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.

FKBP12 contributes to α-synuclein toxicity by regulating the calcineurin-dependent phosphoproteome

“…Both proteins have critical roles in assisting neuronal path finding and branching during regeneration and contribute to presynaptic changes leading to neurotransmitter release, endocytosis, and synaptic vesicle recycling ( 32 , 33 ). Importantly, their activity has been shown to be affected by calcineurin, although the exact mechanism of regulation is not known ( 34 , 35 ). While these proteins do not have homologs in yeast, they share common features with the putative calcineurin- and FKBP12-dependent proteins retrieved from the yeast model.…”

A high-affinity cocaine binding site associated with the brain acid soluble protein 1

“…BASP1 regulates many cellular signaling cascades, which have been implicated as modulators of the neurochemical and behavioral effects of cocaine. For example, BASP1 is phosphorylated by protein kinase C (PKC) and physiologically binds calmodulin ( 44 , 45 ) as well as the calmodulin-dependent phosphatase calcineurin ( 46 ). Likewise, signaling through PKC and calcium/calmodulin-dependent protein kinase II is required for the rewarding effects of cocaine ( 47 – 52 ).…”

Tight binding of NAP-22 with acidic membrane lipids

“…As is shown in a previous report, acetone extracted cholesterol and fatty acids. C/M and C/M/HCl exracted phospholipids such as phodsphatidylethanolamine (PE), phosphatidylcholine (PC), sphingomyelin (SM), and ganglioside [5]. Although, little mobility change of A-NAP-22 was detected in CN-PAGE, large part of NAP-22 moved to the low molecular mass region after the extraction with C/M and C/M/HCl (Fig.…”

“…Since, the mobility pattern of C-NAP-22 after the addition of ganglioside was similar to that of R-NAP-22 and ganglioside, but not PS nor PI, was detected in the lipid extract of R-NAP-22 fraction [5], the effect of four ganglioside species rich in brain was then studied. As is shown in Fig.…”

“…In order to study the latter possibility, successive extraction of lipid from DRM-derived NAP-22 fraction with acetone and chloroform/methanol (C/M) was attempted and the presence of several lipid species was shown in a previous study [5].…”