Molecular consequences of the pathogenic mutation in feline GM1 gangliosidosis

Abstract

G M1 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 G M1 ganglioside. To characterize the genetic mutation responsible for feline G M1 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 G M1 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 G M1 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 G M1 fibroblasts to 18-times normal. β-Galactosidase protein levels in G M1 tissues were normal on Western blots, but immunofluorescence analysis demonstrated that the majority of mutant β-galactosidase protein did not reach the lysosome. Additionally, G M1 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 G M1 gangliosidosis. CIHR Author Manuscript CIHR Author Manuscript CIHR Author ManuscriptThe lysosomal enzyme β-D-galactosidase (βgal, EC 3.2.1.23) cleaves terminal galactose residues from a variety of molecules, including gangliosides G A1 and G M1 . Deficiency of βgal is known to cause two lysosomal storage diseases: G M1 gangliosidosis (neuronopathic) and Morquio B Disease (mucopolysaccharidosis IVB, non-neuronopathic [3,4]. Although the in vivo biochemical effect of the Arg482His mutation often is difficult to discern because it occurs most frequently in compound heterozygotes, patients homozygous for the G1445A substitution present with the infantile (most severe) form of G M1 gangliosidosis [3,7,8]. A similar mutation, Arg482Cys, also produced no residual βgal activity after expression in G M1 gangliosidosis fibroblasts [4].Feline G M1 gangliosidosis, first described in a Siamese cat in 1971 [9], models the juvenile form of the human disease. Onset of clinical neurological disease in affected cats occurs at approximately 3.5 months of age with a fine head or limb tremor. G M1 mutant cats have progressive dysmetria and ambulatory difficulties, with blindness and epileptiform seizures in the terminal disease stage at 9-10 months of age. In the current st...

Diltiazem, a L-type Ca2+ channel blocker, also acts as a pharmacological chaperone in Gaucher patient cells

“…Indirect immunofluorescence and confocal microscopy imaging were performed as previously described [37]. Primary/secondary antibodies and nuclear staining were as previously reported [36].…”

Evaluation of N-nonyl-deoxygalactonojirimycin as a pharmacological chaperone for human GM1 gangliosidosis leads to identification of a feline model suitable for testing enzyme enhancement therapy

“…mutations, used in this study are indicated in Table 1 along with their response to NN-DGJ. Immortalized fibroblasts cell lines from normal (SV3) and GM1 cats (GM1 SV3) were generated in the D. Martin laboratory [28] by calcium phosphate transfection of primary fibroblasts with the plasmid pSV3-DHFR (American Type Culture Collection) containing the large T antigen of Simian Virus 40.…”

“…Indirect immunofluorescence and confocal microscopy imaging for human or feline fibroblasts in the presence or absence of NN-DGJ were performed as previously reported [28].…”

“…GM1 Shiba dogs cannot be used for testing EET-agents, because they are homozygous for a deletion mutation, c.Δ C1647 in the coding region of the protein, which results in no β-Gal (protein or enzyme activity) being made [26,27]. We have recently reported that a naturally occurring feline model of GM1 is caused by a missense mutation [28], p.R483P, which aligns with human p.R482. A similar substitution, p.R482H, has been associated with type 1 GM1 in humans [29], making the results from this model directly applicable to a defined human patient population.…”

“…In the recently published crystal structure of human β-Gal [30], p. Arg482 (a totally buried residue) appears to play an important role in the interdomain interactions within the functional enzyme through the formation of an ion pair with E131 (in the TIM barrel domain), as well as hydrogen bonds with H412, D491 and F492 (within the β-domain 1). The feline β-Gal deficiency disease, initially described in 1971, is a nearly identical replica of human type 2, juvenile GM1 both clinically and biochemically [31], as well as at the molecular level [28].…”