Presence of Ceramidase Activity in Electronegative LDL

Abstract

Ceramide; Sphingomyelinase

Ceramida; Esfingomielinasa

Ceramida; Esfingomielinasa

Electronegative low-density lipoprotein (LDL(−)) is a minor modified fraction of human plasma LDL with several atherogenic properties. Among them is increased bioactive lipid mediator content, such as lysophosphatidylcholine (LPC), non-esterified fatty acids (NEFA), ceramide (Cer), and sphingosine (Sph), which are related to the presence of some phospholipolytic activities, including platelet-activating factor acetylhydrolase (PAF-AH), phospholipase C (PLC), and sphingomyelinase (SMase), in LDL(−). However, these enzymes’ activities do not explain the increased Sph content, which typically derives from Cer degradation. In the present study, we analyzed the putative presence of ceramidase (CDase) activity, which could explain the increased Sph content. Thin layer chromatography (TLC) and lipidomic analysis showed that Cer, Sph, and NEFA spontaneously increased in LDL(−) incubated alone at 37 °C, in contrast with native LDL(+). An inhibitor of neutral CDase prevented the formation of Sph and, in turn, increased Cer content in LDL(−). In addition, LDL(−) efficiently degraded fluorescently labeled Cer (NBD-Cer) to form Sph and NEFA. These observations defend the existence of the CDase-like activity’s association with LDL(−). However, neither the proteomic analysis nor the Western blot detected the presence of an enzyme with known CDase activity. Further studies are thus warranted to define the origin of the CDase-like activity detected in LDL(−).

This research was funded by grants PI13/00364, PI16/00471, FIS PI019/00421, and PI20/00334 from the Instituto de Salud Carlos III, Spanish Ministry of Health (co-financed by the European Regional Development Fund). N.P. is the recipient of FI20/00252 from Instituto de Salud Carlos III. This research was supported by CIBER (Consorcio Centro de Investigación Biomédica en Red) (CB07/08/0016), Instituto de Salud Carlos III, and Ministerio de Ciencia e Innovación and Unión Europea—European Regional Development Fund. CIBERDEM (CB07/08/0016) and CIBERCV (CB16/11/00257) are Instituto de Salud Carlos III Projects. A.A.-S. is member of RETICS INVICTUS PLUS (RD16/0019/0010, the Instituto de Salud Carlos III project). N.P., S.B., N.R., and J.L.S.-Q. are members of the Quality Research Group 2017-SGR-1149 from Generalitat de Catalunya and the Group of Vascular Biology of the Spanish Society of Atherosclerosis.