Characterization Of The Lysosomal Protein Beta-Galactosidase In Gm1-Gangliosidosis Affected Sheep

Masis Isikbay


GM1-Gangliosidosis is an autosomal recessive Lysosomal Storage Disorder that has been identified in humans, sheep, and numerous other mammalian species. GM1 is a fatal disease with no cure that is characterized by acute neurological degeneration. It usually presents with decreased activity in beta-galactosidase, a lysosomal protein coded for by the GLB1 gene. In humans beta-galactosidase normally combines with 2 other proteins, alpha- neuraminidase and cathepsin A to form a Lysosomal Multienzyme Complex that stabilizes its components. It is hypothesized that GM1-affected sheep have a mutation that alters the structure of beta-galactosidase such that it interferes with formation of the complex leading to a unique, secondary deficiency of alpha-neuraminidase. This study utilized molecular techniques to help elucidate the underlying genetic defect that causes GM1 in this ovine model. A 64-kDa beta-galactosidase protein was observed in fibroblast cell homogenates using Western blotting and immunochemiluminescence. No size difference was detected in beta-galactosidase between samples from normal and GM1-affected sheep. RT-PCR was used, after total RNA isolation from normal and GM1-affected sheep cells, to amplify exons 2-16 of the GLB1 transcript. A few possible mutation sites were identified when sequence chromatograms were compared. In addition, what appears to be an alternatively spliced transcript consisting of exons 2-6 and 16 was observed in PCR samples from both normal and GM1-affected sheep. Further analysis is underway to verify these preliminary findings and to sequence the illusive 5` and 3` ends of the ovine GLB1 cDNA.


Lysosomal Storage Disorder; beta-galactosidase; ovine GM1-gangliosidosis

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