Immunocytochemical studies of quaking mice support a role for the myelin-associated glycoprotein in forming and maintaining the periaxonal space and periaxonal cytoplasmic collar of myelinating Schwann cells.

The myelin-associated glycoprotein (MAG) is an integral membrane glycoprotein that is located in the periaxonal membrane of myelin-forming Schwann cells. On the basis of this localization, it has been hypothesized that MAG plays a structural role in (a) forming and maintaining contact between myelinating Schwann cells and the axon (the 12-14-nm periaxonal space) and (b) maintaining the Schwann cell periaxonal cytoplasmic collar of myelinated fibers. To test this hypothesis, we have determined the immunocytochemical localization of MAG in the L4 ventral roots from 11-mo-old quaking mice. These roots display various stages in the association of remyelinating Schwann cells with axons, and abnormalities including loss of the Schwann cell periaxonal cytoplasmic collar and dilation of the periaxonal space of myelinated fibers. Therefore, this mutant provides distinct opportunities to observe the relationships between MAG and (a) the formation of the periaxonal space during remyelination and (b) the maintenance of the periaxonal space and Schwann cell periaxonal cytoplasmic collar in myelinated fibers. During association of remyelinating Schwann cells and axons, MAG was detected in Schwann cell adaxonal membranes that apposed the axolemma by 12-14 nm. Schwann cell plasma membranes separated from the axolemma by distances greater than 12-14 nm did not react with MAG antiserum. MAG was present in adaxonal Schwann cell membranes that apposed the axolemma by 12-14 nm but only partially surrounded the axon and, therefore, may be actively involved in the ensheathment of axons by remyelinating Schwann cells. To test the dual role of MAG in maintaining the periaxonal space and Schwann cell periaxonal cytoplasmic collar of myelinated fibers, we determined the immunocytochemical localization of MAG in myelinated quaking fibers that displayed pathological alterations of these structures. Where Schwann cell periaxonal membranes were not stained by MAG antiserum, the cytoplasmic side of the periaxonal membrane was "fused" with the cytoplasmic side of the inner compact myelin lamella and formed a major dense line. This loss of MAG and the Schwann cell periaxonal cytoplasmic collar usually resulted in enlargement of the 12-14-nm periaxonal space and ruffling of the apposing axolemma. In myelinated fibers, there was a strict correlation between the presence of MAG in the Schwann cell periaxonal membrane and (a) maintenance of the 12-14-nm periaxonal space, and (b) presence of the Schwann cell periaxonal cytoplasmic collar.(ABSTRACT TRUNCATED AT 400 WORDS)

Presence of the myelin-associated glycoprotein correlates with alterations in the periodicity of peripheral myelin.

The myelin-associated glycoprotein (MAG) is an integral membrane protein (congruent to 100,000 mol wt) which is a minor component of purified peripheral nervus system (PNS) myelin. In the present study, MAG was localized immunocytochemically in 1-micrometer thick Epon sections of 7-d and adult rat peripheral nerves, and its localization was compared to that of the major structural protein (Po) of PNS myelin. To determine more precisely the localization of MAG, immunostained areas in 1 micrometer sections were traced on electron micrographs of identical areas from adjacently cut thin sections.l MAG was localized in periaxonal membranes. Schmidt-Lantermann incisures, paranodal membranes, and the outer mesaxon of PNS myelin sheaths. Compact regions of PNS myelin did not react with MAG antiserum. The results demonstrate MAG's presence in "'semi-compact" Schwann cell or myelin membranes that have a gap of 12-14 nm between extracellular leaflets and a spacing of 5 nm or more between cytoplasmic leaflets. In compact regions of the myelin sheath which do not contain MAG, the cytoplasmic leaflets are "fused" and form the major dense line, whereas the extracellular leaflets are separated by a 2.0 nm gap appearing as paired minor dense lines. Thus, it is proposed that MAG plays a role in maintaining the periaxonal space, Schmidt-Lantermann incisures, paranodal myelin loops, and outer mesaxon by preventing "complete" compaction of Schwann cell and myelin membranes. The presence of MAG in these locations also suggests that MAG may serve a function in regulating myelination in the PNS.

Myelin-associated glycoprotein and myelinating Schwann cell-axon interaction in chronic B,B'-iminodipropionitrile neuropathy.

