Myristic acid increases the activity of dihydroceramide Delta4-desaturase 1 through its N-terminal myristoylation.
Dihydroceramide Delta4-desaturase (DES) catalyzes the desaturation of dihydroceramide into ceramide. In mammals, two gene isoforms named DES1 and DES2 have recently been identified. The regulation of these enzymes is still poorly understood. This study was designed to examine the possible N-myristoylation of DES1 and DES2 and the effect of this co-translational modification on dihydroceramide Delta4-desaturase activity. N-MyristoylTransferases (NMT) catalyze indeed the formation of a covalent linkage between myristoyl-CoA and the N-terminal glycine of candidate proteins, as found in the sequence of DES proteins. The expression of both rat DES in COS-7 cells evidenced first that DES1 but not DES2 was associated with an increased dihydroceramide Delta4-desaturase activity. Then, we showed that recombinant DES1 was myristoylated in vivo when expressed in COS-7 cells. In addition, in vitro myristoylation assay with a peptide substrate corresponding to the N-terminal sequence of the protein confirmed that NMT1 has a high affinity for DES1 myristoylation motif (apparent K(m)=3.92 microM). Compared to an unmyristoylable mutant form of DES1 (Gly replaced by an Ala), the dihydroceramide Delta4-desaturase activity of the myristoylable DES1-Gly was reproducibly and significantly higher. Finally, the activity of wild-type DES1 was also linearly increased in the presence of increased concentrations of myristic acid incubated with the cells. These results demonstrate that DES1 is a newly discovered myristoylated protein. This N-terminal modification has a great impact on dihydroceramide Delta4-desaturase activity. These results suggest therefore that myristic acid may play an important role in the biosynthesis of ceramide and in sphingolipid metabolism.
Beauchamp
Erwan
E
Laboratoire de Biochimie, INRA-Agrocampus Rennes, 65 rue de Saint-Brieuc, CS 84215, 35042 Rennes Cedex, France.
Goenaga
Diana
D
Le Bloc'h
Jérome
J
Catheline
Daniel
D
Legrand
Philippe
P
Rioux
Vincent
V
D000409:Alanine / Q000378:metabolism; D020816:Amino Acid Motifs; D000595:Amino Acid Sequence; D019943:Amino Acid Substitution; D000818:Animals; D019556:COS Cells; D002250:Carbon Radioisotopes / Q000378:metabolism; D018929:Cell Culture Techniques; D002522:Chlorocebus aethiops; D003001:Cloning, Molecular; D019295:Computational Biology / Q000379:methods; D018076:DNA, Complementary / Q000235:genetics / Q000302:isolation & purification; D006867:Hydrolases / Q000032:analysis / Q000737:chemistry* / Q000235:genetics / Q000302:isolation & purification / Q000378:metabolism*; D007700:Kinetics; D008969:Molecular Sequence Data; D009097:Multienzyme Complexes; D019814:Myristic Acid / Q000378:metabolism*; D010088:Oxidoreductases / Q000032:analysis / Q000737:chemistry* / Q000235:genetics / Q000302:isolation & purification / Q000378:metabolism*; D010957:Plasmids; D051381:Rats; D011994:Recombinant Proteins / Q000737:chemistry / Q000378:metabolism; D017386:Sequence Homology, Amino Acid; D013379:Substrate Specificity; D014162:Transfection
D002250:Carbon Radioisotopes; D018076:DNA, Complementary; D009097:Multienzyme Complexes; D011994:Recombinant Proteins; D019814:Myristic Acid; C489141:Degs2 protein, mouse; D010088:Oxidoreductases; C527790:Degs1 protein, rat; C527791:Degs2 protein, rat; C108138:dihydroceramide desaturase; D006867:Hydrolases; D000409:Alanine