Susumu Kusunoki

Structures of Sialylated O-Linked Oligosaccharides of Bovine Peripheral Nerve α-Dystroglycan THE ROLE OF A NOVEL O-MANNOSYL-TYPE OLIGOSACCHARIDE IN THE BINDING OF α-DYSTROGLYCAN WITH LAMININ

α-Dystroglycan is a heavily glycosylated protein, which is localized on the Schwann cell membrane as well as the sarcolemma, and links the transmembrane protein β-dystroglycan to laminin in the extracellular matrix. We have shown previously that sialidase treatment, but not N-glycanase treatment, of bovine peripheral nerve α-dystroglycan greatly reduces its binding activity to laminin, suggesting that the sialic acid of O-glycosidically-linked oligosaccharides may be essential for this binding. In this report, we analyzed the structures of the sialylated O-linked oligosaccharides of bovine peripheral nerve α-dystroglycan by two methods. O-Glycosidically-linked oligosaccharides were liberated by alkaline-borotritide treatment or by mild hydrazinolysis followed by 2-aminobenzamide-derivatization. Acidic fractions obtained by anion exchange column chromatography that eluted at a position corresponding to monosialylated oligosaccharides were converted to neutral oligosaccharides by exhaustive sialidase digestion. The sialidases from Arthrobacter ureafaciens and from Newcastle disease virus resulted in the same degree of hydrolysis. The neutral oligosaccharide fraction, thus obtained, gave a major peak with a mobility of 3.8-3.9 glucose units upon gel filtration, and its reducing terminus was identified as a mannose derivative. Based on the results of sequential exoglycosidase digestion, lectin column chromatography, and reversed-phase high-performance liquid chromatography, we concluded that the major sialylated O-glycosidically-linked oligosaccharide of the α-dystroglycan was a novel O-mannosyl-type oligosaccharide, the structure of which was Siaα2-3Galβ1-4GlcNAcβ1-2Man-Ser/Thr (where Sia is sialic acid). This oligosaccharide constituted at least 66% of the sialylated O-linked sugar chains. Furthermore, a laminin binding inhibition study suggested that the sialyl N-acetyllactosamine moiety of this sugar chain was involved in the interaction of the α-dystroglycan with laminin.

Ganglioside composition of the human cranial nerves, with special reference to pathophysiology of Miller Fisher syndrome.

Total ganglioside fractions from the human cranial nerves purified on a Phenyl Sepharose column, were given mild alkaline treatment, after which their composition and amounts of lipid-bound sialic acid were determined by HPTLC-densitometry with resorcinol as the coloring reagent. The total amounts of lipid-bound sialic acid were 156.5 ng/mg of wet tissue in the Ist cranial nerve (olfactory tract) and 131.9 ng/mg in the IInd nerve, greater than the amounts in the other nerves (99.1-120.0 ng/mg). The Ist, IInd, and VIIIth nerves had GM4, but not LM1. It may reflect their histological feature of the central nervous system. The IIIrd, IVth, and VIth nerves, as well as the IInd, had significantly higher percentages of GQ1b (11.6-13.2%) than the other nerves (5.2-8.4%). The high proportion of GQ1b specific to these three cranial nerves involved in the ocular movement lends support to the role of serum anti-GQ1b antibody in the pathogenetic mechanisms of ophthalmoplegia in Miller Fisher syndrome and Guillain-Barré syndrome.

Mutations in COQ2 in familial and sporadic multiple-system atrophy the multiple-system atrophy research collaboration