The myelin-associated glycoprotein (MAG) is a heavily glycosylated integral membrane glycoprotein which is a minor component of isolated rat peripheral nervous system (PNS) myelin. Immunocytochemically MAG has been localized in the periaxonal region of PNS myelin sheaths. The periaxonal localization and biochemical features of MAG are consistent with the hypothesis that MAG plays a role in maintaining the periaxonal space of myelinated fibers. To test this hypothesis, MAG was localized immunocytochemically in 1-micron sections of the L5 ventral root from rats exposed to B,B'-iminodipropionitrile. In chronic states of B,B'-iminodipropionitrile intoxication, Schwann cell periaxonal membranes and the axolemma invaginate into giant axonal swellings and separate a central zone of normally oriented axoplasm from an outer zone of maloriented neurofilaments. Ultrastructurally, the width of the periaxonal space (12-14 nm) in the ingrowths is identical to that found in normally myelinated fibers. These Schwann cell ingrowths which are separated from compact myelin by several micra are stained intensely by MAG antiserum. Antiserum directed against Po protein, the major structural protein of compact PNS myelin, does not stain the ingrowths unless compact myelin is present. These results demonstrate the periaxonal localization of MAG and support a functional role for MAG in maintaining the periaxonal space of PNS myelinated fibers.

Immunocytochemical localization of P0 protein in Golgi complex membranes and myelin of developing rat Schwann cells.

P0 protein, the dominant protein in peripheral nervous system myelin, was studied immunocytochemically in both developing and mature Schwann cells. Trigeminal and sciatic nerves from newborn, 7-d, and adult rats were processed for transmission electron microscopy. Alternating 1-micrometer-thick Epon sections were stained with paraphenylenediamine (PD) or with P0 antiserum according to the peroxidase-antiperoxidase method. To localize P0 in Schwann cell cytoplasm and myelin membranes, the distribution of immunostaining observed in 1-micrometer sections was mapped on electron micrographs of identical areas found in adjacent thin sections. The first P0 staining was observed around axons and/or in cytoplasm of Schwann cells that had established a 1:1 relationship with axons. In newborn nerves, staining of newly formed myelin sheaths was detected more readily with P0 antiserum than with PD. Myelin sheaths with as few as three lamellae could be identified with the light microscope. Very thin sheaths often stained less intensely and part of their circumference frequently was unstained. Schmidt-Lanterman clefts found in more mature sheaths also were unstained. As myelination progressed, intensely stained myelin rings became much more numerous and, in adult nerves, all sheaths were intensely and uniformly stained. Particulate P0 staining also was observed in juxtanuclear areas of Schwann cell cytoplasm. It was most prominent during development, then decreased, but still was detected in adult nerves. The cytoplasmic areas stained by P0 antiserum were rich in Golgi complex membranes.

Endocytic depletion of L-MAG from CNS myelin in quaking mice.

Quaking is an autosomal recessive hypo/dysmyelinating mutant mouse which has a 1-Mbp deletion on chromosome 17. The mutation exhibits pleiotrophy and does not include genes encoding characterized myelin proteins. The levels of the 67-kD isoform of the myelin-associated glycoprotein (S-MAG) relative to those of the 72-kD isoform (L-MAG) are increased in the quaking CNS, but not in other dysmyelinating mutants. Abnormal expression of MAG isoforms in quaking may result from altered transcription of the MAG gene or from abnormal sorting, transport, or targeting of L-MAG or S-MAG. To test these hypotheses, we have determined the distribution of L-MAG and S-MAG in cervical spinal cord of 7-, 14-, 21-, 28-, and 35-d-old quaking mice. In 7-d-old quaking and control spinal cord, L- and S-MAG was detectable in periaxonal regions of myelinated fibers and in the perinuclear cytoplasm of oligodendrocytes. Between 7 and 35 d, L-MAG was removed from the periaxonal membrane of quaking but not control mice. Compared to control mice, a significant increase in MAG labeling of endosomes occurred within oligodendrocyte cytoplasm of 35-d-old quaking mice. S-MAG remained in periaxonal membranes of both quaking and control mice. Analysis of the cytoplasmic domain of L-MAG identifies amino acid motifs at tyrosine 35 and tyrosine 65 which meet the criteria for "tyrosine internalization signals" that direct transmembrane glycoproteins into the endocytic pathway. These results establish that L-MAG is selectively removed from the periaxonal membrane of CNS-myelinated fibers by receptor-mediated endocytosis. The loss of L-MAG from quaking periaxonal membranes results from increased endocytosis of L-MAG and possibly a decrease in L-MAG production.

