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Abstract
The missense mutation, L476P, in the N-acetylgalactosamine 4-sulfatase (4S) gene, has previously been shown to be associated with a severe feline mucopolysaccharidosis type VI (MPS VI) phenotype. The present study describes a second mutation, D520N, in the same MPS VI cat colony, which is inherited independently of L476P and is associated with a clinically mild MPS VI phenotype in D520N/L476P compound heterozygous cats. Biochemical and clinical assessment of L476P homozygous, D520N/L476P compound heterozygous, and D520N homozygous cats demonstrated that the entire range of clinical phenotypes, from severe MPS VI, to mild MPS VI, to normal are clustered within a narrow range of residual 4S activity from 0. 5% to 4.6% of normal levels. When overexpressed in CHO-KI cells, the secreted form of D520N 4S was inactivated in neutral pH conditions. In addition, intracellular D520N 4S protein was rapidly degraded and corresponded to 37%, 14.5%, and 0.67% of normal 4S protein levels in the microsomal, endosomal, and lysosomal compartments, respectively. However, the specific activity of lysosomal D520N 4S was elevated 22. 5-fold when compared with wild-type 4S. These results suggest that the D520N mutation causes a rapid degradation of 4S protein. The effect of this is partially ameliorated as a result of a significant elevation in the specific activity of mutant D520N 4S reaching the lysosomal compartment.
Highlights
The missense mutation, L476P, in the N-acetylgalactosamine 4-sulfatase (4S) gene, has previously been shown to be associated with a severe feline mucopolysaccharidosis type VI (MPS VI) phenotype
Biochemical and clinical assessment of L476P homozygous, D520N/L476P compound heterozygous, and D520N homozygous cats demonstrated that the entire range of clinical phenotypes, from severe MPS VI, to mild MPS VI, to normal are clustered within a narrow range of residual 4S activity from 0.5% to 4.6% of normal levels
This study describes the identification and molecular characterization of a missense mutation (D520N), which causes a rapid lysosomal degradation of feline 4S (f4S) protein but is associated with a significant increase in specific activity
Summary
IDENTIFICATION OF AN N-ACETYLGALACTOSAMINE-4-SULFATASE MUTATION CAUSING INSTABILITY AND INCREASED SPECIFIC ACTIVITY*. A number of colony cats that had no clinical indications of MPS VI had very low or, in some instances, undetectable 4S activity levels in their blood leukocytes despite being genotyped as normal with respect to the L476P mutation. These observations suggested the presence of a second mutation (10). A PCR-based screening method enabled the identification of L476P homozygous cats, D520N/L476P compound heterozygous cats, and D520N homozygous cats in the colony Clinical assessments of these cats have previously demonstrated that the three genotypes are associated with a severe MPS VI phenotype, mild MPS VI phenotype, and normal phenotype, respectively (10, 11). The D520N mutation was engineered into the wild-type f4S cDNA and expressed in CHO-KI cells to determine its effect upon 4S synthesis, specific activity, intracellular processing, and trafficking
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