Abstract
Aspartylglucosaminidase (AGA) is a lysosomal hydrolase that participates in the breakdown of glycoproteins. Defects in the AGA gene result in a lysosomal storage disorder, aspartylglucosaminuria (AGU), that manifests mainly as progressive mental retardation. A number of AGU missense mutations have been identified that result in reduced AGA activity. Human variants that contain either Ser or Thr in position 149 have been described, but it is unknown if this affects AGA processing or activity. Here, we have directly compared the Ser149/Thr149 variants of AGA and show that they do not differ in terms of relative specific activity or processing. Therefore, Thr149 AGA, which is the rare variant, can be considered as a neutral or benign variant. Furthermore, we have here produced codon-optimized versions of these two variants and show that they are expressed at significantly higher levels than AGA with the natural codon-usage. Since optimal AGA expression is of vital importance for both gene therapy and enzyme replacement, our data suggest that use of codon-optimized AGA may be beneficial for these therapy options.
Highlights
Aspartylglucosaminidase (AGA, N4-(β-N-Acetylglucosaminyl)-L-Asparaginase, EC 3.5.1.26) is a lysosomal hydrolase that participates in one of the final steps during the degradation of N-glycosylated proteins
AGA belongs to the group of so-called N-terminal nucleophile (NTN) hydrolases, as the free α-amino group of Thr206 is involved in the catalysis as the base, whereas the OH group of Thr206 functions as a nucleophile during the catalysis [3,6]
The observed higher expression level of the Thr149 AGA at the first glimpse suggests that this variant might be the better option when considering gene therapy or enzyme production for ERT
Summary
Aspartylglucosaminidase (AGA, N4-(β-N-Acetylglucosaminyl)-L-Asparaginase, EC 3.5.1.26) is a lysosomal hydrolase that participates in one of the final steps during the degradation of N-glycosylated proteins. AGA is synthesized as a single-chain precursor molecule that soon after synthesis in the endoplasmic reticulum (ER) homodimerizes and becomes processed into two pro-α and β subunits (Figure 1) [2]. This autocatalytic cleavage takes place between the residues Asp205 and Thr206 and is a prerequisite for the activity of the enzyme [3,4]. AGA belongs to the group of so-called N-terminal nucleophile (NTN) hydrolases, as the free α-amino group of Thr206 is involved in the catalysis as the base, whereas the OH group of Thr206 functions as a nucleophile during the catalysis [3,6]. The members of the NTN hydrolase family, which in addition to AGA include, e.g., the proteasome β subunit and penicillin acylase, show very little similarity at the amino acid sequence level, but they exhibit a highly similar folded structure [6]
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