Studies on the turnover of exogenous mannose-terminal glucocerebrosidase in rat liver lysosomes.

Mannose-terminal glucocerebrosidase prepared by exoglycosidase digestion of human placental glucocerebrosidase is reported effective in the treatment of patients with type 1 Gaucher's disease [Barton et al. (1991); N Engl J Med 324:1464-1470]. However, the amount of enzyme that is necessary for therapeutic effect is much higher than would be predicted from in vitro activity measurements. We have investigated the fate of infused enzyme following intravenous administration in Sprague-Dawley rats. In this model system, the enzyme is rapidly cleared from the plasma compartment by receptor-mediated endocytosis via the mannose-specific receptor present on reticuloendothelial cells. Enzyme activity measured in rat liver biopsy specimens at various times post-infusion revealed a rapid initial loss of approximately one-half of the maximum delivered enzyme in the first hour followed by a slower decay with a half-life of approximately 6-8 h. The loss in enzyme activity is paralleled by a loss in enzyme protein when analyzed by Western blots. There is no evidence for return of enzyme activity or inactive enzyme protein to the plasma. Incomplete integration into the lysosomal membrane was demonstrated by the use of differential extraction of purified rat liver lysosomes to distinguish between lumenal and membrane bound enzyme. Immunoelectron microscopy of rat liver following infusion of mannose-terminal glucocerebrosidase confirmed localization of the delivered enzyme primarily within the lumen of the lysosomes of Kupffer cells and to a lesser extent associated with the lysosomal membrane. Enzyme activity was stable in isolated rat liver lysosomes preloaded with mannose-terminal glucocerebrosidase and incubated in the absence or presence of ATP. Acidification of the lysosomes to pH 3 results in a rapid loss of enzyme activity and protein; however, the relationship between the in vitro loss and the loss in enzyme activity in intact liver is not clear. We conclude from these studies that rapid intracellular degradation of administered glucocerebrosidase is the prime factor responsible for the high dose required for effective treatment of Gaucher's disease.