Aspartylglycosaminuria (AGU), the most common disorder of glycoprotein degradation in humans, is caused by mutations in the gene encoding the lysosomal enzyme glycosylasparaginase (Aga). The resulting enzyme deficiency allows aspartylglucosamine (GlcNAc-Asn) and other glycoasparagines to accumulate in tissues and body fluids, from early fetal life onward. The clinical course is characterized by normal early development, slowly progressing to severe mental and motor retardation in early adulthood. The exact pathogenesis of AGU in humans is unknown and neither therapy nor an animal model for this debilitating and ultimately fatal disease exists. Through targeted disruption of the mouse Aga gene in embryonic stem cells, we generated mice that completely lack Aga activity. At the age of 5-10 months a massive accumulation of GlcNAc-Asn was detected along with lysosomal vacuolization, axonal swelling in the gracile nucleus and impaired neuromotor coordination. A significant number of older male mice had massively swollen bladders, which was not caused by obstruction, but most likely related to the impaired function of the nervous system. These findings are consistent with the pathogenesis of AGU and provide further data explaining the impaired neurological function in AGU patients.

Aspartylglycosaminuria (AGU) is the most common disorder of glycoprotein degradation. AGU patients are deficient in glycosylasparaginase (GA), which results in accumulation of aspartylglucosamine in body fluids and tissues. Human glycosylasparaginase was stably overexpressed in NIH-3T3 mouse fibroblasts, in which the unusual posttranslational processing and maturation of the enzyme occurred in a high degree. The recombinant enzyme was isolated as two isoforms, which were both phosphorylated, and actively transported into AGU fibroblasts and lymphoblasts through mannose-6-phosphate receptor-mediated endocytosis. The rate of uptake into fibroblasts was half-maximal when the concentration of GA in the medium was 5 x 10(-8) M. Immunofluorescence microscopy suggested compartmentalization of the recombinant enzyme in the lysosomes. Supplementation of culture medium with either isoform cleared AGU lymphoblasts of stored aspartylglucosamine when glycosylasparaginase activity in the cells reached 3-4% of that in normal lymphoblasts. A relatively small amount of recombinant GA in the culture medium was sufficient to reverse pathology in the target cells, indicating high corrective quality of the enzyme preparations. The combined evidence indicates that enzyme replacement therapy with the present recombinant glycosylasparaginase might reverse pathology at least in somatic cells of AGU patients.

Glycosylasparaginase is a lysosomal amidase involved in the degradation of glycoproteins. Recombinant human glycosylasparaginase is capable of catalyzing the hydrolysis of the amino acid L-asparagine to L-aspartic acid and ammonia. For the hydrolysis of L-asparagine the K m is 3-4-fold higher and V-mnx 1/5 of that for glycoasparagines suggesting that the full catalytic potential of glycosylasparaginase is not used in the hydrolysis of the free amino acid. L-Asparagine competitively inhibits the hydrolysis of aspartylglucosamine indicating that both the amino acid and glycoasparagine are interacting with the same active site of the enzyme. The hydrolytic mechanism of Lasparagine and glycoasparagines will be discussed.

␤-Aspartyl di-and tripeptides are common constituents of mammalian metabolism, but their formation and catabolism are not fully understood. In this study we provide evidence that glycosylasparaginase (aspartylglucosaminidase), an N-terminal nucleophile hydrolase involved in the hydrolysis of the N-glycosidic bond in glycoproteins, catalyzes the hydrolysis of ␤-aspartyl peptides to form L-aspartic acid and amino acids or peptides. The enzyme also effectively catalyzes the synthesis of ␤-aspartyl peptides by transferring the ␤-aspartyl moiety from other ␤-aspartyl peptides or ␤-aspartylglycosylamine to a variety of amino acids and peptides. Furthermore, the enzyme can use L-asparagine as the ␤-aspartyl donor in the formation of ␤-aspartyl peptides. The data show that synthesis and degradation of ␤-aspartyl peptides are new, significant functions of glycosylasparaginase and suggest that the enzyme could have an important role in the metabolism of ␤-aspartyl peptides.␤-Aspartyl and ␥-glutamyl peptides are normal constituents of mammalian urine (1, 2) and tissues (3). Although ␥-glutamyltransferase (EC 2.3.2.2, GGT) (1) 1 is the key enzyme in the synthesis and hydrolysis of ␥-glutamyl compounds such as glutathione in the ␥-glutamyl cycle (4), little is known about the metabolism of ␤-aspartyl peptides.Glycosylasparaginase (GA; aspartylglucosaminidase;asparagine; ␤-aspartylglucosamine; GlcNAc-Asn) during degradation of glycoproteins. Genetic deficiency of glycosylasparaginase causes a lysosomal storage disease aspartylglycosaminuria (McKusick 208400) that is the most common disorder of glycoprotein degradation in humans and is clinically characterized by severe mental and motor retardation (5, 6).Glycosylasparaginase is a member of the recently described structural superfamily of enzymes termed as N-terminal nucleophile (Ntn) hydrolases (7). The hydrolysis of ␤-aspartylglycosylamines catalyzed by glycosylasparaginase is initiated by the binding of the ␤-aspartyl moiety into the active site of the enzyme through its free ␣-amino and ␣-carboxyl groups (8, 9). The enzyme uses the ␥-hydroxyl and ␣-amino group of its ␤-chain N-terminal threonine as an active site nucleophile and general base in the formation of ␤-aspartyl enzyme, which is subsequently deacylated by water to L-aspartic acid (10, 11). The GA-catalyzed hydrolysis of L-asparagine occurs in a similar manner, resulting in the formation of ␤-aspartyl enzyme and ammonia (12). The mechanism of action of glycosylasparaginase and the structural properties of its substrate-binding site (13) led us to consider that the enzyme might have a role in the metabolism of ␤-aspartyl peptides. In the present study, we demonstrate that synthesis and degradation of ␤-aspartyl peptides are new significant functions of glycosylasparaginase. This suggests that glycosylasparaginase could have an important role in the metabolism of ␤-aspartyl peptides present in body fluids and tissues. EXPERIMENTAL PROCEDURESMaterials-GA was purified to homogeneity from an NIH-3T3 cell line overexpre...

The sequence of hydrolytic reactions in the catabolism of the N-glycosidic oligosaccharide-to-protein region containing 6-linked fucose on the asparagine-linked N-acetylglucosamine may vary from species to species. When alpha-L-fucopyranosyl-(1-->6)-2-acetamido-1-N-(beta-L-aspartyl)-2-deoxy- beta -D-glucopyranosylamine (Fuc-GlcNAc-Asn) was incubated with recombinant human glycosylasparaginase, no hydrolysis of the N-glycosidic bond was detected. After removal of the alpha 1-->6-linked fucose from the compound by alpha-fucosidase, the residual GlcNAc-Asn was rapidly hydrolyzed by glycosylasparaginase. Enzymologically this demonstrates for the first time that the catabolism of Fuc-GlcNAc-Asn in humans occurs via consecutive action of alpha-fucosidase and glycosylasparaginase. The hydrolysis rate of GlcNAc-Asn by glycosylasparaginase remained unaffected in the presence of Fuc-GlcNAc-Asn or several different monosaccharides including fucose. This indicates that any fucose attached alpha 1-->6 to the asparagine-linked N-acetylglucosamine residue prevents the access of the L-asparagine residue of Fuc-GlcNAc-Asn into the deep, funnel-shaped active site of human glycosylasparaginase. These findings explain the accumulation of fucosylated and normal catabolism of nonfucosylated glycoasparagines in fucosidosis.