A two-dimensional protein fragmentation-proteomic study of neuronal ceroid lipofuscinoses: identification and...

The neuronal ceroid lipofuscinoses (NCLs) are a group of neuronal degenerative diseases that primarily affect children. Previously we hypothesized that the similarity of the phenotypes among the variant subtypes of NCL suggests that the NCLs share a common metabolic functional pathway. To test our hypothesis, we have studied several candidate proteins identified using a proteomic approach. We analyzed their differential expression and cataloged their functions and involved pathways. Forty protein peaks, differentially expressed in NCLs, were selected from two-dimensional protein fragmentation (PF2D) maps and twenty-four proteins were identified by MALDI-TOF-MS or LC-ESI-MS/MS. Six proteins were verified by further Western blotting. Our results showed that annexin A1, annexin A2, and vimentin were significantly down-regulated in NCL1, NCL2, NCL3, and NCL8 cells; galectin-1 was down-regulated in NCL1, NCL3, and NCL8 but up-regulated in NCL2 cells; and isoform 5 of caldesmon was up-regulated in all NCL cell types. The histone 2B was down-regulated in NCL3. Functional analysis showed that the differentially expressed proteins identified by PF2D could be grouped into categories of intermediate filaments, cell motility, apoptosis, cytoskeleton, membrane trafficking, calcium binding, nucleosome assembly, pigment granule and cell development. Immunocytochemistry revealed nuclear translocalization of annexin A1 in CLN2-deficient fibroblasts and abnormal distribution of L-caldesmon in cultured CLN1, CLN2, CLN3 and CLN8-deficient fibroblasts. Finding differentially expressed proteins in variant NCLs, which showed disturbances of cytoskeleton, RAGE-dependent cellular pathways and decreased glycolysis provides evidence supporting our hypothesis. These findings may contribute to the discovery of molecular biomarkers and may help further elucidate the pathogenic mechanisms underlying the NCLs.