Epithelial sphingolipid sorting allows for extensive variation of the fatty acyl chain and the sphingosine backbone

Abstract

In kidney MDCK and intestinal Caco-2 epithelial cells, glucosylceramide (GlcCer) and sphingomyelin (SPH) synthesized from the short-chain sphingolipid analogue N-6-[7-nitro-2,1,3-benzoxadiazol-4-yl]aminodecanoyl (C6-NBD)-ceramide are delivered to the cell surface with apical/basolateral polarities of 2-4 and 0.6-0.9 respectively. We have tested how variations in the lipid backbone affect these polarities. First, the C6-NBD moiety was replaced by a bare [14C]octanoyl chain or by the even more bulky fluorophores 8-bimanoylthio-octanoyl (C8-bimane) and 8-diethylaminocoumarin-octanoyl (C8-DECA). In addition, the sphingosine in C6-NBD-ceramide was changed in stereoconfiguration (L-threo) or saturation (dihydro). In all cases, GlcCer and SPH were produced and appeared on the cell surface at 37°C, as assayed by back-exchange. The apical/basolateral polarity of the delivery of GlcCer was variable, but always exceeded 1. GlcCer was apically enriched over SPH (2-6 times for MDCK and 3-9 times for Caco-2). Even GlcCer synthesized from a highly water-soluble truncated ceramide (octanoyl-D-erythro-sphingosine analogue with C8 backbone) was enriched apically by a factor of ≥ 2 both in absolute polarity and compared with SPH. Sphingolipid sorting was quantitatively but not qualitatively affected by dramatic changes in the lipid backbone.