Fate and sorting of acid beta-glucosidase in transgenic mammalian cells

Gaucher disease (GD) is associated with mutations at the acid beta -glucosi dase (GCase) locus and the resultant defective activity of the enzyme produ ct, GCase is a membrane-associated glycoprotein that requires detergents fo r extraction and phospholipid interfaces for full catalytic activity. Norma l human fibroblasts and overexpressing transgenic cell lines mere used to e valuate the intracellular disappearance, degradation, and secretion of huma n GCase, including GD fibroblasts and C2C12 cells transduced with MFG-GCase retrovirus and CHO cells stably transfected with the tetracycline transact ivation conditional expression system (tet-CHO-GCase). Compared to HF, the disappearance of GCase from the transgenic cells was 12-30 times greater, a nd had degradative and secretory components. In tet-CHO-GCase cells the maj ority of GCase was secreted, Intracellular degradation occurred in compartm ents sensitive to monensin and brefeldin A, and the ALLN or leupeptin prote ase inhibitors, i.e., ER, Golgi, and lysosomes, In tet-CHO-GCase cells, GCa se degradation and secretion rates were inversely related to expression lev el, Saponin permeabilization analyses of tet-CHO-GCase cells showed that a majority of GCase was soluble, with a rapid disappearance via secretion and degradation. A progressively increasing proportion of GCase became saponin insoluble with a t(1/2) = 2-3 h. Intracellular saponin-soluble and -insolu ble GCases were degraded with t(1/2) similar to2 and 14 h, respectively. Co nfocal microscopy showed colocalization of glycosylated or unglycosylated G Case with LAMP-2, an integral lysosomal membrane protein, to vesicular bodi es. These studies show that GCase secretion was N-linked glycosylation depe ndent, whereas sorting to and membrane attachment in the lysosome were N-li nked glycosylation independent. (C) 2000 Academic Press.