti-SGPG antibodies may play an important pathogenetic role in this type of chronic neuropathy. © 1996 Wiley-Liss, Inc.

Sensitization of Lewis rats with sulfoglucuronosyl paragloboside: electrophysiological and immunological studies of an animal model of peripheral neuropathy.

Antibodies against sulfoglucuronosyl glycosphingolipids (SGGLs) are known to be present in sera of patients with chronic polyneuropathy associated with IgM paraproteinemia. We recently studied rats sensitized with sulfoglucuronosyl paragloboside (SGPG), a major SGGL species, emulsified with keyhold limpet hemocyanin and Freund's adjuvant. The titer of the IgM class antibodies against SGPG increased up to 1:1,600, while that of the IgG class increased up to 1:800 2 weeks after sensitization. The antibodies showed a high degree of antigenic specificity; no cross-reactivity with other brain glycolipids could be detected. They, however, reacted with human myelin-associated glycoprotein (MAG) by Western blot analysis, but not with rat MAG. These animal models showed minor but clear clinical signs of neuropathy, consisting of mild tail muscle tone loss and walking disabilities. Electrophysiological examination of the sciatic nerves revealed nerve conduction abnormalities which consisted of conduction block and mild decrease in conduction velocity. Thus, our results support the concept that anti-SGPG antibodies may play an important pathogenetic role in this type of chronic neuropathy.

Biosynthesis and regulation of expression of the HNK-1 epitope on myelin-associated glycoprotein in a transfected cell model system.

The HNK-1 antibody recognizes a carbohydrate epitope expressed by many cell adhesion molecules in the nervous system that has been proposed to be an important adhesive determinant. This epitope is particularly prominent on the myelin-associated glycoprotein (MAG) and is related to the antigenic target in an autoimmune mediated demyelinating neuropathy. Elucidation of the mechanisms underlying the biosynthesis and regulation of expression of the HNK-1 epitope is therefore likely to have important functional and clinical implications. In order to investigate its biosynthesis and the regulation of its expression, we have expressed both human and rat MAG in several different cell lines by retroviral infection. These studies indicate that the cellular milieu determines whether the HNK-1 epitope is expressed on the MAG polypeptide and provide an explanation for the significant variation in HNK-1 levels that has been noted in different species. Using a transfected human neuroblastoma line, we have determined that this epitope is present on the fourth and/or fifth immunoglobulin-like domain of rat MAG and that it is added intracellularly, probably in the trans Golgi. Finally we have found that expression of the HNK-1 epitope is increased by activation of different second messenger systems, providing direct evidence that its expression can be regulated independently from that of the MAG polypeptide.

Molecular characterization of the schwann cell myelin protein, SMP: Structural similarities within the immunoglobulin superfamily

The Schwann cell myelin protein (SMP), previously defined in quail and chick by a monoclonal antibody, is in vivo exclusively expressed by myelinating and nonmyelinating Schwann cells and oligodendrocytes. The isolation of the complete nucleotide sequence of SMP is reported here. The predicted polypeptide chain reveals that SMP is a transmembrane molecule of the immunoglobulin superfamily showing sequence similarities with several surface glycoproteins expressed in the nervous and immune systems. In spite of a 43.5% overall sequence identity between rat myelin-associated glycoprotein (MAG) and quail SMP, SMP does not seem to be the avian homolog of MAG, since their expression, regulation, and functions are significantly different. Unusual sequence arrangements shared by SMP, MAG, and two lymphoid antigens suggest the existence of a particular subgroup in the immunoglobulin superfamily.

Preliminary studies on sensitization of Lewis rats with sulfated glucuronyl paragloboside

A large number of patients with peripheral neuropathy and IgM paraproteinemia have IgM monoclonal antibodies which recognize a carbohydrate determinant shared by myelin-associated glycoprotein (MAG) and sulfated glucuronyl glycolipids (SGGLs). There is considerable evidence that these IgM monoclonal antibodies are responsible for demyelination in this disorder. To study the pathogenic role of SGGLs in this type of neuropathy, we sensitized Lewis rats with sulfated glucuronyl paragloboside (SGPG), a major SGGL. Fifty percent of the animals (8/16) developed neurological symptons such as mild to moderate distal tail tone loss, with or without abnormal posture, along with development of anti-SGPG antibodies. These antibodies reacted with SGGLs, but not with rat MAG. Morphological studies showed: (1) axonal change in the lateral aspects of the dorsal columns in the spinal cord; and (2) damage to the endothelial cells in the spinal cord which suggested a breakdown of the blood-brain barrier. There was no obvious change in the peripheral nerve. Since no marked cellular infiltration was detected in these lesions, the clinicopathological findings observed could be induced by humoral mechanism, most likely anti-SGPG antibodies.

