Multiple functions of the myelin-associated glycoprotein MAG (siglec-4a) in formation and maintenance of myelin

The myelin-associated glycoprotein, a minor component of myelin in the central and peripheral nervous system, has been implicated in the formation and maintenance of myelin. Although the analysis of MAG null mutants confirms this view, the phenotype of this mutant is surprisingly subtle. In the CNS of MAG-deficient mice, initiation of myelination, formation of morphologically intact myelin sheaths and to a minor extent, integrity of myelin is affected. In the PNS, in comparison, only maintenance of myelin is impaired. Recently, the large isoform of MAG has been identified as the functionally important isoform in the CNS, whereas the small MAG isoform is sufficient to maintain the integrity of myelinated fibers in the PNS. Remarkably, none of the different defects in the MAG mutant is consistently associated with each myelinated fiber. These observations suggest that other molecules performing similar functions as MAG might compensate, at least partially, for the absence of MAG in the null mutant.     

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 diseases (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 determinants 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.     

Shared antigen between the myelin-associated glycoprotein (MAG) and a cell line from human T cell leukemia (HSB-2)

HSB-2 is a cell line originated from human T-cell leukemia and its membrane antigen is recognized by anti-HNK-1, a monoclonal antibody against human natural killer (NK) cells. Enzyme linked immunosorbent assay (ELISA) revealed that not only anti-HNK-1 but also rabbit polyclonal and human monoclonal anti-myelin-associated glycoprotein (MAG) antibodies were absorbed by cell homogenate of HSB-2. Molt-4 and K-562 cell lines from human T-cell leukemia and human erythroleukemia did not absorb anti-HNK-1 and the anti-MAG antibodies. On the immunoblot of HSB-2 cell homogenate, two major molecules were stained by anti-HNK-1 and anti-MAG antibodies, while Molt-4 and K-562 were not stained by these antibodies. Comparing molecular weights of the two major molecules with those of MAG and derivative of MAG (dMAG), one is larger than MAG and the other is smaller than dMAG. The molecular weights of the two molecules were approximately 130 kDa and 80 kDa. The shared antigen in HSB-2 is not identical with MAG but a part of the molecule in the cells may be shared with a part of MAG molecule.     

Encephalitogenic and neuritogenic T cell responses to the myelin-associated glycoprotein (MAG) in the Lewis rat

Autoimmune responses to the myelin-associated glycoprotein (MAG) are implicated in the immunopathogenesis of both multiple sclerosis (MS) and certain peripheral neuropathies. In this study we demonstrate that T cell responses to defined epitopes of MAG mediate a pathological inflammatory response in the nervous system of the Lewis rat. Peptide-specific T cells were generated against four different MAG epitopes, three of which are common to both L- and S-isoforms of MAG (amino acid (a.a.) sequence: 20-34, 124-137, 354-377) whilst the fourth epitope (a.a. sequence: 570-582) is located in the C-terminal sequence of S-MAG. The adoptive transfer of T cells specific for these epitopes initiated a mild but dose-dependent inflammatory response in the central nervous system (CNS) of naive recipients. Clinical disease was only observed in those animals injected with T cells specific for the a.a. sequence 20-34 (MP1.1), which also initiated an inflammatory response in the peripheral nervous system (PNS). Co-transfer of MP1.1 (a.a. sequence 20-34)-specific T cells with the myelin oligodendrocyte glycoprotein (MOG)-specific monoclonal antibody 8-18C5 enhanced disease severity and induced widespread demyelination in the CNS. In contrast, co-transfer of T cells with the MAG-specific mAb 513 failed to induce demyelination, but had a moderate effect on the local inflammatory response. The ability of MAG to initiate an autoaggressive T cell response in the Lewis rat supports the concept that MAG-specific autoimmune responses may play a role in the pathogenesis of immune mediated diseases of the nervous system in man.     

