Characterisation of a lysosomal sialidase, G9 (NEU)
The G9 gene, located within the MHC class III region of man and mouse, has been shown to encode a 46kDa sialidase, optimally active at pH 4.6. The genomic localization of G9 indicates that it corresponds to the Neu1 gene, which was mapped to the S region of the H-2 complex (the murine MHC class III region) by genetic linkage studies and analysis of Neu1 phenotypes in mice, and has been shown to modulate the sialic acid content of several liver lysosomal enzymes. Three alleles of Neu1 have been defined. The most common allele, Neu1b, is associated with normal levels of liver sialidase activity, whilst a small number of mouse strains, e.g. the SM/J strain, that carry the Neu1a allele, show significantly reduced sialidase activity. In humans, reduced sialidase activity leads to the lysosomal storage disorder, sialidosis, characterised by developmental and neurological abnormalities. Characterisation of the substrate specificity of the G9 sialidase, expressed and purified from Drosophila S2 cells, showed that the enzyme hydrolyses the synthetic substrate 4MU-NANA. However, little or no activity was seen towards a number of natural substrates, including gangliosides, or towards the synthetic substrate, fetuin. It has been established that the G9 sialidase is unique within the sialidase family, in that it requires association with another lysosmal protein protective protein cathepsin A (PPCA) in order to be optimally active. Co-expression of G9 and PPCA in both mammalian and Drosophila expression systems has been attempted in order to accurately characterise the substrate specificity of the G9 sialidase. The reduced sialidase activity within the SM/J mouse makes this a good model to investigate the role of G9 in the immune response. SM/J mice are known to be approximately 20% smaller in size than normal mice, however, it was observed that the thymus size of the SM/J mouse was ~60% smaller than that of an age matched C57BL/6 control. Characterisation of the B- and T-cells of the spleen and thymus of SM/J mice showed variations in the populations of these cells between the two mice strains, with the most significant difference observed in the CD4+ thymic T-cell population. Analysis of antisera taken from SM/J and C57BL/6 control mice after primary and secondary immune responses showed that the levels of antibody isotypes compared in the two strains were similar except for IgG1, which was present at significantly reduced levels in the sera of SM/J mice. Deficiency in hexosaminidase A results in the ganglioside storage disorder, Tay-Sachs disease. HexA-/- mice were generated as a model for Tay-Sachs disease, however, a catabolic bypass, mediated by the lysosomal sialidase, G9, (which occurs in mice but not humans) allows the Tay-Sachs disease model to escape disease onset in the first year of life, in contrast to the progression of the disease in human sufferers. In order to generate a more accurate disease model, the sialidase deficient mouse strain, SM/J has been crossed with the existing Tay-Sachs disease model. This crossing should substantially reduce the sialidase activity that allows the mice to escape onset of Tay-Sachs and thus lead to a disease model more comparable to human Tay-Sachs sufferers.