Glucocerebrosidase deficiency and lysosomal storage of glucocerebroside induced in cultured macrophages

Abstract

A cell culture model simulating the genetic deficiency of glucocerebrosidase has been developed, utilizing macrophages and conduritol B epoxide (CBE), the specific irreversible inhibitor of the enzyme. Rat peritoneal macrophage glucocerebrosidase was completely inhibited when cells were treated with 10 μM CBE for 16 h or 100 μM CBE for 2 h. The t_½ of inactivation was 30 min at 10 μM concentration. When cells were washed free of CBE, the enzyme activity reappeared linearly with time, reaching 50% of control activity 48 h after removal of the inhibitor. CBE-treated macrophages have normal phagocytic activity toward [³H]glycine-coupled latex beads and a normal number of mannose receptors. CBE was found to have no effect on other lysosomal enzymes. When [¹⁴C]glucocerebroside, encapsulated in multilamellar liposomes with α-D -mannopyranoside covalently coupled to the surface, was fed to glucocerebrosidase-depleted macrophages, the radiolabelled glycolipid accumulated and was undegraded. Subcellular fractionation on a Percoll density gradient demonstrated that the stored glucocerebroside in the CBE-treated macrophages was localized in lysosomes.