Previous studies have demonstrated the accumulation of free N- acetylneuraminic acid (NeuAc) in the tissues and fibroblasts of patients suffering from the variants of free NeuAc storage disease, including infantile free NeuAc storage disease and Salla disease. To date, however, the molecular defect has not been discovered in either of these variants, and the effects of NeuAc accumulation on sialoglycoconjugate metabolism and cellular function have not been fully documented. In recent preliminary studies, we have utilized the combined approaches of metabolic labeling, subcellular fractionation, and analysis of subcellular membrane fractions from cultured fibroblasts in studies which have demonstrated a) the lysosomal accumulation of NeuAc (in a unique population of buoyant lysosomes) in affected fibroblasts, and b) the absence of a 62 kDa polypeptide (MP 62) in membranes prepared from lysosome- enriched fractions of metabolically labeled affected fibroblasts. We now propose to utilize cultured fibroblasts from normal and affected individuals in studies designed to determine the molecular defect in infantile NeuAc storage disease which will simultaneously generate new basic information in the areas of lysosome biogenesis and function, NeuAc transport and utilization, and sialoglycoconjugate metabolism. Based on our preliminary observations, four independent but related lines of investigation will be pursued, including: 1) Analysis of the metabolism of exogenous and endogenous sialoglycoconjugates, and utilization and/or accumulation of NeuAc and its metabolites, 2) The detailed qualitative, functional, and metabolic analysis of the contents and membrane components of highly purified lysosomes isolated by subcellular fractionation on Percoll gradients. 3) The detailed analysis of MP 62 from normal lysosomal membranes, and demonstration of its interaction with NeuAc in binding assays or by affinity chromatographic methods, and 4) Demonstration of NeuAc transport in isolated normal lysosomes or lysosomal vesicles (and impaired transport in analogous fractions from affected fibroblasts), the detailed analysis of the transport mechanism, and the reconstitution of the transport system in liposomes.
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