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.     

Molecular specificity of L2 monoclonal antibodies that bind to carbohydrate determinants of neural cell adhesion molecules and their resemblance to other monoclonal antibodies recognizing the myelin-associated glycoprotein

L2 monoclonal antibodies and HNK-1 have been shown to bind to related carbohydrate determinants in the myelin-associated glycoprotein (MAG) and several adhesion molecules of the nervous system including neural cell adhesion molecule (N-CAM), L1 and J1. It is shown here that MAG is the principal component in human white matter binding the L2 antibodies, but the most prominent antigens with the L2 epitopes in human gray matter are of higher Mr. It is also shown that the L2 antibodies resemble HNK-1 in binding to some 19-28 kDa glycoproteins and some sulfated, glucuronic acid-containing sphingoglycolipids of the peripheral nervous system (PNS). In addition, monoclonal and polyclonal antibodies raised to human MAG are shown to cross react with bovine N-CAM due to the presence of common carbohydrate constituents. The results further emphasize the shared antigenicity between MAG, N-CAM and other adhesion molecules. In addition, they demonstrate that the L2 antibodies belong to a family of monoclonal antibodies (including HNK-1, human IgM paraproteins associated with neuropathy, and others) that are characterized by reactivity against carbohydrate determinants shared by human MAG, the 19-28 kDa glycoproteins of the PNS and the sulfated, glucuronic acid-containing sphingoglycolipids of the PNS.     

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.     

Characterization of oligosaccharides that bind to human anti-MAG antibodies and to the mouse monoclonal antibody HNK-1

In some patients with neuropathy and IgM M-proteins the M-proteins bind to a carbohydrate determinant that is shared by the CNS and PNS myelin-associated glycoprotein (MAG) and by several additional glycoproteins and 2 glycolipids in peripheral nerve. The HNK-1 mouse monoclonal antibody binds to the same glycoproteins and glycolipids as well as to a number of other neuronal adhesion molecules and to human natural killer cells. To isolate the epitope-bearing oligosaccharides from their respective glycoproteins we digested delipidated spinal cord and peripheral nerve with pronase. The resulting glycopeptides were fractionated by concanavalin A-Sepharose chromatography to yield tri- and tetraantennary-complex, biantennary-complex and high mannose-type glycopeptides. Glycopeptides bearing the antigenic determinant were identified by their ability to block binding of M-proteins and HNK-1 antibodies to MAG-coated microwells by enzyme-linked immunosorbent assay (ELISA). Blocking activity was detected in the tri- and tetraantennary glycopeptide fraction from both CNS and PNS. The blocking activity was destroyed by pretreatment of the isolated glycopeptides with mild acid hydrolysis. Further fractionation by gel filtration chromatography indicated that the reactive glycopeptides from peripheral nerve and spinal cord eluted in the same position. The data suggest that CNS and PNS MAG and other peripheral nerve glycoproteins share similar oligosaccharides, and that the M-proteins and HNK-1 bind to the same structures.     

Sulfated glucuronyl glycolipids reacting with anti-myelin-associated glycoprotein monoclonal antibodies including IgM paraproteins in neuropathy: species distribution and partial characterization of epitopes

It was recently established that anti-myelin associated glycoprotein (MAG) IgM paraproteins associated with neuropathy and a substantial number of experimentally produced rat and mouse monoclonal antibodies that react with MAG (e.g. HNK-1) also bind to some sulfated glucuronic acid-containing sphingoglycolipids of human peripheral nerve. A species study revealed that these glycolipids could be detected readily by TLC overlay experiments in the acidic glycolipid fractions from human, monkey, bovine, cat and dog peripheral nerve. The glycolipids were also present in the nerves of rat, mouse, rabbit, guinea pig and chicken, but their concentration was about an order of magnitude lower. These antigenic glycolipids were present in the purified myelin fraction from cat nerve, but their level was not enriched over that in whole homogenate. Partial characterization of the epitopes in the glycolipids was accomplished by comparing binding of the human and experimental monoclonal antibodies to sulfated glucuronyl paragloboside (SGPG), to the desulfated lipid (GPG), and to the methyl ester of the desulfated lipid (MeGPG). All of the human, mouse and rat antibodies reacted with the intact SGPG, but none exhibited binding to MeGPG indicating that either the sulfate or the free carboxyl group on SGPG was required for reactivity. Five out of 11 human IgM paraproteins retained partial and variable reactivity with GPG showing that the sulfate was not absolutely required for binding, while the other 6 did not react with GPG. These results demonstrate idiotypic heterogeneity among the IgM paraproteins. Only 1 of 14 monoclonal antibodies produced experimentally in mice or rats retained reactivity with GPG.(ABSTRACT TRUNCATED AT 250 WORDS)     

