The Deubiquitinating Enzyme Assays Of Ubiquitin Specific Protease26(USP26) Mutants

Objective：The ubiquitin specific protease, usp26, is an X-linked genelocated at Xq26.2. It spans2794bp and comprises a single exon (OnlineMendelian Inheritance in Man (OMIM)300309). Its protein USP26consistsof913amino acids with a predicted molecular weight of104KDa. TheUsp26gene was first reported by Wang et al., who isolated it from mousespermatogonia. Its expression was reported to be testis-specific in mice andhumans. USP26belongs to a large family of deubiquitinating enzymes(DUBs), which can act at various points in the Ub pathway includingpolyubiquitin chain processing, cleaving ubiquitin (Ub) from Ub-conjugatedprotein substrates to rescue substrate from degradation, or by removingresidual Ub to assist in proteasome degradation, and thereby regulating thelocalization and activity of this substrate. However, the biological mechanismand molecular basis of USP26is still under study. Resently, many studiessuggested that usp26single nucleotide polymorphism associated with maleinfertility. Several sequence changes in the usp26gene were detected amonginfertile men. Dirac AM et.al reported the regulatory function of USP26depends on its USP activity in androgen receptor signaling. Whether thosemutations confer a different enzyme property or efficacy of USP26is a key toexplain and rationalize their associations with male infertility. The aim of thisassay was to detect the deubiquitinating enzyme activity of USP26and theimpact of the mutations on USP26enzyme activity.Methods：1Plasmid construction. The DNA of Usp26(pGEFP-Usp26) waskindly provided by Dr. Annette M.G. Dirac. Using pGEFP-Usp26as atemplate, Usp26was add to BamHI site by PCR using forward primer5’-GGATCCATGGCTGCCCTATTCCTAC-3’(introducing a BamHI site) and reverse primer5’-GGATCCTTATTCCTTCTGAAGGGTCTCC-3’(introducing a BamHI site). The amplified cDNA was subcloned in-frame with GST into the expression vector pGEX-6p-1at the BamHI siteto generate pGEX-Usp26. And pAC-T7-Usp26was produced byinserting a T7promoter from BglII and HindIII fragment of pET-3d (apBR322Amprreplicon) into pACYC184(Cmrreplicon) at its BamHI andHindIII site. Then, the complete coding sequence of Usp26was insertedinto pAC-T7plasmid at the BamHI site. Those plasmids were confirmedby DNA sequencing.2Site-directed mutagenesis. Site-directed mutageneses of thosemutations (468G>T，1274C>T，2144A>T，2204T>G，2239A>T,and CS mutant,911G>C) were carried out. Using pGEX-Usp26as atemplate, the PCR reaction was performed at95°C for30s,55°C for1min, and68°C for10min for a total of18cycles. Mutations wereconfirmed by DNA sequencing.3Deubiquitinating enzyme assays. The deubiquitinating enzymeactivity was detected by USP cleavage assay using glutathione S-transferase-Ub52(GST-Ub52) and ubiquitin-β-galactosidase (Ub-Met-β-gal) fusion protein as model substrate. Plasmids pACYC-usp46(pACYC184Cmrreplicon) containing usp46, and pGEX-6p-1were usedas positive control and negative control, respectively. For cleavage of theGST-Ub52substrate, E. coli strain BL21(DE3) cells harboring pGEX-Ub52were further transformed with either pAC-T7-Usp26or pACYC-usp46, and cells harboring both plasmids were grown on LB added withampicillin and chloramphenicol. Single colony was inoculated in samemedium and diluted the next day and grown until they reached an OD600of1.0. Cultures were then induced with0.1mM IPTG for4h. Solubleprotein extracts were prepared after sonication of the cell lysate. GSTfusion proteins and their cleavage products were purified by GSH-Sepharose Resin and detected by10%SDS PAGE. The other modelsubstrate, Ub-Met-β-gal, is a fusion protein in which the ubiquitin was fused to the N-terminus of β-galactosidase. Plasmids pGEX-usp46(pBR322Amprreplicon) containing the GST-USP46, and pAC-T7vector,were used as positive control and negative control, respectively. E. coliBL21(DE3) cells harboring pAC-M-β-gal were transformed with pGEX-Usp26or pGEX-usp46, and colonies resistant to both ampicillin andchloramphenicol were grown-up. Protein expression was induced byIPTG, and total protein extracts were analyzed by Western blotting withanti-β-galactosidase purified monoclonal antibody and anti-mouse IgG.The resulting bands were analyzed by Odyssey V3.0software.Results:1The DNA sequencing result suggest that site-directed mutagenesis of468G>T,1274C>T,2144A>T,2204T>G,2239A>T and911G>C weresuccessful changed.2The enzymatic activity of USP26to cleave ubiquitinated protein, GST-Ub52and Ub-Met-β-gal, was investigated using the co-expression system to E.coli. After induced by IPTG, GST-Ub52was cleaved to a product of theexpected size of36KDa by co-expression of USP46. The wild-type USP26resulted in the same cleavage product with USP46, and at an increasedefficiency. As expected, when the active site Cys has been substituted with aSer residue (C304S), the activity of USP26disappeared. Thus, USP26possessed deubiquitinating enzyme activity.3We detected the five mutations of usp26,468G>T,1274C>T,2144A>T,2204T>G, and2239A>T. Interestingly, the mutants of USP26have thesame activity as the wild type. For cleavage of the Ub-Met-β-gal substrate,USP46and the empty expression vector were used as a positive control andnegative control. Conforming to the GST-Ub52assay, mutants of USP26possessed the same deubiquitinating enzyme activity as the wild type. Takenall together, this result suggests that mutations of usp26,468G>T，1274C>T，2144A>T，2204T>G， and2239A>T, do not influence thedeubiquitinating enzyme activity of USP26. Conclusions:1The five SNPs site-directed mutagenesis of usp26were carried outsuccessfully.2We confirm the validity of the assay system and reveal that USP26hasdeubiquitinating enzyme activity detected by USP cleavage assay using eitherUb-Met-b-gal or GST-Ub52as model substrates.3The USP26wild type has deubiquitinating enzyme activity. When theactive site Cys has been substituted with a Ser residue (C304S), the activity ofUSP26disappeared.4The five SNP of usp26,468G>T,1274C>T,2144A>T,2204T>G,2239A>T, do not influence deubiquitinating enzyme activity of USP26.