Microtubule-associated protein 1B: a neuronal binding partner for myelin-associated glycoprotein.

Myelin-associated glycoprotein (MAG) is expressed in periaxonal membranes of myelinating glia where it is believed to function in glia-axon interactions by binding to a component of the axolemma. Experiments involving Western blot overlay and coimmunoprecipitation demonstrated that MAG binds to a phosphorylated neuronal isoform of microtubule-associated protein 1B (MAP1B) expressed in dorsal root ganglion neurons (DRGNs) and axolemma-enriched fractions from myelinated axons of brain, but not to the isoform of MAP1B expressed by glial cells. The expression of some MAP1B as a neuronal plasma membrane glycoprotein (Tanner, S.L., R. Franzen, H. Jaffe, and R.H. Quarles. 2000. J. Neurochem. 75:553-562.), further documented here by its immunostaining without cell permeabilization, is consistent with it being a binding partner for MAG on the axonal surface. Binding sites for a MAG-Fc chimera on DRGNs colocalized with MAP1B on neuronal varicosities, and MAG and MAP1B also colocalized in the periaxonal region of myelinated axons. In addition, expression of the phosphorylated isoform of MAP1B was increased significantly when DRGNs were cocultured with MAG-transfected COS cells. The interaction of MAG with MAP1B is relevant to the known role of MAG in affecting the cytoskeletal structure and stability of myelinated axons.

Induction of experimental ataxic sensory neuronopathy in cats by immunization with purified SGPG.

IgM paraproteins in about 50% of the patients with neuropathy associated with IgM gammopathy react with carbohydrate moieties in myelin-associated glycoprotein (MAG) and in sulfated glucuronic glycolipids (SGGLs) in human peripheral nerves. However, the role of anti-MAG/SGGL antibodies in the pathogenesis of neuropathy remains unclear. In order to induce an animal model of neuropathy associated with anti-MAG/SGGL antibodies, cats were immunized with sulfoglucuronyl paragloboside (SGPG). All four cats immunized with SGPG developed clinical signs of sensory neuronopathy within 11 months after initial immunization, characterized by unsteadiness, falling, hind limb weakness and ataxia. In two cats the ataxia and hind limb paralysis were so severe that the animals had to be euthanized. Pathological examination revealed sensory ganglionitis with inflammatory infiltrates in the dorsal root ganglia. No overt signs of pathology were noted in the examined roots or nerves. High titer anti-SGPG/MAG antibodies were detected in all 4 cats immunized with SGPG but not in 3 control cats. Our data demonstrate that immunization of cats with SGPG induced anti-SGPG antibodies and sensory neuronopathy clinically resembling the sensory ataxia of patients with monoclonal IgM anti-MAG/SGPG antibodies. This study suggests that these anti-MAG/SGPG antibodies play a role in the pathogenesis of this neuropathy.

Glycopeptide fractions prepared from purified central and peripheral rat myelin.

Myelin was purified from rat brain and sciatic nerve after in vivo labeling with [3H]fucose and [14C]glucosamine to provide a radioactive marker for glycoproteins. The glycoproteins in the isolated myelin were digested exhaustively with pronase, and glycopeptides were isolated from the digest by gel filtration on Bio-Gel P-10. The glycopeptides from brain myelin separated into large and small molecular weight fractions, whereas the glycopeptides of sciatic nerve myelin eluted as a single symmetrical peak. The large and small glycopeptide fractions from central myelin and the single glycopeptide fraction from peripheral myelin were analyzed for carbohydrate by colorimetric and gas liquid chromatographic techniques. The glycopeptides from brain myelin contained 2.4 micrograms of neutral sugar and 0.59 micrograms of sialic acid per mg total myelin protein, whereas sciatic nerve myelin glycopeptides contained 10 micrograms of neutral sugar and 3.8 micrograms of sialic acid per mg total protein. Similarly, the gas-liquid chromatographic analyses showed that the glycopeptides from peripheral myelin contained 4- to 7-fold more of each individual per mg total myelin protein than those from central myelin. Most of the sialic acid and galactose in the glycopeptides from central myelin were in the large molecular weight fraction, and the small molecular weight glycopeptides contained primarily mannose and N-acetylglucosamine. The considerably higher content of glycoprotein-carbohydrate in peripheral myelin supports the results of gel electrophoretic studies, which indicate that the major protein in peripheral myelin is glycosylated while the glycoproteins in purified central myelin are quantitatively minor components.

