Radiolabeling and Isolation of Thy-1 Active Glycolipids from Murine Brain and Lymphoma Cell Lines

Abstract

Abstract Murine Thy-1 active glycolipids were isolated and characterized by radiolabeling, thin layer chromatography (TLC), and an immune response-plaque forming cell (PFC) assay. Brain gangliosides were labeled in vivo by intracranial injection of [1-14C]N-acetylmannosamine (ManNAc). Gangliosides were extracted, separated by one and two dimensional TLC, and visualized by autoradiography. The gangliosides isolated from AKR (Thy-1.1) and ICR (Thy-1.2) mice were assayed for Thy-1.1 and 1.2 activity with an anti-Thy-1 PFC assay. One Thy-1.1 active compound was identified out of about 15 AKR gangliosides. None of the AKR gangliosides had Thy-1.2 activity in the PFC assay. Similarly, one Thy-1.2 active ganglioside was identified out of the ICR gangliosides and none of these had Thy-1.1 activity. Thus the brain gangliosides demonstrated allogeneic specificity for Thy-1 antigen. BW5147 (Thy-1.1) and S49.1 (Thy-1.2) murine lymphoma cell lines that express Thy-1 were also examined for Thy-1 active glycolipids. The cell lines were incubated in vitro with either [1-14C]palmitate or [1-14C]-ManNAc, and the glycolipids were extracted, separated by two dimensional TLC, and visualized by autoradiography. A Thy-1.2 active compound was identified from the S49.1 cells. None of the BW5147 glycolipids had Thy-1.2 activity and none of the S49.1 glycolipids had Thy-1.1 activity. Neuraminidase and mild HC1 treatment of Thy-1 active glycolipids abrogated the subsequent PFC response. The Thy-1 glycolipids bound to DEAE cellulose as expected for gangliosides. We therefore conclude that Thy-1 antigenicity is associated with gangliosides of mouse brain and lymphoma cells.