The Niemann–Pick C1 protein in feline fibroblasts William S. Garver, a, * Kyra Somers, b Kumar Krishnan, a Thomas Mitchell, b Randall A. Heidenreich, a and Mary Anna Thrall b a Department of Pediatrics, Section of Medical and Molecular Genetics, Arizona Health Sciences Center, The University of Arizona, Tucson, AZ 85724, USA b Department of Microbiology, Immunology and Pathology, Pathology Building, College of Veterinary Medicine and Biological Sciences, Colorado State University, Fort Collins, CO 80523, USA Received 1 February 2002; accepted 1 March 2002 Abstract 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 con- ducted 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 pre- immune 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 sum- mary, 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. Ó 2002 Elsevier Science (USA). All rights reserved. Keywords: Niemann–Pick type C; Niemann–Pick C1; Feline; Fibroblasts 1. Introduction Niemann–Pick type C (NPC) disease is a rare inher- ited metabolic disorder characterized by hepatospleno- megaly, progressive neurodegeneration, and storage of lipids such as cholesterol and glycosphingolipids in most tissues [1,2]. The concentration of cholesterol in tissues increases as a function of age, except for the brain where there is a simultaneous loss of cholesterol from myelin of the central nervous system [3]. It is therefore believed that while there is a loss of cholesterol from myelin, the accumulation of cholesterol in neurons and glial cells of the central nervous system is associated with neurode- generation [4]. Recent studies using the feline model for NPC have also found that glycosphingolipids may also have an important role in the pathogenesis of NPC disease [5]. At the cellular level, cholesterol has been shown to accumulate within late endosomes/lysosomes and trans-cisternae of the Golgi apparatus, subsequently delaying cholesterol transport to other cellular com- partments involved in maintaining cholesterol homeo- stasis [6–8]. The human, mouse, and feline genes responsible for the first complementation group of NPC have been identified and shown to encode the Niemann–Pick C1 (NPC1) protein [9–11]. The NPC1 protein contains several structural motifs required for regulating mobi- lization of late endosomal/lysosomal cholesterol. Among the structural motifs is a unique cysteine-rich NPC1 domain that contains a leucine zipper, a sterol- sensing domain that is homologous to 3-hydroxy-3- methylglutaryl coenzyme A (HMG-CoA) reductase and Molecular Genetics and Metabolism 76 (2002) 31–36 www.academicpress.com * Corresponding author. Fax: +520-626-7407. E-mail address: wgarver@peds.arizona.edu (W.S. Garver). 1096-7192/02/$ - see front matter Ó 2002 Elsevier Science (USA). All rights reserved. PII:S1096-7192(02)00015-X