A novel approach for the topographical localization of glycolipids on the cell surface

In this study we have developed a prototype system for distinguishing between the topographical distribution of glycolipids versus glycoproteins on the ultrastructural level. Direct modification of membrane-based sialic acids with biotin groups labels both glycolipids and glycoproteins. In this case, subsequent ultrastructural localization of biotinylated sites would not discern between these two classes of glycoconjugate in an unambiguous manner. When biotinylated cells are fixed prior to interaction with ferritin-conjugated avidin, the mean distance of marker molecules from the membrane bilayer is 8.0 nm. In contrast, if the cells are allowed to cap through the action of ferritin-avidin conjugates on unfixed cells, the average distance (13.0 nm) of the marker molecules appears even more distant from the membrane on the capped portion of the cell (uropod), whereas those on the head region are positioned in close proximity to the bilayer (3.7 nm). In order to exclusively label cell surface glycolipids on the ultrastructural level, bovine brain gangliosides were biotinylated in vitro and the haptenized gangliosides were incorporated into intact cells. In this case, marker molecules denoting the incorporated gangliosides were found in relatively close juxtaposition to the membrane surface, in a manner strikingly similar to the labeling pattern of the head region on capped cells. These results support the concept that, in the native state, the carbohydrate portion of glycolipids is positioned closer to the membrane bilayer than that of glycoproteins.