Purification and properties of human hepatic aspartylglucosaminidase.
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Aspartylglucosaminidase (EC 3.5.1.26), the lysosomal enzyme which hydrolyzes the N-acetylglucosamine-asparagine linkages in glycoproteins, was purified from human liver to homogeneity. The purification procedure included chromatography on DEAE-cellulose and concanavalin A-Sepharose, gel filtration on Sephadex G-200, and high performance liquid chromatography. The purified enzyme had a final specific activity of 1,200,000 units/mg of protein, a pH optimum of 6.1, a pI of 5.7, a Vmax of 1,240,000 units/mg, and a Km of 1.25 mM toward a natural substrate, aspartylglucosamine. The purified enzyme was remarkably thermostable, retaining 90% of initial activity after 1 h at 60 degrees C. The molecular weight of the native enzyme was estimated to be 80,000 by gel filtration and 84,000 by analytical polyacrylamide gel electrophoresis. Under denaturing conditions, the molecular weight was 76,000, indicating that the native enzyme was a monomer. Amino acid composition revealed only 2 methionine residues/enzyme molecule. (+info)
Finger clubbing and aspartylglucosamine excretion in a laxative-abusing patient.
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A young woman with a previous history of anorexia nervosa presented with severe finger clubbing. Urine samples intermittently contained significant amounts of aspartylglucosamine. Liver biopsy showed abnormal cytoplasmic inclusions in phagocytic cells. The patient was found to be abusing senna laxative. (+info)
Co-localization of hydrolytic enzymes with widely disparate pH optima: implications for the regulation of lysosomal pH.
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Lysosomes are traditionally defined by their acidic interior, their content of degradative 'acid hydrolases', and the presence of distinctive membrane proteins. Terminal degradation of the N-linked oligosaccharides of glycoproteins takes place in lysosomes, and involves several hydrolases, many of which are known to have acidic pH optima. However, a sialic acid-specific 9-O-acetyl-esterase and a glycosyl-N-asparaginase, which degrade the outer- and inner-most linkages of N-linked oligosaccharides, respectively, both have pH optima in the neutral to alkaline range. By immunoelectron microscopy, these enzymes co-localize in lysosomes with several conventional acid hydrolases and with lysosomal membrane glycoproteins. Factors modifying the pH/activity profiles of these enzymes could not be found in lysosomal extracts. Thus, the function of the enzymes with neutral pH optima must depend either upon their minimal residual activity at acidic pH, or upon the possibility that lysosomes are not always strongly acidic. Indeed, when lysosomes are marked in living cells by uptake of fluorescently labeled mannose 6-phosphorylated proteins, the labeled organelles do not all rapidly accumulate Acridine Orange, a vital stain that is specific for acidic compartments. One plausible explanation is that lysosomal pH fluctuates, allowing hydrolytic enzymes with a wide range of pH optima to efficiently degrade macromolecules. (+info)
Localization of the disulfide bond involved in post-translational processing of glycosylasparaginase and disrupted by a mutation in the Finnish-type aspartylglycosaminuria.
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The heavy chain of human glycosylasparaginase (N4-(beta-N-acetylglucosaminyl)-L-asparaginase (EC 3.5.1.26)) has five cysteinyl residues (Cys-61, Cys-64, Cys-69, Cys-163, and Cys-179). A Cys-163 to serine substitution due to a point mutation in the glycosylasparaginase gene causes the most common disorder of glycoprotein degradation, the Finnish-type aspartylglycosaminuria. To localize the potential disulfide bonds, the isolated heavy chain of human leukocyte glycosylasparaginase was treated with the enzyme alpha-chymotrypsin, and the resulting peptides were separated by high performance liquid chromatography prior to and after reduction and S-carboxymethylation. The peptide containing the Cys-163 residue and the peptide to which it is connected with a disulfide were structurally characterized by mass spectrometry. The disulfide bond crucial for catalytic activity, subunit processing, and biological transport of glycosylasparaginase was located close to the carboxyl terminus of the heavy chain at positions 163 and 179. (+info)
Immediate interaction between the nascent subunits and two conserved amino acids Trp34 and Thr206 are needed for the catalytic activity of aspartylglucosaminidase.
