Infusion of alpha-galactosidase A reduces tissue globotriaosylceramide storage in patients with Fabry disease.

Fabry disease is a lysosomal storage disorder caused by a deficiency of the lysosomal enzyme alpha-galactosidase A (alpha-gal A). This enzymatic defect results in the accumulation of the glycosphingolipid globotriaosylceramide (Gb(3); also referred to as ceramidetrihexoside) throughout the body. To investigate the effects of purified alpha-gal A, 10 patients with Fabry disease received a single i.v. infusion of one of five escalating dose levels of the enzyme. The objectives of this study were: (i) to evaluate the safety of administered alpha-gal A, (ii) to assess the pharmacokinetics of i.v.-administered alpha-gal A in plasma and liver, and (iii) to determine the effect of this replacement enzyme on hepatic, urine sediment and plasma concentrations of Gb(3). alpha-Gal A infusions were well tolerated in all patients. Immunohistochemical staining of liver tissue approximately 2 days after enzyme infusion identified alpha-gal A in several cell types, including sinusoidal endothelial cells, Kupffer cells, and hepatocytes, suggesting diffuse uptake via the mannose 6-phosphate receptor. The tissue half-life in the liver was greater than 24 hr. After the single dose of alpha-gal A, nine of the 10 patients had significantly reduced Gb(3) levels both in the liver and shed renal tubular epithelial cells in the urine sediment. These data demonstrate that single infusions of alpha-gal A prepared from transfected human fibroblasts are both safe and biochemically active in patients with Fabry disease. The degree of substrate reduction seen in the study is potentially clinically significant in view of the fact that Gb(3) burden in Fabry patients increases gradually over decades. Taken together, these results suggest that enzyme replacement is likely to be an effective therapy for patients with this metabolic disorder.

Proteolytic activity of purified avian sarcoma and leukemia virus NC-PR protein expressed in Escherichia coli.

Processing of the internal structural and enzymatic proteins of retroviruses occurs during or shortly after budding and is accomplished by the viral protease (PR), which belongs to the large family of aspartic proteases. It is not known how the activity of PR is regulated so that proteolysis occurs at this time. Cellular aspartic proteases are synthesized as zymogens with short N-terminal extensions that are proteolytically removed to generate the free active enzyme. In the avian sarcoma and leukosis viruses (ASLV), PR is expressed as the carboxy-terminal domain of the Gag polyprotein, which thus has a structure analogous to such a zymogen. We have investigated the enzymatic properties of ASLV PR when it is part of a longer protein, NC-PR, serving as a model for Gag. This protein represents about one-third of Gag and consists of the nucleocapsid (NC) domain fused to the N-terminus of PR. NC-PR and derivatives of NC-PR were expressed in bacterial cells and purified. In short-term assays, these fusion proteins lacked measurable protease activity toward an exogenous substrate prepared by in vitro translation. In contrast to PR, which is a homodimer, NC-PR migrated as a monomer both by glycerol gradient sedimentation and by gel filtration chromatography. Thus the NC domain appears to inhibit enzymatic activity by altering the dimerization potential of the PR domains. However, upon long incubations NC-PR was found to cleave itself to generate free and fully active PR, implying that dimerization was not prevented entirely. On the basis of these results, we hypothesize that the Gag protein in vivo is also incompletely active as a protease, because upstream portions of Gag interfere with proper interaction of the PR domains. The eventual dimerization, perhaps triggered by other events, then could lead to a cascade whereby PR is proteolytically freed from Gag and thereby gains enzymatic activity.