Myelin-associated glycoprotein (MAG) distribution in human central nervous tissue studied immunocytochemically with monoclonal antibody

Recent biochemical data show that myelin-associated glycoprotein (MAG) is the antigen for a monoclonal antibody found in sera of patients with IgM paraproteinemia and neuropathy (Braun et al. 1982). Immunoreactivity of this antibody with CNS has not been described. To study this, monoclonal anti-MAG was used in the avidin-biotin-peroxidase complex method (Hsu et al. 1981) to immunostain paraffin and epon sections of human CNS. Well characterized polyclonal MAG antiserum (Quarles et al. 1981) was employed in comparison tests. In paraffin sections of developing CNS, both monoclonal and polyclonal MAG antisera stained oligodendroglia and myelin. In adult CNS, periaxonal regions of myelin sheaths were immunostained in paraffin sections and semithin epon sections treated with monoclonal and polyclonal anti-MAG. In electron-microscopic experiments that included milder pretreatment of epon thin sections and more precise reaction product localization, entire thickness of myelin sheaths were immunostained. Thus, in electron micrographs, monoclonal and polyclonal anti-MAG immunoreactivity also have the same localization. In other electron-microscopic experiments, the same reaction product localization was observed with antiserum to myelin basic protein (MBP), a known constituent of compact myelin. Thus, results with this monoclonal anti-MAG provide important new evidence to support the localization of MAG in compact CNS myelin. Our data also suggest that monoclonal antibodies against MAG will be useful in studies of the pathogenesis of multiple sclerosis and other demyelinating diseases.     

The human anti-myelin-associated glycoprotein IgM system

Two mouse monoclonal antiidiotypic antibodies that react with human monoclonal IgM antibodies with specificity for myelin-associated glycoprotein (MAG) have been used to study the immunological specificity of the reported cross-reactions involving the anti-MAG IgM. Both of the antiidiotypic antibodies are shown to react with the combining site of their respective idiotypic IgM and to inhibit the reaction between IgM and MAG. Using these antiidiotypic antibodies as "surrogate" antigen, we have demonstrated immune cross-reactivity between MAG, a human peripheral nerve glycolipid, and a low-molecular-weight protein of human peripheral nerve myelin. In addition, we have used the two antiidiotypic antibodies to conduct a search for evidence of shared idiotypy among 34 different neuropathy-associated paraproteins. Our results provide no evidence for a neuropathy-associated idiotype, suggesting a degree of polymorphism in the human anti-MAG IgM system.     

Anti-MAG IgM paraproteins from some patients with polyneuropathy associated with IgM paraproteinemia also react with sulfatide

Sera from five of 11 patients with neuropathy associated with IgM paraproteinemia (NAIgMPP) and reactivity against myelin-associated glycoprotein (MAG) also had elevated levels of IgM against sulfatide. Although three patients had anti-sulfatide IgM titers of less than or equal to 1:1000, two patients had titers of greater than or equal to 1:50,000. Absorption of patient serum with sulfatide revealed that anti-MAG IgM paraproteins in two patients with high titer anti-sulfatide IgM crossreacted with sulfatide. Our study is the first to show that some anti-MAG IgM paraproteins crossreact with sulfatide, a major acidic glycolipid of myelin. Moreover, some patients with NAIgMPP have polyclonal anti-sulfatide IgM in addition to anti-MAG IgM paraproteins. Therefore, sulfatide may be a target antigen in some patients with NAIgMPP.     

Anti-myelin associated glycoprotein antibodies recognize HNK-1 epitope on CNS

Antibodies to myelin-associated glycoprotein (MAG) are associated with demyelinating polyneuropathy and are specific for the HNK-1 epitope. To test if anti-MAG IgM recognize HNK-1 on CNS, sera from 20 patients and 238 controls were tested on rat slices by indirect immunofluorescence (IIF). IgM from anti-MAG positive patients, but not from control sera, stained rat brain with perineuronal or neuropil pattern, depending on the CNS region. IIF titers significantly correlated with ELISA anti-MAG titers. The staining of patients' sera were inhibited by mouse anti-HNK-1 monoclonal antibody. Our results demonstrate that anti-MAG IgM recognizes HNK-1 outside the peripheral nerve myelin carriers.     

