G(M1)-ganglioside degradation and biosynthesis in human and murine G(M1)-gangliosidosis.

BACKGROUND: Gangliosides are building blocks of cell membranes and their biosynthesis and degradation have been extensively studied in the past. Regulation of the metabolism of these glycolipids controls fundamental cell functions. G(M1)-gangliosidosis, a neurodegenerative glycosphingolipid storage disease, is caused by deficiency of lysosomal beta-galactosidase with consequent disruption of the normal degradative pathway of G(M1)-ganglioside. We studied the impact of G(M1)-ganglioside accumulation on its biosynthetic enzyme in cells and tissues from human patients and from the G(M1)-gangliosidosis mouse model. METHODS: We tested the qualitative and quantitative pattern of gangliosides by thin layer chromatography and N-acetylneuraminic acid dosage, respectively. Regulation of G(M1)-ganglioside biosynthesis was evaluated by G(M1) synthase assay in human and murine samples. RESULTS: G(M1)-ganglioside accumulation has an inhibitory effect on the human but not on the mouse G(M1) synthase. We present evidence that G(M1) synthase activity in human and murine cells are regulated by different mechanisms. CONCLUSIONS: Alternative pathways in the mouse may account for these results and possibly explain some of the phenotypical differences between the human and mouse forms of this disorder.

Determinant factors of spectrum of missense variants in mucopolysaccharidosis IVA gene.

Design of efficient treatment strategies for diseases requires clarification of the nature of each mutation causing the disease. In this study, we have investigated three factors to correctly predict the correlation between genotype and phenotype on N-acetylgalactosamine-6-sulfate sulfatase (GALNS) gene responsible for one of lysosomal storage diseases, known as mucopolysaccharidosis IVA (MPS IVA); (i) evolutionary conservation of amino acid residues among family proteins, (ii) conservativeness of amino acid changes in GALNS, and (iii) structural conservation of amino acid residue. The results showed that (i) the likelihood of a missense variant causing MPS IVA was directly correlated with the level of evolutionary conservation and inversely correlated with conservativeness but not correlated with the structural conservation, (ii) the disease-causative mutations were 9 times more likely to be located on the 'highly conserved' residues than the polymorphisms, (iii) the likelihood of 'non-conservative' amino acid changes in missense mutations was 6.8 times higher than those in the polymorphisms, (iv) the degree of evolutionary conservation was nearly as predictive in phenotype as that of conservativeness of amino acid changes, and (v) the combination of the two factors, evolutionary conservation and conservativeness, provides a better association between missense variants and clinical severity with higher sensitivity (83.5-88.9%) and specificity (71.4-88.3%), than that obtained by either factor alone. These findings suggest that the combination of evolutionary conservation and conservativeness is a useful tool to predict the effect of each mutation on the clinical phenotype and can be applied to the analysis of phenotype/genotype relation in other genetic diseases.

Development of MPS IVA mouse (Galnstm(hC79S.mC76S)slu) tolerant to human N-acetylgalactosamine-6-sulfate sulfatase.

Mucopolysaccharidosis IVA (MPS IVA) is an autosomal recessive disease caused by N-acetylgalactosamine-6-sulfate sulfatase (GALNS) deficiency. In recent studies of enzyme replacement therapy for animal models with lysosomal storage diseases, cellular and humoral immune responses to the injected enzymes have been recognized as major impediments to effective treatment. To study the long-term effectiveness and side effects of therapies in the absence of immune responses, we have developed an MPS IVA mouse model, which has many similarities to human MPS IVA and is tolerant to human GALNS protein. We used a construct containing both a transgene (cDNA) expressing inactive human GALNS in intron 1 and an active site mutation (C76S) in adjacent exon 2 and thereby introduced both the inactive cDNA and the C76S mutation into the murine Galns by targeted mutagenesis. Affected homozygous mice have no detectable GALNS enzyme activity and accumulate glycosaminoglycans in multiple tissues including visceral organs, brain, cornea, bone, ligament and bone marrow. At 3 months, lysosomal storage is marked within hepatocytes, reticuloendothelial Kupffer cells, and cells of the sinusoidal lining of the spleen, neurons and meningeal cells. The bone storage is also obvious, with lysosomal distention in osteoblasts and osteocytes lining the cortical bone, in chondrocytes and in the sinus lining cells in bone marrow. Ubiquitous expression of the inactive human GALNS was also confirmed by western blot using the anti-GALNS monoclonal antibodies newly produced, which resulted in tolerance to immune challenge with human enzyme. The newly generated MPS IVA mouse model should provide a good model to evaluate long-term administration of enzyme replacement.