Alex Quakenbush


Spring 2015

Faculty Advisor

Ami Ahern-Rindell


College of Arts and Sciences




Lysosomal Storage Disorders are a class of inherited metabolic conditions that result from alterations in the function of lysosomal enzymes.One example is GM1 Gangliosidosis (GM1), a disorder in which the activity of β-galactosidase is deficient resulting in neurodegeneration and early death.The enzyme, β-gal, is a member of the Lysosomal Multienzyme Complex (LMC), which transports proteins to the lysosome and enables various functions.LMC members include β-gal, α-neuraminidase and the Protective Protein Cathepsin A (PPCA).In a unique ovine model of GM1, there is a primary deficiency in the activity of β- galactosidase and a secondary deficiency in α-neuraminidase activity.The cause of the secondary deficiency in α-neuraminidase activity, which is not seen in any other animal model of GM1, is currently unknown.The α-neuraminidase protein is coded for by the NEU1 gene and is, a glycohydrolitic enzyme that is active in the lysosome.The secondary deficiency of α- neuraminidase seen in our inbred sheep may be due to a polymorphism in NEU1, which if taken in conjunction with the recently identified putative disease-causing mutation in the gene that codes for β-galactosidase (GLB1), may disrupt the ability of α-neuraminidase to properly bind to the other components in the LMC.This would prevent α-neuraminidase from properly trafficking to the lysosome and becoming activated.This project compares the exon sequence from inbred sheep affected with this unique form of GM1 to the sequence from sheep from the inbred flock that are not affected, looking for potential differences between the two sequences.The sequence comparison between affected and normal sheep may reveal differences that contribute to this secondary α-neuraminidase activity deficiency seen in our sheep model.Whole genomic DNA was isolated from normal and affected sheep.PCR was performed to amplify individual exons with specifically designed primers.Products were visualized through gel 5 electrophoresis and sent to Eurofins Genomics for sequencing of the amplicons.Analysis of sequence was done using the Sequencher program and BLAST from NCBI.Results indicate no confident or impactful NEU1 sequence differences between the normal and affected sheep or between these sequences and the reference sequence from GenBank.The lack of differences in the coding sequence for NEU1 may indicate the secondary deficiency of α-neuraminidase results instead from a polymorphic change in the gene coding for PPCA and that this is what prohibits proper formation of the LMC.Alternatively, the missense mutation identified in the GLB1 gene may alter the ß-galactosidase protein sufficiently that it impairs the overall structure of the LMC resulting in the secondary deficiency of α-neuraminidase.


Genetics, Experimental; Lysosomal storage diseases

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