The Impacts Of Two Nonsense Mutations And Six Missense Mutations On Enzymatic Activity Of USP26

Objective：Ubiquitin specific protease26gene (Usp26) is located onXq26.2including2794bp. It encodes amino acids and the expectedmolecular weight of the corresponding protein is104KDa. Studies haveshown that there are many abnormal genes leading to male sterility on the Xchromosome, and one of them is Usp26which is specifically expressed intesticular tissue. In2005, Stouffs et al. first reported the correlation betweengene mutation of Usp26and dyszoospermia, and the following studies putforward that Usp26gene mutations could lead to male infertility. While someother studies supported the opposite opinion. However, most conclusionscome from the epidemiological researches which belong to the method ofobservation, in addition, the biological function of Usp26remains to beelucidated. Thus, we analyze the enzyme activity of USP26between wild-typeand variants from molecular perspective. It has reported more than20genemutations of Usp26, of which15mutations have been successfullyconstructed and been tested the effects on the activity of USP26. In order tofurther confirm the effects of the remaining eight reported mutations on theactivity of USP26, this study constructed the eight mutant expression plasmidsand detected the effects of eight mutations on USP26using the establishedubiquitin enzyme activity detection system.Methods：1Plasmid construction of pGEX-Usp26. Using pGEX-Usp26as template,site-directed mutagenesis was carried out to generate pGEX-Usp26(520G>T,565G>T,1037T>A,1498G>A,1549C>T,1609C>G,2182A>T and2195T>C) mutant plasmids. Mutations were confirmed by DNA sequencing.2Plasmid construction of pAC-T7-Usp26. The pAC-T7-Usp26mutationswere produced by inserting the complete coding sequence of Usp26from pGEX-Usp26into pAC-T7plasmid at the BamH I site, and confirmed byrestriction enzymes digestion of Sal I and Pst I.3Using Ub-Met-β-gal and GST-Ub52as model substrates, the USPcleavage assay was conducted to assess enzymatic activity of the mutations.And the results were detected by Odyssey Infrared Imaging System.Results:1Sequencing results confirmed that the inserted fragment with2794bpwas the coding regions of human Usp26gene, and the corresponding expectedlocis got changed resulting in amino acid changes E174X (X：nonsensemutation), E189X, L346H, E500K, P517S, Q537E, I728F and F732S.2Using restriction enzymes Sal I and Pst I identified the constructedmutant plasmids of pAC-T7-Usp26and produced two fragments with the sizeof1.5Kb and5.7Kb, respectively. Thus, the mutant plasmids ofpAC-T7-Usp26were constructed successfully.3The missense mutations (1037T>A,1498G>A,1549C>T,1609C>G,2182A>T and2195T>C) effects on enzyme activity: These six mutationshave the same activity compared with the wild type, detected by USP cleavageassay using both Ub-Met-β-gal and GST-Ub52as substrates.4The nonsense mutations (520G>T and565G>T) effects on enzymeactivity: These two mutations still have the activity of deubiquitinating. While,they should not have the activity in theory as they both encode thetranscription products with only522bp and567bp (not including the activecenter of enzyme). The possible cause of this phenomenon is that in someprokaryotes even if there is termination codon, the encoding process still couldcontinue if there is initiation codon in the following sequence.5Constructing transcription terminal mutations520*G>T（522bp）,565*G>T（567bp）artificially, both mutants couldn’t ubiquitinate model substrateUb-Met-β-gal and GST-Ub52, and to further verified the520G>T and565G>T mutations could lead to the lost enzyme activity of USP26if they couldresult in transcription termination. Conclusions:1We have successfully constructed two nonsense mutations (520G>Tand565G>T) and six missense mutations (1037T>A,1498G>A,1549C>T,1609C>G,2182A>T and2195T>C) of Usp26.2The missense mutations (1037T>A,1498G>A,1549C>T,1609C>G,2182A>T and2195T>C) do not influence the deubiquitinating enzymeactivity of USP26in this two kinds of model substrate detection system.3Limited by prokaryotic expression system the two nonsense mutations(520G>T and565G>T) also have no effect on the activity of USP26.