The lysosome, a specialized sub-cellular catabolic organelle, contains a variety of hydrolytic enzymes responsible for the degradation of a wide range of biomolecules. Many inherited disorders of lysosomal function occur as a result of mutations in the genes for these lysosomal acid hydrolases. Since the protein content of the lysosomes is determined by both intracellular and extracellular protein trafficking, it may be possible to target constitutive lysosomal proteins to the particle from an exogenous pool. By utilizing the normal biosynthetic and translocation mechanisms and the capacity for receptor mediated endocytosis, lysosomal enzymes may be replaced. Ideally, suitable engineering of the enzyme molecule permits binding to endocytic receptors (lectins) and high efficiency translocation to the lysosome where the enzyme maintains activity for a suitably long duration. Because lysosomal storage of glucocerebroside occurs only in tissue macrophages in Gaucher disease, glucocerebrosidase was chosen to serve as a prototype for the study of enzyme replacement. This protein for which the gene has now been isolated and characterized was purified to homogeneity and extensively characterized with respect to amino acid sequence, complete oligosaccharide structure, biosynthesis and translocation in order to evaluate its potential for these studies. Procedures were developed for the large scale enzymatic modification of the carbohydrate portion of glucocerebrosidase to take advantage of naturally occurring lectin receptors on the membranes of reticuloendothelial cells. By complete characterization of glucocerebrosidase with respect to carbohydrate structure and composition, it became possible to develop new analytical methods and adapt old ones to permit quantitative measurement of the extent of the modification of the oligosaccharide without loss of enzymatic activity. The efficiency of the delivery of glucocerebrosidase to reticuloendothelial cells in rats has been studied to predict the therapeutic potential of the treatment for Gaucher disease. In addition, cell culture models using animal and human macrophages have been developed for use in this evaluation.
