The biosynthesis of a mannolipid containing a metabolite of retinoic acid by 3T12 mouse fibroblasts.

Retinol and retinoic acid (RA) increase the adhesive properties of spontaneously transformed mouse fibroblasts (BALB/c 3T12-3 cells) and the incorporation of [2-3H]mannose into cellular glycoconjugates. Therefore we searched for a mannolipid of retinoic acid similar to mannosylretinylphosphate (MRP) in these cells. Radioactively labeled RA was incorporated into a compound of chromatographic characteristics similar to those of standard MRP. This metabolite contained the same 3H:14C ratio as the precursor [11, 12-3H, 15-14C]retinoic acid, demonstrating that no decarboxylation had occurred. A doubly labeled mannolipid was obtained from cells incubated with [2-3H]mannose and [15-14C]retinoic acid. This mannolipid was readily cleaved by mild acid, yielding [3H]mannosephosphate and a compound that migrated as standard anhydroretinol on thin layer of silica gel in toluene:chloroform:methanol (4:1:1) at Rf 0.93. Standard all trans-MRP yields all-trans-anhydroretinol under these conditions. An ion pair reverse phase HPLC system was developed to further characterize the mannolipids obtained from retinol and retinoic acid in 3T12 cells. [15-3H]Retinol and [15-14C]retinoic acid were incorporated into mannolipids that cochromatographed upon HPLC with standard MRP. The mixture of the [15-3H]retinol and [15-14C]retinoic acid derived mannolipids was subjected to mild acid hydrolysis, after purification by HPLC. Nearly 100% of the compounds was hydrolyzed, yielding all-trans-[3H]anhydroretinol and a 14C]labeled product which was eluted from HPLC as a slightly more polar compound than all-trans-anhydroretinol. The retinoic acid-derived mannolipid (MXP) represented approximately 4% of the total radioactivity in the methanolic extract of 3T12 cells incubated for 20 hours in the presence of labeled retinoic acid. However, if the cells were incubated for an additional 20 hours in the absence of the radioactive precursor, MXP represented 40% of the total extracted radioactivity. These results demonstrate that 3T12 cells synthesize mannosylretinylphosphate from retinol and a mannosylretinoidphosphate (MXP) from retinoic acid. These results exclude the possibility of a reduction of retinoic acid to retinol, but suggest that a closely related compound is formed from RA and that this retinol-like compound (X) is incorporated into MXP.