Analysis of the feline immune response to human myelin-associated glycoprotein

Elucidation of the pathogenesis of demyelinating peripheral neuropathy associated with myelin-associated glycoprotein (MAG) binding IgM paraproteins requires an in vivo animal model of the syndrome. Multiple immunizations of cats with MAG in Freund's adjuvant did not produce an antibody response but four immunizations with MAG-iscom (Morein, B. et al. (1984) Nature, 308: 457-460) did induce IgM antibodies which bound to human MAG and cat peripheral nerve myelin. Despite the presence of antibody for a 13-month period, no neuropathy developed. At necropsy, the peripheral nerves were ultrastructurally normal and no antibody was detectable in the endoneurium. A competitive ELISA indicated that the cat and human IgM antibodies recognized different epitopes.     

Characterization of HNK-1 bearing glycoproteins in human peripheral nerve myelin.

The HNK-1 carbohydrate epitope, which is shared by several members of the immunoglobulin gene super-family, is also the target epitope for IgM anti-MAG autoantibodies in patients with demyelinating neuropathy. By Western blot analysis, there are 7 HNK-1 immunoreactive glycoproteins in human peripheral nerve myelin, two of which have previously been identified as the myelin associated glycoprotein (MAG) and the P0 glycoprotein. In this study, the remaining HNK-1 bearing glycoprotein bands were characterized by immunoblot and NH2-terminal sequence analysis, and were all identified as degradation products or aggregates of the Po glycoprotein. MAG and P0 are therefore the only HNK-1 bearing glycoproteins in human peripheral nerve myelin.     

Human monoclonal IgM autoantibodies with restricted antigenic specificity for myelin express unrelated idiotypes

Idiotype-specific polyclonal antisera were prepared against myelin-binding human IgM paraproteins with specificity for the myelin-associated glycoprotein (MAG). Eight anti-idiotypic sera (6 against one monoclonal IgM and 2 against another) were tested by gel precipitation, immunoradiometric and ELISA assays for binding to the myelin-binding IgM paraproteins from 12 patients with demyelinating peripheral neuropathy. Anti-idiotypic antibodies bound only to the MAG-specific IgM used for immunization; there was no evidence of idiotypic cross-reactivity between the different IgM paraproteins.     

Microheterogeneity of anti-myelin-associated glycoprotein antibodies

Antibodies to the myelin-associated glycoprotein (MAG) are implicated in the pathogenesis of an acquired demyelinating polyneuropathy. We studied IgM affinity to MAG in 18 patients with anti-MAG antibodies. Binding of sera was tested for anti-MAG immunoreactivity in central nervous system (CNS) by ELISA and in CNS and peripheral nervous system (PNS) by Western blot analysis. Furthermore, immunohistochemical characterization of IgM binding on sural nerve tissue was investigated using the indirect peroxidase method. Western blot analysis revealed that all sera detected MAG in central myelin, but only eight in peripheral myelin. Anti-MAG-IgM-ELISA-titers correlated significantly (p<0.0001) with PNS-Western blot results. By indirect immunoperoxidase immunohistochemistry, 12 sera stained myelin sheaths, while 6 sera showed no staining. These results demonstrate considerable variations in antibody binding strength to MAG between PNS myelin and CNS myelin. The relevance of these differences for the pathogenesis of the neuropathy and clinical impairment remains to be demonstrated.     

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.     

Neuropathy and anti-MAG antibodies without detectable serum M-protein

Anti-MAG IgM antibodies were detected by ELISA in a patient with slowly progressive peripheral neuropathy. Serum IgM content was normal, and no M-protein was detected by serum protein electrophoresis, immunoelectrophoresis, or immunostaining. By immunoblot analysis, the anti-MAG antibodies were IgMk; they reacted with human and bovine MAG but not with mouse MAG. The data suggest that there was an anti-MAG IgM M-protein in concentration too low to be detected by conventional techniques. Tests for anti-MAG antibodies should be done in patients with slowly progressive neuropathy of unknown etiology, even in the absence of detectable serum M-protein.     

Cytomegalovirus is not associated with IgM anti-myelin-associated glycoprotein/sulphate-3-glucuronyl paragloboside antibody-associated neuropathy.

