CLN3 disease process: missense point mutations and protein depletion in vitro

• Published in: European journal of paediatric neurology Vol. 5; pp. 81 - 88
• Main Authors: Golabek, Adam A., Kida, Elizabeth, Walus, Mariusz, Kaczmarski, Wojciech, Wujek, Piotr, Wisniewski, Krystyna E.
• Format: Journal Article
• Language: English
• Published: England Elsevier Ltd 2001
• Subjects: Antisense Elements (Genetics); Antisense experiments; Cell culture; Child; CLN3; Humans; In Vitro Techniques; Kidney - cytology; Membrane Glycoproteins; Microscopy, Confocal; Molecular Chaperones; Mutation, Missense; Mutations; Neuroblastoma; Neuronal Ceroid-Lipofuscinoses - genetics; Neuronal Ceroid-Lipofuscinoses - metabolism; Neurons - cytology; Neurons - enzymology; Overexpression; Proteins - analysis; Proteins - genetics; Proteins - metabolism; Transfection; Tumor Cells, Cultured; CLN3; Mutations; Cell culture; Overexpression; Antisense experiments
• ISSN: 1090-3798; 1532-2130
• DOI: 10.1053/ejpn.2000.0440

Although the CLN3 gene associated with the disease process in subjects with the juvenile form of neuronal ceroid lipofuscinosis was discovered in 1995, our knowledge of the physiological function of its gene product, CLN3 protein, is still incomplete. To gain more insight into the structural properties and function of CLN3 protein we studied at present: i) how the naturally occurring point mutations Arg334Cys and Leu101Pro affect the biological properties of CLN3 protein, and ii) whether depletion of CLN3 protein synthesis by using an antisense approach induces a distinct phenotype in cells of neuronal origin in vitro. Here we report that although both CLN3 mutant proteins are targeted to lysosomes, thus similar to wild-type CLN3 protein, they are devoid of the biological activity of wild-type CLN3 protein such as its effect on lysosomal pH or intracellular processing of amyloid- β protein precursor and cathepsin D in vitro. The Leu101 Pro mutation affected significantly the maturation and stability of CLN3 protein. The Arg334Cys mutation influenced mildly the maturation and turnover of CLN3 protein, but at the same time abolished the function of CLN3 protein in vitro, which suggests that the Arg334 may constitute a part of the active site of CLN3 protein. In addition, we show that depletion of CLN3 protein synthesis in human neuroblastoma cells in vitro induces outgrowth of long cellular processes and formation of cellular aggregates and affects the viability of these cells. This finding suggests that CLN3 protein is implicated in biological processes associated with the differentiation of cells of neuronal origin.