BACKGROUND Multiple-system atrophy is an intractable neurodegenerative disease characterized by autonomic failure in addition to various combinations of parkinsonism, cerebellar ataxia, and pyramidal dysfunction. Although multiple-system atrophy is widely considered to be a nongenetic disorder, we previously identified multiplex families with this disease, which indicates the involvement of genetic components. METHODS In combination with linkage analysis, we performed whole-genome sequencing of a sample obtained from a member of a multiplex family in whom multiple-system atrophy had been diagnosed on autopsy. We also performed mutational analysis of samples from members of five other multiplex families and from a Japanese series (363 patients and two sets of controls, one of 520 persons and one of 2383 persons), a European series (223 patients and 315 controls), and a North American series (172 patients and 294 controls). On the basis of these analyses, we used a yeast complementation assay and measured enzyme activity of parahydroxybenzoate-polyprenyl transferase. This enzyme is encoded by the gene COQ2 and is essential for the biosynthesis of coenzyme Q10. Levels of coenzyme Q10 in lymphoblastoid cells and brain tissue were measured on high-performance liquid chromatography. RESULTS We identified a homozygous mutation (M78V-V343A/M78V-V343A) and compound heterozygous mutations (R337X/V343A) in COQ2 in two multiplex families. Furthermore, we found that a common variant (V343A) and multiple rare variants in COQ2, all of which are functionally impaired, are associated with sporadic multiple-system atrophy. The V343A variant was exclusively observed in the Japanese population. CONCLUSIONS Functionally impaired variants of COQ2 were associated with an increased risk of multiple-system atrophy in multiplex families and patients with sporadic disease, providing evidence of a role of impaired COQ2 activities in the pathogenesis of this disease. (Funded by the Japan Society for the Promotion of Science and others.).

Ganglioside complexes as new target antigens in Guillain-Barré syndrome.

Antibodies specific for a complex of gangliosides GD1a and GD1b (GD1a/GD1b) were found in sera from eight of 100 patients with Guillain–Barré syndrome (GBS) by the use of enzyme‐linked immunosorbent assay and thin‐layer chromatogram immunostaining. Those sera also had antibody activities to such ganglioside complexes as GD1a/GM1, GD1b/GT1b, and GM1/GT1b but had little or no reactivity to the each isolated antigen. Clustered epitopes of the ganglioside complex in the plasma membrane may be targeted by such an antibody, and interaction between the antibody and ganglioside complex may induce the neuropathy. Ann Neurol 2004

Anti‐GQ1b IgG antibody is associated with ataxia as well as ophthalmoplegia

Close association between the increase in anti‐GQ1b immunoglobulin G (IgG) antibody and ophthalmoplegia in Miller Fisher syndrome (MFS) and Guillain–Barré syndrome (GBS) has been reported. We investigated whether anti‐GQ1b IgG antibody also is associated with ataxia, another of the MFS triad. Of 149 patients who had anti‐GQ1b IgG antibody without profound weakness, 144 showed ophthalmoplegia (120 showed both ophthalmoplegia and ataxia; 24, ophthalmoplegia without ataxia). In contrast, five showed ataxia without ophthalmoplegia. Some large neurons of the dorsal root ganglia were immunostained with anti‐GQ1b monoclonal antibody. Anti‐GQ1b IgG antibody may thus be associated with ataxia as well as ophthalmoplegia. Ataxia may be due to its binding to a subset of primary sensory neurons.

Efficacy of the anti–IL-6 receptor antibody tocilizumab in neuromyelitis optica A pilot study

Objective: To evaluate the safety and efficacy of a humanized anti–interleukin-6 receptor antibody, tocilizumab (TCZ), in patients with neuromyelitis optica (NMO). Methods: Seven patients with anti–aquaporin-4 antibody (AQP4-Ab)-positive NMO or NMO spectrum disorders were recruited on the basis of their limited responsiveness to their current treatment. They were given a monthly injection of TCZ (8 mg/kg) with their current therapy for a year. We evaluated the annualized relapse rate, the Expanded Disability Status Scale score, and numerical rating scales for neurogenic pain and fatigue. Serum levels of anti-AQP4-Ab were measured with AQP4-transfected cells. Results: Six females and one male with NMO were enrolled. After a year of TCZ treatment, the annualized relapse rate decreased from 2.9 ± 1.1 to 0.4 ± 0.8 (p < 0.005). The Expanded Disability Status Scale score, neuropathic pain, and general fatigue also declined significantly. The ameliorating effects on intractable pain exceeded expectations. Conclusion: Interleukin-6 receptor blockade is a promising therapeutic option for NMO. Classification of evidence: This study provides Class IV evidence that in patients with NMO, TCZ reduces relapse rate, neuropathic pain, and fatigue.

