Abstract
Secretory, membrane, and lysosomal proteins undergo covalent modifications and acquire their secondary and tertiary structure in the lumen of the endoplasmic reticulum (ER). In order to pass the ER quality control system and become transported to their final destinations, many of them are also assembled into oligomers. We have recently determined the three-dimensional structure of lysosomal aspartylglucosaminidase (AGA), which belongs to a newly discovered family of homologous amidohydrolases, the N-terminal nucleophile hydrolases. Members of this protein family are activated from an inactive precursor molecule by an autocatalytic proteolytic processing event whose exact mechanism has not been thoroughly determined. Here we have characterized in more detail the initial events in the ER required for the formation of active AGA enzyme using transient expression of polypeptides carrying targeted amino acid substitutions. We show that His124 at an interface between two heterodimers of AGA is crucial for the thermodynamically stable oligomeric structure of AGA. Furthermore, the side chain of Thr206 is essential both for the proteolytic activation and enzymatic activity of AGA. Finally, the proper geometry of the residues His204-Asp205 seems to be crucial for the activation of AGA precursor polypeptides. We propose here a reaction mechanism for the activation of AGA which could be valid for homologous enzymes as well.
Highlights
Secretory, membrane, and lysosomal proteins undergo covalent modifications and acquire their secondary and tertiary structure in the lumen of the endoplasmic reticulum (ER)
In the case of AGA and penicillin acylase activation, the precursor polypeptide chain is cleaved into two polypeptide subunits of the active protein, whereas in proteasome and glutamine 5-phosphoribosyl-1-pyrophosphate amidotransferase, the activation results in the removal of a propeptide
We observed that there were two clusters in the three-dimensional structure of the native human AGA in which amino acids show high conservation (Fig. 3, B and C). One of these regions is located on the interface between the two ␣ dimers, and another is located on the active site area, suggesting functionally essential roles for these areas of the molecule. These regions were selected as targets to in vitro mutagenesis followed by functional analysis of polypeptides coded by the mutagenized cDNAs
Summary
N-terminal nucleophile hydrolase; AGA, aspartylglucosaminidase; CST, castanospermine; ER, endoplasmic reticulum; PAGE, polyacrylamide gel electrophoresis; WT, wild type. The N-terminal residue, which is exposed upon the proteolytic cleavage of the precursor polypeptide, has been shown to be essential for the enzymatic activity in the Ntn hydrolases. This residue is threonine in AGA and proteasome -subunit, serine in penicillin acylase, and cysteine in glutamine 5-phosphoribosyl-1-pyrophosphate amidotransferase and glucosamine-6-phosphate synthase. All of these residues can function as a catalytic nucleophile and are located at the beginning of a -strand. By using site-directed mutagenesis we wanted to 1) characterize the dimerization of AGA and 2) define the amino acid residues critical for the autocatalytic activation mechanism itself
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