CLN5 and CLN8 protein association with ceramide synthase: Biochemical 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: Actins - chemistry; Actins - metabolism; Amino Acid Sequence; Animals; Cell Line; Ceramide synthase; Chromosome Mapping; CLN5p; CLN8p; CLN9p; Female; Fibroblasts - chemistry; Fibroblasts - metabolism; Histones - genetics; Histones - metabolism; Homozygote; Humans; Lysosomal Membrane Proteins; Male; Membrane Proteins - chemistry; Membrane Proteins - genetics; Membrane Proteins - metabolism; Mice; Molecular Sequence Data; Neuronal ceroid lipofuscinosis; Neuronal Ceroid-Lipofuscinoses - genetics; Neuronal Ceroid-Lipofuscinoses - metabolism; Proteomics - methods; Sequence Analysis, DNA; Sphingosine N-Acyltransferase - chemistry; Sphingosine N-Acyltransferase - metabolism; Vimentin - genetics; Vimentin - metabolism
• 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 CLN9 variant, are reclassified as CLN5 disease. CLN5‐deficient (CLN5−/−) fibroblasts demonstrate adhesion defects, increased growth, apoptosis, and decreased levels of ceramide, sphingomyelin, and glycosphingolipids. The CLN8 protein (CLN8p) corrects growth and apoptosis in CLN5−/− cells. Related proteins containing a Lag1 motif (CerS1/2/4/5/6) partially corrected these deficits, with CerS1, which is primarily expressed in brain, providing the best complementation, suggesting CLN5p activates CerS1 and may co‐immunoprecipitate with it. CLN8p complements CLN5‐deficient cells, consolidating the interrelationship of CLN5p/CLN8p, whose potential roles are explored as activators of (dihydro)ceramide synthases. Homozygosity mapping using microarray technology led to identification of CLN5 as the culprit gene in previously classified CLN9‐defective cases. Similar to CLN5−/−cells, ceramide synthase activity, C16/C18:0/C24:0/C24:1 ceramide species, measured by MS is decreased in CLN8−/− cells. Comparison of normal versus CLN5−/− cell CerS1‐bound proteins by immunoprecipitation, differential gel electrophoresis, and MS revealed absence of γ‐actin in CLN5−/− cells. The γ‐actin gene sequence is normal in CLN5−/− derived DNA. The γ‐actin‐bound proteins, vimentin and histones H2Afz/H3F3A/Hist1H4, were absent from the γ‐actin protein complex in CLN5−/− cells. The function of CLN5p may require vimentin and the histone proteins to bind γ‐actin. Defective binding could explain the CLN5−/− cellular phenotype. We explore the role of the CLN5/CLN8 proteins in ceramide species specific sphingolipid de novo synthesis, and suggest that CLN5/CLN8 proteins are more closely related than previously believed.