LASS5 is a bona fide dihydroceramide synthase that selectively utilizes palmitoyl-CoA as acyl donor.
We demonstrated recently (Riebeling, C., Allegood, J.C., Wang, E., Merrill, A. H. Jr., and Futerman, A. H. (2003) J. Biol. Chem. 278, 43452-43459) that upon over-expression in human embryonic kidney cells, longevity assurance gene homolog 5 (LASS5, previously named TRH4) elevates the synthesis of (dihydro)ceramides selectively enriched in palmitic acid. To determine whether LASS5 is a bona fide dihydroceramide synthase or, alternatively, whether it modifies an endogenous dihydroceramide synthase, we over-expressed LASS5 with a hemagglutinin (HA) tag at the C terminus, solubilized it using digitonin, and purified it by immunoprecipitation. Solubilized LASS5-HA displays the same fatty acid selectivity as the membrane-bound enzyme. After elution from agarose beads, only one band could be detected by SDS-PAGE, and its identity was confirmed to be LASS5 by mass spectrometry. Dihydroceramide synthase activity of the eluted LASS5-HA protein was totally dependent on exogenously added phospholipids. Moreover, eluted LASS5-HA was highly selective toward palmitoyl-CoA as acyl donor and was inhibited by the (dihydro)ceramide synthase inhibitor, fumonisin B1. This study identifies LASS5 as a genuine dihydroceramide synthase and demonstrates that mammalian dihydroceramide synthases do not require additional subunits for their activity.
Lahiri
Sujoy
S
Department of Biological Chemistry, Weizmann Institute of Science, Rehovot 76100, Israel.
Futerman
Anthony H
AH
D002460:Cell Line; D002518:Ceramides / Q000378:metabolism*; D005786:Gene Expression Regulation; D006388:Hemagglutinins; D006801:Humans; D047468:Immunoprecipitation; D007668:Kidney / Q000166:cytology; D013058:Mass Spectrometry; D008565:Membrane Proteins / Q000378:metabolism*; D010088:Oxidoreductases / Q000378:metabolism*; D010171:Palmitoyl Coenzyme A / Q000378:metabolism*; D051049:Sphingosine N-Acyltransferase
D002518:Ceramides; D006388:Hemagglutinins; D008565:Membrane Proteins; D010171:Palmitoyl Coenzyme A; D010088:Oxidoreductases; C473786:dihydroceramide synthase; C493790:CERS1 protein, human; C505722:CERS5 protein, human; D051049:Sphingosine N-Acyltransferase