DELIVERY OF CYTOSOLIC LIVER ARGINASE INTO THE MITOCHONDRIAL MATRIX SPACE - A POSSIBLE NOVEL SITE FOR GENE REPLACEMENT THERAPY

As a toxic metabolic byproduct in mammals, excess ammonia is converted into urea by a series of five enzymatic reactions in the liver that c onstitute the urea cycle. A portion of this cycle takes place in the m itochondria, while the remainder is cytosolic. Liver arginase (L-argin ine ureahydrolase, AI) is the fifth enzyme of the cycle, catalyzing th e hydrolysis of arginine to ornithine and urea within the cytosol. Pat ients deficient in this enzyme exhibit hyperargininemia with episodic hyperammonemia and long-term effects of mental retardation and spastic ity. However, the hyperammonemic effects are not so catastrophic in ar ginase deficiency as compared to other urea cycle defects. Earlier stu dies have suggested that this is due to the mitigating effect of a sec ond isozyme of arginase (AII) expressed predominantly in the kidney an d localized within the mitochondria. In order to explore the curious d ual evolution of these two isozymes, and the ways in which the intrigu ing aspects of AII physiology might be exploited for gene replacement therapy of AI deficiency, the cloned cDNA for human AI was inserted in to an expression vector downstream from the mitochondrial targeting le ader sequence for the mitochondrial enzyme ornithine transcarbamylase and transfected into a variety of recipient cell types. AI expression in the target cells was confirmed by northern blot analysis, and compe tition and immunoprecipitation studies showed successful translocation of the exogenous AI enzyme into the transfected cell mitochondria. St ability studies demonstrated that the translocated enzyme had a longer half-life than either native cytosolic AI or mitochondrial AII. Incub ation of the transfected cells with increasing amounts of arginine pro duced enhanced levels of mitochondrial AI activity, a substrate-induce d effect that we have previously seen with native AII but never AI. Al ong with exploring the basis biological questions of regulation and su bcellular localization in this unique dual-enzyme system, these result s suggest that the mitochondrial matrix space may be a preferred site for delivery of enzymes in gene replacement therapy.