Functional Analysis of the Ser149/Thr149 Variants of Human Aspartylglucosaminidase and Optimization of the Coding Sequence for Protein Production

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.

Keywords: aspartylglucosaminidase; aspartylglucosaminuria; gene therapy; lysosomal storage disorder; lysosomes; single nucleotide polymorphism.

Figure 1

Processing and activation of aspartylglucosaminidase (AGA). AGA is synthesized in the ER as a 346 amino acid (aa) polypeptide from which 23 residues of the signal peptide are removed. AGA contains two N-glycosylation sites at Asn38 and Asn308 [5]. Very soon after synthesis in the ER, two AGA precursors homodimerize, inducing an autocatalytic cleavage of both precursors N-terminally to Thr206 into 27 kDa pro-α and 17 kDa β subunits. After transport to lysosomes, the pro-α is C-terminally cleaved into 24 kDa mature α subunit, whereas processing of the β subunit gives rise to the 14 kDa β’ subunit. Neither of these lysosomal processing steps displays an effect on the enzyme activity.

Figure 2

Single nucleotide polymorphism rs2228119 (NM_000027.3:c.446C>G - p.(Thr149Ser) results in amino acid variation Ser vs. Thr at position 149 (base and amino acid variation in red) of the human AGA enzyme.

Figure 3

Processing and activity of Strep-tagged AGA variants. SNP149-AGA variants with C-terminal Twin-strep-tag were transiently expressed in (a) HEK293T or (b) HeLa cells. Empty pExpr-IBA103 served as control. Western Blot with anti-AGA antibody shows correct processing of all constructs; (c,d) Western Blot signals were quantified and normalized to renilla luciferase activity to correct for transfection efficiency; (e,f) AGA activity was measured in the same cell lysates as used for Western Blot and normalized to renilla luciferase activity; (g,h) AGA activity was normalized to AGA protein amount. n ≥ 4, shown as mean ± SD. Statistical analysis by unpaired t-test (c,d,g,h) and One-Way ANOVA (e,f). Values of p < 0.05 were considered significant (*) and values of p < 0.01 very significant (**).

Figure 4

Comparison of processing and activity of the optimized and natural AGA variants. SNP149-AGA variants were transiently expressed in (a) HEK293T or (b) HeLa cells. Empty pcDNA3 plasmid served as a control. Western Blot with anti-AGA antibody shows correct processing of all constructs, with higher expression level of codon-optimized constructs; (c,d) Western Blot signals were quantified and normalized to renilla luciferase activity to correct for transfection efficiency; (e,f) AGA activity was measured in the same cell lysates as used for Western Blot. AGA activity was normalized to renilla luciferase activity; (g,h) AGA activity was normalized to AGA protein amount. n = 5, shown as mean ± SD. Statistical analysis by One-Way ANOVA. Values of p < 0.01 were considered very significant (**) and p < 0.001 extremely significant (***).

Figure 5

Activity of the optimized and natural AGA variants in control and patient fibroblasts. The Thr/Ser149 AGA variants were transiently expressed in wildtype (WT) and AGU_Fin-major (AGU-Fin) fibroblasts. The pcDNA3 plasmid without insert served as a control. AGA activity was measured and normalized to total protein amount of the lysates. Normalized AGA activity in wildtype fibroblasts transfected with the natural AGA variant Ser149 was set as 100%. n = 4, shown as mean ± SD. Statistical analysis by Two-Way ANOVA. * vs. pcDNA3, # vs. WT. Values of p < 0.05 were considered significant (* or #) while values of p < 0.01 were considered very significant (**) and p < 0.001 extremely significant (*** or ###).

Figure 6

Transfection with the optimized and natural AGA variants in aspartylglucosaminuria (AGU) patient fibroblasts improves lysosomal morphology. The Thr/Ser149 AGA variants were transiently expressed in AGU_Fin-major (AGU-Fin) fibroblasts. Uppermost row: WT and AGU-Fin fibroblasts transfected with pCDNA3; Middle and lowermost row: AGU-Fin fibroblasts transfected with the indicated constructs. The cells were stained with Lysotracker-Red and the nuclei were visualized with 4′,6-diamidin-2-phenylindol (DAPI). Scale bar 20 µm.