The neuronal ceroid lipofuscinoses are pediatric neurodegenerative diseases with common clinical features. Of the nine clinical variants (CLN1-CLN9), six have been genetically identified. Most variant Show more
The neuronal ceroid lipofuscinoses are pediatric neurodegenerative diseases with common clinical features. Of the nine clinical variants (CLN1-CLN9), six have been genetically identified. Most variants manifest cell death and dysregulated sphingolipid metabolism, suggesting the proteins defective in these disorders may interact along one pathway. NCL patient-derived cell lines exhibit cell growth and apoptotic defects that reverse following transfection with the wild-type gene. The membrane-bound proteins CLN3, CLN6, and CLN8 complement each other, as do CLN1 and CLN2 proteins, with respect to growth and apoptosis. The CLN2 protein also corrects growth and apoptosis in CLN3-, CLN6-, and CLN8-deficient cell lines. Neither CLN1-deficient nor CLN2-deficient growth defects are corrected by CLN3, CLN6, and CLN8 proteins. CLN2, CLN3, CLN6, and CLN8 proteins co-immunoprecipitate and co-localize to early and/or recycling endosomes and lipid rafts. Additionally, CLN2p and CLN1p co-immunoprecipitate. The work presented supports interactions between NCL proteins occurring at multiple points along one pathway. Show less
Juvenile neuronal ceroid lipofuscinosis (JNCL) is an autosomal recessively inherited lysosomal storage disease involving a mutation in the CLN3 gene. The sequence of CLN3 was determined in 1995; howev Show more
Juvenile neuronal ceroid lipofuscinosis (JNCL) is an autosomal recessively inherited lysosomal storage disease involving a mutation in the CLN3 gene. The sequence of CLN3 was determined in 1995; however, the localization of the CLN3 gene product (Cln3p) was not confirmed. In this study, we investigated endogenous Cln3p using two peptide antibodies raised against two distinct epitopes of murine Cln3p. Identification of the liver 60 kDa protein as Cln3p was ascertained by amino acid sequence analysis using tandem mass spectrometry. Liver Cln3p was predominantly localized in the lysosomal membranes, not in endoplasmic reticulum (ER) or Golgi apparatus. As the tissue concentration of brain Cln3p was much lower than that of liver Cln3p, it could be detected only after purification from brain extract using anti-Cln3p IgG Sepharose. The apparent molecular masses of liver Cln3p and brain Cln3p were determined to be about 60 kDa and 55 kDa, respectively. Both brain and liver Cln3p were deglycosylated by PNGase F treatment to form polypeptides with almost the same molecular mass (45 kDa). However, they were not affected by Endo h treatment. In addition, it was also elucidated that the amino terminal region of Cln3p faces the cytosol. Show less