[35-S]sulfate incorporation into myelin glycoproteinsmi=entral nervous system.

The in vivo incorporation of [35-S]sulfate and [3H]fucose into rat brain myelin was investigatedmmost of the 35S in the myelin was in sulfatide, but about 4% was associated with the residual proteins after chloroform/methanol extraction. Polyacrylamide gel electrophoresis of these proteins indicated that the major 35-S-labeled component corresponded to the major fucose-labeled glycoproteinmthe labeling of this predominant glycoprotein with sulfate was more selective than with fucose, since there was relatively little incorporation of sulfate into some of the minor fucose-labeled glycoproteins. There was little or no 35-S associated with proteolipid or basic protein on polyacrylamide gels. The fucose-labeled glycoproteins were converted to glycopeptides by pronase digestion and separated into two major classes by gel filtration on Sephadex-G-50. Only the higher molecular weight class contained significant amounts of 35-S. The association of 35-S with the glycopeptides was not due to binding of sulfatide or free inorganic sulfate. The results indicate that the predominant myelin-associated glycoprotein in rat brain is sulfated.

[35-S]sulfate incorporation into myelin clycoproteins; II. Peripheral nervous tissue.

The in vivo incorporation of [35-S]sulfate, [3-H]fucose and [3-H]leucine into sciatic nerve myelin was investigatedmpolyacrylamide gel electrophoresis of thr proteins indicated that the 35-S-labeling of proteins occurred almost exclusively in the major myelin protein; A smaller myelin glycoprotein migrating just ahead of the major one was labeled with [3-H]fucose but did not incorporate 35-S to a detectable extent. There was little or no 35-S associated with basic proteins on polyacrylamide gels when the proteins were extracted with chloroform/methanol; Fucose-labeled myelin glycoproteins were converted to glycopeptides by pronase digestion; The glycopeptides gave a single peak on tsephadex G-50 in which the 3-H and 35-S coincided. The association of 35-S with glycopeptides was not caused by binding of sulfatide or free inorganic sulfate. This study shows that the major myelin protein in the sciatic nerve of the rat is glycosylated and sulfated.

Purification and partial characterization of the myelin-associated glycoprotein from adult rat brain.

The myelin-associated glycoprotein was purified from rat central nervous system myelin by selective extraction with lithium diiodosalicylate-phenol followed by gel filtration on a column of Sepharose CL-6B. Amino acid analysis of the purified glycoprotein revealed an excess of acidic over basic amino acids and a relatively high content of nonpolar residues. On the basis of weight, the molecule is about one-third carbohydrate consisting of 5% fucose, 23% mannose, 20% galactose, 34% N-acetylglucosamine, and 18% N-acetylneuraminic acid.

Hypomyelination in copper-deficient rats. Prenatal and postnatal copper replacement.

Copper deficiency induced by a low copper diet in three generations of rats was associated with substantial reductions in the yield of myelin (56%), brain weight (11%), and body weight (43%) in F2 generation rat pups nursed by their own copper-deficient mothers. The composition of the purified myelin was not different from that of controls in the content of individual proteins, lipids, 2',3'-cyclic nucleotide 3'-phosphohydrolase (CNP) activity, or GM1 ganglioside. The major myelin-associated glycoprotein (mGP) was consistently shifted slightly toward higher apparent molecular weight in the copper-deficient animals. Postnatal copper replacement by a foster mother produced a normal yield of myelin per gram of brain tissue, but failed to reverse the deficiency of brain and body growth. After copper replacement in a copper-deficient mother's diet prior to conception, a subsequent litter showed correction of all abnormalities found in her previous litters. The results suggest that copper is essential for myelin formation and general growth during critical periods in development.

Molecular mimicry in chronic inflammatory demyelinating polyneuropathy and melanoma.

Polyclonal immunoglobulin M antibodies to the monosialoganglioside GM2, sulfoglucuronyl glycolipids, and sulfatide were detected by thin-layer chromatography and enzyme-linked immunosorbent assay in the serum of a patient with melanoma and chronic inflammatory demyelinating polyneuropathy. Both the patient's serum and polyclonal antibodies against GM2 reacted strongly with a biopsy of melanomatous tissue from the patient, suggesting a process of molecular mimicry.

Immunocytochemical localization of the myelin-associated glycoprotein. Fact or artifact?