Characterization of the antigen determinant on HSB-2 cells shared with myelin-associated glycoprotein (MAG) using monoclonal antibodies

The existence of cross-antigenicity between myelin-associated glycoprotein (MAG) and natural killer cells has been reported previously. In this study, we have characterized the antigenic determinant on HSB-2 cells which is shared with MAG using two types of mouse monoclonal anti-MAG antibodies, one recognizing the peptide molecule (IgG-P) and the other recognizing the carbohydrate molecule of MAG (IgM-C). Enzyme-linked immunosorbent assay (ELISA) revealed that IgM-C was absorbed by cell homogenate of HSB-2, and some bands were stained by IgM-C on the immunoblot of HSB-2 cell homogenate, while IgG-P was not absorbed by HSB-2 cell homogenate and no band was stained by IgG-P on the immunoblot of HSB-2 cell homogenate. Therefore it is suggested that the shared antigenic determinant between MAG and HSB-2 cells is not in the peptide molecule but in the carbohydrate molecule.     

Characterization of the antigenic determinant on HSB-2 cells shared with myelin-associated glycoprotein (MAG) using monoclonal antibodies

The existence of cross-antigenicity between myelin-associated glycoprotein (MAG) and natural killer cells has been reported previously. In this study, we have characterized the antigenic determinant on HSB-2 cells which is shared with MAG using two types of mouse monoclonal anti-MAG antibodies, one recognizing the peptide molecule (IgG-P) and the other recognizing the carbohydrate molecule of MAG (IgM-C). Enzyme-linked immunosorbent assay (ELISA) revealed that IgM-C was absorbed by cell homogenate of HSB-2, and some bands were stained by IgM-C on the immunoblot of HSB-2 cell homogenate, while IgG-P was not absorbed by HSB-2 cell homogenate and no band was stained by IgG-P on the immunoblot of HSB-2 cell homogenate. Therefore it is suggested that the shared antigenic determinant between MAG and HSB-2 cells is not in the peptide molecule but in the carbohydrate molecule.     

NK cells in patients with peripheral neuropathy and IgM monoclonal protein reacting with the myelin-associated glycoprotein (MAG)

We studied Leu 7+ cell distribution and natural killer (NK) activity in the peripheral blood of six patients who had peripheral neuropathy and monoclonal IgM protein directed against myelin-associated glycoprotein (anti-MAG IgM). We did not find any difference between patients and control subjects (healthy or polyneuropathic, some with IgM monoclonal paraprotein but without anti-MAG activity). The presence of autologous sera did not interfere with these results. We noted an increase in Leu 11+ cell percentages after pre-incubation of the patient cells with autologous sera but the phenotypes of cells from control subjects did not change after incubation with autologous or patient sera.     

Occurrence of myelin-associated glycoprotein (MAG)-like immunoreactivity in some nervous, endocrine, and immune-related cells of the rat

The occurrence and distribution of myelin-associated glycoprotein (MAG)-like immunoreactivity was investigated in the rat using a polyclonal antibody to MAG purified from rat brain. In the nervous system, MAG immunoreactivity was found in the periaxonal portion of the myelinated fibers and in a small number of oligodendroglia in the cortex, hippocampus, and the spinal cord. The sheath of Schwann cells in unmyelinated fibers and satellite cells in the spinal ganglia were also immunoreactive for MAG. In the endocrine system, the noradrenaline-containing cells in the adrenal medulla and some endocrine cells in the duodenum showed MAG immunoreactivity. In the immune system, numerous reticular cells with slender cytoplasmic processes, which formed a dense network, were immunopositive for MAG within the germinal center in the lymph nodes and spleen. In the thymus, a number of epithelial reticular cells within the medulla showed variation in staining intensity. These findings provide new information on the wide distribution of MAG immunoreactivity in the nervous, endocrine, and immune systems, and may contribute to the further understanding of the biological roles of this protein.     

Binding properties of liposomes containing the myelin-associated glycoprotein MAG to neural cell cultures.

