Molecular consequences of the pathogenic mutation in feline GM1 gangliosidosis

GM1 gangliosidosis is an inherited, fatal neurodegenerative disease caused by deficiency of lysosomal β-d-galactosidase (EC 3.2.1.23) and consequent storage of undegraded GM1 ganglioside. To characterize the genetic mutation responsible for feline GM1 gangliosidosis, the normal sequence of feline β-galactosidase cDNA first was defined. The feline β-galactosidase open reading frame is 2010 base pairs, producing a protein of 669 amino acids. The putative signal sequence consists of amino acids 1–24 of the β-galactosidase precursor protein, which contains seven potential N-linked glycosylation sites, as in the human protein. Overall sequence homology between feline and human β-galactosidase is 74% for the open reading frame and 82% for the amino acid sequence. After normal β-galactosidase was sequenced, the mutation responsible for feline GM1 gangliosidosis was defined as a G to C substitution at position 1448 of the open reading frame, resulting in an amino acid substitution at arginine 483, known to cause GM1 gangliosidosis in humans. Feline β-galactosidase messenger RNA levels were normal in cerebral cortex, as determined by quantitative RT-PCR assays. Although enzymatic activity is severely reduced by the mutation, a full-length feline β-galactosidase cDNA restored activity in transfected GM1 fibroblasts to 18-times normal. β-Galactosidase protein levels in GM1 tissues were normal on Western blots, but immunofluorescence analysis demonstrated that the majority of mutant β-galactosidase protein did not reach the lysosome. Additionally, GM1 cat fibroblasts demonstrated increased expression of glucose-related protein 78/BiP and protein disulfide isomerase, suggesting that the unfolded protein response plays a role in pathogenesis of feline GM1 gangliosidosis.