Schindler disease: the molecular lesion in the alpha-N-acetylgalactosaminidase gene that causes an infantile neuroaxonal dystrophy.

Abstract

Schindler disease is a recently recognized infantile neuroaxonal dystrophy resulting from the deficient activity of the lysosomal hydrolase, a-N-acetylgalctosaminidase (a-GalNAc). The recent isolation and expression of the full-length cDNA encoding a-GalNAc facilitated the identification of the molecular lesions in the affected brothers from family D, the first cases described with this autosomal recessive disease. Southern and Northern hybridization analyses of DNA and RNA from the affected homozygotes revealed a grossly normal aGalNAc gene structure and normal transcript sizes and amounts. Therefore, the a-GalNAc transcript from an affected homozygote was reverse-transcribed, amplified by the polymerase chain reaction (PCR), and sequenced. A single G to A transition at nucleotide 973 was detected in multiple subclones containing the PCR products. This point mutation resulted in a glutamic acid to lysine substitution in residue 325 (E325K) of the a-GalNAc polypeptide. The base substitution was confirmed by dot blot hybridization analyses of PCR-amplified genomic DNA from family members with allele-specific oligonucleotides. Furthermore, transient expression of an a-GalNAc construct containing the E325K mutation resulted in the expression of an immunoreactive polypeptide which had no detectable a-GalNAc activity. (J. Clin. Invest. 1990Invest. .86:1752Invest. -1756

Glycoproteomics Analysis of Human Liver Tissue by Combination of Multiple Enzyme Digestion and Hydrazide Chemistry

“…34 The deficiency of lysosomal enzyme may cause lysosomal storage disease which is fatal to human of all ages. For example, Schindler disease is an infantile neuroaxonal dystrophy resulting from the deficient activity of the lysosomal hydrolase, R-N-acetylgalactosaminidase (R-NAGA), 35 which is identified with two N-glycosylation sites on Asn 177 and Asn 201 in this study. Glycosylation is largely restricted to the addition of complex glycans to proteins or lipids localized on the cell surface or within lumenal compartments of subcellular organelles, such as the endoplasmic reticulum (ER), Golgi apparatus, endosomes, or lysosomes.…”

Lysosomal alpha-N-acetylgalactosaminidase deficiency, the enzymatic defect in angiokeratoma corporis diffusum with glycopeptiduria.

“…Notably, transient expression of the mutant enzyme in COS-l cells resulted in the production ofan immunologically detectable enzyme protein evidencing the fact that the enzyme was synthesized, but was markedly unstable (21). Future identification of the molecular lesion causing a-N-acetylgalactosaminidase deficiency in the proband with angiokeratoma and expression ofthe mutant protein may provide insight into the biochemical nature of the enzymatic defect.…”

“…Recently, the molecular nature of the lesion causing the infantile form of a-N-acetylgalactosaminidase has been shown to be a missense mutation in codon 325 of exon 8 of the a-Nacetylgalactosaminidase gene that alters a glutamic acid to a lysine residue (21,22). Notably, transient expression of the mutant enzyme in COS-l cells resulted in the production ofan immunologically detectable enzyme protein evidencing the fact that the enzyme was synthesized, but was markedly unstable (21).…”

The molecular lesion in the alpha-N-acetylgalactosaminidase gene that causes angiokeratoma corporis diffusum with glycopeptiduria.

“…Presumably, the mature glycopeptide is dimerized to form the 117-kD active form in the Golgi. Recently, the molecular lesion in the consanguineous German brothers with the infantile-onset form was identified as a single G to A transition at nucleotide (nt) 973 which predicted a glutamic acid to lysine substitution at residue 325 (designated E325K) in the a-GalNAc polypeptide (14). Cultured fibroblasts from the infantile-onset patients, who were homoallelic for the E325K mutation, had < 1% normal a-GalNAc activity and no detectable immunoreactive enzyme protein.…”

“…The sequence was confirmed by dideoxy sequencing, and then the mutant cDNA was subcloned into the eukaryotic expression vector p91023(B) (31) kindly provided by Dr. R. J. Kaufman (Genetics Institute, Cambridge, MA). This expression construct was designated p91-R329W, and the previously constructed expression vectors for the normal and E325K alleles (14) were designated p91-AGB and p91-E325K, respectively. For transient expression studies, each construct was transfected into COS-1 cells (32), harvested at 72 h after transfection, and then assayed for activity and immunoreactive protein as previously described (7).…”