Immune staining for the myelin-associated glycoprotein (MAG) by the peroxidase-antiperoxidase technique in vibratome, paraffin and Epon sections of peripheral nerve resulted in a periaxonal ring of immune reaction product and an absence of staining over compact myelin. The thickness of the periaxonal rings of staining in Epon sections were similar when axons were surrounded by single Schwann cell processes and when they were surrounded by compact myelin sheaths. Thicker rings of periaxonal staining were present when axons were surrounded by multiple layers of uncompacted Schwann cell membranes. When swelling of the Schwann cell periaxonal cytoplasm separated the periaxonal Schwann cell membrane from compact myelin by distances that could be readily resolved in the light microscope, immune staining for MAG was found over the periaxonal Schwann cell membrane but not over compact myelin. Biochemical experiments in which myelin or MAG was treated with sodium ethoxide and hydrogen peroxide, under conditions similar to those used for etching Epon sections prior to immune staining, showed that the immune reactivity of MAG was stable to these treatments. Finally, a pre-embedding technique for immune staining at the electron-microscopic level showed MAG reaction product on the cytoplasmic side of Schwann cell periaxonal membranes and the membranes of Schmidt-Lanterman incisures and paranodal loops. These results confirm and strengthen the evidence from previous reports indicating that MAG is confined to periaxonal membranes, Schmidt-Lanterman incisures, paranodal loops, and the outer mesaxon of myelinating Schwann cells and is absent from compact myelin. These results are discussed in reference to a recent report apparently showing the presence of MAG in compact CNS myelin. Based on the data presented and discussed in this paper, it is concluded that MAG is located in specific non-compact regions of central and peripheral myelin sheaths and not in compact myelin lamellae.

Multiple antibodies to nerve glycoconjugates in a patient with neuropathy and monoclonal IgA gammopathy.

Several immunological abnormalities were found in a patient with mixed axonal and demyelinating neuropathy in association with a prominent monoclonal IgA lamda band in his serum. He had antibodies to the major LM1 ganglioside of human nerve that were restricted to the IgA class, but they were polyclonal and distinct from the major monoclonal IgA component. He also had a low level of a monoclonal IgM lamda antibody to the myelin-associated glycoprotein and sulfate-3-glucuronyl paragloboside that was not detected by routine immunofixation. The observations made on this complex patient, are presented in the context of emphasizing that immunological reactivities to neural antigens that are not due to the principal monoclonal antibody may be present in patients with neuropathy in association with gammopathy.

The species distribution of nervous system antigens that react with anti-myelin-associated glycoprotein antibodies.

The reactivity of monoclonal and polyclonal antibodies directed against human central nervous system (CNS) myelin-associated glycoprotein (MAG) was investigated in a number of animal species. The antibodies included mouse monoclonal antibodies obtained by immunization with human MAG; HNK-1, a mouse monoclonal antibody raised against a human lymphoblastoma and used to identify a subset of lymphocytes with natural killer function; human IgM paraproteins associated with neuropathy; and polyclonal antibodies obtained from rabbits immunized with rat or human MAG. Following polyacrylamide gel electrophoresis of CNS and peripheral nervous system (PNS) tissue from human, bovine, cat, rabbit, guinea pig, rat, mouse, frog, gold fish and chicken, proteins were electrophoretically transferred onto nitrocellulose. The immune-staining of electroblots showed distinct interspecies variation in the reactivity of the antibodies with MAG. In addition, the species distribution of several low molecular weight glycoproteins present in PNS tissue that cross-react with anti-MAG antibodies was determined. These low molecular weight antigens are not present in CNS homogenates or in purified human CNS myelin. It was also shown that IgM from a patient with peripheral neuropathy and paraproteinemia associated with anti-MAG antibodies recognized these low molecular weight antigens. The results suggest that IgM paraproteins, HNK-1 and some mouse monoclonal antibodies react with carbohydrate determinants shared by MAG and several lower molecular weight glycoproteins present only in human, bovine, cat and chicken PNS. Rabbit polyclonal anti-rat MAG antisera and mouse monoclonal antibodies reacting with peptide epitopes of MAG are much more specific for detecting MAG than antibodies reacting with carbohydrate epitopes of human MAG. The results are discussed in relation to human demyelinating peripheral neuropathy associated with IgM paraproteinemia.

Variability in the binding of anti-MAG and anti-SGPG antibodies to target antigens in demyelinating neuropathy and IgM paraproteinemia.

