engleski

The effect of ganglioside composition on activity, expression and submembrane localization of plasma membrane Ca2+ ATPase isoforms in mouse brain

Gangliosides are sialic acid-containing glycosphingolipids that reside in the outer leaflet of the plasma membrane, with their localization being more robust in lipid rafts (LR). Gangliosides are known to modulate the structure, function and localization of membrane proteins, including the plasma membrane Ca2+ ATPase (PMCA), thus having an impact on various signaling pathways. PMCA plays an important role in the maintenance of levels of intracellular Ca2+, essential to the functioning of neurons, with its activity and expression varying during ageing and neurodegeneration. In this study investigated the effect of ganglioside composition on PMCA activity, expression and submembrane localization of PMCA isoforms, using St8sia1 null mice with impaired synthesis of gangliosides. Wild type (WT) and null mice littermates were sacrificed, brain tissue neuroanatomically dissected and cortical and cerebellar homogenates prepared. PMCA activity was measured spectrophotometrically. Protein expression of PMCA 1, 2 and 4 isoforms in homogenates was analyzed by Western blotting. LR and non-raft (nLR) fractions from cortices and cerebella were isolated by ultracentrifugation in discontinuous sucrose gradients, and submembrane localization of PMCA isoforms analyzed by Western blotting. Data revealed statistically lower PMCA activity in cortices of null mice compared to the WT mice. Total protein amount of PMCA1 and PMCA4 isoforms was statistically lower in cortices of St8sia1 null mice compared to WT mice, whilst the expression of all three isoforms was unvaried in the cerebella. Analysis of submembrane localization was shown not to be statistically different between cortical LR and nLR fractions of null and WT mice, albeit there is statistically lower amount of all three isoforms in cerebellar LR fractions of null mice compared to their WTs. These results led us to hypothesize that alterations in ganglioside composition might contribute to loss of PMCA activity or expression, related to ageing and neurodegeneration.

glycosphingolipids ; PMCA ; lipid rafts ; St8sia1 null

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