Monoclonal antibodies reactive with the HNK-1 epitope of myelin-associated glycoprotein (MAG) and the sulphate-3-glucuronyl paragloboside (SGPG)-like glycolipids are often found in the serum of patients with IgM paraprotein-associated demyelinating neuropathy. The presence of such antibodies in patients with chronic polyneuropathy has recently been associated with evidence of active cytomegalovirus (CMV) infection by the polymerase chain reaction. We wished to test these findings and examined sera from patients with MAG-reactive or MAG-nonreactive paraproteinemic neuropathy and patients with paraproteinemia only for the presence of CMV DNA and anti-CMV antibodies. CMV DNA was not detected in sera from any patient group. Furthermore, anti-CMV antibody prevalence was normal and similar in all 3 groups. We therefore report no evidence of an association between CMV infection and anti-MAG/SGPG antibodies associated with paraproteinemic peripheral neuropathy.     

Confocal microscopic localization of anti-myelin-associated glycoprotein autoantibodies in a patient with peripheral neuropathy initially lacking a detectable IgM gammopathy

We report here on a patient with anti-myelin-associated glycoprotein (MAG) neuropathy in whom examination of a sural nerve biopsy by multichannel confocal microscopy showed a partly overlapping distribution of MAG and IgM deposits in myelinated fibers. Our data demonstrate that MAG in Schmidt-Lanterman incisures and paranodal loops, as well as some additional HNK-1-positive components of the basal lamina, are the major targets of the anti-MAG monoclonal IgM autoantibodies in this neuropathy in vivo. Perforation of the basal lamina can allow the penetration and binding of anti-MAG IgM inside myelinated fibers. Our results support and extend the notion that the production of monoclonal anti-MAG IgM may be antigenically driven by MAG molecules and that this process may occur in the immunologically privileged environment of the nerve prior to the appearance of a genuine gammopathy in serum. Received: 12 May 1997 / Revised: 13 August 1997 / Revised, accepted: 19 November 1997     

Reactivity with the peripheral myelin glycoprotein P0 in serum from patients with monoclonal IgM gammopathy and polyneuropathy

The major glycoprotein P0 from human and bovine peripheral nerves carries the L2/HNK-1 and L3 carbohydrate epitopes and is recognized by serum from patients with IgM gammopathy and polyneuropathy. Only serum from patients with reactivity toward the myelin-associated glycoprotein (MAG) was reactive with P0, while serum that did not react with MAG also did not recognize P0. Furthermore, the neural adhesion molecules L1, N-CAM, and J1 were also recognized by the serum that reacted with MAG, while the L3 carbohydrate-carrying cell adhesion molecule AMOG was not recognized. These observations indicate a restricted specificity in carbohydrate reactivity of IgM paraproteins and implicate yet another and, for the first time, peripheral myelin-specific glycoprotein in the pathogenesis of demyelinating neuropathy.     

Polyneuropathy attributes: a comparison between patients with anti-MAG and anti-sulfatide antibodies

Thirty-two patients with a peripheral neuropathy and paraproteinemia were tested for IgM antibodies against myelin-associated protein (MAG) and sulfatide by means of enzyme-linked immunosorbent assay. Nine patients (28%) had increased anti-sulfatide IgM antibodies and showed a chronic, slowly progressive, distally pronounced, and symmetric polyneuropathy with sensory to sensory-motor impairment, ataxia, hyporeflexia, and axonal involvement in electrophysiological studies. Ten patients (31%) with increased anti-MAG antibodies had a similar, homogeneous polyneuropathy syndrome but presented with demyelinating features. A weak crossreactivity between anti-MAG and anti-sulfatide antibodies was present in only three patients. In conclusion, although the two neuropathy groups clearly differed in their electrophysiological features, their clinical presentation was rather similar. Received: 19 July 1999 / Received in revised form: 23 December 1999 / Accepted: 2 May 2000     

The neuropathy of plasma cell dyscrasia: binding of IgM M-proteins to peripheral nerve glycolipids

In some patients with neuropathy and plasma cell dyscrasia, the M-proteins bind to peripheral nerves. Binding of M-proteins to peripheral nerve glycolipids was examined by immunostaining after thin-layer chromatography. The IgM from 16 patients with anti-MAG M-proteins bound to the same two glycolipid bands in peripheral nerve. The IgM that bound to the glycolipids had the same idiotype as the anti-MAG M-protein, indicating that it was the M-protein that bound to both glycolipids. The reactive glycolipids did not contain sialic acid and were not gangliosides. No immunostaining of peripheral nerve glycolipids was observed with IgM from patients with neuropathy and IgM M-proteins that did not bind to MAG, and the anti-MAG antibodies did not bind to brain glycolipids. Anti-MAG M-proteins probably bind to the same or closely related carbohydrate determinants that are shared by a number of glycoproteins and glycolipids of peripheral nerve.