Effects of A6E Mutation on Protein Expression and Supramolecular Assembly of Yeast Asparagine Synthetase

Abstract

Asparagine synthetase deficiency (ASD) has been found to be caused by certain mutations in the gene encoding human asparagine synthetase (ASNS). Among reported mutations, A6E mutation showed the greatest reduction in ASNS abundance. However, the effect of A6E mutation has not yet been tested with yeast asparagine synthetase (Asn1/2p). Here, we constructed a yeast strain by deleting ASN2 from its genome, introducing the A6E mutation codon to ASN1, along with GFP downstream of ASN1. Our mutant yeast construct showed a noticeable decrease of Asn1p(A6E)-GFP levels as compared to the control yeast expressing Asn1p(WT)-GFP. At the stationary phase, the A6E mutation also markedly lowered the assembly frequency of the enzyme. In contrast to Asn1p(WT)-GFP, Asn1p(A6E)-GFP was insensitive to changes in the intracellular energy levels upon treatment with sodium azide during the log phase or fresh glucose at the stationary phase. Our study has confirmed that the effect of A6E mutation on protein expression levels of asparagine synthetase is common in both unicellular and multicellular eukaryotes, suggesting that yeast could be a model of ASD. Furthermore, A6E mutation could be introduced to the ASNS gene of acute lymphoblastic leukemia patients to inhibit the upregulation of ASNS by cancer cells, reducing the risk of developing resistance to the asparaginase treatment.