Sterol carrier protein-2 expression alters sphingolipid metabolism in transfected mouse L-cell fibroblasts.
The influence of sterol carrier protein-2 (SCP-2) on the cellular metabolism of sphingolipids was examined in control mouse L-cells and stably transfected clones expressing the protein SCP-2. Three approaches were used to examine for differences; (1) compositional analysis of endogenous sphingolipid classes, (2) metabolism of NBD-ceramide, and (3) live cell labelling via endocytic uptake of BODIPY-sphingomyelin. SCP-2 over expression significantly altered the endogenous levels of both neutral and acidic sphingolipid classes. Among the neutral sphingolipids, expression of SCP-2 induced a 1.7-fold increase in the level of lactosylceramide (LacCer, p < 0.05) and a similar fold decrease in the level of the higher-order neutral glycosylceramides (p < 0.05). Among the acidic sphingolipids, SCP-2 resulted in a 5.2-fold decrease in the endogenous plasma membrane level of ganglioside GM1 (p < 0.03). Incubation of both control and transfected cell lines with NBD-ceramide resulted in the rapid establishment of a steady-state distribution of NBD-labelled sphingomyelin (NBD-SM) and glucosylceramide (NBD-GlcCer). In the SCP-2 expressing clones the conversion of NBD-Cer to NBD-GlcCer was 30% lower during incubation periods between 5 and 30 min (p < 0.025). Inspection of the cells by fluorescence microscopy after incubation with BODIPY labelled sphingomyelin (BODIPY-SM) revealed similar punctuated patterns with no distinguishable differences between the cell types. These results imply that SCP-2 plays a role in modulating enzymatic steps involved in metabolism of sphingolipid homeostasis.
Milis
Daniel G
DG
Department of Chemistry, Luther College, Decorah, Iowa, 52101-1045, USA.
Moore
Messiah K
MK
Atshaves
Barbara P
BP
Schroeder
Friedhelm
F
Jefferson
John R
JR
D009327:4-Chloro-7-nitrobenzofurazan / Q000031:analogs & derivatives / Q000493:pharmacokinetics / Q000494:pharmacology; D000818:Animals; D001692:Biological Transport; D002352:Carrier Proteins / Q000378:metabolism*; D002462:Cell Membrane / Q000378:metabolism; D002518:Ceramides / Q000494:pharmacology; D005347:Fibroblasts / Q000378:metabolism*; D005677:G(M1) Ganglioside / Q000031:analogs & derivatives / Q000378:metabolism; D005963:Glucosylceramides / Q000493:pharmacokinetics; D006706:Homeostasis; D007739:L Cells; D051379:Mice; D008856:Microscopy, Fluorescence; D013107:Sphingolipids / Q000378:metabolism*; D013109:Sphingomyelins / Q000493:pharmacokinetics; D014162:Transfection
D002352:Carrier Proteins; D002518:Ceramides; D005963:Glucosylceramides; C041065:N-4-nitrobenzo-2-oxa-1,3-diazoleaminocaproyl sphingosylphosphorylcholine; D013107:Sphingolipids; D013109:Sphingomyelins; C502266:ganglioside GM1alpha; C072087:sterol carrier proteins; D005677:G(M1) Ganglioside; C037974:N-(7-(4-nitrobenzo-2-oxa-1,3-diazole))-6-aminocaproyl sphingosine; D009327:4-Chloro-7-nitrobenzofurazan