The myelin-associated glycoprotein MAG is a neural cell adhesion molecule which belongs to the immunoglobulin superfamily and the carbohydrate based L2/HNK-1 family of adhesion molecules. In this study we further characterize the adhesive properties of MAG. MAG incorporated into liposomes bound to cultured peripheral and central nervous system neurons known to be myelinated in vivo. Expression of the neuronal MAG receptor(s) on spinal cord neurons increased with time in culture and correlated with the time of active myelination of these neurons in vivo. MAG bound only poorly if at all to cerebellar neurons which are not myelinated in vivo and not to cultured oligodendrocytes or Schwann cells. A low level of MAG binding to astrocytes or fibroblast-like cells that was MAG antibody inhibitable could also be observed. The adhesion molecules L1 and N-CAM, two other members of the immunoglobulin superfamily, were not found to be the neuronal receptors for MAG. RGD containing peptides did not inhibit binding of MAG-liposomes to neurons. The soluble form of MAG which contains most, if not all, of the extracellular domain of the molecule and binds to collagen, did not interfere with the binding of MAG-liposomes to neurons. Conversely, MAG-liposomes did not bind to collagen, suggesting that MAG shows different binding properties as an integral membrane protein than as a fragment containing the extracellular domain of the molecule.     

Immunocytochemical observations on the distribution of myelin-associated glycoprotein and myelin basic protein in multiple sclerosis lesions

To study the distribution of myelin-associated glycoprotein (MAG) in human nervous tissue and in multiple sclerosis (MS) lesions, we used paraffin sections and our modification of the peroxidase-antiperoxidase technique. Sections of MS lesions also were treated with antiserum to basic protein (BP) and with histological stains for axons and myelin sheaths. In tissue from normal developing central nervous system, oligodendroglia, their processes, and wwly formed myelin sheaths were intensely stained by MAG antiserum. In adults, MAG was found in periaxonal regions of myelinated fibers of the central and peripheral nervous system. The most striking finding in MS lesions was the extension of decreased MAG immunostaining into white matter that appeared normal when treated with BP antiserum or luxol fast blue. In acute early MS lesions the decrease in MAG immunostaining extended far beyond the margin of acute demyelination, where the BP staining of degenerating sheaths often was increased. In chronic inactive plaques, this decrease in periaxonal MAG immunostaining was limited to relatively few fibers in a thin rim around each lesion. These observations suggest that in MS, immunoreactivity of periaxonal MAG is altered before myelin breakdown begins. Early in degeneration, myelin sheaths and their fragments often were more intensely stained by BP antiserum than normal sheaths; later the staining intensity decreased. In shadow plaques, BP antiserum stained some oligodendroglia. Their appearance and location among thinly myelinated axons suggested that these oligondendroglia were forming new sheaths around previously demyelinated axons.     

Carbohydrate moieties of myelin-associated glycoprotein, major glycoprotein of the peripheral nervous system myelin and other myelin glycoproteins potentially involved in cell adhesion.

The myelin-associated glycoprotein (MAG) and the major glycoprotein of the peripheral nervous system myelin (P0) are two members of the family of cell adhesion molecules (CAMs). A role in cell adhesion of the carbohydrate moiety of these molecules has been attributed to the presence of N-glycans bearing the HNK-1 carbohydrate epitope. On the other hand, it has been suggested that these glycoproteins could be ligands of an endogenous mannose-binding lectin present in myelin, the cerebellar soluble lectin (CSL). In order to further document the heterogeneity of the glycans of these two CAMs, we have used several probes: an anti-carbohydrate antibody of the HNK-1 type, called Elec-39, the plant lectin concanavalin A (ConA), and the endogenous lectin CSL involved in myelin compaction. This study shows that CSL binds to a small proportion of the polypeptide chains of MAG found in adult CNS of rats and man and the polypeptide chains of P0 molecules from adult human and rat sciatic nerve. For MAG from adult rat brain, the binding of CSL is restricted to glycans of polypeptide chains which could be separated from the others according to their solubility properties. These MAG molecular entities react also with the Elec-39 antibody and with ConA. These results confirm that P0 and MAG are heterogeneous in their carbohydrate moieties.(ABSTRACT TRUNCATED AT 250 WORDS)     

Encephalitogenic and immunogenic potential of myelin-associated glycoprotein (MAG), oligodendrocyte-specific glycoprotein (OSP) and 2',3'-cyclic nucleotide 3'-phosphodiesterase (CNPase) in ABH and SJL mice.

