title: Ganglioside contained in the neuronal tissue-enriched acidic protein of 22 kDa (NAP-22) fraction prepared from the detergent-resistant membrane microdomain of rat brain inhibits the phosphatase activity of calcineurin.
abstract: Neurons have well-developed membrane microdomains called "rafts" that are recovered as a detergent-resistant membrane microdomain fraction (DRM). Neuronal tissue-enriched acidic protein of 22 kDa (NAP-22) is one of the major protein components of neuronal DRM. To determine the cellular function of NAP-22, interacting proteins were screened with an immunoprecipitation assay, and calcineurin (CaN) was detected. Further studies with NAP-22 prepared from DRM and CaN expressed in bacteria showed the binding of these proteins and a dose-dependent inhibitory effect of the NAP-22 fraction on the phosphatase activity of CaN. On the other hand, NAP-22 expressed in bacteria showed low binding to CaN and a weak inhibitory effect on phosphatase activity. To solve this discrepancy, identification of a nonprotein component that modulates CaN activity in the DRM-derived NAP-22 fraction was attempted. After lyophilization, a lipid fraction was extracted with chloroform/methanol. The lipid fraction showed an inhibitory effect on CaN without NAP-22, and further fractionation of the extract with thin-layer chromatography showed the presence of several lipid bands having an inhibitory effect on CaN. The mobility of these bands coincided with that of authentic ganglioside (GM1a, GD1a, GD1b, and GT1b), and authentic ganglioside showed an inhibitory effect on CaN. Treatment of lipid with endoglycoceramidase, which degrades ganglioside to glycochain and ceramide, caused a diminution of the inhibitory effect. These results show that DRM-derived NAP-22 binds several lipids, including ganglioside, and that ganglioside inhibits the phosphatase activity of CaN.
authors.affiliation: Department of Biology, Graduate School of Science, Kobe University, Kobe, Japan.
authors.forename: Yuumi
authors.initials: Y
authors.lastname: Kobayashi
authors.affiliation: Department of Biology, Graduate School of Science, Kobe University, Kobe, Japan.
authors.forename: Ronan
authors.initials: R
authors.lastname: da Silva
authors.affiliation: Division of Biochemistry and Cellular Biology, National Institute of Neuroscience, National Center of Neurology and Psychiatry, Kodaira, Japan.
authors.forename: Haruko
authors.initials: H
authors.lastname: Kumanogoh
authors.affiliation: Bioscience and Biotechnology Center, Nagoya University, Nagoya, Japan.
authors.forename: Shinji
authors.initials: S
authors.lastname: Miyata
authors.affiliation: Bioscience and Biotechnology Center, Nagoya University, Nagoya, Japan.
authors.forename: Chihiro
authors.initials: C
authors.lastname: Sato
authors.affiliation: Bioscience and Biotechnology Center, Nagoya University, Nagoya, Japan.
authors.forename: Ken
authors.initials: K
authors.lastname: Kitajima
authors.affiliation: Department of Biotechnology and Life Science, Tokyo University of Agriculture and Technology, Koganei, Japan.
authors.forename: Shun
authors.initials: S
authors.lastname: Nakamura
authors.affiliation: Department of Biology, Graduate School of Science, Kobe University, Kobe, Japan.
authors.forename: Mistuhiro
authors.initials: M
authors.lastname: Morita
authors.affiliation: Department of Biology, Graduate School of Science, Kobe University, Kobe, Japan.
authors.forename: Fumio
authors.initials: F
authors.lastname: Hayashi
authors.affiliation: Department of Biology, Graduate School of Science, Kobe University, Kobe, Japan.
authors.forename: Shohei
authors.initials: S
authors.lastname: Maekawa
mesh_terms: D000818:Animals; D001921:Brain / Q000166:cytology*; D019703:Calcineurin / Q000378:metabolism*; D002148:Calmodulin-Binding Proteins / Q000737:chemistry / Q000378:metabolism*; D002478:Cells, Cultured; D002855:Chromatography, Thin Layer; D003598:Cytoskeletal Proteins / Q000737:chemistry / Q000378:metabolism*; D003902:Detergents / Q000494:pharmacology; D005732:Gangliosides / Q000737:chemistry / Q000378:metabolism*; D006026:Glycoside Hydrolases / Q000494:pharmacology; D047468:Immunoprecipitation; D050356:Lipid Metabolism / Q000187:drug effects; D021962:Membrane Microdomains / Q000187:drug effects / Q000378:metabolism*; D009419:Nerve Tissue Proteins / Q000737:chemistry / Q000378:metabolism*; D009474:Neurons / Q000378:metabolism / Q000648:ultrastructure; D010744:Phosphoric Monoester Hydrolases / Q000378:metabolism; D051381:Rats; D017208:Rats, Wistar
chemical_list: D002148:Calmodulin-Binding Proteins; D003598:Cytoskeletal Proteins; D003902:Detergents; D005732:Gangliosides; D009419:Nerve Tissue Proteins; C066853:Basp1 protein, rat; D019703:Calcineurin; D010744:Phosphoric Monoester Hydrolases; D006026:Glycoside Hydrolases; C050427:endoglycoceramidase
keywords: NAP-22; calcineurin; ganglioside; lipid raft; neuron; synapse