The overall goal of this research is to characterize the biochemistry and molecular biology of two key enzymes involved in the lysosomal degradation of Asparagine-linked glycoproteins: Glycosylasparaginase Chitobiase. This work is medically important because genetic deficiency of the asparaginase in humans results in a lysosomal storage disease, Aspartylglucosaminuria(AGU). AGU occurs almost exclusively in the Finnish people, and we have recently cloned both the normal and Finnish AGU defective cDNAs which encode the asparaginase associated with this disorder. The asparaginase consists of an alpha and beta subunit, and both are encoded by the single cDNA. This single gene structure means that the subunits are formed post-translationally by proteolysis. We have found the AGU genotype to consist of two nucleotide changes which result in AGU asparaginase having two amino acid substitutions. It appears that these alterations disallow the cleavage of the nascent polypeptide into its alpha/beta subunits. We will determine whether either one or both amino acid changes are necessary for the loss of enzyme activity in the AGU form of asparaginase. This experiment will be done by transfecting cells separately with genetically engineered cDNAs that contain only one or the other of the two mutations, and then testing whether normal or aberrant levels of enzyme activity are induced. The active site region of the asparaginase will be located by labeling it with radioactive inhibitor or substrate to help understand the mechanism of the enzyme and to relate this to its genetic loss from AGU patients. Two non-Finnish American AGU patient's genes will also be characterized. The research will determine the asparaginase gene sequences in both normal and Finnish-AGU human cell lines. Ibis sequence information will be used to design a specific DNA probe to detect the Finnish AGU genotype in patients and heterozygote carriers. The second lysosomal enzyme to be studied, chitobiase, is not associated with human pathology. This glycosidase, however, is not expressed in a single phylogenetic branch that includes the carnivores and ungulates. A major question to be explained about chitobiase is this unique evolutionary occurrence in nature. There are multiple chitobiase mRNAs in humans and rats, and these appear to have been formed by alternative splicing of the primary chitobiase gene transcript in these species. A model whereby alternative splicing has caused the loss of enzyme expression in those animals lacking chitobiase will be tested. The number and quality of chitobiase-related mRNAs in human and bovine will be determined and compared. Genomic DNA for the chitobiase locus in these species will be cloned and sequenced and the exon-intron arrangement will be characterized. These experiments should prove or disprove the model of alternative splicing having caused evolutionary elimination of this enzyme from ungulate/carnivore animals. This study of chitobiase genes should provide new insights into how the glycoprotein degradative pathway evolved, may show mechanisms by which aberrant versions have arisen to cause human lysosomal storage diseases, and could characterize basic concepts in molecular evolution.

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National Institute of Diabetes and Digestive and Kidney Diseases (NIDDK)
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Pathobiochemistry Study Section (PBC)
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University of South Alabama
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Aronson Jr, N N (1999) Aspartylglycosaminuria: biochemistry and molecular biology. Biochim Biophys Acta 1455:139-54
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