Gaucher disease (GD) is associated with mutations at the acid beta-glucosidase (GCase) locus and the resultant defective activity of the enzyme product. GCase is a membrane-associated glycoprotein that requires detergents for extraction and phospholipid interfaces for full catalytic activity. Normal human fibroblasts and overexpressing transgenic cell lines were used to evaluate the intracellular disappearance, degradation, and secretion of human GCase, including GD fibroblasts and C2C12 cells transduced with MFG-GCase retrovirus and CHO cells stably transfected with the tetracycline transactivation conditional expression system (tet-CHO-GCase). Compared to HF, the disappearance of GCase from the transgenic cells was 12-30 times greater, and had degradative and secretory components. In tet-CHO-GCase cells the majority of GCase was secreted. Intracellular degradation occurred in compartments sensitive to monensin and brefeldin A, and the ALLN or leupeptin protease inhibitors, i.e., ER, Golgi, and lysosomes. In tet-CHO-GCase cells, GCase degradation and secretion rates were inversely related to expression level. 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 -insoluble GCases were degraded with t(1/2) approximately 2 and 14 h, respectively. Confocal microscopy showed colocalization of glycosylated or unglycosylated GCase with LAMP-2, an integral lysosomal membrane protein, to vesicular bodies. These studies show that GCase secretion was N-linked glycosylation dependent, whereas sorting to and membrane attachment in the lysosome were N-linked glycosylation independent.
Copyright 2000 Academic Press.