Glycosylasparaginase-catalyzed synthesis and hydrolysis of beta-aspartyl peptides.

beta-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 beta-aspartyl peptides to form L-aspartic acid and amino acids or peptides. The enzyme also effectively catalyzes the synthesis of beta-aspartyl peptides by transferring the beta-aspartyl moiety from other beta-aspartyl peptides or beta-aspartylglycosylamine to a variety of amino acids and peptides. Furthermore, the enzyme can use L-asparagine as the beta-aspartyl donor in the formation of beta-aspartyl peptides. The data show that synthesis and degradation of beta-aspartyl peptides are new, significant functions of glycosylasparaginase and suggest that the enzyme could have an important role in the metabolism of beta-aspartyl peptides.

Recombinant human glycosylasparaginase catalyzes hydrolysis of L-asparagine.

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 Km is 3-4-fold higher and Vmax 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 L-asparagine and glycoasparagines will be discussed.

Influence of L-fucose attached alpha 1-->6 to the asparagine-linked N-acetylglucosamine on the hydrolysis of the N-glycosidic linkage by human glycosylasparaginase.

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.

A mouse model for the human lysosomal disease aspartylglycosaminuria.

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.

Enzymatic synthesis of the N-glycosidic bond by beta-aspartylation of glycosylamines.

Glycosylasparaginase (EC 3.5.1.26) is an amidase, which cleaves the N-glycosidic linkage during glycoprotein degradation leading to the liberation of L-aspartic acid from various glycoasparagines. In this work we demonstrate that glycosylasparaginase is also capable of catalyzing the synthesis of the N-glycosidic bond by N-beta-aspartylation of beta-glycosylamine using 1-amino-N-acetylglucosamine as the nucleophile and L-aspartic acid beta-methyl ester as the beta-aspartyl donor. Kinetic studies indicated that beta-glycosylamine has 1390-fold higher reactivity than water in the de-beta-aspartylation of the beta-aspartylenzyme, indicative of the presence of a beta-glycosylamine binding sub-site at the substrate binding site of glycosylasparaginase. The reaction can be applied to glycosylaparaginase-catalyzed biosynthesis of novel glycoasparagines.

Recombinant glycosylasparaginase and in vitro correction of aspartylglycosaminuria.

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.