title: Large-scale purification and preliminary x-ray diffraction studies of human aspartylglucosaminidase.
abstract: Aspartylglucosaminidase (AGA) is a lysosomal asparaginase that takes part in the ordered degradation of glycoproteins and a deficiency of which results in a lysosomal accumulation disease aspartylglucosaminuria in human. The mature enzyme consists of 24-kDa and 17-kDa subunits, which are both heterogeneously glycosylated. Activation of the enzyme from a single precursor polypeptide into two subunits is accomplished in the endoplasmic reticulum (ER). The relative lack of this proteolytic capacity in several tested high-producing expression systems has complicated the production of active recombinant enzyme in high quantities, which would be an alternative for purification of this molecule for crystallization. Consequently, the AGA enzyme has to be purified directly from cellular or tissue sources for crystallographic analysis. Here we describe a large-scale purification method to produce milligram amounts of homogeneous AGA from human leukocytes. The purified AGA enzyme represents a heterogeneous pool of molecules not only due to glycosylation, but also heterogeneity at the polypeptide level, as demonstrated here. We were able to isolate a homogeneous peptide pool that was successfully crystallized and preliminary X-ray data collected from the crystals. The crystals diffract well to 2.0 angstroms and are thus suitable for determination of the crystal structure of AGA.
authors.affiliation: National Public Health Institute, Department of Human Molecular Genetics, Helsinki, Finland.
authors.forename: R
authors.initials: R
authors.lastname: Tikkanen
authors.forename: J
authors.initials: J
authors.lastname: Rouvinen
authors.forename: A
authors.initials: A
authors.lastname: Törrönen
authors.forename: N
authors.initials: N
authors.lastname: Kalkkinen
authors.forename: L
authors.initials: L
authors.lastname: Peltonen
mesh_terms: D001227:Aspartylglucosylaminase / Q000737:chemistry*; D015153:Blotting, Western; D018360:Crystallography, X-Ray; D006801:Humans; D007962:Leukocytes / Q000201:enzymology; D011487:Protein Conformation; D011499:Protein Processing, Post-Translational
chemical_list: D001227:Aspartylglucosylaminase