title: N-terminal deletion of peptide:N-glycanase results in enhanced deglycosylation activity.
abstract: Peptide:N-glycanase catalyzes the detachment of N-linked glycan chains from glycopeptides or glycoproteins by hydrolyzing the beta-aspartylglucosaminyl bond. Peptide:N-glycanase in yeast binds to Rad23p through its N-terminus. In this study, the complex formed between Peptide:N-glycanase and Rad23p was found to exhibit enhanced deglycosylation activity, which suggests an important role for this enzyme in the misfolded glycoprotein degradation pathway in vivo. To investigate the role of this enzyme in this pathway, we made stepwise deletions of the N-terminal helices of peptide:N-glycanase. Enzymatic analysis of the deletion mutants showed that deletion of the N-terminal H1 helix (Png1p-DeltaH1) enhanced the deglycosylation activity of N-glycanase towards denatured glycoproteins. In addition, this mutant exhibited high deglycosylation activity towards native glycoproteins. Dynamic simulations of the wild type and N-terminal H1 deletion mutant implied that Png1p-DeltaH1 is more flexible than wild type Png1p. The efficient deglycosylation of Png1p-DeltaH1 towards native and non-native glycoproteins offers a potential biotechnological application.
authors.affiliation: State Key Laboratory of Microbial Technology, Shandong University, Jinan, China.
authors.forename: Shengjun
authors.initials: S
authors.lastname: Wang
authors.forename: Fengxue
authors.initials: F
authors.lastname: Xin
authors.forename: Xiaoyue
authors.initials: X
authors.lastname: Liu
authors.forename: Yuxiao
authors.initials: Y
authors.lastname: Wang
authors.forename: Zhenyi
authors.initials: Z
authors.lastname: An
authors.forename: Qingsheng
authors.initials: Q
authors.lastname: Qi
authors.forename: Peng George
authors.initials: PG
authors.lastname: Wang
mesh_terms: D001709:Biotechnology; D004268:DNA-Binding Proteins / Q000378:metabolism; D006031:Glycosylation; D006863:Hydrogen-Ion Concentration; D007700:Kinetics; D008958:Models, Molecular; D043524:Peptide-N4-(N-acetyl-beta-glucosaminyl) Asparagine Amidase / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism*; D011485:Protein Binding; D017433:Protein Structure, Secondary; D012260:Ribonucleases / Q000378:metabolism; D012441:Saccharomyces cerevisiae / Q000201:enzymology; D029701:Saccharomyces cerevisiae Proteins / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism*; D017384:Sequence Deletion; D013329:Structure-Activity Relationship; D020349:Surface Plasmon Resonance
chemical_list: D004268:DNA-Binding Proteins; C490846:RAD23 protein, S cerevisiae; D029701:Saccharomyces cerevisiae Proteins; D012260:Ribonucleases; C029600:ribonuclease B; C468820:PNG1 protein, S cerevisiae; D043524:Peptide-N4-(N-acetyl-beta-glucosaminyl) Asparagine Amidase