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Sterol carrier protein-2 expression alters sphingolipid metabolism in transfected mouse L-cell fibroblasts

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Abstract

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.

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Abbreviations

SCP-2:

sterol carrier protein-2

SAP:

sphingolipid activator protein

BSA:

bovine serum albumin

DF-BSA:

defatted BSA

HMEM:

hepes buffered minimum essential media

PBS:

phosphate buffered saline

BODIPY:

boron dipyrromethenedi-fluoride

NDB-:

N-[7-(4-nitrobenzo-2-oxa-1,2-diazole)]-6-aminocaproyl-, NDB-Cer, N-[7-(4-nitrobenzo-2-oxa-1,2-diazole)]-6-aminocaproyl-D-erythro-sphingosine

Cer:

ceramide

GlcCer:

glucosylceramide

CoA:

co-enzyme A

CGT:

ceramide glucosyltransferase

Gal:

galactose

GalCer:

galactosylceramide

Glc:

glucose

GlcCer:

glucosylceramide

GSL:

glycosphingolipid

LacCer:

lactosylceramide

SL:

sphingolipid

SM:

sphingomyelin

TLC:

thin layer chromatography

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Authors and Affiliations

  1. Department of Chemistry, Luther College, Decorah, Iowa, 52101-1045

    Daniel G. Milis, Messiah K. Moore & John R. Jefferson

  2. Department of Physiology and Pharmacology, Texas A&M University, TVMC, College Station, Texas, 77843-4466

    Barbara P. Atshaves & Friedhelm Schroeder

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  1. Daniel G. Milis
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  2. Messiah K. Moore
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  3. Barbara P. Atshaves
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  4. Friedhelm Schroeder
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  5. John R. Jefferson
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Correspondence to John R. Jefferson.

Additional information

Portions of this work were presented in abstract at the 2004 annual meeting of the American Society of Biochemistry and Molecular Biology, Boston Mass FASEB J, 18(8): C271, 2004

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Milis, D.G., Moore, M.K., Atshaves, B.P. et al. Sterol carrier protein-2 expression alters sphingolipid metabolism in transfected mouse L-cell fibroblasts. Mol Cell Biochem 283, 57–66 (2006). https://doi.org/10.1007/s11010-006-2270-1

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  • DOI: https://doi.org/10.1007/s11010-006-2270-1

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