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. Issue 9 (18th May 2015)

• Record Type: Journal Article
• Authors: Kobayashi, Yuumi; da Silva, Ronan; Kumanogoh, Haruko; Miyata, Shinji; Sato, Chihiro; Kitajima, Ken; Nakamura, Shun; Morita, Mistuhiro; Hayashi, Fumio; Maekawa, Shohei
• Is Part Of: Journal of neuroscience research. Volume 93:Issue 9(2015)
• Page Start: 1462
• Page End: 1470
• Publication Date: 2015-05-18
• Subjects: neuron -- lipid raft -- synapse -- calcineurin -- NAP‐22 -- ganglioside; Neurobiology -- Periodicals; 612
• DOI: 10.1002/jnr.23599
• Languages: English
• ISSNs: 0360-4012

Abstract

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. © 2015 Wiley Periodicals, Inc. Abstract : The fraction of neuron‐enriched protein NAP‐22 (also called BASP1 or CAP‐23) prepared from Triton‐insoluble low‐density fraction of rat brain contained several lipids. Ganglioside contained in the fraction was found to inhibit the enzymatic activity of CaN in a dose‐dependent manner (open bars, 20 μM; solid bars, 100 μM).