Subcellular localization of glycosphingolipids in human neutrophils

The subcellular distribution of five major glycosphingolipids (GSLs) in human neutrophils was analyzed. The neutrophils were isolated from the blood of six donors and subdivided in three fractions containing the cell membranes, and the primary and the secondary granules, respectively. The separation was confirmed with antibodies detecting established subcellular fraction-specific molecules. The two main neutral GSLsGalbeta1-4Glcbeta1-1'Cer (lactosylceramide, LacCer) and nLc4Cer (paragloboside, PG) and the three gangliosides IV3NeuAcnLc4Cer (2-3SPG), IV6NeuAcnLc4Cer (2-6SPG), and VI3NeuAcnLc6Cer (2-3SnHC) were quantitated using the immunochemical digoxigenin (DIG) staining procedure. Secondary granules contained the highest amount of these GSLs. They are followed by the primary granules and the cell membranes. Based on this quantitation, we conclude that the majority of the GSLs of neutrophils occur intracellularly. These findings are in striking contrast to the general assumption of GSLs being mainly concentrated in the cell membrane.