The sphingosine 1-phosphate breakdown product, (2E)-hexadecenal, forms protein adducts and glutathione conjugates in vitro

Abstract

Sphingosine 1-phosphate (S1P), a bioactive lipid involved in various physiological processes such as cell proliferation and apoptosis, can be irreversibly cleaved by the S1P lyase, yielding phosphoethanolamine and (2E)-hexadecenal (2EHD). The latter metabolite, an α,β-unsaturated fatty aldehyde, may be susceptible to nucleophilic attack by cellular biomolecules. Hence, we studied whether 2EHD forms reaction products with glutathione (GSH) and proteins in vitro. Using liquid chromatography-tandem mass spectrometry (LC-MS/MS) and stable isotopically labeled reference material, we identified a total of nine novel reaction products of 2EHD in a cell-free approach: two GSH conjugates and seven L-amino acid adducts. Both GSH conjugates were also found in HepG2 cell lysates incubated with 2EHD. Likewise, we detected 4 out of 7 amino acid adducts released from the model protein BSA and proteins extracted from HepG2 cells. Hereby, the 2EHD Michael adduct with L-histidine proved to be the most prominent adduct. Most interestingly, inhibition of the enzymatically driven oxidative degradation of 2EHD resulted in increased levels of both GSH conjugates and protein adducts in HepG2 cell lysates. Hence, our data provide new insights into sphingolipid metabolism and will be useful to investigate certain disorders linked to an impaired fatty aldehyde metabolism in more detail.

Divergent Role of Sphingosine 1-Phosphate in Liver Health and Disease

“…Additionally, it can be speculated that an increased expression of S1P lyase is accompanied by an enhanced formation of the cleavage product hexadecenal. As it has been indicated that hexadecenal is a reactive molecule that can form adducts with cellular nucleophiles such as liver proteins, further studies are of interest investigating whether the protein adduct formation of hexadecenal is connected with a dysfunction of hepatic liver proteins [ 19 ].…”

“…The terminal degradation of sphingolipids is initiated by an irreversible cleavage of S1P via S1P lyase, leading to the formation of hexadecenal and phosphoethanolamine. The aldehyde can be further metabolized to palmitoyl CoA, however it has been indicated that hexadecenal is an active compound that can form adducts with cellular nucleophiles such as proteins and glutathione [ 18 , 19 ].…”

S1P Lyase Regulation of Thymic Egress and Oncogenic Inflammatory Signaling

“…Furthermore, trans-2-hexadecenal reacts readily with deoxyguanosine and DNA to produce the diastereomeric cyclic 1,N(2)-deoxyguanosine adducts 3-(2-deoxy- β -d-erythro-pentofuranosyl)-5,6,7,8-tetrahydro-8R-hydroxy-6R-tridecylpyrimido[1,2-a]purine-10(3H)one and 3-(2-deoxy- β -d-erythro-pentofuranosyl)-5,6,7,8-tetrahydro-8S-hydroxy-6S-tridecylpyrimido[1,2-a]purine-10(3H)one [ 91 ]. Trans-2-hexadecenal forms Michael adduct with glutathione and with seven amino acids [ 92 ]. It also binds directly to Bax, leading to a conformational change in Bax, resulting in its activation, which induces apoptosis ( Figure 1(b) ) [ 24 , 25 ].…”

2‐Hexadecenal inhibits growth of C6 glioma cells

“…2 Little is known about the biological role of 2HD. 4,5 We were the first to discover a new nonenzymatic pathway of 2HD formation from sphingolipids under the action of gamma radiation and UV light. 3 It has been reported that 2HD forms adducts with DNA and conjugates with glutathione.…”

“…3 It has been reported that 2HD forms adducts with DNA and conjugates with glutathione. 4,5 We were the first to discover a new nonenzymatic pathway of 2HD formation from sphingolipids under the action of gamma radiation and UV light. We have established that when reactive oxygen All authors contributed equally to this work.…”