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Aspartylglucosaminidase (AGA, EC 3.5.1.26) is a dimeric lysosomal hydrolase involved in the degradation of glycoproteins. The synthesized precursor polypeptide of AGA is rapidly activated in the endoplasmic reticulum by proteolysis into two subunits. Expression of the alpha- and beta-subunits of AGA in separate cDNA constructs showed that independently folded subunits totally lack enzyme activity, and even when co-expressed in vitro they fail to produce an active heterodimer of the enzyme. Both of the subunits are required for the enzyme activity, and the immediate interaction of the subunits in the endoplasmic reticulum is necessary for the correct folding of the dimeric enzyme molecule. The specific amino acid residues essential for the active site of the AGA enzyme were further analyzed by site-directed mutagenesis and in vitro expression of mutagenized constructs. Replacement of Thr206, the most amino-terminal residue of the beta-subunit, with Ser resulted in a complete loss of enzyme activity without influencing intracellular processing or transport of the mutant polypeptide to the lysosomes. Analogously, replacement of the most amino-terminal tryptophan, Trp34 with Phe or Ser in the alpha-subunit, resulted in a totally inactive enzyme without influencing the intracellular processing or stability of the polypeptide. These results suggest that the catalytic center of this amidase is formed by the interaction of the amino-terminal parts of two subunits and requires both Trp34 in the alpha-subunit and Thr206 in the beta-subunit. (+info)
Recombinant glycosylasparaginase and in vitro correction of aspartylglycosaminuria.
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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. (+info)
Human leucocyte glycosylasparaginase is an alpha/beta-heterodimer of 19 kDa alpha-subunit and 17 and 18 kDa beta-subunit.
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Human lysosomal glycosylasparaginase (AGA; EC 3.5.1.26) consists of two glycosylated subunits, alpha and beta. Treatment with 3% SDS at 45 degrees C as part of a new purification scheme did not affect enzyme activity, but the alpha-subunit migrated an apparent 19 kDa peptide on SDS/PAGE instead of as a 24 kDa peptide, as observed without this SDS treatment. The N-terminal sequence was similar to that of the 24 kDa form, and, after reversed-phase h.p.l.c., the 19 kDa form was transformed to an apparent 24 kDa peptide on SDS/PAGE, indicating that their primary structures were identical. As the molecular mass of the alpha-subunit deduced from its cDNA was 19.5 kDa, the variation might be due to incomplete SDS coating of the 24 kDa form. This was confirmed by the tendency of the 24 kDa variant to polymerize even in the presence of SDS. The molecular mass of the beta-subunit was 17 and 18 kDa in accordance with previous reports. Chemical cross-linking with 1-ethyl-3-(3-dimethylaminopropyl)carbodi-imide resulted in the appearance of a 38 kDa peptide on SDS/PAGE which reacted with both the subunit-specific antisera on Western-blot analysis. On SDS/PAGE at pH 10.2 the active enzyme migrated as an apparent 43 kDa peptide. These results indicate that native human glycosylasparaginase is a heterodimer. (+info)
Aspartylglucosaminuria in northern Norway: a molecular and genealogical study.
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Aspartylglucosaminuria (AGU, McKusick 208400) is an autosomal recessive lysosomal storage disorder. Ninety percent of all patients are from Finland and only sporadic cases have been reported from elsewhere. In northern Norway, however, nine patients from seven families have been diagnosed with AGU. All these Norwegian patients were homozygous for the most prevalent Finnish AGU mutation (AGUFin) and show the polymorphism uniquely associated with AGUFin in Finland. Genealogical investigation of nine parents proved Finnish ancestry in all pedigrees. Therefore, AGU in Norway most likely resulted from immigration of Finnish carriers. These Finnish immigrants originated mostly from the Tornio valley area in northern Finland in a continuous immigration movement from 1700 to 1900. The majority settled in the western part of northern Norway, leading to a "cluster" of AGU in that particular area. The Finnish immigrants intermixed considerably with Lapps and these two ethnic origins should thus be considered as high risk groups for AGUFin in northern Norway. (+info)