title: CLN5 and CLN8 protein association with ceramide synthase: biochemical and proteomic approaches.
abstract: 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.
authors.affiliation: Department of Pediatric, American University of Beirut, Beirut, Lebanon.
authors.forename: Saria El
authors.initials: SE
authors.lastname: Haddad
authors.forename: Marwan
authors.initials: M
authors.lastname: Khoury
authors.forename: Mohammad
authors.initials: M
authors.lastname: Daoud
authors.forename: Rami
authors.initials: R
authors.lastname: Kantar
authors.forename: Hayat
authors.initials: H
authors.lastname: Harati
authors.forename: Talal
authors.initials: T
authors.lastname: Mousallem
authors.forename: Oscar
authors.initials: O
authors.lastname: Alzate
authors.forename: Brian
authors.initials: B
authors.lastname: Meyer
authors.forename: Rose-Mary
authors.initials: RM
authors.lastname: Boustany
mesh_terms: D000199:Actins / Q000737:chemistry / Q000378:metabolism; D000595:Amino Acid Sequence; D000818:Animals; D002460:Cell Line; D002874:Chromosome Mapping; D005260:Female; D005347:Fibroblasts / Q000737:chemistry / Q000378:metabolism; D006657:Histones / Q000235:genetics / Q000378:metabolism; D006720:Homozygote; D006801:Humans; D051907:Lysosomal Membrane Proteins; D008297:Male; D008565:Membrane Proteins / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D051379:Mice; D008969:Molecular Sequence Data; D009472:Neuronal Ceroid-Lipofuscinoses / Q000235:genetics / Q000378:metabolism; D040901:Proteomics / Q000379:methods*; D017422:Sequence Analysis, DNA; D051049:Sphingosine N-Acyltransferase / Q000737:chemistry / Q000378:metabolism*; D014746:Vimentin / Q000235:genetics / Q000378:metabolism
chemical_list: D000199:Actins; C113332:CLN5 protein, human; C121767:CLN8 protein, human; C509977:CLN9 protein, human; D006657:Histones; D051907:Lysosomal Membrane Proteins; D008565:Membrane Proteins; D014746:Vimentin; C493790:CERS1 protein, human; D051049:Sphingosine N-Acyltransferase