ABSTRACT
Niemann-Pick C (NPC) disease is an autosomal recessive neurovisceral lysosomal storage disorder that results in defective intracellular transport of cholesterol. The major form of human NPC (NPC1) has been mapped to chromosome 18, the NPC1 gene (NPC1) has been sequenced and several mutations have been identified in NPC1 patients. A feline model of NPC has been characterized and is phenotypically, morphologically, and biochemically similar to human NPC1. Complementation studies using cultured fibroblasts from NPC affected cats and NPC1 affected humans support that the gene responsible for the NPC phenotype in this colony of cats is orthologous to human NPC1. Using human-based PCR primers, initial fragments of the feline NPC cDNA were amplified and sequenced. From these sequences, feline-specific PCR primers were generated and designed to amplify six overlapping bands that span the entire feline NPC1 open reading frame. A single base substitution (2864G-C) was identified in NPC1 affected cats. Obligate carriers are heterozygous at the same allele and a PCR-based assay was developed to identify the geneotype of all cats in the colony. The mutation results in an amino acid change from cysteine to serine (C955S). Several of the mutations identified in people occur in the same region. Marked similarity exists between the human and feline NPC1 cDNA sequences, and is greater than that between the human and murine NPC1 sequences. The human cDNA sequence predicts a 1278aa protein with a lysosomal targeting sequence, several trans-membrane domains and extensive homology with other known mediators of cholesterol homeostasis.
Subject(s)
Cat Diseases/genetics , Mutation , Niemann-Pick Diseases/genetics , Amino Acid Sequence , Amino Acid Substitution , Animals , Base Sequence , Carrier Proteins/genetics , Cats , DNA Mutational Analysis , Deoxyribonucleases, Type II Site-Specific/genetics , Disease Models, Animal , Heterozygote , Intracellular Signaling Peptides and Proteins , Membrane Glycoproteins/genetics , Molecular Sequence Data , Niemann-Pick C1 Protein , Sequence Homology, Amino AcidABSTRACT
Niemann-Pick type C (NPC) disease is a rare inherited metabolic disorder characterized by hepatosplenomegaly, progressive neurodegeneration, and storage of lipids such as cholesterol and glycosphingolipids in most tissues. The current study was conducted to characterize the Niemann-Pick C1 (NPC1) protein in feline fibroblasts. This was accomplished by generating rabbit polyclonal antibodies against a peptide corresponding to amino acids 1256-1275 of the feline NPC1 protein. The results obtained using immunoblot analysis identified two major proteins that migrated at approximately 140 and 180 kDa. These two proteins were absent when immunoblots were incubated in the presence of feline NPC1 antibody and immunizing peptide, or preimmune serum. Fluorescence microscopy of feline fibroblasts incubated with the feline NPC1 antibody revealed granular staining within the perinuclear region of the cell. This granular staining was diminished when feline fibroblasts were incubated in the presence of feline NPC1 antibody and immunizing peptide, or was completely absent when feline fibroblasts were incubated in the presence of preimmune serum. Additional studies using double-labeled fluorescence microscopy indicated that feline NPC1 partially colocalized with markers for late endosomes/lysosomes, endoplasmic reticulum, and microtubules, but not the trans-Golgi network. In summary, the results presented in this report demonstrate that the NPC1 protein in feline fibroblasts has a similar distribution as that previously described for human and murine fibroblasts.