Synthetic peptides of myelin-associated glycoprotein (MAG), oligodendrocyte-specific glycoprotein (OSP) and 2',3'-cyclic nucleotide 3'-phosphodiesterase (CNPase) were screened for their ability to induce experimental allergic encephalomyelitis (EAE) in ABH (H-2A(g7)) and SJL (H-2(s)) mice.The use of overlapping 16mer MAG peptides identified residues 97-112 as a T-cell and encephalitogenic epitope in ABH mice which induced clinical and histological signs of acute EAE. Immunization of SJL mice with MAG peptides failed to induce disease whereas immunization of SJL mice with synthetic peptides of OSP induced major T-cell responses to OSP 73-88 and 81-96. Another epitope, OSP 57-72, that induced EAE, failed to induce T-cell responses in mice immunised with peptides based on the whole sequence supporting a role for cryptic epitopes. In comparison, whilst immunization of ABH mice with OSP revealed two immunodominant T-cell epitopes (49-64 and 137-152), an encephalitogenic epitope was not identified. Similarly, immunization of both SJL and ABH mice with CNPase peptides induced T-cell responses to several epitopes. However, these were not encephalitogenic.This study is the first to identify an encephalitogenic epitope of MAG and immunodominant epitopes of MAG, OSP and CNPase in SJL and ABH mice. The ability of both cryptic and noncryptic peptide epitopes of these myelin antigens to initiate EAE suggests that mice at least are not tolerant to some regions of MAG and OSP and that such specific autoimmune responses may play an important role in the pathogenesis of immune-mediated neurological diseases such as multiple sclerosis.     

A quantitation of myelin-associated glycoprotein and myelin basic protein loss in different demyelinating diseases

The loss of myelin-associated glycoprotein (MAG) and myelin basic protein (MBP) was compared by quantitative immunocytochemistry in demyelinating lesions of measles encephalomyelitis (ME), multiple sclerosis (MS), and progressive multifocal leukoencephalopathy (PML). Serial sections from paraffin-embedded tissue were reacted with antisera for MAG and MBP, and areas of staining loss were compared morphometrically. Lesions in ME showed MAG loss equal to that of MBP, lesions of PML showed MAG loss greater than that of MBP, and MS lesions showed a mixture of patterns. These data demonstrate distinctive patterns of MAG and MBP loss in these three diseases.     

The distribution of myelin-associated glycoprotein and myelin basic protein in actively demyelinating multiple sclerosis lesions

Active plaques from 4 patients with multiple sclerosis were examined for myelin-associated glycoprotein (MAG) and myelin basic protein (MBP) using the peroxidase-antiperoxidase (PAP) immunocytochemical procedure applied to paraffin sections. MBP loss was intimately related to the presence of infiltrating macrophages which appeared to remove MBP-positive fragments directly off intact myelin sheaths. Phagocytosis of MAG-positive myelin sheaths was also observed. These findings support previous morphological studies that suggest that phagocytosis by macrophages of myelin attached to axons is an important mechanism of demyelination in multiple sclerosis.     

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.     