Densitometry of immunostained Western blots or thin layer chromatograms and enzyme-linked immunosorbent assays (ELISAs) were used to compare the relative strengths of IgM binding to myelin-associated glycoprotein (MAG), P0 glycoprotein, peripheral myelin protein-22 (PMP-22), sulfate-3-glucuronyl paragloboside (SGPG), and other potential target antigens in a series of eleven patients with sensory or sensorimotor demyelinating neuropathy and IgM paraproteinemia. The IgM from all patients exhibited reactivity with both MAG and SGPG, and there was a statistically significant correlation between the overlay assays and ELISAs for measuring the strength of IgM binding to MAG and to SGPG. However, the data revealed variations in the relative strengths with which the antibodies bound to the potential target antigens and heterogeneity in their fine specificities. First, there was a poor correlation between the strength of binding to MAG and to SGPG, respectively. Second, reactivity with MAG or SGPG in a few of the patients was only detected by one of the two assay systems. Third, about one-third of the patients' IgM absolutely required the sulfate on SGPG for reactivity, whereas the others retained some reactivity after removal of the sulfate. Fourth, IgM from two of the patients exhibited unusually strong reactivity with the proteins of compact myelin, P0 and PMP22. These relative differences in strengths of antibody binding to the potential antigens were compared with the patients' clinical presentations and with their responses to intravenous immunoglobulin (IVIg) therapy in a clinical trial in which they participated. For the most part, these variations did not correlate with clinical presentation, which was relatively homogeneous in this series of patients. However, an inverse relationship was noted between degree of reactivity to MAG by ELISA and response to IVIg. Two of the patients who responded had only mild elevations of IgM antibodies to nerve glycoconjugates and exhibited some unusual immunochemical and clinical characteristics in comparison to the other patients. The results demonstrate differences in the relative strengths with which anti-MAG and anti-SGPG IgM antibodies from different patients bind to potential neural target antigens which may affect pathogenic mechanisms and response to therapy.

Glycoconjugates in nervous tissue and small cell lung cancer share immunologically cross-reactive carbohydrate determinants.

Small cell lung cancer (SCLC) is a bronchogenic carcinoma of neuroectodermal origin that expresses a variety of nervous system markers characteristic of neuroendocrine cells. In addition, SCLC cell lines and biopsies have been shown immunocytochemically to express an antigen recognized by HNK-1, a mouse monoclonal antibody which recognizes a surface antigen on natural killer cells and on the myelin-associated glycoprotein (MAG) and other nervous system glycoconjugates. Immunoblot data are presented which identify 2 groups of HNK-1-reactive plasma membrane glycoproteins with Mrs of about 80 000 and 130 000, respectively, from several SCLC cell lines. Using antibodies to MAG carbohydrate and protein determinants as probes, it is shown that the SCLC glycoproteins reacting with HNK-1 do not appear to share structural similarity with MAG apart from carbohydrate determinants. Using similar techniques with a panel of polyclonal antibodies, data are shown indicating that there is no cross-reactivity of SCLC proteins with other myelin proteins including P0, P1, P2, proteolipid protein and myelin basic protein. A possible role of the carbohydrate antigen in mediating nervous system disease associated with SCLC is suggested.

Specificity of human IgM monoclonal antibodies from patients with peripheral neuropathy.

Some patients with peripheral neuropathy and gammopathy have IgM monoclonal antibodies that react with the myelin-associated glycoprotein (MAG), some 20-26 kDa glycoproteins present only in the peripheral nervous system (PNS), and some acidic glycolipids that are also PNS-specific. This communication describes an investigation of 18 patients with IgM paraproteinemia and neuropathy to test for the presence of antibodies that react with each of these components. Eleven patients had IgM that reacted with MAG, and in all cases the IgM also reacted with the lower Mr glycoproteins and the acidic glycolipids that are specific for the PNS. With respect to the other 7 patients that did not react with MAG, in no instance did immune-staining of electroblots reveal the presence of reactivity with the 20-26 kDa glycoproteins of the PNS or with any other protein antigen in the PNS or central nervous system (CNS). However, these 7 patients fell into 3 categories with regard to reactivity with acidic glycolipids: three reacted with the acidic glycolipid fraction of both PNS and CNS tissue; two reacted with the acidic glycolipid fraction of the PNS but not the CNS; and two showed no reactivity with the acidic glycolipids from either PNS or CNS.

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.