Lysosomal aspartylglucosaminidase is processed to the active subunit complex in the endoplasmic reticulum.

Abstract

Aspartylglucosaminidase (AGA) is a lysosomal enzyme, the deficiency of which leads to a human storage disease, aspartylglucosaminuria (AGU). Although numerous mutations have been identified in AGU patients, elucidation of the molecular pathogenesis of the disease has been hampered by the missing information on the cellular events resulting in the maturation and activation of the enzyme. Here we used the expression of in vitro mutagenized constructs of the AGA cDNA to define three specific proteolytic trimming steps resulting in mature AGA. Removal of the signal peptide is immediately followed by proteolytic cleavage of the precursor into two subunits and results in biologically active enzyme already in the endoplasmic reticulum. This early activation has not previously been described for lysosomal enzymes. The subsequent lysosomal trimming does not influence the enzymatic activity of AGA. It consists only of a single proteolytic cleavage which removes 10 amino acids from the C‐terminal end of the larger subunit, in contrast to the multistep lysosomal processing observed in several other hydrolases.

Activation of Glycosylasparaginase

“…Since glycosylasparaginase does not possess protease activity per se, the activation cannot be an autocatalytic process. It is a spontaneous intramolecular reaction with the key Based on the data presented here and in other published studies (5,6), we propose the following model of autoproteolysis of glycosylasparaginase, illustrated in Fig. 7.…”

“…In human glycosylasparaginase, no histidine residues are involved in enzyme activity (6). The pH dependence of glycosylasparaginase activity is not consistent with histidine as a proton acceptor-donor.…”

“…It is known that in the human enzyme only His-204 in the above sequence is involved in activation (6). We replaced the corresponding histidine residue (His-150) in the MBP-bacterial enzyme fusion with serine by site-directed mutagenesis and examined its expression in E. coli.…”

Purification and crystallization of precursors and autoprocessed enzymes of Flavobacterium glycosylasparaginase: an N-terminal nucleophile hydrolase

“…Cleavage is essential for GA hydrolase activity and occurs at the amide bond in the consensus site between residues Asp151 and Thr152 (Ikonen et al, 1993;Fisher et al, 1993;Tarentino et al, 1995). This conserved threonine is thought to play central roles in both hydrolase activity (Kaartinen et al, 1991;Fisher et al, 1993) and autoproteolysis (Guan et al, 1996).…”

Adenovirus-mediated gene transfer results in decreased lysosomal storage in brain and total correction in liver of aspartylglucosaminuria (AGU) mouse

“…The intracellular processing and transport of AGA has been studied in detail. 6,7 AGA is synthesized as a precursor polypeptide which becomes proteolytically activated in the endoplasmic reticulum resulting in ␣ and ␤ subunits. The mature enzyme is an ␣ 2 ␤ 2 heterotetramer, which is also partially secreted and endo-cytosed by recipient cells by mannose-6-phosphate receptor.…”