Distribution of papovavirus, myelin-associated glycoprotein, and myelin basic protein in progressive multifocal leukoencephalopathy lesions

To study how viruses interact with oligodendroglia and produce demyelination, we immunostained paraffin and epon sections of lesions from patients with progressive multifocal leukoencephalopathy (PML) with antisera to papovaviruses, oligodendroglial myelin-associated glycoprotein (MAG), and myelin basic protein (MBP) according to the peroxidase-antiperoxidase method. In paraffin sections from a rapidly progressive case of PML, hyperimmune JC virus antiserum stained single oligodendroglia which were located in white matter that appeared normal histologically and stained normally with MAG and MBP antisera. In zones surrounding areas of demyelination, virus containing oligodendroglia were most numerous and MAG staining of periaxonal regions was decreased, but there was little change in MBP staining. In demyelinated regions, both MAG and MBP staining were severely altered; also there was much less JC virus staining. In tissue from three other chronic cases, viral antiserum stained fewer oligodendrocytes and the differences in MAG and MBP staining were much less striking. In epon sections from two biopsies of central nervous system tissue, we studied the electron microscopic appearance of oligodendroglia that also had been stained by JC virus antiserum. Virions were present in all nuclei and in some cytoplasmic regions. The results suggest that changes in MAG distribution are useful indicators of early oligodendroglial abnormalities which can cause myelin breakdown.     

Comparative binding of murine and human monoclonal antibodies reacting with myelin-associated glycoprotein to myelin and human lymphocytes

Human monoclonal IgM antibodies present in the blood of some patients with peripheral neuropathy and murine hybrid IgM antibodies C5 and C6, raised against myelin-associated glycoprotein, and HNK-1, raised against the human T cell line HSB-2, all bind to the carbohydrate moiety of myelin-associated glycoprotein. The relative avidity of the monoclonal antibodies was HNK-1 greater than C5/C6 much greater than human IgM, as determined in a competitive binding radioimmunoassay. HNK-1 bound myelin equally well at incubation temperatures between 4 degrees C and 37 degrees C; the human antibodies bound significantly only at 4 degrees C; and C6 bound best at 4 degrees C, less strongly at 20 degrees C and did not bind at 37 degrees C. All of the antibodies bound to a band corresponding to myelin-associated glycoprotein on immunoblots of human CNS myelin proteins in addition to several other antigens. Flow cytometric studies revealed that the murine but not the human antibodies bind to peripheral blood lymphocytes. Taken together, these data suggest that the antibodies probably recognize the same epitope but bind with different avidity.     

Myelin-associated glycoprotein (MAG) in the CNS of adult shiverer (Shi/Shi) mice

Brain fractions of adult control (+/+) and shiverer (Shi/Shi) mice were investigated by sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) and immunoblotting. Immunostaining with specific antisera against rat brain myelin-associated glycoprotein (MAG) was detected at about the 96-kD region of gels in electrophoresed samples of the total homogenate, low-speed supernatant fraction, and low- and high-speed sedimentable portions of brain from +/+ mice. Reduced immunostaining was observed in the corresponding samples of brain fractions from Shi/Shi mice. The cerebrum, cerebellum, and medulla of +/+ and Shi/Shi mice were examined immunocytochemically for MAG on paraffin-embedded sections. Periaxonal immunostaining for MAG was observed in all the regions and the highest concentrations were in the corpus callosum, in the central cores of cerebellar folia, and in the medulla. Patterns of distribution were similar in +/+ and Shi/Shi mice, although the density of immunostaining around individual axons and the number of immunostained axons were significantly reduced in Shi/Shi mice. In addition, the three brain regions of Shi/Shi mice exhibited oligodendrocyte-like cells that contained immunostaining for MAG in the cytoplasm and periphery of their perikarya. This type of immunostained cell was not observed in +/+ mice. In this study, immunoblotting with brain fractions and immunocytochemistry revealed strong evidence for reduced concentrations of MAG in the CNS of Shi/Shi mice compared to control mice. In addition, there is immunocytochemical evidence for abnormal accumulation of MAG in perikarya of oligodendroglial-like cells, suggesting the possibility of a transport block for myelin proteins in the shiverer mutant.