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CLN5 and CLN8 protein association with ceramide synthase: biochemical and proteomic approaches.

Publication ,  Journal Article
Haddad, SE; Khoury, M; Daoud, M; Kantar, R; Harati, H; Mousallem, T; Alzate, O; Meyer, B; Boustany, R-M
Published in: Electrophoresis
December 2012

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.

Duke Scholars

Published In

Electrophoresis

DOI

EISSN

1522-2683

Publication Date

December 2012

Volume

33

Issue

24

Start / End Page

3798 / 3809

Location

Germany

Related Subject Headings

  • Vimentin
  • Sphingosine N-Acyltransferase
  • Sequence Analysis, DNA
  • Proteomics
  • Neuronal Ceroid-Lipofuscinoses
  • Molecular Sequence Data
  • Mice
  • Membrane Proteins
  • Male
  • Lysosomal Membrane Proteins
 

Citation

APA
Chicago
ICMJE
MLA
NLM
Haddad, S. E., Khoury, M., Daoud, M., Kantar, R., Harati, H., Mousallem, T., … Boustany, R.-M. (2012). CLN5 and CLN8 protein association with ceramide synthase: biochemical and proteomic approaches. Electrophoresis, 33(24), 3798–3809. https://doi.org/10.1002/elps.201200472
Haddad, Saria El, Marwan Khoury, Mohammad Daoud, Rami Kantar, Hayat Harati, Talal Mousallem, Oscar Alzate, Brian Meyer, and Rose-Mary Boustany. “CLN5 and CLN8 protein association with ceramide synthase: biochemical and proteomic approaches.Electrophoresis 33, no. 24 (December 2012): 3798–3809. https://doi.org/10.1002/elps.201200472.
Haddad SE, Khoury M, Daoud M, Kantar R, Harati H, Mousallem T, et al. CLN5 and CLN8 protein association with ceramide synthase: biochemical and proteomic approaches. Electrophoresis. 2012 Dec;33(24):3798–809.
Haddad, Saria El, et al. “CLN5 and CLN8 protein association with ceramide synthase: biochemical and proteomic approaches.Electrophoresis, vol. 33, no. 24, Dec. 2012, pp. 3798–809. Pubmed, doi:10.1002/elps.201200472.
Haddad SE, Khoury M, Daoud M, Kantar R, Harati H, Mousallem T, Alzate O, Meyer B, Boustany R-M. CLN5 and CLN8 protein association with ceramide synthase: biochemical and proteomic approaches. Electrophoresis. 2012 Dec;33(24):3798–3809.
Journal cover image

Published In

Electrophoresis

DOI

EISSN

1522-2683

Publication Date

December 2012

Volume

33

Issue

24

Start / End Page

3798 / 3809

Location

Germany

Related Subject Headings

  • Vimentin
  • Sphingosine N-Acyltransferase
  • Sequence Analysis, DNA
  • Proteomics
  • Neuronal Ceroid-Lipofuscinoses
  • Molecular Sequence Data
  • Mice
  • Membrane Proteins
  • Male
  • Lysosomal Membrane Proteins