Soluble Recombinant Expression And Characterization Of Three Eukaryotic Protein Glycoamidases

Glycoamidase(EC 3.5.1.52),whose formal name is peptide-N4-(N-acetyl-?-glucosaminyl)asparagine amidase(PNGase),or N-glycanase(N-glycanase).PNGase hydrolyzes the linkage between N-acetylglucosamine and the asparagine residue in the N-glycan glycopeptide or glycoprotein,releasing the oligosaccharide chain while leaving Asn residue on the peptide or protein.Glycoamidases are widely exist among eukaryotic cells,especially around the cytoplasmic endoplasmic reticulum.It plays a role in distinguishing between folded and misfolded glycoproteins.Glycosidases can synergize with secreted protein degradation systems in eukaryotic cells in order to achieve deglycosylation of misfolded glycoproteins synthesized in the endoplasmic reticulum in the cytoplasm.Among the identified eukaryotic PNGase homologous proteins,sequence analysis revealed the presence of a catalytic triad composed of Cys,His,and Asp,which is shared by members of the transglutaminase-like superfamily.Its members exhibit different catalytic activities,including transglutaminase,protease and PNGase.All these catalytic activities involve the formation or hydrolysis of amide bonds.As a typical representative of eukaryotic PNGase,yeast PNGase(yPnglp)is thoroughly studied.The researchers conducted in-depth studies on its structure,function,and association with other proteins,and the researchers carried out recombinant expression of E.coli and renaturation of inclusion bodies to obtain recombinant yPnglp with in vitro activity.Meanwhile,some researchers found that PNGase from Arabidopsis(AtPnglp)was expressed in E.coli as inclusion body,and showed TGase activity after renaturation.However,other reports indicated that AtPnglp could compensate for the influence of yPnglp knock-out in yeast,which implied that it has PNGase activity in vivo.In order to study this contradictive phenomenon of AtPnglp and further our understanding on eukaryotic PNGase,we adopted a combined strategy of vector selection,condon optimization and intein-fusing,obtained soluble expression of AtPng1p in E.coli cytoplasm.Base on previously method,using denatured RNase B as a substrate,the deglycosylation activity of AtPnglp was successfully verified.The optimal reaction condition factors,including temperature,pH,and buffer component were further explored.Additionally,we were unable to confirm the transglutaminase activity of recombinant AtPnglp,indicating the natural activity of AtPnglp as PNGase both in vivo and in vitro.Previously yPng1p has also been recombinantly expressed in E.coli but as inclusion bodies.After refolding,recombinant yPnglp exhibited fine acidity and may hold potential usage in glycoprotein analysis.However,the refolding of inclusion bodies was inefficient.Based on our experience in AtPnglp study,the soluble expression and efficient purification of yPnglp and CrPnglp from Chlamydomonas reinhardtii was readily achieved.Then,the PNGase activity of recombinant yPnglp and CrPng1p was further confirmed.The displayed adaptability of our strategy on soluble expression of eukaryotic PNGase may facilitate the biochemical characterization and the biotechnological applications of eukaryotic PNGases.