Gene defects responsible for the three major forms of the neuronal ceroid lipofuscinoses (NCLs), autosomal-recessive, neurodegenerative disorders of childhood, have been identified recently. Two of these genes, associated with the infantile (CLN1) and classical late infantile (CLN2) forms of NCL, code for lysosomal enzymes: palmitoyl-protein thioesterase and carboxypeptidase, respectively. The CNL3 gene, associated with juvenile NCL, the most common form of NCL in the United States, encodes a protein of predicted 438 amino acid residues. Until the present, 23 disease-associated mutations have been found in the CLN3 gene, including the 1.02-kb common deletion. The function of the CLN3 gene product is still unknown. Hydrophobicity plots indicate that CLN3 protein, in contrast to soluble lysosomal enzymes, is a highly hydrophobic transmembrane protein. Our preliminary date indicate that this protein is a resident of lysosomal membrane, has a long turnover rate, is phosphorylated, and shows extensive N-glycosylation of the complex type that demonstrates cell-type-specific differences. The selective vulnerability of particular cells to the CLN3 gene defect in spite of the widespread distribution of the storage suggests that CLN3 is vital for the metabolism of only specific cell types. Thus, this project proposal focuses on experimental verification of two working hypotheses predicting that i) the selective vulnerability of cells to the CLN3 gene defect is related to the different metabolism of CLN3 protein in particular cell types and ii) the selective vulnerability to the CLN3 gene defect is caused by the specific metabolic requirements of susceptible cells. As a first approach to elucidate these phenomena, we intend to address the following issues: i) whether the metabolism of CLN3 protein differs in various cell types and how disease-associated mutations affect this process and ii) how disease-associated mutations affect the function of lysosomes. The properties of CLN3 protein will be exploited in mammalian expression systems by expressing CLN3 protein alone, and CLN3 protein tagged with the green fluorescent protein and FLAG epitope. The biological consequences of disease-associated mutations for the biogenesis and integrity of lysosomes will be evaluated by using comparative morphological and biochemical studies of lymphocytic cell lines and cultured skin fibroblasts from healthy subjects and affected individuals also by experimentally modeling the lysosomal storage. These studies will be done by using immunoprecipitation, Western blotting, immunofluorescence, immunoelectron microscopy, protein sequencing, and subcellular fractionation techniques. We expect that the results of these studies will allow us to gain insight into the role of CLN3 protein in lysosomal function and to define how disease-associated mutations of CLN3 protein affect this process. These data also will help to clarify the role of the endosomal-lysosomal membrane in cell biology, in general.
