Biochemical and molecular genetic studies of human alpha-N-acetylgalactosaminidase and Schindler disease.
Item
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Title
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Biochemical and molecular genetic studies of human alpha-N-acetylgalactosaminidase and Schindler disease.
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Identifier
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AAI9108187
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identifier
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9108187
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Creator
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Wang, Anne May.
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Contributor
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Adviser: Robert J. Desnick
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Date
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1990
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Language
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English
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Publisher
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City University of New York.
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Subject
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Biology, Molecular | Biology, Genetics | Health Sciences, Pathology
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Abstract
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Human {dollar}\alpha{dollar}-{dollar}N{dollar}-acetylgalactosaminidase ({dollar}\alpha{dollar}-GalNAc; E.C. 3.2.1.49), the lysosomal glycohydrolase that cleaves {dollar}\alpha{dollar}-{dollar}N{dollar}-acetylgalactosaminyl moieties from glycoconjugates, is encoded by a gene on chromosome 22q13 {dollar}\to{dollar} ter. Deficient enzymetic activity results in Schindler disease, an autosomal recessive disorder with increased urinary excretion of glycopeptides containing {dollar}\alpha{dollar}-{dollar}N{dollar}-acetylgalactosaminyl moieties. Two clinical phenotypes have been described. Type I is an infantile form of neuroaxonal dystrophy, whereas Type II is characterized by angiokeratoma corporis diffusum with no neurological involvement. In these studies, a full-length {dollar}\alpha{dollar}-GalNAc cDNA was characterized. The {dollar}\alpha{dollar}-GalNAc sequence encoded 411 amino acids. Functional integrity of the cDNA was demonstrated by transient expression. Northerns revealed two transcripts of 3.6 and 2.2 kb. The {dollar}\alpha{dollar}-GalNAc cDNA had 46.9% amino acid homology with human {dollar}\alpha{dollar}-galactosidase A ({dollar}\alpha{dollar}-Gal A) cDNA suggesting the evolutionary relatedness of these genes. Isolation of {dollar}\alpha{dollar}-GalNAc cDNA facilitated characterization of the molecular lesions in the first affected homozygotes with Schindler disease. These lesions were determined by the PCR-amplification and sequencing of {dollar}\alpha{dollar}-GalNAc cDNA from affected homozygotes with Type I and II disease. Type I disease, a single G to A transition at nucleotide 973 resulted in a glutamic acid to lysine substitution in residue 325 of the {dollar}\alpha{dollar}-GalNAc polypeptide. In Type II a single C to T transition was identified at nucleotide 985 which resulted in an arginine to tryptophan substitution at position 329. These base substitutions were confirmed by dot-blot analyses. Transient expression of {dollar}\alpha{dollar}-GalNAc constructs containing these transitions resulted in the expression of polypeptide with no detectable {dollar}\alpha{dollar}-GalNAc activity. The isolation of an {dollar}\alpha{dollar}-GalNAc cDNA facilitated the characterization of the {dollar}\alpha{dollar}-GalNAc structural gene. The {dollar}\alpha{dollar}-GalNAc gene was {dollar}\sim{dollar}14 kb with nine exons. The 5{dollar}\sp\prime{dollar} flanking sequence revealed two Sp1 and one GC-box promotor elements. Six Alu-repetitive elements were identified in the reverse orientation. Comparison of the {dollar}\alpha{dollar}-GalNAc gene with the {dollar}\alpha{dollar}-Gal A gene revealed homologous exonic placement of {dollar}\alpha{dollar}-GalNAc introns 2-7 with {dollar}\alpha{dollar}-Gal A introns 1-6. Predicted amino acid homology among {dollar}\alpha{dollar}-GalNAc exons 2-7 with {dollar}\alpha{dollar}-Gal A exons 1-6 ranged from 46.2% to 62.7%. In contrast, there was little similarity between {dollar}\alpha{dollar}-Gal A exon 7 and {dollar}\alpha{dollar}-GalNAc exons 8 and 9 (15.8% homology). These studies suggest that these genes are evolutionarily related and arose through duplication and divergence from a common ancestral genes.
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Type
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dissertation
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Source
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PQT Legacy CUNY.xlsx
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degree
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Ph.D.
