CLN 5 and CLN 8 protein association with ceramide synthase: B iochemical and proteomic approaches

• Published in: Electrophoresis 2012-12, Vol.33 (24), p.3798-3809
• Main Authors: Haddad, Saria El, Khoury, Marwan, Daoud, Mohammad, Kantar, Rami, Harati, Hayat, Mousallem, Talal, Alzate, Oscar, Meyer, Brian, Boustany, Rose‐Mary
• Format: Article
• Language: English
• Subjects: Apoptosis; Cellular; Complement; Electrophoresis; Genes; Histones; Patients; Proteins
• ISSN: 0173-0835; 1522-2683
• DOI: 10.1002/elps.201200472

Four patients with juvenile neuronal ceroid lipofuscinoses, a childhood neurodegenerative disorder that was previously described as CLN 9 variant, are reclassified as CLN 5 disease. CLN 5‐deficient ( CLN 5 −/− ) fibroblasts demonstrate adhesion defects, increased growth, apoptosis, and decreased levels of ceramide, sphingomyelin, and glycosphingolipids. The CLN 8 protein ( CLN 8p) corrects growth and apoptosis in CLN 5 −/− cells. Related proteins containing a L ag1 motif ( C er S 1/2/4/5/6) partially corrected these deficits, with C er S 1, which is primarily expressed in brain, providing the best complementation, suggesting CLN 5p activates C er S 1 and may co‐immunoprecipitate with it. CLN 8p complements CLN 5‐deficient cells, consolidating the interrelationship of CLN 5p/ CLN 8p, whose potential roles are explored as activators of (dihydro)ceramide synthases. Homozygosity mapping using microarray technology led to identification of CLN 5 as the culprit gene in previously classified CLN 9‐defective cases. Similar to CLN 5 −/− cells, ceramide synthase activity, C 16/ C 18:0/ C 24:0/ C 24:1 ceramide species, measured by MS is decreased in CLN 8 −/− cells. Comparison of normal versus CLN 5 −/− cell C er S 1‐bound proteins by immunoprecipitation, differential gel electrophoresis, and MS revealed absence of γ‐actin in CLN 5 −/− cells. The γ‐actin gene sequence is normal in CLN 5 −/− derived DNA . The γ‐actin‐bound proteins, vimentin and histones H 2 A fz/ H 3 F 3 A / H ist1 H 4, were absent from the γ‐actin protein complex in CLN 5 −/− cells. The function of CLN 5p may require vimentin and the histone proteins to bind γ‐actin. Defective binding could explain the CLN 5 −/− cellular phenotype. We explore the role of the CLN 5/ CLN 8 proteins in ceramide species specific sphingolipid de novo synthesis, and suggest that CLN 5/ CLN 8 proteins are more closely related than previously believed.