Abstract
Aspartylglucosaminidase (AGA) is a lysosomal enzyme involved in the degradation of N-linked glycoproteins in lysosomes. AGA is synthesized as an inactive precursor molecule, which is rapidly activated in the endoplasmic reticulum by a proteolytic cleavage into alpha- and beta-subunits. We have recently determined the three-dimensional structure of AGA and shown that it is a globular molecule with a heterotetrameric (alphabeta)2 structure. On the basis of structural and functional analyses, AGA seems to be the first mammalian protein belonging to a newly described protein family, the N-terminal nucleophile hydrolases. Because the activation of the prokaryotic members of the N-terminal nucleophile hydrolase family seems to be triggered by the assembly of the subunits, we have studied the initial folding and oligomerization of AGA and provide evidence that dimerization of two precursor molecules in the endoplasmic reticulum is a prerequisite for the activation of AGA. To gain further information on the structural determinants influencing the early folding of AGA, we used site-specific mutagenesis of cysteine residues to define the role of intrachain disulfide bridges in the folding and activation of the enzyme. The N-terminal disulfide bridges in both the alpha- and beta-subunits seem to have only a stabilizing role, whereas the C-terminal disulfide bridge in both subunits evidently plays an important role in the early folding and activation of AGA.
Highlights
Aspartylglucosaminidase (AGA) is a lysosomal enzyme step is much slower than that of the ␣-subunit. Neither of these involved in the degradation of N-linked glycoproteins in lysosomal trimming steps affects the catalytic activity of AGA
Because the activation of the prokaryotic members of the N-terminal nucleophile hydrolase family seems to be triggered by the assembly of the subunits, we have studied the initial folding and oligomerization of AGA and provide evidence that dimerization of two precursor molecules in the endoplasmic reticulum is a prerequisite for the activation of AGA
SDS-PAGE analysis of the labeled polypeptides showed that after a 1-h chase period, the 42-kDa precursor of the wild-type AGA was cleaved into the 27-kDa pro-␣- and 17-kDa -subunits, and after a 6-h chase the pro-␣-subunit was almost completely processed into the 24-kDa ␣-subunit (Fig. 2)
Summary
Aspartylglucosaminidase (AGA) is a lysosomal enzyme step is much slower than that of the ␣-subunit. Because the activation of the prokaryotic members of the N-terminal nucleophile hydrolase family seems to be triggered by the assembly of the subunits, we have studied the initial folding and oligomerization of AGA and provide evidence that dimerization of two precursor molecules in the endoplasmic reticulum is a prerequisite for the activation of AGA. The substitution of cysteine 163 by serine causes a loss of a disulfide bridge in the ␣-subunit, resulting in a failure in both the folding and proteolytic activation of the precursor molecule. On the basis of the crystal structure, AGA was enzyme that hydrolyzes the amide bond between asparagine suggested to be the first mammalian protein belonging to a and N-acetylglucosamine as a final step in the ordered degra- newly described protein family, the N-terminal nucleophile dation of glycoproteins in lysosomes (1).
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