Transport and processing of β-hexosaminidase in normal and mucolipidosis-II cultured fibroblasts. Effect of monensin and nigericin

Abstract

The univalent-cation ionophores monensin (4.0 .mu.M) and nigericin (0.5 .mu.M) inhibited the abnormal excretion of .beta.-hexosaminidase from mucolipidosis-II cultured fibroblasts by 62 and 76%, respectively, with a corrresponding intracellular accumulation of the enzyme. As shown by lectin binding, the enzyme which accumulated in monensin-treated cells did not contain galactose residues, whereas the corresponding enzyme from nigericin-treated cells was galactosylated. Monensin probably acts at an early point in the process of hydrolase glycosylation, and nigericin acts later, both presumably within the Golgi region, allowing the accumulation of different glycosylated forms of the enzyme. The intra- and extra-cellular distribution of .beta.-hexosaminidase in ionophore-treated normal cells was essentially unchanged, whereas concanavalin A precipitability of excreted enzyme was increased and its ability to be taken up by deficient fibroblasts was decreased. The bivalent-cation ionophore A23187 (1 .mu.M) reduced .beta.-hexosaminidase excretion from mucolipidosis-II cells by 82%, and by 96% when used with EGTA [ethylene glycol bis(.beta.-aminoethyl ether-)-N,N,N'',N''-tetraacetic acid] (1 mM). However, there was no intracellular accumulation of enzyme, suggesting that the effect of this ionophore was restricted to the inhibition of synthesis. It therefore appears that the actual transport of .beta.-hexosaminidase in mucolipidosis-II cells is affected by univalent-cation ionophores in a selective manner. Individual ionophores could be used to identify the sites of hydrolase oligosaccharide processing in the Golgi region by causing intermediate glycosylated forms of the transported hydrolase to accumulate in a specific Golgi compartment preceding the blocking site of the ionophore.