The impacts of nineteen mutations on the enzymatic activity of USP26

Abstract

Objective: The association between mutations in the USP26 gene and male infertility has been studied intensively. However, the biological function of the mutant proteins remains to be elucidated. To confirm the effects of the reported mutations, we analyse the enzyme activity of USP26 between the wild-type and the variants from a molecular perspective.

Methods: Using pGEX-USP26 as a template, site-directed mutagenesis was conducted to generate nineteen USP26 mutant plasmids. Using Ub-Met-β-gal and GST-Ub52 as model substrates, a USP cleavage assay was conducted to assess the enzymatic activities of the mutants.

Results: The enzyme activity of the Q156H mutant disappeared, but the other 18 mutants had the same activity as the wild type. E174# and E189# were terminal mutants, but they still had the same activity as the wild type. When we constructed the transcription terminal mutants E174#(1-522 bp), E174#(523-2742 bp), E189#(1-567 bp) and E189#(568-2742 bp) artificially, the enzyme activity of these four mutants disappeared.

Conclusions: We have successfully constructed nineteen mutants of USP26. The enzyme activity of the Q156H mutant disappeared, but the enzyme activities of the other 18 mutants were the same as that of the wild type.

Keywords: Deubiquitinating enzyme activity; Gene expression; Site-directed mutagenesis; Ubiquitin-proteasome protease (USP26).