LARGE-SCALE PURIFICATION AND PRELIMINARY-X-RAY DIFFRACTION STUDIES OFHUMAN ASPARTYLGLUCOSAMINIDASE

Aspartylglucosaminidase (AGA) is a lysosomal asparaginase that takes p art in the ordered degradation of glycoproteins and a deficiency of wh ich results in a lysosomal accumulation disease aspartylglucosaminuria in human, The mature enzyme consists of 24-kDa and 17-kDa subunits, w hich are both heterogeneously glycosylated, Activation of the enzyme f rom a single precursor polypeptide into two subunits is accomplished i n the endoplasmic reticulum (ER). The relative lack of this proteolyti c capacity in several tested high-producing expression systems has com plicated the production of active recombinant enzyme in high quantitie s, which would be an alternative for purification of this molecule for crystallization, Consequently, the AGA enzyme has to be purified dire ctly from cellular or tissue sources for crystallographic analysis, He re we describe a large-scale purification method to produce milligram amounts of homogeneous AGA from human leukocytes. The purified AGA enz yme represents a heterogeneous pool of molecules not only due to glyco sylation, but also heterogeneity at the polypeptide level, as demonstr ated here, We were able to isolate a homogeneous polypeptide pool that was successfully crystallized and preliminary X-ray data collected fr om the crystals, The crystals diffract well to 2.0 Angstrom and are th us suitable for determination of the crystal structure of AGA. (C) 199 6 Wiley-Liss, Inc.