N-Gly col ylneuraminic Acid-Containing GM 1 zyxw - Is a New Molecule for Serum Antibody in Guillain-Bard Syndrome zyxw J Masaaki Odaka, MD,* Nobuhiro Yuki, MD, PhD,* Hiide Yoshino, MD, PhD,? Takeshi Kasama, PhD,$ Shizuo Handa, MD, PhD,O Fumitoshi Irie, PhD,? Yoshio Hirabayashi, PhD,? Akemi Suzuki, MD, PhD,II Koichi Hirata, MD, PhD* To clarify the pathogenesis of Guillain-Barrt syndrome (GBS) after parented injections of bovine brain gan- gliosides, we searched for new molecules in bovine brain gangliosides recognized by sera from GBS patients. Gangliosides fractionated in a Q-Sepharose column were used zyxwvutsr as the antigens, and the binding of serum IgG or IgM was examined by thin-layer chromatography/im- munostaining. Fourteen of 175 serum samples from the patients reacted with the monosialoganglioside fraction 2. In the neutral solvent system, a band in this frac- tion migrated with N-acetylneuraminic acid-containing GM1 [GMl(NeuAc)], whereas in the alkaline solvent system it migrated slower. This suggested that the band was N-glycolylneuraminic acid-containing GM1 [GMl(NeuGc)]. In both solvent systems, its mobility was almost the same as that of authentic GMl(NeuGc) from mouse liver. Secondary ion mass spectrometry showed that the ganglioside's structure was consistent with that of GMl(NeuGc). IgG anti-GMl(NeuGc) anti- bodies in sera from the GBS patients were significantly absorbed by GMl(NeuAc), indicative that the anti- GMl (NeuGc) antibodies cross-react with GM1(NeuAc). N-Glycolylneuraminic acid-containing gangliosides are zyxwvu so highly immunogenic in humans that the injec- tion of GMl(NeuGc) could induce the production of zyxwvu I& anti-GM1 (NeuGc) antibody, which cross-reacts with GM1(NeuAc) . From the *Department of Neurology, Dokkyo Universiry School of Medicine, Tochigi; and ?Department of Neurology, Kohnodai Hos- pital, National Center of Neurology and Psychiatry, Chiba; SInstru- mental Analysis Research Center for Life Science, and SDeparrment of Biochemistry, Faculty of Medicine, Tokyo Medical and Dental University, Tokyo; SLaboratory for Cellular Glycobiology, Frontier Research Program, Institute of Physical and Chemical Research (RIKEN), Saitama; and "Metabolism Section, Tokyo Metropolitan Institute of Medical Science, Tokyo, Japan. Received Nov 14, 1997, and in revised form Jan 15, 1998. Ac- cepted for publication Jan 16, 1998. Address correspondence to Dr Yuki, Department of Neurology, Dokkyo University School of Medicine, Kitakobayashi 880, Mibu, Shimotsuga, Tochigi 321-0293, Japan. Odaka M, Yuki N, Yoshino H, Kasama T, Handa S, hie F, Hirabayashi Y, Suzuki A, Hirata K. N-Glycolylneuraminic acid-containing GM1: a new molecule for serum antibody in Guillain-Bar& syndrome. Ann Neurol 1 9 9 8 4 3 ~ 2 9 - 834 Gangliosides extracted from bovine brain tissue have been widely administered to patients with various neu- rological disorders. Reports of patients who developed Guillain-Bard syndrome (GBS) after ganglioside ad- ministration have increased. ls2 Sera from patients with GBS frequently have antibodies that react with the ma- jor bovine brain gangliosides GM1, GM2, GDla, GDlb, GTlb, and GQlb.3-5 Some GBS patients har- bor serum antibodies to GalNAc-GDla or GMlb, mi- nor gangliosides in bovine We searched for other gangliosides in bovine brain ganglioside mixtures that would be recognized by serum antibodies from GBS patients. We then examined whether they could be target molecules, immunogens, ot both. Materials and Methods zyxw Thin-Layer zyxwv Chromatography/Imrnunostaining of Bovine Brain Gangliosides with Sera fiom GBS Patients Total bovine brain gangliosides were fractionated in a Q-Sepharose column into 13 fractions as described previ- 0us1y.'~ Each fraction was then separated by thin-layer chro- matography (TLC). Solvent system zyxw A chloroform/methanol/ 12 mM magnesium chloride in water (5:4:1, by volume) and solvent system B: chloroform/methanol/ 12 mM magne- sium chloride in wated2.5 M ammonia in water (50:40:7:3, by volume) were the developers. Fraction 1 contained N-acetyheuraminic acid-containing GM1 [GMl (NeuAc)]; fractions 3, 4, and 5, GDlb(NeuAc); fractions 6, 7, 8, 9, and 10, GDla(NeuAc); fractions 11 and 12, GTlb(NeuAc); and fraction 13, GQlb(NeuAc) (Table 1). Serum samples from 175 patients with GBS were ob- tained within 4 weeks of the onset of neurological symp- toms. All the patients met the criteria for GBS." Immuno- staining on the TLC plates was done as reported elsewhere.8 The plates were overlaid with the patients' sera (1:500 dilu- tion), followed by peroxidase-conjugated anti-human y-chain or p-chain specific antibodies (1 : 1,000 dilution; Dako, Glostrup, Denmark). Detection of N-Glycolylneurarninic Acid-Containing GMl [GMl (NeuGc)] in the Human Nervous System by Using Cholera Toxin and Anti-GMl (NeuGc) Antibody Anterior and posterior horns, as well as anterior and poste- rior roots, obtained from a human spinal cord, were dis- sected at autopsy. Each monosialoganglioside fraction was prepared as described previously. l2 The monosialoganglioside fractions were developed on a TLC plate with alkaline sol- vent system B. GMl(NeuAc) and GMl(NeuGc) were de- Copyright zyxwvu 0 1998 by the American Neurological Association zyx 829