Anti-Gal-C antibodies in GBS subsequent to mycoplasma infection: Evidence of molecular mimicry

The authors previously reported the presence of antibody against galactocerebroside (Gal-C) in sera from patients with Guillain–Barré syndrome subsequent to Mycoplasma pneumoniae infection. Anti-Gal-C antibody activities in these sera were inhibited specifically by the M. pneumoniae reagent. A rabbit anti-Gal-C antibody recognized several glycolipids in M. pneumoniae. These data show that a Gal-C-like structure is present in M. pneumoniae, indicative of molecular mimicry between a major myelin glycolipid, Gal-C, and M. pneumoniae.

GM1b is a new member of antigen for serum antibody in Guillain-Barré syndrome.

Serum antibody from some patients with Guillain-Barre syndrome recognized an antigen of a minor component in human brain monosialoganglioside fraction. We purified that antigen, which migrated at a position slightly lower than that of GM1 on a thin-layer chromatogram (TLC), by using Iatrobeads column chromatography and preparative TLC. Structural analyses, including fast atom bombardment mass spectrometry, showed it to be GM1b. An enzyme-linked immunosorbent assay (ELISA) using purified GM1b showed that anti-GM1b antibody was present in 22 of 104 cases tested. No anti-GM1b antibody was present in the sera from control patients with other diseases or from the normal controls. Four sera recognized only GM1b among the 11 ganglioside antigens tested. The other 18 sera had antibodies to other antigens, most of which shared no terminal epitope with GM1b. Eight of nine sera samples with anti-GalNAc-GD1a antibody also had anti-GM1b antibody. Antibody to a minor monosialoganglioside, GM1b, was found to be a useful diagnostic marker for Guillain-Barre syndrome. Further study is needed to determine whether this antibody plays a role in the pathogenetic mechanism of the syndrome. NEUROLOGY 1996;47: 237-242

Anti-ganglioside complex antibodies associated with severe disability in GBS

Ganglioside complexes (GSCs) are known as target antigens in Guillain-Barré syndrome (GBS). To elucidate the clinical importance of the anti-GSC antibodies in GBS, we investigated serum antibodies to GSCs containing two of the gangliosides, GM1, GD1a, GD1b and GT1b, and analyzed clinical features of anti-GSC-positive GBS patients. Thirty-nine (17%) of 234 GBS patients had IgG anti-GSC antibodies. Anti-GSC-positive GBS had antecedent gastrointestinal infection and lower cranial nerve deficits more frequently than control GBS. The presence of antibody specificity to GD1a/GD1b and/or GD1b/GT1b was significantly associated with severe disability and a requirement for mechanical ventilation.

Anti-neurofascin antibody in patients with combined central and peripheral demyelination

Objectives: We aimed to identify the target antigens for combined central and peripheral demyelination (CCPD). Methods: We screened target antigens by immunohistochemistry and immunoblotting using peripheral nerve tissues to identify target antigens recognized by serum antibodies from selected CCPD and chronic inflammatory demyelinating polyradiculoneuropathy (CIDP) cases. We then measured the level of antibody to the relevant antigen in 7 patients with CCPD, 16 patients with CIDP, 20 patients with multiple sclerosis, 20 patients with Guillain-Barré syndrome, 21 patients with other neuropathies, and 23 healthy controls (HC) by ELISA and cell-based assays using HEK293 cells. Results: At the initial screening, sera from 2 patients with CCPD showed cross-like binding to sciatic nerve sections at fixed intervals, with nearly perfect colocalization with neurofascin immunostaining at the node and paranode. ELISA with recombinant neurofascin revealed significantly higher mean optical density values in the CCPD group than in other disease groups and HC. Anti-neurofascin antibody positivity rates were 86% in patients with CCPD, 10% in patients with multiple sclerosis, 25% in patients with CIDP, 15% in patients with Guillain-Barré syndrome, and 0% in patients with other neuropathies and HC. The cell-based assay detected serum anti-neurofascin antibody in 5 of 7 patients with CCPD; all others were negative. CSF samples examined from 2 patients with CCPD were both positive. In anti-neurofascin antibody–positive CCPD patients, including those with a limited response to corticosteroids, IV immunoglobulin or plasma exchange alleviated the symptoms. Conclusion: Anti-neurofascin antibody is frequently present in patients with CCPD. Recognition of this antibody may be important, because patients with CCPD who are antibody positive respond well to IV immunoglobulin or plasma exchange.