Molecular cloning of human myelin‐associated glycoprotein

The nucleotide sequence for human myelin-associated glycoprotein (MAG) and its deduced amino acid sequence, obtained by analysis of two overlapping cDNA clones isolated from a human brain cDNA library, is presented and compared to that reported for rat MAG. The sequence provides an open reading frame of 1,878 nucleotides encoding a peptide of 626 amino acids with a calculated molecular weight of 69.1 kD. It is 89% homologous in nucleotide sequence to the large isoform of rat MAG, with 95% homology in the amino acid sequence. It contains 9 potential glycosylation sites, one more than in rat, and shares other key features with rat MAG, including 5 immunoglobulin-like regions of internal homology, an RGD sequence, and potential phosphorylation sites. Its structure appears to be highly conserved in evolution, possibly suggesting a close interdependence between its structure and function. The human gene is located on the proximal long arm of chromosome 19 (19q12----q13.2).

Antibodies to Myelin-Associated Glycoprotein (MAG) in the cerebrospinal fluid of multiple sclerosis patients

Cerebrospinal fluids (CSF) and sera from patients with multiple sclerosis (MS), other neurological disease (ONDs) and healthy controls were tested for antibodies to myelin-associated glycoprotein (MAG) by several different assays. Using a very sensitive, solid-phase radioimmunoassay with radioiodinated protein A, a statistically significant elevation of anti-MAG antibodies was detected in MS CSFs in comparison to those from ONDs and healthy controls. The antibodies reacted with human MAG, but not with rat MAG, and appeared to be directed towards carbohydrate dterminants in the glycoprotein. The CSFs from high IgG producers had significantly greater anti-MAG antibody levels than those from low IgG producers, even though the assays were done on CSF samples that had been normalized to the same IgG concentration, the elevated antibodies were not detected when the same samples were tested with a liquid-phase radioimmunoassay or an enzyme-linked immunosorbent assay, and the antibodies in the MS CSF also could not be detected by Western blotting. An elevated level of antibodies was not found in sera from MS patients by any of the assays, possibly because these samples gave higher and more variable background. The results suggest that there is a low level of humoral immunity to MAG in MS patients that can only be detected by the most sensitive assays. This weak immune response to MAG may be secondary to the demyelinating process, but could play a role in the progression of the disease.

The myelin-associated glycoprotein gene: Mapping to human chromosome 19 and mouse chromosome 7 and expression in quivering mice

Myelin-associated glycoprotein (MAG), a membrane glycoprotein of 100 kDa, is thought to be involved in the process of myelination. A cDNA encoding the amino-terminal half of rat MAG has recently been isolated and sequenced. We have used this cDNA in Southern blot analysis of DNA from 32 somatic cell hybrids to assign the human locus for MAG to chromosome 19 and the mouse locus to chromosome 7. Since the region of mouse chromosome 7-known to contain several other genes that are homologous to genes on human chromosome 19-also carries the quivering ( qv) locus, we considered the possibility that a mutation in the MAG gene could be responsible for this neurological disorder. While MAG-specific DNA restriction fragments, mRNA, and protein from qv qv mice were apparently normal in size and abundance, we have not ruled out the possibility that qv could be caused by a point mutation in the MAG gene.

Mouse monoclonal and rabbit polyclonal antibodies prepared to human myelin-associated glycoprotein also react with glycosphingolipids of peripheral nerve

A panel of mouse monoclonal antibodies and rabbit polyclonal antisera that were raised to myelin-associated glycoprotein (MAG) were screened for reactivity with acidic glycolipids from brain and peripheral nerve by enzyme-linked immunosorbent assay (ELISA) and/or a thin-layer chromatogram overlay technique. Seven out of 7 mouse monoclonal antibodies that recognize carbohydrate epitopes in human MAG also reacted with acidic glycolipids from human and cat peripheral nerve, while monoclonal antibodies that react with polypeptide epitopes on MAG did not react with these glycolipids. Rabbit anti-human MAG antisera also strongly reacted with the glycolipids from peripheral nerve, while rabbit antisera raised to rat MAG did not. None of the antibodies reacted with similar glycolipid fractions prepared from adult human brain. Overlay of thin-layer chromatograms revealed that all the mouse and rabbit antibodies showing reactivity with peripheral nerve glycolipids were binding to the same two sphingoglycolipids that react with human anti-MAG IgM paraproteins in neuropathy and with HNK-1 (anti-Leu-7), a mouse IgM monoclonal antibody that identifies a subset of human lymphocytes with natural killer function. Thus, the carbohydrate epitope(s) in MAG which is shared with nerve acidic glycolipids appears to be highly immunogenic in mice and rabbits. Further, it is clear that the antibodies that react with the carbohydrate moieties of human MAG cannot be used as specific probes for this glycoprotein.