Abstract
Ceramide produced at the endoplasmic reticulum (ER) is transported to the lumen of the Golgi apparatus for conversion to sphingomyelin (SM). N-(3-Hydroxy-1-hydroxymethyl-3-phenylpropyl)dodecanamide (HPA-12) is a novel analog of ceramide. Metabolic labeling experiments showed that HPA-12 inhibits conversion of ceramide to SM, but not to glucosylceramide, in Chinese hamster ovary cells. Cultivation of cells with HPA-12 significantly reduced the content of SM. HPA-12 did not inhibit the activity of SM synthase. The inhibition of SM formation by HPA-12 was abrogated when the Golgi apparatus was made to merge with the ER by brefeldin A. Moreover, HPA-12 inhibited redistribution of a fluorescent analog of ceramide, N-(4,4-difluoro-5,7-dimethyl-4-bora-3a,4a-diaza-s-indacene-3-pentanoyl)-d-erythro-sphingosine (C(5)-DMB-Cer), from intracellular membranes to the Golgi region. Among four stereoisomers of the drug, (1R,3S)-HPA-12, [corrected] which resembles natural ceramide stereochemically, was found to be the most active, although (1R,3S)-HPA-12 [corrected] did not affect ER-to-Golgi trafficking of protein. Interestingly, (1R,3S)-HPA-12 [corrected] inhibited conversion of ceramide to SM little in mutant cells defective in an ATP- and cytosol-dependent pathway of ceramide transport. These results indicated that (1R,3S)-HPA-12 [corrected] inhibits ceramide trafficking from the ER to the site of SM synthesis, possibly due to an antagonistic interaction with a ceramide-recognizing factor(s) involved in the ATP- and cytosol-dependent pathway.
Highlights
Sphingolipids are ubiquitous constituents of membrane lipids in mammalian cells and play important roles in cell growth, differentiation, and apoptosis [1,2,3]
Fluorescence Microscopy—Chinese hamster ovary (CHO) cells grown on glass coverslips (22-mm diameter) in 35-mm dishes were incubated in 1 ml of F12 medium containing 1 M of C5-DMB-Cer or C6-NBD-Cer complexed with 1 M BSA at 4 °C for 30 min, washed with 1 ml of F12 medium three times and incubated in 1 ml of Nutridoma medium in the presence or absence of 2.5 M HPA-12 at 4 °C for 15 min
We showed that (1R,3R)-HPA-12, a novel analog of Cer, inhibits ATP-dependent transport of Cer from the endoplasmic reticulum (ER) to the site of SM synthesis without appreciable inhibition of the main pathway of ER-to-Golgi trafficking of glycoproteins
Summary
Ceramide; ER, endoplasmic reticulum; SM, sphingomyelin; GlcCer, glucosylceramide; CHO, Chinese hamster ovary; HPA, N-(3-hydroxy-1-hydroxymethyl-3-phenylpropyl)alkanamide; C6-NBD-Cer, 6-[N-(7-nitrobenzo-2-oxa-1,3-diazol-4-yl)amino]caproyl-D-erythro-sphingosine; C5-DMB-Cer, N-(4,4-difluoro-5,7dimethyl-4-bora-3a,4a-diaza-s-indacene-3-pentanoyl)-D-erythro-sphingosine; C16-Cer, N-palmitoyl-D-sphingosine; PBS, phosphate-buffered saline; BFA, brefeldin A; GM3, N-acetylneuraminyl lactosylceramide; PLAP, human placental alkaline phosphatase; PLAP-HA, a chimera of human placental alkaline phosphatase with influenza hemagglutinin; Endo H, endoglycosidase H; SMase, sphingomyelinase; D-e-MAPP, (1S,2R)-D-erythro-2-(N-myristoylamino)-1-phenyl-1-propanol; BSA, bovine serum albumin; NBS, newborn bovine serum; PC, phosphatidylcholine; GPI, glycosylphosphatidylinositol; DMB, 4,4-difluoro-5,7-dimethyl-4-bora-3a,4a-diaza-s-indacene. A stereoisomer having the 1R,3R configuration was found to be the most active among the four stereoisomers of HPA-12
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