Publications

• Aspartylglucosaminuria: cDNA encoding human aspartylglucosaminidase and the missense mutation causing the disease.

  Ikonen E., Baumann M., Groen K., Syvaenen A.-C., Enomaa N., Halila R., Aula P., Peltonen L.

  We have isolated a 2.1 kb cDNA which encodes human aspartylglucosaminidase (AGA, E.C. 3.5.1.26). The activity of this lysosomal enzyme is deficient in aspartylglucosaminuria (AGU), a recessively inherited lysosomal accumulation disease resulting in severe mental retardation. The polypeptide chain ... >> More

  We have isolated a 2.1 kb cDNA which encodes human aspartylglucosaminidase (AGA, E.C. 3.5.1.26). The activity of this lysosomal enzyme is deficient in aspartylglucosaminuria (AGU), a recessively inherited lysosomal accumulation disease resulting in severe mental retardation. The polypeptide chain deduced from the AGA cDNA consists of 346 amino acids, has two potential N-glycosylation sites and 11 cysteine residues. Transient expression of this cDNA in COS-1 cells resulted in increased expression of immunoprecipitable AGA protein. Direct sequencing of amplified AGA cDNA from an AGU patient revealed a G----C transition resulting in the substitution of cysteine 163 with serine. This mutation was subsequently found in all the 20 analyzed Finnish AGU patients, in the heterozygous form in all 53 carriers and in none of 67 control individuals, suggesting that it represents the major AGU causing mutation enriched in this isolated population. Since the mutation produces a change in the predicted flexibility of the AGA polypeptide chain and removes an intramolecular S-S bridge, it most probably explains the deficient enzyme activity found in cells and tissues of AGU patients. << Less

  EMBO J. 10:51-58(1991) [PubMed] [EuropePMC]

• Characterization of the mutation responsible for aspartylglucosaminuria in three Finnish patients. Amino acid substitution Cys163-->Ser abolishes the activity of lysosomal glycosylasparaginase and its conversion into subunits.

  Fisher K.J., Aronson N.N. Jr.

  The mutation that causes a deficiency of the lysosomal amidase, glycosylasparaginase, has been characterized in fibroblasts from three Finnish patients diagnosed with aspartylglucosaminuria (AGU). The polymerase chain reaction was used to amplify the glycosylasparaginase protein coding sequence fr ... >> More

  The mutation that causes a deficiency of the lysosomal amidase, glycosylasparaginase, has been characterized in fibroblasts from three Finnish patients diagnosed with aspartylglucosaminuria (AGU). The polymerase chain reaction was used to amplify the glycosylasparaginase protein coding sequence from the three AGU patients in order to compare them to the normal sequence from a full-length human placenta cDNA clone HPAsn.6 (Fisher, K.J., Tollersrud, O.K., and Aronson, N.N., Jr. (1990) FEBS Lett. 269, 440-444). Two base changes were found to be common to all three Finnish AGU patients, a G482----A transition that results in an Arg161----Gln substitution and a G488----C transversion that causes Cys163----Ser. Detection of both point mutations from PCR-amplified cDNA or genomic DNA was facilitated by their creation of new endonuclease restriction sites. Expression studies in COS-1 cells revealed only the Cys163----Ser mutation caused a deficiency of glycosylasparaginase activity. This same substitution also prevented the normal posttranslational processing of the precursor glycosylasparaginase polypeptide into its alpha and beta subunits. Cell-free expression of the single-chain glycosylasparaginase precusor did not produce an active enzyme, suggesting that post-translational generation of subunits may be required for catalytic activity. << Less

  J. Biol. Chem. 266:12105-12113(1991) [PubMed] [EuropePMC]

• Crystal structure of glycosylasparaginase from Flavobacterium meningosepticum.

  Xuan J., Tarentino A.L., Grimwood B.G., Plummer T.H. Jr., Cui T., Guan C., van Roey P.

  The crystal structure of recombinant glycosylasparaginase from Flavobacterium meningosepticum has been determined at 2.32 angstroms resolution. This enzyme is a glycoamidase that cleaves the link between the asparagine and the N-acetylglucosamine of N-linked oligosaccharides and plays a major role ... >> More

  The crystal structure of recombinant glycosylasparaginase from Flavobacterium meningosepticum has been determined at 2.32 angstroms resolution. This enzyme is a glycoamidase that cleaves the link between the asparagine and the N-acetylglucosamine of N-linked oligosaccharides and plays a major role in the degradation of glycoproteins. The three-dimensional structure of the bacterial enzyme is very similar to that of the human enzyme, although it lacks the four disulfide bridges found in the human enzyme. The main difference is the absence of a small random coil domain at the end of the alpha-chain that forms part of the substrate binding cleft and that has a role in the stabilization of the tetramer of the human enzyme. The bacterial glycosylasparaginase is observed as an (alphabeta)2-tetramer in the crystal, despite being a dimer in solution. The study of the structure of the bacterial enzyme allows further evaluation of the effects of disease-causing mutations in the human enzyme and confirms the suitability of the bacterial enzyme as a model for functional analysis. << Less

  Protein Sci. 7:774-781(1998) [PubMed] [EuropePMC]

• Crystallographic snapshot of a productive glycosylasparaginase-substrate complex.

  Wang Y., Guo H.C.

  Glycosylasparaginase (GA) plays an important role in asparagine-linked glycoprotein degradation. A deficiency in the activity of human GA leads to a lysosomal storage disease named aspartylglycosaminuria. GA belongs to a superfamily of N-terminal nucleophile hydrolases that autoproteolytically gen ... >> More

  Glycosylasparaginase (GA) plays an important role in asparagine-linked glycoprotein degradation. A deficiency in the activity of human GA leads to a lysosomal storage disease named aspartylglycosaminuria. GA belongs to a superfamily of N-terminal nucleophile hydrolases that autoproteolytically generate their mature enzymes from inactive single chain protein precursors. The side-chain of the newly exposed N-terminal residue then acts as a nucleophile during substrate hydrolysis. By taking advantage of mutant enzyme of Flavobacterium meningosepticum GA with reduced enzymatic activity, we have obtained a crystallographic snapshot of a productive complex with its substrate (NAcGlc-Asn), at 2.0 A resolution. This complex structure provided us an excellent model for the Michaelis complex to examine the specific contacts critical for substrate binding and catalysis. Substrate binding induces a conformational change near the active site of GA. To initiate catalysis, the side-chain of the N-terminal Thr152 is polarized by the free alpha-amino group on the same residue, mediated by the side-chain hydroxyl group of Thr170. Cleavage of the amide bond is then accomplished by a nucleophilic attack at the carbonyl carbon of the amide linkage in the substrate, leading to the formation of an acyl-enzyme intermediate through a negatively charged tetrahedral transition state. << Less

  J. Mol. Biol. 366:82-92(2007) [PubMed] [EuropePMC]